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Cytological aspects of seasonal changes in the mesophyll chlorenchyma cells of Pinus Contorta dougl... 1977

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CYTOLOGICAL ASPECTS OF SEASONAL CHANGES IN THE MESOPHYLL CHLORENCHYMA CELLS OF PINUS CONTORTA Dougl. ex Loud ssp LATIFOLIA (Engelm, ex Wats) IN RELATION TO FROST HARDINESS by Roger M i c h a e l Shaneman B . S c , U n i v e r s i t y o f B r i t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFI L.LMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (the^Depiartmentv.ofo.iB^tariy,)';"--',^ r; We a c c e p t t h i s t h e s i s as ,conforming t o the r e q u i red s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA August 1977 Roger M i c h a e l Shaneman, 1977 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depa r tmen t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l no t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depa r tmen t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date ABSTRACT The n e e d l e c hlorenchyma o f P i n u s c o n t o r t a Dougl. ex Loud spp. Lat? - f o l i a (Engelm. ex Wats) was f i x e d on s i t e i n the s u b - a l p i n e f o r e s t s o f the s o u t h e r n i n t e r i o r p l a t e a u o f B r i t i s h C o l u m b i a , i n o r d e r t o c o n d u c t c o m p a r a t i v e o b s e r v a t i o n s by l i g h t and e l e c t r o n m i c r o s c o p y on t h e c h l o r e n - chymal c y t o l o g y i n summer and w i n t e r . M a t e r i a l f i x e d i n the growing s e a - son (summer) de m o n s t r a t e d a c h l o r e n c h y m a l u l t r a s t r u c t u r e s i m i l a r t o t h a t o f o t h e r mature h i g h e r p l a n t c h l o r e n c h y m a l t i s s u e s . M a t e r i a l f i x e d i n mid w i n t e r ( u s u a l l y a t below f r e e z i n g t e m p e r t u r e s ) r e v e a l e d e x t e n s i v e changes i n the s t r u c t u r a l and p o s i t i o n a l a s p e c t s o f most c y t o p l a s m i c com- po n e n t s . Some o f t h e most d r a m a t i c changes i n the chlorenchyma c y t o p l a s m i n the w i n t e r s t a t e a r e as f o l l o w s : c h l o r o p l a s t s became i r r e g u l a r l y shaped and clumped t o g e t h e r u s u a l l y i n c e r t a i n s p e c i f i c a r e a s o f the e e l 1; c l o s e x a s - s o c i a t i o n s form between the o u t e r membranes o f a d j a c e n t c h l o r o p l a s t s , t h e s e a r e a s may l e a d t o the a p p a r e n t f u s i o n o f some c h l o r o p l a s t s . The c y t o - plasm became m a s s i v e l y v a c u o l a t e d , p a r t i c u l a r l y i n a r e a s removed from t h e n u c l e u s o r the c h l o r o p l a s t clumps. The e x t e n t o f t h i s v a c u o l a t i o n can be deduced from the use o f the a d j e c t i v e "foamy" t o d e s c r i b e the extent'.and degree o f t h e s e v a c u o l a t e d c y t o p l a s m i c a r e a s . A s s o c i a t e d w i t h the c y t o - p l a s m i c w i n t e r v a c u o l e s were h i g h l y o s m i o p h i l i c b o d i e s . A d i s t i n c t s e a - s o n a l c y c l e o f c y t o p l a s m i c o i l r e s e r v e s was a l s o noted 1 , The r e a c t i o n o f the w i n t e r c h l o r e n c h y m a l c y t o p l a s m t o e x t e n t i v e m a n i p u l a t i o n i n f i x a t i v e o s m o t i c p o t e n t i a l was r e m a r k a b l y c o n s e r v a t i v e . T h i s o b s e r v a t i o n a l l o w s i n f e r e n c e s t o be made about the p e r m e a b i l i t y c h a r a c t e r i s t i c s o f w i n t e r c y t o p l a s m i c membranes. i i i In o r d e r t o v e r i f y the o b s e r v e d w i n t e r changes, c h e m i c a l f i x a t i o n s t u d i e s were supplemented by o b s e r v a t i o n s on m a t e r i a l c o l l e c t e d , t r a n s - p o r t e d , s t o r e d , s e c t i o n e d and o b s e r v e d by l i g h t m i c r o s c o p y i n the f r o - zen and unthawed, s t a i n e d o r f i x e d s t a t e . T h i s work was c o n d u c t e d w i t h s p e c i a l l y d e s i g n e d t r a n s p o r t d e v i c e s and a c r y o m i c r o t o m e and c r y o m i c r o s - cope. O b s e r v a t i o n s were a l s o c o n d u c t e d i n summer and w i n t e r on the c h l o - renchyma o f P i n u s a l b i c a u l i s Engelm and Tsuga m e r t e n s i a n a (Bong.) C o r r . c o l l e c t e d near t i m b e r l i n e a t the W h i s t l e r mountain s k i r e s o r t n i n e t y m i l e s n o r t h o f Vancouver. O b s e r v a t i o n s from the c r y o m i c r o s c o p y o f P i n u s c o n t o r t a n e e d l e s i n d i c a t e t h a t t h e s e a s o n a l changes a r e not a r t i f a c t s o f c h e m i c a l f i x a t i o n p r o c e d u r e s . O b s e r v a t i o n s on the n e e d l e s o f t h e two c o n i f e r s p e c i e s c o l l e c t e d a t W h i s t l e r mountain i n d i c a t e t h a t the d e t a i l - ed s e a s o n a l o b s e r v a t i o n s on P i n u s c o n t o r t a may a l s o a p p l y t o t h e n e e d l e c h l o r e n c h y m a o f o t h e r c o n i f e r s p e c i e s . A p o s s i b l e s p r i n g t r a n s i t i o n a l s t a g e between t h e w i n t e r and summer u l t r a s t r u c t u r a l appearance o f P i n u s c o n t o r t a chlorenchyma i s d i s c u s s e d . R e l a t e d e x p e r i m e n t s were c o n d u c t e d t o i n d i c a t e what e f f e c t s the n e e d l e age o f f i e l d t r e e s had on winter-summer c o m p a r i s o n s , and what e f f e c t . dro u g h t s t r e s s and a r t i f i c i a l h a r d e n i n g and f r e e z i n g had on c h l o r e n c h y m a l u l t r a s t r u c t u r e . The d r o u g h t and h a r d e n i n g - f r e e z i n g e x p e r i m e n t s were c o n - d u c t e d on l a b o r a t o r y grown t r e e s i n c o n t r o l l e d e n v i r o n m e n t chambers. No c l e a r r e l a t i o n s h i p between f r o s t h a r d i n e s s and drought s t r e s s was e s t a - b l i s h e d . E x p e r i m e n t s on the a r t i f i c i a l inducement o f f r o s t h a r d i n e s s i n s e e d l i n g t r e e s were not c o n s i d e r e d s u c c e s s f u l . i v TABLE OF CONTENTS PAGE LIST OF PLATES AND FIGURES v i \ LIST OF TABLES x i i ACKNOWLEDGEMENTS x i i i INTRODUCTION 1 MATERIALS AND METHODS I L i g h t and T r a n s m i s s i o n E l e c t r o n M i c r o s c o p y P r e p a r a t i o n P r o c e d u r e s . 11 II S c a n n i n g E l e c t r o n M i c r o s c o p y P r o c e d u r e s 16 I I I E x p e r i m e n t a l F i x a t i o n s and P r o c e d u r e s . 17 IV E x p e r i m e n t s on S e e d l i n g s R a i s e d i n the Growth Chamber.... 19 RESULTS I S t r u c t u r e o f Needle T i s s u e s and Chlorenchyma C e l l s 21 II The C y t o l o g y o f the Chlorenchyma i n the Summer C o n d i t i o n . 29 A L i g h t M i c r o s c o p y 29 B U l t r a s t r u c t u r a l Changes i n the Chlorenchyma R e l a t e d t o t h e A g i n g o f t h e Ne e d l e 31 C O b s e r v a t i o n s on S p e c i f i c O r g a n e l l e s : E l e c t r o n M i c r o s c o p y 35 D E x p e r i m e n t s on Summer N e e d l e s . 43 I I I Autumnal Changes i n the U l t r a s t r u c t u r e o f the ChLor.enchyma 45 IV The Chlorenchyma i n the W i n t e r C o n d i t i o n . . . 46 A L i g h t M i c r o s c o p y 46 B O b s e r v a t i o n s on the W i n t e r C o n d i t i o n : E l e c t r o n M i c r o s c o p y 49 V PAGE C W i n t e r M a t e r i a l F i x e d w i t h D i f f e r i n g F i x a t i v e S o l u t i o n s 58 D E x p e r i m e n t s w i t h W i n t e r Needles F r o z e n and Thawed Under F i e l d and L a b o r a t o r y C o n d i t i o n s 60 V V e r n a l Changes i n the U l t r a s t u r c t u r e o f the Chlorenchyma. 6k VI E x p e r i m e n t s on P i n e S e e d l i n g s i n C o n t r o l l e d E n v i r o n m e n t s . 68 A A r t i f i c i a l F r o s t H a r d e n i n g : U l t r a s t r u c t u r a l O b s e r v a t i o n s 68 B Summer C o n d i t i o n T r ees Exposed t o Drought: Ul t r a s t r u c t u r a l O b s e r v a t i o n s 70 DISCUSSION I D i s c u s s i o n o f T e c h n i c a l Problems and P r o c e d u r e s 72 II D i s c u s s i o n o f Seasonal Changes i n the C y t o p l a s m o f Needle Chlorenchyma 85 I I I D i s c u s s i o n o f P o s s i b l e F r e e z i n g Mechanisms i n the Needle Chlorenchyma 111 IV D i s c u s s i o n o f E x p e r i m e n t s i n C o n t r o l l e d Environment Chambers Mk SUMMARY 127 APPENDIX I " S p e c i f i x S t a i n . S o l u t i o n s 137 APPENDIX II C r y o s e c t i o n i n g and L i g h t M i c r o s c o p y o f F r o z e n T i s s u e 138 A C r y o s e c t i o n i n g 138 B Cryomi c r o s c o p y 140 PLATES AND EXPLANATIONS LEGEND 11,2 PLATES AND EXPLANATIONS ^hk BIBLIOGRAPHY 1 67 v i LIST OF PLATES AND FIGURES PLATE I F i g u r e 1 Needle C e l l s and T i s s u e s PAGE 1M> PLATE II The Shape and S e c t i o n a l I n t e r p r e t a t i o n s o f a G e n e r a l i z e d 145 P i n e M e s o p h y l l c e l l . F i g u r e 2 The shape o f the chlorenchyma c e l l : d r a w i n g o f th e t h r e e - d i m e n s i o n a l s u r f a c e o f a t y p i c a l p i n e c hlorenchyma c e l l . F i g u r e 3 Drawing o f the s e c t i o n a l appearance o f the c e l l c u t i n a p l a n e p a r a l l e l t o f a c e A. F i g u r e 4 Drawing o f t h e s e c t i o n a l appearance o f the c e l l c u t i n a p l a n e p a r a l l e l t o f a c e C. F i g u r e 5 Drawing o f an a l t e r n a t e s e c t i o n a l appearance o f the c e l l c u t i n a p l a n e p a r a l l e l t o f a c e C. F i g u r e 6 Drawing o f t h e s e c t i o n a l appearance o f the c e l l c u t i n a p l a n e p a r a l l e l t o f a c e B. PLATE M l S c a n n i n g E l e c t r o n M i c r o s c o p y o f M e s o p h y l l Chlorenchyma. F i g u r e 7 T r a n s v e r s e v i e w o f chlorenchyma near one o f the n e e d l e c o r n e r s . F i g u r e 8 Two c e l l s showing f a c e s A and C. F i g u r e 9 D e t a i l o f t r a b e c u l a r wal1 i n f o l d i n g s . 146 PLATE IV L i g h t M i c r o s c o p y - Summer Chlorenchyma. 147 F i g u r e 10 C e l l photographed i n the f r o z e n c o n d i t i o n . F i g u r e 11 A f r o z e n c e l l t h a t has been a l l o w e d t o thaw. F i g u r e 12 C e l l s t a i n e d f o r t a n n i n s . F i g u r e 13 C e l l s t a i n e d w i t h Sudan B l a c k B. F i g u r e 14 High m a g n i f i c a t i o n o f a c e l l s t a i n e d w i t h Sucan B l a c k B. V I I PAGE PLATE V The Summer - W i n t e r L i g h t M i c r o s c o p y : C y t o l o g y o f Tusga 148 mertens i ana. F i g u r e 15 Low m a g n i f i c a t i o n o f the ch l o r e n c h y m a . F i g u r e 16 High m a g n i f i c a t i o n o f p e r i p h e r a l c y t o p l a s m show- ing c h l o r o p l a s t a l i g n m e n t and s t r u c t u r e . F i g u r e 17 L.M. o f w i n t e r T. m e r t e n s i a n a chlorenchyma c y - to p l a s m . F i g u r e 18 L.M. o f w i n t e r T. m e r t e n s i a n a chlorenchyma c y o - t o p l a s m . PLATE VI E l e c t r o n M i c r o s c o p y o f Summer Chlorenchyma. F i g u r e 19 P a r t s o f f o u r summer c e l l s . F i g u r e 20 C h l o r o p l a s t and c y t o p l a s m f r o m a v e r y young (2 mm) n e e d l e . F i g u r e 21 C h l o r o p l a s t and m i t o c h o n d r i a from a 2 y e a r o l d n e e d l e . 149 PLATE VII E l e c t r o n M i c r o s c o p y o f Summer Chlorenchyma: C o n t i n u e d . F i g u r e 22 The c y t o p l a s m near t h e n u c l e u s . F i g u r e 23 O s m i o p h i l i c b o d i e s . F i g u r e 24 O s m i o p h i l i c body and membranous f o r m a t i o n , F i g u r e 25 A d i c t y o s o m e and c y t o p l a s m i c v e s i c u l a t i o n , 150 PLATE V I M E l e c t r o n M i c r o s c o p y o f Summer Chlorenchyma: C o n t i n u e d . F i g u r e 26 D i c t y o s o m a l a c t i v i t y . F i g u r e 27 D i c t y o s o m a l a c t i v i t y n e ar t h e c e l l w a l l . F i g u r e 28 M u l t i - l o b e d m i t o c h o n d r i a . F i g u r e 29 O i l body a s s o c i a t e d w i t h m i t o c h o n d r i a . 151 PLATE IX E l e c t r o n M i c r o s c o p y o f Summer Chlorenchyma: C o n t i n u e d . F i g u r e 30 E l e c t r o n m i c r o g r a p h o f p a r t o f a n u c l e u s . F i g u r e 31 E l e c t r o n m i c r o g r a p h o f the n u c l e o l u s . F i g u r e 32 L i g h t p h o t o m i c r o g r a p h o f the n u c l e a r a r e a . PLATE X L i g h t M i c r o s c o p y o f W i n t e r T i s s u e . F i g u r e 33 A f r o z e n c e l l . F i g u r e 3k T a n n i n s p e c i f i c s t a i n i n g . F i g u r e 35 1 Aim p l a s t i c s e c t i o n showing c y t o p l a s m i c d e t a i Is F i g u r e 36 Sudan B l a c k B s t a i n i n g . PLATE XI t o XVII show E.M. s t u d i e s o f the w i n t e r c h l o r e n c h y m a : PLATE XI E l e c t r o n M i c r o s c o p y o f W i n t e r Chlorenchyma. F i g u r e 37 T r a n s v e r s e s e c t i o n o f s e v e r a l w i n t e r c e l l s . PLATE XII E l e c t r o n M i c r o s c o p y o f W i n t e r Chlorenchyma: C o n t i n u e d . F i g u r e 38 A narrow l o n g i t u d i n a l s e c t i o n : o f a w i n t e r c e l l . F i g u r e 39 A wide l o n g i t u d i n a l s e c t i o n o f a w i n t e r c e l l . PLATE X I I I E l e c t r o n M i c r o s c o p y o f W i n t e r Chlorenchyma: c o n t i n u e d . F i g u r e kQ P a r t o f a t r a n s e c t i o n o f a w i n t e r c e l l above the t a n n i n v a c u o l e . F i g u r e kl W i n t e r c y t o p l a s m i c v a c u o l i z a t i o n between c h l o - r o p l a s t clumps. F i g u r e kl D e t a i l s o f a c h l o r o p l a s t clump. PLATE XIV E l e c t r o n M i c r o s c o p y o f W i n t e r Chlorenchyma: C o n t i n u e d . F i g u r e k3 D e t a i l s o f an o b l i q u e s e c t i o n o f the f u s e d c h l o - r o p l a s t o u t e r membranes and o f a " d o u b l e " c h l o - r o p l a s t . I x F i g u r e 44 C h l o r o p l a s t o f an o l d e r n e e d l e . F i g u r e 45 D e t a i l s o f the r e g i o n s o f adherance between the o u t e r c h l o r o p l a s t membranes. F i g u r e 46 High m a g n i f i c a t i o n o f i n s e r t ' i n f i g u r e 45. F i g u r e 47 D e t a i l s o f w i n t e r c h l o r o p l a s t . PAGE 157 PLATE XV E l e c t r o n M i c r o s c o p y o f W i n t e r Chlorenchyma: c o n t i n u e d . F i g u r e 48 An a r e a i n two c e l l s showing d e t a i l s o f c y t o - p l a s m i c v a c u o l e s and o s m i o p h i l i c b o d i e s . 158 PLATE XVI E l e c t r o n M i c r o s c o p y o f W i n t e r Chlorenchyma: C o n t i n u e d . 159 F i g u r e 49 D e t a i l s o f o s m i o p h i l i c b o d i e s and c y t o p l a s m i c v a c u o l e s . - F i g u r e 50 D e t a i l s o f o s m i o p h i l i c b o d i e s and c y t o p l a s m i c v a c u o l e s . F i g u r e 51 D e t a i l s o f o s m i o p h i l i c b o d i e s and c y t o p l a s m i c v a c u o l a r p o s i t i o n r e l a t i v e t o c h l o r o p l a s t clumps and the c e l l w a l l . F i g u r e 52 D e t a i l s o f o s m i o p h i l i c b o d i e s and v a c u o l a r po- s i t i o n r e l a t i v e t o the c e l l w a l l i n a s e c t i o n t a n g e n t i a l t o the c e l l . PLATE XVII E l e c t r o n M i c r o s c o p y o f W i n t e r Chlorenchyma: C o n t i n u e d . 160 F i gure 53 M i t o c h o n d r i a l s u b s t r u c t u r e . F i g u r e 54 D i c t y o s o m a l s t r u c t u r e . F i g u r e 55 L.M. p l a s t i c s e c t i o n o f the w i n t e r n u c l e u s . F i gure 56 W i n t e r n u c l e a r e n v e l o p e and n u c l e o l a r s t r u c t u r e F i gure 57 S t r u c t u r e o f the w i n t e r n u c l e u s . PLATE X V I I I E l e c t r o n M i c r o s c o p y o f the W i n t e r Chlorenchyma; C o n t i n u e d . 161 and W i n t e r V a r i a t i o n s i n P r e p a r a t i v e P r o c e d u r e s . F i g u r e 58 L a r g e w i n t e r o i l body and a s s o c i a t e d m i t o c h o n - d r i a . X F i g u r e 59 D i f f e r i n g w i n t e r f i x a t i o n s i n a t l e a s t f o u r c e l l s . F i g u r e 60 W i n t e r f i x a t i o n i n sea w a t e r ( e l e c t r o l y t e ) f i x a t i v e . F i g u r e 61 W i n t e r f i x a t i o n i n sea w a t e r ( e l e c t r o l y t e ) f i xa t i v e . F i g u r e 62 W i n t e r u l t r a s t r u c t u r e o f a needle' exposed t o -196°C. PAGE -161 PLATE XIX E l e c t r o n M i c r o s c o p y o f t h e A p r i l 27th 1973 F i x a t i o n . 162 F i g u r e 63 S t r u c t u r a l and p o s i t i o n a l f e a t u r e s o f c h l o r o - p l a s t . F i g u r e 64 High m a g n i f i c a t i o n o f f i g u r e 63 i n s e r t o f the r e g i o n between c h l o r o p l a s t s . F i g u r e 65 P l a s t o g l o b u l i c l u s t e r s i n i r r e g u l a r c h l o r o - p l a s t s . F i g u r e 66 The n u c l e a r r e g i o n o f the c y t o p l a s m . F i g u r e 67 View o f an a r e a o f c y t o p l a s m . PLATE XX E l e c t r o n M i c r o s c o p y o f Chlorenchyma: S p r i n g F i x a t i o n and 163 M a t e r i a l P r e p a r e d by V a r i o u s P r o c e d u r e s . F i g u r e 68 D e t a i l s o f an a g g r e g a t e o f o s m i o p h i l i c m a t e r - i a l and an o i l body i n the s p r i n g c y t o p l a s m . F i g u r e 69 The n u c l e a r r e g i o n o f a 3 y e a r o l d summer e e l 1 . F i g u r e 70 P a r t o f a 5 y e a r o l d summer c e l l f i x e d w i t h the a c r o l e i n f i x a t i v e . F i g u r e 71 C h l o r o p l a s t d e t a i l s from a t h r e e y e a r o l d a c r o - l e i n f i x e d e e l 1. F i g u r e 72 C y t o p l a s m i c (?) d e t a i l s o f a summer c e l l expo- sed t o -18 °C. PLATE XXI S t u d i e s on P i n u s c o n t o r t a S e e d l i n g s S u b j e c t e d t o V a r i o u s T r eatments i n C o n t r o l l e d Environment Chambers. F i g u r e 73 D e t a i l s o f t a n n i n , c y t o p l a s m i c v a c u o l e s and o s m i o p h i l i c b o d i e s i n a r t i f i c i a l l y hardened s e e d l i ngs. 164 x i F i g u r e 7k Two c h l o r o p l a s t s p o s s i b l y f u s e d from a r t i - f i e a l l y hardened s e e d l i n g s . F i g u r e 75 S e e d l i n g s compared a f t e r f r o s t e x p o s u r e , hard- ened s e e d l i n g s and c o n t r o l s e e d l i n g . F i g u r e 76 C h l o r o p l a s t d e t a i l s from drought exposed seed- 1 i n g . F i g u r e 77 P e r i n u c l e a r c y t o p l a s m i c d e t a i l s from d r o u g h t exposed s e e d l i n g . PAGE 164 PLATE XXII A p p a r a t u s f o r Frozen S e c t i o n O b s e r v a t i o n . F i g u r e 78 -Cryomicrotbme equipment. F i g u r e 79 C r y o m i c r o s c o p e equipment. 165 PLATE XXI I I F i g u r e 80 Diagramof t h e c r y o m i c r o s c o p e s t a g e , 166 x i i PAGE LIST OF TABLES TABLE I F i x a t i o n Dates and Times 13 ACKNOWLEDGEMENTS i w i s h t o thank Dr. Thana B i s a l p u t r a f o r s u g g e s t i n g t h i s s t u d y and f o r h i s p a t i e n c e , a d v i c e , s u p p o r t and g u i d a n c e t h r o u g h t h e l e n g t h o f t h i s s t u d y . I w i s h t o thank Dr. D. M u l l i c k f o r g i v i n g me t h e i n i t i a l i n t e r e s t i n c r y o b i o l o g y . I would a l s o l i k e t o thank my w i f e V i v i a n a , Mr. D. Walker and Mr. R. J e f f e r d whose a s s i s t a n c e and encouragement a l l o w e d me t o c o m p l e t e t h i s work. F i n a l l y , I am i n d e b t e d t o M a n a l t a Coal L t d . f o r a l l o w i n g me t h e l e a v e o f absence n e c e s s a r y t o co m p l e t e t h i s work. 1 INTRODUCTION A l l n o n - t r o p i c a l c o n t i n e n t a l l i f e on t h i s p l a n e t must make some deg- ree o f a d a p t i o n t o a t l e a s t t h e s e a s o n a l p r e s e n c e o f low t e m p e r a t u r e s w h i c h would cause t h e c e l l u l a r w a t e r o f m e t a b o l i e a l l y a c t i v e c e l l s t o f r e e z e . In the m a j o r i t y o f h i g h e r o r g a n i s m s i n a grow i n g and m e t a b o l i c a l l y a c t i v e s t a t e , t h e f r e e z i n g o f wa t e r w i t h i n the c y t o p l a s m i s f a t a l (Burke e t a l . , 1976 and Tumanov, 1967)- Two broad c l a s s e s o f a d a p t i v e mechanisms appear t o have e v o l v e d t o p r e v e n t t h e f r e e z i n g o f c e l l w a t e r . The f i r s t mechanism i s termed the p r i n c i p l e o f f r o s t a v o i d a n c e . In t h i s p r i n c i p l e , t h e f r e e z - in g o f c e l l u l a r w a t e r i s p r e v e n t e d by a number o f means, some o f w h i c h a r e : the m a i ntenance o f i n t e r n a l t i s s u e t e m p e r a t u r e s a t a h i g h enough l e v e l t o a v o i d f r e e z i n g (as i n t h e case o f homeothermic a n i m a l s ) ; t h e b u i l d up o f c r y o p r o t e c t i v e s u b s t a n c e s t o h i g h enough l e v e l s w i t h i n c e l l t i s s u e s (as in t h e c a s e o f some i n s e c t l a r v a e and t r e e buds) t o p r e v e n t i c e f o r m a t i o n ; by p o s s e s s i n g a s u r v i v a l phase i n a l i f e c y c l e c h a r a c t e r i z e d by t h e r e - d u c t i o n o f wa t e r c o n t e n t i n t h e c e l l s t o l e v e l low enough t o p r e v e n t i n - t r a c e l l u l a r i c e c r y s t a l i z a t i o n (such as t h e seeds o r s p o r e s o f annual p l a n t s ) ; o r by c r e a t i n g c o n d i t i o n s w h i c h a l l o w c e l l w a t e r t o s u p e r - c o o l . These f r o s t a v o i d a n c e mechanisms have been d e s c r i b e d i n d e t a i l by L e v i t t (1972). Woody h i g h e r p l a n t s m a i n t a i n t h e i r c o m p e t i t i v e advantage { i n p a r t ) by m a i n t a i n i n g and b u i l d i n g upon l a r g e l i v i n g t i s s u e masses from y e a r t o y e a r . In t e m p e r a t e , h i g h l a t i t u d e and a l t i t u d e zone s p e c i e s , t h i s r e q u i r e s t h e s u r v i v a l o f a s i g n i f i c a n t p o r t i o n o f t h e l i v i n g c e l l mass t h r o u g h t h e f r o s t 2 season. Such organisms are examples of the second major type of adapt i ve mechanism t o f r o s t , that of f r o s t t o l e r a n c e . F ros t t o l e r a n t p l a n t s enable v i t a l c e l l s to s u r v i v e the w i n t e r season by p repar ing c e l l s s t r u c t u r a l l y and c h e m i c a l l y in such a manner that they are a b l e to t o l e r a t e i ce forma- t i o n in t h e i r t i s s u e s . The v i t a l ee l Is are! a b l e , to survive-'• f r o s t - : r e l a t e d s t r e s s by c o n t r o l l e d e x t r a c y t o p l a s m i c i ce fo rmat ion w i t h f r e e c e l l water being re leased from dehydrat ing and p lasmolyz ing c e l l s . There i s a general agreement in recent l i t e r a t u r e w i t h regard to phy- s i c a l s t r e s s f a c t o r s r e l a t e d to the fo rmat ion of i ce in l i v i n g t i s s u e s which must be t o l e r a t e d by f r o s t hardy p l a n t s (see reviews by Mazur, 1970t and W e i s e r , 1970). These f a c t o r s a r e : 1) The c o n c e n t r a t i o n and in some cases p o s s i b l y the p r e c i p i t a t i o n or g e l l i n g of c e l l s o l u t e s due to a g r e a t l y reduced so l vent v o l - ume. 2) Changes in i o n i c strength, , pH, and osmot ic e q u i l i b r i u m in the c e l l i n t e r i o r and e x t e r i o r . 3) The c o l l a p s e and severe p l a s m o l y s i s of the c e l l as c e l l water moves to s p e c i f i c i ce n u c l e a t i o n s i t e s . h) The macromolecular c o r r e l a t i o n of the above th ree p o i n t s would r e s u l t in the loss of membrane semipermeabi1ity and the denatu r - ing of c e l l p r o t e i n s whose s t r u c t u r e was dependent on c e l l water and/or the normal e q u i l i b r i u m of c e l l s o l u t e s . The above s t r e s s f a c t o r s are i n t e r n a l s t r e s s e s to the c e l l caused by the f r e e z i n g of c e l l u l a r water o u t s i d e the plasma membrane. In a t tempt ing 3 t o u n d e r s t a n d t h e f r o s t h a r d i n e s s p r o c e s s e s o f woody p l a n t s , e x p l a n a t i o n s o f how t h e c e l l t o l e r a t e s and a m e l i o r a t e s t h e above s t r e s s e s a r e r e q u i r e d . The a b i l i t y t o s u r v i v e f r o s t r e l a t e d s t r e s s e s i s a s e a s o n a l l y a c q u i r - ed p r o p e r t y i n woody p l a n t s . T i s s u e s i n e x t r e m e l y hardy p l a n t s cannot t o - l e r a t e even a few degrees o f f r o s t d u r i n g the a c t i v e growth phase o f t h e p l a n t , but can t o l e r a t e t e m p e r a t u r e s as low as -196°C when t h e y a r e p r e - pared f o r s e a s o n a l f r e e z i n g ( S a k a i , 1966 and 1973; and K r a s a v t s e v , 1973). The g e n e r a l f a c t o r s i n v o l v e d i n the p r o c e s s o f d e v e l o p i n g maximum h a r d i n e s s l e v e l s i s f a i r l y w e l l u n d e r s t o o d and t h i s p r o c e s s has been d i - v i d e d i n t o s e v e r a l phases by Tumanov (1967) and Weiser (1970). These phases can be g e n e r a l i z e d i n t o two main s t a g e s . The f i r s t s t a g e i n v o l v e s t h e d e t e c t i o n o f t h e impending c o l d season by s h o r t e r p h o t o p e r i o d s and c o o l e r d a i l y minimum t e m p e r a t u r e s a n d ^ p o s s i b l y o t h e r f a c t o r s ; t h e c e s s a - t i o n o f t i s s u e g r o w t h , c o u p l e d w i t h an i n c r e a s e i n c e r t a i n t y p e s o f b i o - c h e m i c a l a c t i v i t y . The c e s s a t i o n o f t i s s u e growth i s c o n s i d e r e d ( p a r t i c - u l a r l y by R u s s i a n a u t h o r s (Tumanov e_t_ a_l_. , 1973)) t o be a s p e c i a l i z e d , f r o s t h a r d i n e s s dependent form o f p l a n t dormancy. The d e t e c t i o n mechanism i s t r a n s l a t e d i n t o a complete s u c c e s s i o n o f growth and d i f f e r e n t i a t i o n i n t h e p l a n t t i s s u e . T h i s t r a n s l a t i o n may be by a hormonal f a c t o r ( I r v i n g and Lanphear, 1967). A l t h o u g h s t a g e one i s a dormancy s t a g e i n terms o f growth c e s s a t i o n , k i . e . , c e l l d i v i s i o n and d i f f e r e n t i a t i o n i n t o c e l l s o f s p e c i a l i z e d f u n c t i o n ; c e l l s d e v e l o p i n g f r o s t h a r d i n e s s may become more a c t i v e m e t a b o l i c a l l y than t h e y were d u r i n g p a r t s o f th e g r o w i n g season ( S i m i n o v i t c h et_ aj_. , 1968). T h i s a c t i v i t y i s i n i t i a t e d by a marked i n c r e a s e i n r i b o s o m a l RNA (Gusta and W e i s e r , 1972) and c e r t a i n p r o t e i n s ( C l e m e n t s , 1938; S i m i n o v i t c h e t a l . , 1968; Pomeroy e t a l . , 1970;and Gusta and W e i s e r , 1972). Other c e l l c ons- t i t u e n t s a l s o i n c r e a s e , some o f w h i c h a r e : s o l u b l e s u g a r s ( P a r k e r , 1956; Heber, 1959 and Pomeroy e_t aj_. , 1970), l i p i d s , p a r t i c u l a r l y p h o s p h o l i p i d s ( S i m i n o v i t c h et_ a_l_. , 1975) and u n s a t u r a t e d f a t t y a c i d s (Wi 11 emot e t a l . , 1977)- In c o n t r a s t , t h e s t a r c h c o n t e n t o f c o n i f e r n e e d l e s a p p ears t o d e c l i n e w i t h t h e approach o f maximum f r o s t h a r d i n e s s ( C l e m e n t s , 1938 ;.: P a r k e r , 1956 and L i t t l e , 1970). The b i o c h e m i c a l changes appear t o be h i g h l y v a r i a b l e i n d i f f e r e n t p l a n t organs and between d i f f e r e n t p l a n t s p e c i e s o r even v a r i e t i e s ( S m i t h , 1968). D e s p i t e t h e e x t e n t o f c e l l u l a r a c t i v i t y , t h e h a r d i n e s s o f p l a n t s i n s t a g e one i s s t i l l r e l a t i v e l y low (W e i s e r , 1970). Stage one o f f r o s t h a r d i n e s s development i s o f i n d e t e r m i n a t e l e n g t h . But because c e l l u l a r b i o c h e m i c a l t r a n s f o r m a t i o n s a r e i n v o l v e d , t h e r e i s p r o b a b l y a minimum ti m e p e r i o d i n v o l v e d . In t h e n a t u r a l s t a t e , t h e b i o - c h e m i c a l t r a n s f o r m a t i o n s o f s t a g e one seem t o o c c u r o v e r s e v e r a l weeks ( S i m i n o v i t c h et_ a j _ . ; 1968 and W e i s e r , 1970). Stage two. o f f r o s t h a r d i n e s s development o c c u r s o v e r a r e l a t i v e l y s h o r t t i m e ( s e v e r a l days o r l e s s , S a k a i , 1966), and does not r e q u i r e o r e l i c i t any s u b s t a n t i a l m e t a b o l i c r e s p o n s e from the l i v i n g c e l l . I n s t e a d , 5 s t a g e two a p p ears t o i n v o l v e a m e c h a n i c a l a c c o m m o d a t i o n o f t h e c y t o p l a s m t o i n c r e a s i n g amounts o f c e l l u l a r d e h y d r a t i o n due t o e x t e r n a l i c e f o r m a t - i o n . Stage two r e q u i r e s r e d u c t i o n o f t i s s u e t e m p e r a t u r e s by i n c r e m e n t a l s t e p s below f r e e z i n g w i t h p r e c i s e l y d e f i n a b l e t e m p e r a t u r e d r o p s b e f o r e maximum h a r d i n e s s l e v e l s a r e o b t a i n e d . Thus t h i s s t a g e r e q u i r e s expo- s u r e t o moderate f r e e z i n g t e m p e r a t u r e s t o e n a b l e a p o t e n t i a l l y hardy c e l l t o s u r v i v e e x t r e m e l y low t e m p e r a t u r e s . T h i s p r o c e s s has been e x t e n s i v e l y s t u d i e d by S a k a i (1966 and 1973) and K r a s a v t s e v (1973). As an example o f t h i s s t a g e o f f r o s t a c c l i m a t i o n , S a k a i d e m o n s t r a t e d t h e n e c e s s i t y o f ex- p o s i n g a t w i g ( a f t e r the c o m p l e t i o n o f s t a g e one ) t o s e v e r a l hours a t -30°C b e f o r e t h e t w i g was a b l e t o t o l e r a t e -196°C ( S a k a i , 1973). W h i l e s t a g e one i s an a c t i v e m e t a b o l i c s t a g e , s t a g e two a p p ears t o i n v o l v e p h y s i c a l changes i n t h a t t h e p r o g r e s s i v e e x p o s u r e t o lower temp- e r a t u r e s may i n v o l v e p h y s i c a l d e f o r m a t i o n s and r e a rrangements o f c y t o p l a s m - i c components i n such a manner as t o a l l o w i c e t o l e r a n c e ( W e i s e r , 1970). W e i s e r p o i n t s o u t t h a t some enzymic a c t i v i t y and m e t a b o l i c c o n t r o l may s t i l l be o p e r a t i n g d u r i n g s t a g e two, i n d i c a t i n g t h a t s t a g e s one and two a r e not c o m p l e t e l y s e p a r a b l e i n t i m e . The c y t o l o g i c a l m a n i f e s t a t i o n o f s t a g e two i s t h e p l a s m o l y s i s o f t h e c e l l caused by d e h y d r a t i o n as w a t e r i s l o s t t o the e x t r a c e l l u l a r i c e nu- c l e a t i o n s i t e s (see S c a r t h and L e v i t t (1937) f© r a- r e v i e w o f c r y o m i c r o s - c o p i c s t u d i e s o f f r o z e n c e l l s ) . The R u s s i a n a u t h o r s b e l i e v e t h e i c e plasm- o l y s i s i s so e x t e n s i v e t h a t i t caused plasmodesmatal c y t o p l a s m i c s t r a n d s t o break and w i t h d r a w from t h e c e l l w a l l (Genkel e t a l . , 1971). 6 The e x a c t l o c a t i o n o f i c e i s a m a t t e r o f c o n t r o v e r s y and appears t o o c c u r i n t h e e x t r a c e l l u l a r spaces i f p r e s e n t , o r i n t h e i n t r a m u r a l space around t h e p l a s m o l y z e d p r o t o p l a s t ( S c a r t h and L e v i t t , 1937)- T h e r e i s gen- e r a l agreement t h a t i c e f o r m a t i o n w i t h i n t h e c y t o p l a s m i s f a t a l i n f r o s t hardy c e l l s (Tumanov, 1967; W e i s e r , 1970; S a k a i , 1973 and Burke e t a l . , 1976). But i t i s d e b a t a b l e whether i c e f o r m a t i o n can o c c u r i n t h e main v a c u o l e w i t h o u t c a u s i n g s e r i o u s damage ( P a r k e r , 1963). U l t r a s t r u c t u r a l l o c a l i z a t i o n o f i c e has been a t t e m p t e d but i t has been c o n f i n e d t o t i s s u e c o o l e d a t u n n a t u r a l l y h i g h c o o l i n g r a t e s ( S a k a i and O t s u k a , 1967), o r i n an i m a l t i s s u e w i t h no n a t u r a l f r o s t r e s i s t a n c e mechanisms, and a g a i n a t u n n a t u r a l l y h i g h r a t e s o f c o o l i n g (Rapatz e t a l . , 1963 and N e i , 1976). I t has been d e t e r m i n e d by e x t e n s i v e c o l o r i m e t r y s t u d i e s t h a t i n hardy p l a n t s a t below f r e e z i n g t e m p e r a t u r e s , an e q u i l i b r i u m i s m a i n t a i n e d between c y t o p l a s m i c f r e e w a t e r and e x t r a c e l l u l a r i c e n u c l e a t i o n s i t e s ( W e i s e r , 1970). T h i s e q u i l i b r i u m would s h i f t i n d i r e c t p r o p o r t i o n t o changes i n tem p e r a t - u r e ; i . e . as t h e t e m p e r a t u r e d e c r e a s e s , f r e e w a t e r l e a v e s t h e p r o t o p l a s t and m i g r a t e s t o t h e i e e n u c l e a t i o n s i t e s ; and as t h e t e m p e r a t u r e r i s e s , t h e i c e m e l t s and t h e p r o t o p l a s t b e g i n s t o r e h y d r a t e . There a r e two i m p o r t a n t p h y s i c a l p r o p e r t i e s o f a hardy p l a n t c e l l t h a t r e l a t e s t o t h i s e q u i l i b r i u m between t h e i c e phase and t h e c y t o p l a s m i c f r e e w a t e r phase. F i r s t l y , t he hardy p r o t o p l a s t must r e l e a s e w a t e r as i t i s thermodynami- c a l l y r e q u i r e d t o p r e v e n t i n t e r c e l l u l a r f r e e z i n g ( W e i s e r , 1970). T h i s i m p l i e s t h a t r e s i s t a n t c e l l s a r e permeable t o wat e r ( S c a r t h and L e v i t t , 7 1937 and McKe n z i e , 1971*). S e c o n d l y , t h e w a t e r must be r e l e a s e d s l o w l y so t h a t i c e f o r m a t i o n i s not a sudden e x p l o s i v e o c c u r r e n c e !.bu't a s l o w and c o n t r o l l e d e v e n t . T h i s a ppears t o be t h e r e s u l t o f an i n c r e a s e i n e r y o p r o t e c t i v e s u b s t a n c e s , such as s u g a r s and h y d r o p h i l i c polymers t h a t w i l l r e l e a s e f r e e w a t e r o n l y g r a d - u a l l y a t p r o g r e s s i v e l y lower t e m p e r a t u r e s ( W e i s e r , 1970). The p h y s i c a l s t a t e o f some o f t h e s e h y d r o p h i l i c polymers may be i n t h e g e l form ( P a r k - e r , 1958 and Tumanov, 19^7)- C y t o l o g i c a l s t u d i e s on f r o s t r e s i s t a n t c e l l s i n t h e l i t e r a t u r e a r e o f t e n c o n f u s i n g and c o n t r a d i c t o r y when compared w i t h each o t h e r due t o th e l a r g e v a r i e t y o f org a n i s m s s t u d i e d (which may p o s s e s s d i f f e r i n g f r o s t r e s i s t a n t mechanisms), and t h e r e i s a l s o a f r e q u e n t f a i l u r e i n t h e l i t e r - a t u r e t o c o r r e l a t e c y t o l o g i c a l i n t e r p r e t a t i o n s w i t h t h e degree o f f r o s t h a r d i n e s s i n the ob s e r v e d c e l l s , o r w i t h t h e e x t e r n a l e n v i r o n m e n t a l temp- e r a t u r e s t o w h i c h t h e y have been exposed. One example o f c y t o l o g i c a l d i f f e r e n c e s between d i f f e r e n t s p e c i e s i n - v o l v e s t h e tendency f o r c h l o r o p l a s t s t o s w e l l and d i s p e r s e randomly w i t h i n the e n t i r e p r o t o p l a s t i n w i n t e r wheat (Chein andWu, 1965) ,whereas i n P i n u s s p e c i e s , they tend t o clump t o g e t h e r i n s p e c i f i c a r e a s o f t h e c y t o p l a s m ( H o l z e r , 1958). S i m i l a r l y , t h e main c e l l v a c u o l e becomes s u b d i v i d e d i n t o nu- merous s m a l l v a c u o l e s i n ca m b i a l and se c o n d a r y woody t i s s u e o f s e v e r a l p l a n t s ( S i m i n o v i t c h e_t aj_. , 1968; Robards et^ aJL , 1969; M i a , 1970 and 1972; Murman- i s , 1970 and I t o h , 1971). Yet i n c o n i f e r n e e d l e s , t h e main v a c u o l e remains i n t a c t t h r o u g h o u t t h e w i n t e r (Lewis and T u t t l e , 1923; H o l z e r , 1958, P a r k e r 8 and P h i l p o t t , 1961 and 1963; H a r r i s 1971. and Chabot and Chabot, 1975). One b a s i c c y t o l o g i c a l f e a t u r e w h i c h appears t o be a p p l i c a b l e t o most r e c e n t c y t o l o g i c a l work i s t h e co n c e p t o f c y t o p l a s m i c a u g m e n t a t i o n w h i c h was d i s c u s s e d by S c a r t h and L e v i t t (1937), but r e f i n e d and p l a c e d i n t o a more modern c o n t e x t by S i m i n o v i t c h et_ aj_. (1968). C y t o p l a s m i c augmentat- ion r e f e r s t o t h e i n c r e a s e d volume and d e n s i t y o f t h e c y t o p l a s m ( a t t h e l i g h t m i c r o s c o p y l e v e l ) o f many f r o s t hardy c e l l s i n w i n t e r . S i m i n o v i t c h et_ aj_. (1968) s u g g e s t s t h a t t h e o b s e r v e d i n c r e a s e s o f c e r t a i n b i o c h e m i c a l compounds i n s t a g e one o f f r o s t h a r d i n e s s development may acc o u n t f o r t h i s o b s e r v a t i o n . Examples o f the p r o l i f e r a t i o n o f b i o c h e m i c a l components o f c y t o p l a s m i c s t r u c t u r e s would be the v e r y s u b s t a n t i a l s t a g e one i n c r e a s e o f membrane p h o s p h o l i p i d s ( S i m i n o v i t c h e t a l . , 1975)- An h i s t o r i c a l c y t o l o g i c a l d i s p u t e t h a t has o n l y r e c e n t l y been s e t t l e d was c o n c e r n e d w i t h t h e f a t e o f t h e c o n i f e r c h l o r o p l a s t s . H a b e r l a n d t (1876) n o t i c e d a c l u m p i n g o f c o n i f e r c h l o r o p l a s t s a f t e r f r o s t e x p o s u r e . H o l z e r (1958) c o n f i r m e d t h e s e o b s e r v a t i o n i n s t u d i e s conducted on P i n u s s p e c i e s . In c o n t r a s t , Lewis and T u t t l e (1923) and Zacharawa (1929) proposed t h a t t h e c h l o r o p l a s t s were d e s t r o y e d d u r i n g t h e w i n t e r i n c o n i f e r s . In P i c e a g l a u c a , Lewis and t u t t l e (1923) o b s e r v e d t h e c l u m p i n g f o l l o w e d by d i s i n - t e g r a t i o n o f c h l o r o p l a s t s w i t h p r o g r e s s i v e l y s e v e r e f r o s t s . T h i s i d e a o f w i n t e r c h l o r o p l a s t d e s t r u c t i o n was w i d e l y c i t e d i n the l i t e r a t u r e , p a r - t i c u l a r l y i n r e l a t i o n t o t h e c h l o r o p l a s t s i n t h e n e e d l e s o f e v e r g r e e n con- i f e r s . Some papers i n s u p p o r t o f t h e c o n c e p t have appeared q u i t e r e c e n t - l y ( G e r h o l d , 1959; and P e r r y and B a l d w i n , 1966). 9 A s u b s t a n t i a l body o f l i t e r a t u r e has been w r i t t e n on f r o s t r e s i s t a n c e i n p l a n t s . A l l e n and Herman (1970 e s t i m a t e d t h a t by 1971, more than 4,000 papers on t h e s u b j e c t had been w r i t t e n . Review papers on t h e s u b j e c t i n - c l u d e Maxinov (1929); S c a r t h and L e v i t t (1937); Clements (1938 ); L e v i t t (1941); P a r k e r (1963); Tumanov (1967); O l i e n (1967); Mazur (1970); A l l e n and Hermann (1971); Meryman. (1971) , L i t v a n (1972); Tumanov e_t a_j_. , (1973) and Burke e_t a_]_. , (1976). Symposia and books on c r y o b i o l o g y w h i c h c o n t a i n - ed s e c t i o n s d i r e c t l y r e l a t e d t o p l a n t f r o s t h a r d i n e s s i n c l u d e works e d i t e d by: L e v i t t (1956), Meryman (1966) and T r o s h i n (1967). L e v i t t (1972) has atte m p t e d t o s t a n d a r d i z e n o m e n c l a t u r e and d e f i n e t h e major parameters i n - v o l v e d i n p l a n t e n v i r o n m e n t a l s t r e s s e s , e s p e c i a l l y t h o s e r e l a t e d t o f r o s t h a r d i n e s s and/or s e n s i t i v i t y . The work i s an e x c e l l e n t and i m p o r t a n t r e - f e r e n c e on t h e t o p i c o f p l a n t h a r d i n e s s . The p r e s e n t s t u d y has dem o n s t r a t e d t h a t a fundamental p o s i t i o n a l and s t r u c t u r a l rearrangement o f c y t o p l a s m i c components i n p i n e chlorenchyma o c c u r s d u r i n g w i n t e r . O b s e r v a t i o n s were made u s i n g both l i g h t and e l e c t - ron m i c r o s c o p e s . T h i s work was o r i g i n a l l y based s o l e l y on o b s e r v a t i o n s on P i n u s c o n t o r t a s sp l a t i f o l i a , b ut i t was expanded t o i n c l u d e o b s e r v a t i o n s on s e a s o n a l changes i n P i n u s a l b i c a u l i s and Tsuga m e r t e n s i a n a . S i m i l a r changes were o b s e r v e d i n a l l t h r e e s p e c i e s . S t u d i e s were a l s o c o n d u c t e d on the a r t i f i c i a l f r o s t h a r d e n i n g o f p i n e s e e d l i n g s i n c o n t r o l l e d e n v i r o n m e n t a l c o n d i t i o n s , a l t h o u g h t h e s e e x p e r i m e n t s were l a r g e l y u n s u c c e s s f u l . The p o s s i b i l i t y o f a r e l a t i o n s h i p between drought e x p o s u r e and f r o s t r e s i s t a n c e was a l s o i n v e s t i g a t e d . 10 B e s i d e s d i r e c t c o m p a r i s o n s between summer and w i n t e r c y t o p l a s m i c s t a t e s , t h e summer chlorenchyma o f n e e d l e s o f v a r y i n g ages was a l s o s t u d i - ed i n o r d e r t o a s c e r t a i n t h a t f a c t o r s not r e l a t e d t o s e a s o n a l e n v i r o n m e n t - a l f e a t u r e s , such as s e n e s c e n c e , were not i n v o l v e d w i t h t h e summer-winter c o m p a r i s o n s . A c y t o l o g i c a l c o n d i t i o n t h a t may r e p r e s e n t a s p r i n g o r an i n t e r m e d i a t e c o n d i t i o n between t h e summer c y t o p l a s m i c s t a t e and t h e w i n t - e r c y t o p l a s m was a l s o i d e n t i f i e d . T e c h n i c a l d i f f i c u l t i e s were e x p e r i e n c e d i n o b t a i n i n g adequate u l t r a - s t r u c t u a l f i x a t i o n s o f p i n e c h l o r e n c h y m a . The m o d i f i c a t i o n s o f s t a n d a r d u l t r a s t r u c t u r a l t e c h n i q u e s d e v e l o p e d i n t h i s work t o a c h i e v e adequate f i x - a t i o n s s h o u l d a s s i s t f u t u r e works on c o n i f e r t i s s u e . O b s e r v a t i o n s on c h e m i c a l l y f i x e d and p r e p a r e d m a t e r i a l were supplement- ed w i t h o b s e r v a t i o n s on m a t e r i a l i n u n f i x e d , u n s t a i n e d , f r o z e n s t a t e . The d e t a i l s o f t h i s p r o c e d u r e and the c o n s t r u c t i o n o f a v e r y s i m p l e cryotome and c r y o m i c r o s c o p e t o conduct t h e s e o b s e r v a t i o n s i s a l s o d e s c r i b e d . 11 MATERIALS AND METHODS I. L i g h t and Transmi ss i o n E l e c t r o n M i c r o s c o p y P r e p a r a t i o n P r o c e d u r e s Young t r e e s ( f i v e t o f i f t e e n y e a r s o f age) o f P i n u s c o n t o r t a Dougl. ex Loud ssp l a t i f o l i a Engelm., ( i d e n t i f i e d per C r i t c h f i e l d , .1957), were chosen f o r t h i s p r o j e c t . The t r e e s from w h i c h n e e d l e s were s t u d i e d were c o l l e c t e d i n two l o c a t i o n s : A l l i s o n Pass l o c a t e d on the c o a s t a l d i v i d e i n t h e n o r t h e r n Cascade m o u n t a i n s , and the Sunday Summit p l a t e a u r e g i o n about t w e n t y - f o u r m i l e s t o t h e n o r t h - e a s t . Both s i t e s a r e appr o - x i m a t e l y f o u r thousand f e e t above sea l e v e l . The A l l i s o n Pass s i t e , be- cause o f c o a s t a l i n f l u e n c e s , has a more moderate mean a v e r a g e tempera- t u r e and considerably more annual p r e c i p i t a t i o n than t h e Sunday Summit l o - c a t i o n . Both s i t e s have a pronounced d r y p e r i o d w h i c h u s u a l l y l a s t from l a t e J u l y t o e a r l y O c t o b e r . In bo t h a r e a s , t h e s e l e c t e d t r e e s (presumab- l y seeded from a d j a c e n t mature t r e e s ) a r e i n s u n l i t c l e a r i n g s , and grow in w e l l d r a i n e d g l a c i a l g r a v e l s o i l s . In t h e A l l i s o n Pass r e g i o n s t a n d s of P i n u s c o n t o r t a o f v a r i o u s ages a r e mixed w i t h A b i e s a m a b i l i s . The Sun- day Summit s i t e i s surrounded by an a p p a r e n t l y pure s t a n d o f P i n u s c o n t o r - t a t r e e s a p p r o x i m a t e l y t h i r t y y e a r s o l d . F i v e t r e e s a t each s i t e were p e r m a n e n t l y l a b e l l e d and used f o r a l l e x p e r i m e n t s . Only n e e d l e s from the upper branches o f t h e s o u t h s i d e o f t h e t r e e were p r o c e s s e d f o r m i c r o s c o p i c o b s e r v a t i o n s . N e e d l e s t h a t showed no c h l o r o t i c a r e a s were used, and most f i x a t i o n s were c a r r i e d o ut w i t h i n an hour b e f o r e o r a f t e r s u n r i s e . In a l l c a s e s , t h e f i r s t 5 mm o f t i s s u e from t h e t i p o f each n e e d l e was d i s c a r d e d and o n l y t h e n e x t 10 mm p o r t i o n was used i n f i x a t i o n s . F or w i n t e r f i x a t i o n s t i s s u e s were f i x e d i n t h e p r i m a r y f i x a t i v e o f g l u t a r a l d e h y d e f o r m a l i n s o l u t i o n a t -k°C (when t h e a i r t e m p e r a t u r e was below t h e f r e e z i n g p o i n t ) . A l l n e e d l e s were c u t t r a n s v e r s e l y i n t o s e c t i o n s 0.5 mm t h i c k i n a pool o f p r i m a r y f i x a t i v e s o l u t i o n . They were then t r a n s f e r r e d t o v i a l s c o n t a i n i n g s i m i l a r f i x a t i v e a t t h e t e m p e r a t u r e s mentioned above and f i x e d f o r two t o t w e l v e h o u r s . The w i n t e r f i x a t i o n t e m p e r a t u r e s were m a i n t a i n e d by a s a l t - w a t e r - i c e b a t h and t h e summer f i x a t i o n t e m p e r a t u r e was r e g u l a t e d by a w a t e r b a t h . S p e c i f i c d e t a i l s o f the t i m e and c o n d i - t i o n s o f each f i x a t i o n a r e g i v e n i n T a b l e I. Two p r i m a r y f i x a t i v e s o l u t i o n s were used. The most used p r i m a r y f i x a t i v e i s made up by m i x i n g f r e s h l y made 25% f o r m a l d e h y d e (from p a r a - f o r m a l d e h y d e ) and 25% p u r e g l u t a r a l d e h y d e ( E l e c t r o n M i c r o s c o p y S c i e n c e s , F o r t Washington PA) w i t h a 0.1 M Sodium C a c o d y l a t e b u f f e r a t pH 6.8. The f i n a l c o n t e n t o f the f i x a t i v e s o l u t i o n c o n t a i n s : 0.5% f o r m a l d e h y d e , 2.0% g l u t a r a l d e h y d e and the f o l l o w i n g a d d i t i v e s : k% s u c r o s e , 0.0001% Kodak P h o t o f l o , and 0.01% C a C ^ - The o s m o t i c p r e s s u r e o f t h i s f i x a t i v e i s a p p r o x i m a t e l y 700 mOsm. The second p r i m a r y f i x a t i v e ( t h e a c r o l e i n f i x a t i v e ) , was a s o l u t i o n o f 2% a c r o l e i n and 2% g l u t a r a l d e h y d e b u f f e r e d i n t h e same manner as the s t a n d a r d f i x a t i v e . In c o n t r a s t t o t h e f i r s t method, t h e a c r o l e i n f i x a t i o n s were c a r r i e d o u t a t room t e m p e r a t u r e f o r Table I Collecting Times and Conditions of P. contorta Needles Season Year Month Day Time of Day Location fr Air Temp. (°C) Fixation Used Fixation Temp. (°C) it Needle Ages (Months) 0 - June l 5 t Allison Pass Sunday Summlt Summer 1972 Aug. 20 Dawn 12 12 S 4 3. 15 Fall 1972 Nov. 26 Dawn -4 - S 5 6 Winter Jan. 5 Dawn -14 -16 S -4 8, 20, 32, 56 1 :00 PH -8 -8 S -If 8 Feb. 9 Dawn -11 Some of this Hater- Ia1 collected for freezer storage In laboratory -15 S -4 9 Spring 1973 Mar. . 30 Dawn -3 -6 S -4 & +4 10 2:00 PH - 5 s 4 10,22 Apr. 27 Dawn 2 5 s I* 11 Summer 1973 June . 6 Dawn 6 s it 0. 12 36 29 Dawn 7 8 S & A 4 1. 37, 61 Summer 1973 July 21 Dawn - 14 S fr A 4 2, 14 Key ^o Symbols: S Standard fixation (Glut. fr Para.) A Acrolein fixation 1i h o u r s . A f t e r p r i m a r y f i x a t i o n , t h e m a t e r i a l was t a k e n t h r o u g h two c h a n g e s i n t h e b u f f e r w h i l e t h e t e m p e r a t u r e was r a i s e d s l o w l y t o room t e m p e r a - t u r e . A l l m a t e r i a l was p o s t - f i x e d i n \% osmium t e t r o x i d e b u f f e r e d w i t h 0.1 M sod ium c a c o d y l a t e b u f f e r w i t h no a d d i t i v e . W i n t e r m a t e r i a l was p o s t - f i x e d f o r two t o s i x h o u r s and m a t e r i a l f r o m o t h e r s e a s o n s was p o s t - f i x e d f o r s i x t e e n h o u r s , ( p o s t f i x a t i o n s were c a r r i e d o u t a t room t e m p e r a t u r e ) . M a t e r i a l was washed t w i c e i n d i s t i l l e d w a t e r , and d e h y d r a t e d i n a g r a d e d m e t h a n o l s e r i e s . M e t h a n o l was r e p l a c e d by a c e t o n e o v e r s e v e r a l c h a n g e s . Graded a c e t o n e and S p u r r ' s res in( l969) m i x t u r e s were used f o r i n f i l t r a t i o n o f p l a s t i c . I t i s i m p o r t a n t t h a t t h e l a s t m i x t u r e w i t h t h e h i g h c o n c e n t r a t i o n o f p l a s t i c be l e f t o v e r n i g h t . A f t e r two c h a n g e s i n p u r e p l a s t i c o f two h o u r s e a c h , m a t e r i a l was t r a n s f e r r e d i n t o f r e s h embedding m e d i a i n a a luminum b o a t s and p o l y m e r i z e d i n a vacuum o v e n f o r 2 h o u r s a t 50°C and 12 h o u r s a t 70°C. T e c h n i c a l d i f f i c u l t i e s were e x p e r i e n c e d w i t h t r i m m i n g and s e c t i o n - i n g . I t was n e c e s s a r y t o t r i m b l o c k s f o r s e c t i o n i n g w i t h t h e h y p o d e r - m i s e i t h e r d i s s e c t e d f r o m t h e t i s s u e o r a s f a r removed as p o s s i b l e f r o m t h e t o p o f t h e p y r a m i d i n o r d e r t o p r e v e n t t e a r i n g o f t h e t i s s u e . F i n - a l t r i m m i n g o f t h e b l o c k s was c a r r i e d o u t u s i n g g l a s s k n i v e s t o o b t a i n smooth s u r f a c e s on t h e b l o c k f a c e and s i d e s . I t was f o u n d t h a t t h e e x - treme hardness o f the c o n t e n t s o f the main c e l l u l a r v a c u o l e s o f t e n caused t h e s h a t t e r i n g o f s e c t i o n s d u r i n g m i c r o t o m i n g . The b e s t r e s u l t s were o b t a i n e d by u s i n g g l a s s k n i v e s and c u t t i n g o n l y one s e c t i o n on each a r e a o f the k n i f e edge. S i l v e r - g o l d s e c t i o n s (800-1000 A t h i c k ) f o r e l e c t r o n m i c r o s c o p y c o u l d be o b t a i n e d r e a d i l y , w h i l e 2 - s e c t i o n s f o r l i g h t m i c r o s c o p y were much more d i f f i c u l t t o o b t a i n . A l l s e c t i o n s were c u t on a R e i c h e r t OmU-3 u l t r a - m i c r o t o m e , and u l t r a - t h i n s e c t i o n s were c o l l e c t e d on copper g r i d s c o a t e d w i t h Formvar f i l m s . These s e c t i o n s were s t a i n e d i n u r a n y l a c e t a t e f o r f o r t y f i v e m i n u t e s f o l l o w e d by twenty minutes i n l e a d c i t r a t e ( R e y n o l d s , 1963). They were examined w i t h a Z e i s s EM 9A o r 9S e l e c t r o n m i c r o s c o p e For 1 i g h t m i c r o s c o p y , 5,/um s e c t i o n s were mounted o n t o g l a s s s l i d e s and s t a i n e d f o r 5 minutes i n t o l u i d i n e b l u e (0.05% a g ) . F r e s h mater- i a l was c u t i n t o 15/um t o 2Qum s e c t i o n s on a R i c h e r t CO^ gas e x p a n s i o n cryotome. S e c t i o n s were i m m e d i a t e l y f i x e d w i t h " 5%' f o r m a l d e h y d e f o r a f u r t h e r 10 m i n u t e s i n e t h a n o l i c Sudan b l a c k B. Tan n i n s t a i n i n g was c a r r i e d o ut by p l a c i n g l o n g i t u d i n a l l y - c u t n e e d l e s i n t h e s t a i n - f o r m - a l d e h y d e m i x t u r e o f Johansen (19^0) (see a p p e n d i x A) and then c r y o - s e c t i o n e d by method o u t l i n e d above. P h o t o m i c r o g r a p h s were t a k e n w i t h a Z e i s s p h o t o m i c r o s c o p e . 16 I |je Scanning E l e c t r o n M i c r o s c o p y P r o c e d u r e s Mature one y e a r o l d n e e d l e s were c o l l e c t e d from t h r e e and f o u r y e a r o l d s a p l i n g s grown o u t d o o r s f o r two y e a r s p r i o r t o s a m p l i n g a t t h e Un- i v e r s i t y o f B r i t i s h C olumbia. N e e d l e s were p l a c e d i n p e t r i d i s h e s c o n t a i n i n g 5% f r e s h l y p r e p a r e d f o r m a l i n i n t h e b u f f e r and a d d i t i v e s o l u t i o n used w i t h t h e p r i m a r y f i x - a t i v e s o l u t i o n f o r t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y . The t i p and bottom 1 cm o f t h e n e e d l e s were d i s c a r d e d and t h e r e m a i n i n g c e n t r a l p o r t i o n . of the n e e d l e was c u t l o n g i t u d i n a l l y i n t o two p a r t s . Each l o n g i t u d i n a l p a r t o f t h e n e e d l e was th e n c u t t r a n s v e r s e l y i n t o two o r t h r e e segments about 1 cm i n l e n g t h . A f t e r a one hour p r i m a r y f i x a t i o n , n e e d l e segments were p l a c e d i n \% osmium t e t r o x i d e i n d i s t i l l e d w a t e r a t k°C. The osmium t e t r o x i d e s o l u t i o n was then changed a f t e r 2k hours t o f r e s h f i x a t i v e and l e f t f o r . a n o t h e r 2k hours a t k°C. Needle segments were then washed i n d i s t i l l e d w a t e r f o r t h i r t y m i n u t e s , f o u r t i m e s . N e e d l e s were then d r i e d by c r i t i c a l p o i n t method. D r i e d n e e d l e seg- ments were then h e l d a t each end w i t h f o r c e p s and bent u n t i l t h e y broke i n h a l f . The broken segments were then c u t 2 mm below t h e breakage p o i n t and mounted on a specimen h o l d e r w i t h s i l v e r amalgam w i t h t h e broken f a c e f a c i n g up ( t h e t r a n s v e r s e f a c e o f t h e n e e d l e ) . T i s s u e was then c o a t e d w i t h g o l d and o b s e r v e d on a Cambridge s c a n n i n g e l e c t r o n m i s c r o s c o p e . 17 M l . E x p e r i m e n t a l F i x a t i o n s and P r o c e d u r e s Three . f i x a t i v e s o l u t i o n s were used t o s t u d y t h e re s p o n s e o f w i n t e r t i s s u e u l t r a s t u r c t u r e t o o s m o t i c s t r e s s and t o d e t e r m i n e whether t h e unsual c y t o p l a s m i c a p p e a r a n c e o f t h e w i n t e r t i s s u e may be p a r t i a l l y due t o f i x a t i o n a r t i f a c t s . These s o l u t i o n s were as f o l l o w s : 1. A p r i m a r y f i x a t i o n s o l u t i o n c o n t a i n i n g t h e same f i x a t i v e as d e s c r i b e d p r e v i o u s l y ( 1 % g l u t a r a l d e h y d e and 0.5% f o r m a l d e h y d e ) , but w i t h o u t any a d d i t i o n a l c h e m i c a l s . The o s m o t i c p r e s s u r e o f t h i s s o l u t i o n was a p p r o x i m a t e l y k00% mOsm. 2. A m i x t u r e o f 33% sea water and 66% b u f f e r s o l u t i o n c o n t a i n i n g t h e same g l u t a r a l d e h y d e and f o r m a l d e h y d e c o n c e n t r a t i o n s as i n th e s t a n d a r d p r i m a r y f i x a t i v e . The o s m o t i c p r e s s u r e o f t h i s f i x a t i o n s o l u t i o n was g r e a t e r than 1050 mOsm. 3- A n o n - e l e c t r o l y t e s o l u t i o n c o n s i s t i n g o f t h e s t a n d a r d p r i m a r y f i x a t i v e s o l u t i o n but w i t h t h e a d d i t i o n o f 10% i n s t e a d o f k% s u c r o s e . The o s m o t i c p r e s s u r e o f t h i s s o l u t i o n was 800 mOsm. Some n i n e - m o n t h - o l d l o n g s h o o t s c o m p l e t e w i t h n e e d l e s were removed from t r e e s a t t h e c o l l e c t i n g s i t e s i n w i n t e r a t an a i r t e m p e r a t u r e o f -11°C. The s h o o t s were m a i n t a i n e d a t -5°C o r lower w h i l e t h e y were brought t o Vancouver by p l a c i n g them i n j a r s capped w i t h cheese c l o t h i n a b r i n e i c e b a t h . Ini'the l a b o r a t o r y - , t h e y were s t o r e d i n a commercial f r e e z e r a t -18°C. The n e e d l e s can be s t o r e d i n t h i s way f o r up t o two months b e f o r e b e i n g f i x e d f o r e l e c t r o n m i c r o s c o p y i n a manner s i m i l a r t o t h e w i n t e r f i x a t i o n s i n t h e f i e l d . 18 M a t e r i a l kept i n t h e f r e e z e r and summer n e e d l e s were p l a c e i n l i q - u i d n i t r o g e n u n t i l a l l b o i l i n g stopped and then f i x e d by t h e same p r o c e - d u r e used f o r summer n e e d l e s i n t h e f i e l d . To d e t e r m i n e whether f r e e z e r s t o r e d l o n g s h o o t s were v i a b l e , t h e s h o o t s were removed from t h e f r e e z e r , wrapped i n aluminum f o i l and p l a c e d i n a r e f r i g e r a t o r f o r 12 hours a t k°C t o s l o w l y thaw. The base o f t h e "l o n g shoot was then f r e s h l y c u t and p l a c e d i n d i s t i l l e d w a t e r . The c u t - t i n g s were p l a c e d e i t h e r i n a growth chamber w i t h a 16 h r . o r 2k h r . l i g h t c y c l e and a t e m p e r a t u r e c y c l e r a n g i n g from k t o 12° C, o r o u t d o o r s ( d u r i n g t h e s p r i n g ) w i t h an a p p r o x i m a t e t e m p e r a t u r e f l u c t u a t i o n range o f 0°C t o 15°C. The c h o i c e o f p l a c i n g s h o o t s o u t d o o r s was made o n l y when a growth chamber w i t h t h e l i g h t and t e m p e r a t u r e ranges d e s c r i b e d above was not a v a i l a b l e . A f t e r one week o f l i g h t e x p o s u r e and above f r e e z i n g t e m p e r a t u r e s , n e e d l e s were removed from t h e shoot a t t h e end o f a l i g h t c y c l e and f r e e hand s e c t i o n s were c u t . S e c t i o n s were immersed i n IKI s o l u t i o n f o r s e v e r a l m i n u t e s and ob- s e r v e d . The p r e s e n c e o f h i g h l y r e f r a c t i l e c h l o r o p l a s t s c o n t a i n i n g l a r g e d a r k s t a r c h b o d i e s was i n t e r p r e t e d t o i n d i c a t e v i a b l e m a t e r i a l . In o r d e r t o v e r i f y t h a t t h e c y t o p l a s m i c appearance o f t h e w i n t e r c e l l s was not an a r t i f a c t o f c h e m i c a l f i x a t i o n o r o f h y d r a t i n g o r thaw- ing t h e t i s s u e , a p r o c e d u r e was d e v e l o p e d f o r t h e o b s e r v a t i o n o f f r o z e n c e l l s from t h e f i e l d w h i c h had never been thawed. The p r o c e d u r e and nec- e s s a r y m o d i f i c a t i o n s t o t h e s t a n d a r d cryotome and l i g h t m i c r o s c o p e a r e o u t l i n e d i n Appendix I I . See a l s o p l a t e XXIV. 19 IV. E x p e r i m e n t s on S e e d l i n g s R a i s e d i n the Growth Chamber Cones from the c o l l e c t i n g s i t e s were opened by s c o r c h i n g them w i t h a Bunsen b u r n e r . The seeds were s t r a t i f i e d f o r t h r e e weeks on m o i s t f i l - t e r paper i n t h e d a r k a t 4°C, and then p l a n t e d i n sandy s o i l i n 2 g a l l o n p o t s . Growth was a c c e l e r a t e d by p l a c i n g the p l a n t s i n a growth chamber under c o n t i n u o u s i l l u m i n a t i o n u n t i l t h e f i e l d e q u i v a l e n t o f two t o t h r e e y e a r o l d p l a n t s b e a r i n g mature n e e d l e s were o b t a i n e d . S e e d l i n g s w i t h mature n e e d l e s were then p l a c e d i n a 12 h r . l i g h t and 12 h r . d a r k n e s s r e - gime and a 12 h r . t e m p e r a t u r e c y c l e o f 20°C f o l l o w e d by 12°C i n a growth chamber. To d e t e r m i n e the e f f e c t o f dro u g h t on the mesophyl1 c e l l u l t r a s t r u - c t u r e , two p l a n t s were d e p r i v e d o f wa t e r f o r t w e n t y - f i v e days i n the grow- th chamber. D u r i n g t h i s t i m e , t h e y were s u b j e c t t o a 16/18 hour temper- a t u r e c y c l e o f 25°C and 15°C At the end o f t h i s p e r i o d , n e e d l e s were f i x e d a c c o r d i n g t o the g e n e r a l p r o c e d u r e f o r summer f i x a t i o n . F r o s t h a r d i n e s s was i n t r o d u c e d a r t i f i c i a l l y by p l a c i n g two p l a n t s (grown from seed i n 2 g a l l o n p o t s o u t d o o r s ) t h a t had j u s t c ompleted a c y c l e o f l o n g s h o o t and n e e d l e e l o n g a t i o n i n a growth chamber f o r f o u r weeks. An e i g h t hour p h o t o p e r i o d and t e m p e r a t u r e o f 10°C were used f o r the f i r s t week. The same c o n d i t i o n s were m a i n t a i n e d d u r i n g subsequent weeks e x c e p t t h a t the t e m p e r a t u r e was lowered t o , and c o n t i n u o u s l y kept a t k°C. A f t e r t h i s p e r i o d , the p l a n t s were t a k e n o u t o f the growth cham- ber and p l a c e i n a commercial r e f r i g e r a t o r a t -3°C f o r 2, 3 and k hours 20 e v e r y o t h e r day f o r s i x d a y s . Care was t a k e n t o move the s e e d l i n g s t o the r e f r i g e r a t o r o n l y ' d u r i n g the l i g h t c y c l e o f the growth p e r i o d . One day i n the chamber a t k °C was a l l o w e d between each day o f the f r o s t ex- posure t r e a t m e n t . The p l a n t s were then placed.in t h e r e f r i g e r a t o r f o r a f i n a l s i x hour t r e a t m e n t and t r a n s f e r r e d t o a commercial f r e e z e r i n an i n s u l a t e d box. There was no l i g h t i n g i n t h e r e f r i g e r a t o r o r the f r e e z e r . The p l a n t s were p l a c e d i n t h e f r e e z e r f o r e i g h t hours a t -18°C. The n e e d l e s were then removed and f i x e d a t below f r e e z i n g t e m p e r a t u r e s f o l - l o w i n g t h e normal w i n t e r f i x a t i o n p r o c e d u r e . D u r i n g b o t h t h e r e f r i g e r - a t o r and f r e e z e r t r e a t m e n t s , the a p p l i a n c e s ' were opened e v e r y t h i r t y m i n u t e s f o r a few seconds t o a l l o w gas exchange t o o c c u r . T h i s caused o n l y a b r i e f t e m p e r a t u r e f l u c t u a t i o n i n the r e f r i g e r a t o r and none i n the f r e e z e r . The p l a n t s r e c e i v e d no water d u r i n g t h e l a s t week i n t h e k°C regime and d u r i n g subsequent h a r d e n i n g a t lower t e m p e r a t u r e s . Two p l a n t s , one from t h e d r o u g h t e x p e r i m e n t . w h i c h was not watered f o r 30 days and a w e l l w a t e r e d t r e e from t h e growth chamber under t h e 12 h r . t e m p e r a t u r e a n d . l i g h t c y c l e were t a k e n t h r o u g h t h e h a r d e n i n g r e - gime d e s c r i b e d above. The f i n a l s t a g e o f h a r d e n i n g i n v o l v e d p l a c i n g t h e p l a n t s i n a commercial f r e e z e r a t -18°C f o r e i g h t hours f o l l o w e d by f i x - a t i o n i n a s i m i l a r manner t o t h a t d e s c r i b e d above. Some o f t h e n e e d l e s o f t h e s e t r e e s were r a i s e d t o room t e m p e r a t u r e ~ a f t e r the f r e e z e r t r e a t - ment and f i x e d as d e s c r i b e d i n t h e p r o c e d u r e f o r t h e f i x a t i o n o f summer n e e d l e s . 21 RESULTS I. S t r u c t u r e o f Needle T i ssues and Chiorenchyma C e l 1 s . The n e e d l e i s a complex s t r u c t u r e a n a t o m i c a l l y . The g e n e r a l nomen- c l a t u r e and arrangement o f c e l l s and t i s s u e s i s d e s c r i b e d w i t h r e f e r e n c e t o f i g u r e 1, wh i c h i s a d a r k f i e l d p h o t o g r a p h o f a p o r t i o n o f an u n s t a i n e d t r a n s v e r s e s e c t i o n o f a f r e s h n e e d l e . A t h i c k c u t i c l e (C and a r r o w h e a d ) , c o v e r s a la y e r , o f s m a l l e p i d e r m a l c e l l s ( E ) , w h i c h a r e c u b o i d a l . The t h i n - w a l l e d hypodermal c e l l s o f t h e so c a l l e d " w a t e r - l a y e r " (WL, Shaw, 1914), l i e i m m e d i a t e l y beneath the e p i - d e r m i s . The second hypodermal l a y e r c o n s i s t i n g o f l a r g e r e l o n g a t e d f i b e r c e l l s (TH), r u n n i n g p a r a l l e l t o t h e a x i s o f t h e n e e d l e . In P i n u s a l b i c a l - j_s_ n e e d l e s , t h e r e i s no " w a t e r - l a y e r " and the s c i e r e n c h m a t o u s hypodermal l a y e r s a r e s e v e r a l c e l l s t h i c k . The m e s o p h y l l chlorenchyma (M) 1ies between the dermal l a y e r s and the endodermis ( E n ) . In P i n u s c o n t o r t a ssp, l a t i f o l i a specimens c o l l e c t e d f o r t h i s work, t h e r e a r e two r e s i n d u c t s (Rd) l o c a t e d e n t i r e l y w i t h i n the m e s o p h y l l . Both appear t o run the l e n g t h o f the n e e d l e between the endodermis and the " c o r n e r s " o f the n e e d l e . The r e s i n d u c t s were s u r - rounded by a l a y e r o f t h i n - w a l l e d s e c r e t o r y c e l l s ( t h e s e c r e t o r y e p i t h e - l i u m ( S E ) ) , w h i c h i s i n t u r n s u r r o u n d e d by a r i n g o f f i b e r c e l l s (FC) o f t e n s e v e r a l c e l l s t h i c k . * " c o r n e r s " - W i t h r e s p e c t t o t h e h a l f - c i r c l e t r a n s e e t i o n a l ; . appearance o f the n e e d l e s o f t h i s s p e c i e s ; the term would mean t h e s u r f a c e a r e a where t h e f l a t s i d e ( a d a x i a l s u r f a c e ) meets t h e c u r v e d s i d e ( a b a x i a l s u r f a c e ) o f t h e n e e d l e . 22 "There a r e u s u a l l y two l a y e r s o f chlorenchyma c e l l s on t h e a d a x i a l and a b a x i a l s i d e s o f the n e e d l e i n t h e median r e g i o n s . Most s e c t i o n s i n t h i s work were c u t from t h i s r e g i o n f o r l i g h t and e l e c t r o n m i c r o s c o p y , more s p e c i f i c a l l y , f rom t h e a r e a o u t l i n e d by t h e r e c t a n g l e i n f i g u r e 1 . T h i s i s a r e g i o n on e i t h e r s i d e o f the median l o n g i t u d i n a l p l a n e i n the a b a x i a l s i d e o f t h e n e e d l e . The c hlorenchyma c e l l s a r e c h a r a c t e r i z e d by t h e i r c r e n u l a t e d appear- ance. T h i s f e a t u r e , w h i c h i s a d i s t i n c t i v e c h a r a c t e r i s t i c o f most s p e c i e s o f P i n u s , i s t h e r e s u l t o f numerous i n v a g i n a t i o n s o f -the p r i m a r y c e l l w a l l . These s t r u c t u r e s have been a s s i g n e d v a r i o u s names , but i n t h i s work t h e term ' t r a b i c u l a e ' ( B o l d , 1957) w i l l be used. The c hlorenchyma c e l l s i n the c o r n e r s ( l a t e r a l p o r t i o n s ) o f the nee- d l e and about t h e r e s i n d u c t s a r e c i r c u l a r o r o v o i d i n shape i n t r a n s v e r s e s e c t i o n s w h i l e t he c e l l s i n t h e me d i a l r e g i o n examined i n t h i s work as d e s c r i b e d above a r e r e c t a n g u l a r i n t r a n s e c t i o n a l . . ' p r o f i l e . In t h e c i r - c u l a r o r o v o i d c e l l s , t he t r a b i c u l a e a r e s h o r t and i n v a g i n a t e w i t h u n i f o r m s p a c i n g from t h e c e l l w a l l s whose p l a n e s a r e normal t o t h e p l a n e o f a n e e d l e t r a n s e c t i o n . . The c e l l w a l l s l y i n g p a r a l l e l t o the p l a n e o f n e e d l e t r a n s e c t i o n s : have no t r a b i c u l a e . The t h r e e d i m e n t i o n a l shape o f c h l o r e n c h y m a l c e l l s i n the median ab- a x i a l and a d a x i a l m e s o p h y l l i s shown i n f i g u r e 2. The g e n e r a l i z e d model was r e c o n s t r u c t e d from t h e s t u d y o f s e r i a l s e c t i o n s and l a t e r c o n f i r m e d ' See Esau (1965) and M i r o v (1967) 23 by e v i d e n c e from s c a n n i n g e l e c t r o n m i c r o s c o p y . The t h r e e d i m e n s i o n a l shape o f t h e c e l l a p p r o x i m a t e s t h e g e o m e t r i c shape d e s c r i b e d as a r i g h t a n g l e d p a r a l l e l e p i p e d . T h i s shape has s i x s i d e s w i t h o p p o s i t e s i d e s b e i n g p a r a - l l e l t o each o t h e r and h a v i n g t h e same a r e a . Three d i f f e r i n g s u r f a c e s o r s i d e s o f t h e c e l l a r e shown i n f i g u r e 2 and a r e l a b e l l e d w a l l s u r f a c e s A, B and C. Each w a l l s u r f a c e w i l l have an i d e n t i c a l o p p o s i t e s u r f a c e not shown i n f i g u r e 2. These s u r f a c e s a r e a l s o termed s u r f a c e s A, B and C i n t h i s work. The w a l l s u r f a c e s l a b e l l e d A i n f i g u r e 2 c o r r e s p o n d s t o t h e w a l l s u r - f a c e s o f a c e l l i n t h e p l a n e o f n e e d l e t r a n s e c t i o n . Note t h e p e r i p h e r a l p a r t s o f t h e c e l l f a c e A c o n t a i n c l e f t s i n t h e c e l l s u r f a c e caused by t h e i n v a g i n a t i o n o f t r a b i c u l a e from t h e c e l l w a l l f a c e s l a b e l l e d B and C. No t r a b i c u l a e were o b s e r v e d i n p i n e c h l o r e n c h y m a on t h e c e l l f a c e s i n t h e t r a n s v e r s e p l a n e o f t h e n e e d l e ( f a c e A ) . A c e l l v e r y s i m i l a r t o t h e d i a - gram i n f i g u r e 2 i s shown l a b e l l e d A i n f i g u r e s 1 and 8. The w a l l s u r f a c e s l a b e l l e d "B" i n f i g u r e 2 l i e s i n t h e r a d i a l l o n g - i t u d i n a l p l a n e s o f t h e n e e d l e . In t h e median c h l o r e n c h y m a , t h e s e w a l l s have a s h o r t " t r a b i c u l a e i n v a g i n a t i n g from t h e i r s u r f a c e . The w a l l s u r - f a c e s l a b e l l e d "C" l i e i n t h e t a n g e n t i a l p l a n e s o f the n e e d l e . In t h e o u t e r m o s t c h l o r e n c h y m a l m e s o p h y l l t i e r o f c e l l s , one c e l l f a c e "C" i s a p p r e s s e d t o t h e hypodermal s c l e r e n e h y m a and c o n v e r s e l y , one c e l l f a c e "C" S h o r t and l o n g t r a b i c u l a e r e f e r t o t h e r e l a t i v e d i s t a n c e t h e t r a b i c u - l a r w a l l s t r u c t u r e p e n e t r a t e s t h e c e l l i n t e r i o r . 2h a t t a c h e s t o t h e endodermis i n the innermost t i e r o f n e e d l e c h l o r e n c h y m a l c e l l s . Throughout t h i s work, t h e d i f f e r i n g w a l l s u r f a c e s o f t h e c h l o r - enchymal c e l l w i l l be r e f e r e d t o as f a c e A - the two c e l l w a l l s l y i n g i n the t r a n s v e r s e p i a n e o f t h e n e e d l e ; f a c e B - the two c e l l w a l l s l y i n g i n the r a d i a l p l a n e s o f the n e e d l e ; and f a c e C - the end w a l l s o f the c e l l . The t r a b i c u l u m appears t o be b a s i c a l l y a s h e l f - l i k e i n g r o w t h o f the c e l l w a l l p e r p e n d i c u l a r t o the c e l l s u r f a c e . For a l m o s t the e n t i r e l e n g t h o f t h e i n v a g i n a t i o n , the p r i m a r y w a l l s a r e a p p r e s s e d t o each o t h e r on e i t h e r s i d e o f a m i d d l e . l a m e l l a . At t h e end o f each t r a b i c u l u m , the w a l l f o l d s back on i t s e l f s i m i l a r t o a ' h a i r p i n t u r n ' ( f i g u r e 3 ) . The m i d d l e l a m e l l a o f t e n s e p a r a t e s a t t h e t i p o f the t r a b i c u l u m f o r m i n g a s m a l l ex- t r a c e l l u l a r s p a c e . The s t u d y o f s e r i a l s e c t i o n s i n d i c a t e s t h a t t h i s ex- t r a c e l l u l a r space i s o f t e n c o n t i n u o u s w i t h t h e l a r g e e x t r a c e l l u l a r a i r spaces about t h e e e l 1. The arrangement o f the c h l o r e n c h y m a l a i r spaces and the chlorenchyma c e l l s i n P i n u s c o n t o r t a i s i n a s p e c i f i c manner. The a i r spaces o c c u r next t o the c e l l s u r f a c e s A and e x t e n d as narrow unbroken passages from t h e hypodermis t o t h e endodermis i n t h e t r a n s v e r s e p l a n e o f t h e n e e d l e . The chlorenchyma c e l l s a r e a r r a n g e d i n the same manner so t h a t c e l l s u r - f a c e s B and C a r e c o n t i g u o u s w i t h o t h e r m e s o p h y l 1 i c , hypodermal o r endo- dermal c e l l s . Thus i n n e e d l e l o n g i t u d i n a l s e c t i o n s , the m e s o p h y l l c e l l s f o r m s h e e t s o f s i n g l e c e l l s e x t e n d i n g from t h e endodermis t o t h e hypo- dermis a l t e r n a t i n g w i t h hoi low passages o f e x t r a c e l l u l a r s p a c e s . T h i s 25 o r g a n i z a t i o n b r e a k s down o n l y under t h e rows o f stomata w h i c h run l o n g i - t u d i n a l l y a l m o s t t h e l e n g t h o f the n e e d l e . Beneath t h e stomata a r e sub- s t o m a t a l a i r spaces w h i c h form l o n g i t u d i n a l c o n n e c t i o n s between t h e c h l o r - enchymal a i r s p a c e s . When f i x e d and c r i t i c a l p o i n t d r i e d n e e d l e segments were broken open f o r s c a n n i n g e l e c t r o n m i c r o s c o p y i n t h e t r a n s v e r s e p l a n e o f the nee- d l e , t he me s o p h y l l chlorenchyma broke a p a r t a l o n g t h e t r a s v e r s e a i r s p a c e s . T h i s exposed an i n t a c t s h e e t o f chlorenchyma c e l l s l y i n g i n t h e t r a n s - v e r s e p l a n e o f the n e e d l e w i t h v e r y l i t t l e damage t o the exposed t r a n s - v e r s e e e l 1 w a l l f a c e s . F i g u r e 7 i n d i c a t e s t h a t t h e r e were few a r e a s o f attachment between the c e l l w a l l s o f s e p a r a t e t r a n s v e r s e , sheets^ of.meso- phyl1 e e l 1s. When t h e me s o p h y l l chlorenchyma f r a c t u r e d i n t h e l o n g i t u d i n a l p l a n e o f the n e e d l e , the m e s o p h y l l chlorenchyma c e l l s u s u a l l y b roke a p a r t a t the j u n c t i o n between the end w a l l o f t h e a d j a c e n t c e l l s . In t h i s c a s e , i n t a c t c e l l w a l l s were not u s u a l l y o b s e r v e d . I n s t e a d , fragments o f c e l l w a l l s remained a t t a c h e d t o a d j a c e n t c e l l s o r p i e c e s o f the e e l l...wal;l;s-'-includ- i n g remnants o f the p r o t o p l a s t were broken o f f ( f i g u r e 8 ) . In t h i n s e c t - i o n s o f t h e j u n c t i o n a l r e g i o n s between t h e end w a l l s o f a d j a c e n t m e s o p h y l l c e l l s , t he m i d d l e l a m e l l a and numerous p r i m a r y p i t f i e l d s a r e o b s e r v e d . Thus t h e s c a n n i n g e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s appears t o i n d i c a t e t h a t i n t h e t r a n s v e r s e p l a n e o f the n e e d l e , the n e e d l e m e s o p h y l l c o n s i s t s o f s i n g l e t i e r o r s h e e t o f m e s o p h y l l c e l l s e x t e n d i n g from t h e endodermis 26 to the hypodermis. W i t h i n each t r a n s v e r s e ^ t i e r , i n d i v i d u a l c e l l s are f i r m l y at tached to each o ther w i t h middle l a m e l l a between adjacent end w a l l s and r a d i a l c e l l w a l l s . Pr imary p i t f i e l d s and piasmodesmata in the end w a l l s of mesophyll c e l l s p rov ide cont inuous c y t o p l a s m i c connect ions between the endodermis to the hypodermis through the t r a n s v e r s e mesophyl1 c e l l s h e e t s . The complex t r a b i c u l a r i n f o l d i n g s of the c e l l w a l l of the c h l o r e n - chyma r e s u l t in s e c t i o n a l p r o f i l e s which are d i f f i c u l t to i n t e r p r e t . F i g - ures 3 to 6 of p l a t e II show how s e c t i o n s of the g e n e r a l i z e d c e l l in f i g - ure 2 would look i f s e c t i o n e d in severa l d i f f e r e n t p l a n e s . When the needle is sec t ioned t r a n s v e r s e l y , the chlorenchyma eel 1s are cut in the plane of face A ( f i g u r e 2). S e c t i o n s in t h i s plane show the c e l l in the t r a n s v e r s e p lane of the needle and the s h e l f - l i k e t r a b i c u l a e are viewed from the s i d e ( i . e . , at r i g h t angles to the w a l l from which they i n v a - g i na te , :f igure 3) • If c e l l s are sec t ioned in planes which extend in the same d i r e c t i o n as the l o n g i t u d i n a l a x i s of the n e e d l e , i t can be seen that the c h l o r e n - chyma c e l l s are not near l y as t h i c k in the n e e d l e ' s l o n g i t u d i n a l p lane as they are wide or long in the t r a n s v e r s e plane ( f i g u r e s k to 6). When the c e l l i s sec t ioned in the same plane as the n e e d l e ' s s u r - face ( i . e . , a t a n g e n t i a l c u t ) , the c e l l w i l l be cut in the same plane as the s u r f a c e C in f i g u r e 2. If t h i s s e c t i o n i s cut near the c e l l s u r - f a c e , then the t r a b i c u l a e w i l l be cut in the s e c t i o n plane and the s e c - t i o n would have a p r o f i l e s i m i l a r to that shown in f i g u r e k which cou ld 27 be c o n f u s e d as t h r e e s e p a r a t e c e l l s ( t h e t r a b i c u l a r w al1s a r e i d e n t i c a l w i t h t h e appearance o f t h e w a l l s o f two i m m e d i a t e l y a d j a c e n t c e l l s ) . I f the s e c t i o n i s c u t deeper i n t o t h e c e l l and a p p r o a c h i n g t h e e q u a t o r i a l p l a n e , t he s e c t i o n may be below t h e p o i n t o f maximal t r a b i c u l a r i n v a g - i n a t i o n and t h e s e c t i o n would have a p r o f i l e s i m i l a r t o t h a t shown i n f i g u r e 5 i n w h i c h no p a r t s o f t h e t r a b i c u l a e a r e seen. When t h e chlorenchyma c e l l i s s e c t i o n e d i n t h e r a d i a l p l a n e s / o f t h e n e e d l e , t h e s e c t ion would be p a r a l l e l t o t h e p l a n e o f w a l l s u r f a c e B o f * f i g u r e 2. F i g u r e 6 r e p r e s e n t s a s e c t i o n i n t h i s p l a n e c l o s e t o s u r f a c e B t o make i t appear as i f t h e s h o r t t r a b i c u l a e d i s s e c t t he c e l l i n t o two p o r t i o n s . The two l o n g t r a b i c u l a e w h i c h i n v a g i n a t e from f a c e C i n f i g u r e 2 can be viewed i n two d i f f e r e n t p l a n e s as shown i n f i g u r e s 3 and k. When v i e w - ed i n t h e s e two s e c t i o n a l p l a n e s , i t can be seen t h a t t h e two t r a b i c u l a e d i v i d e top a r e a s o f t h e c e l l i n t e r i o r i n t o t h r e e l a r g e l o b e s . The open s i d e o f two o f t h e l o b e s i s p a r t i a l l y r e s t r i c t e d t o the c e l l i n t e r i o r by the smal1 t r a b i c u l a e i n v a g i n a t i n g i n t o t h e c e l l from s u r f a c e B. In f i g u r e 2, t h e c e l l i s shown t o be s u b d i v i d e d i n t o s i x l o b e s by s i x l o n g and s h o r - t r a b i c u l a e . The shape o f m e s o p h y l l chlorenchyma c e l l w a l l s i n P i n u s a l b i c a u l i s n e e d l e s c o l l e c t e d a t W h i s t l e r mountain were v e r y s i m i l a r t o t h o s e des- c r i b e d f o r P i n u s c o n t o r t a . S e c t i o n s o f m e s o p h y l l from Tsuga m e r t e n s i a n a 28 c o l l e c t e d from W h i s t l e r mountain r e v e a l e d a m e s o p h y l l chlorenchyma con- s i s t i n g o f c y l i n d r i c a l c e l l s w i t h rounded ends. The long a x i s o f t h e c e l l s were a r r a n g e d r a d i a l l y from t h e endodermis t o t h e hypodermis. No e v i d e n c e o f t r a b e c u l a r i n f o l d i n g were found i n chlorenchyma o f t h i s c o n i f e r s p e c i e s . The r e l a t i o n s h i p between m e s o p h y l l c e l l s and the i n t e r c e l l u l a r a i r spaces was not a s - u n i f o r m ;as' i n -the Tsuga mesophyl 1 . The a i r spaces formed a more anastomosing system about i n d i v i d u a l c e l l s r a t h e r than b e i n g l a r g e l y con- f i n e d t o t h e t r a n s v e r s e p l a n e o f t h e n e e d l e . As i n P i n u s c o n t o r t a , the p r i m a r y p i t f i e l d s , plasmodesmata and t h e a r e a s o f w a l l t o w a l l c o n t a c t between m e s o p h y l l c e l l s and m e s o p h y l l endodermal c e l l s o r m e s o p h y l l hypoder- mal c e l l s was l a r g e l y c o n f i n e d t o t h e end w a l l s o f t h e c y l i n d r i c a l c e l l s . Below the m e s o p h y l l i n P i n u s c o n t o r t a was;a w e l l d e f i n e d endodermis c o n s i s t i n g o f a s i n g l e l a y e r o f c l o s e l y packed c e l l s w h i c h a r e o v o i d i n t r a n s e c t i o n , ( f i g u r e 1 ) . W i t h i n t h e endodermal l a y e r and s u r r o u n d i n g t h e v a s c u l a r b u n d l e s (VB) i s t h e t r a n s f u s i o n t i s s u e , w h i c h i s u n i q u e l y gymno- spermous (Esau, 1965). I t c o n s i s t s o f i r r e g u l a r l y shaped t r a n s f u s i o n t r a - c h e i d s (TT), w i t h s e c o n d a r y w a l 1 s c o n t a i n i n g numerous b o r d e r e d p i t s , and l a r g e l i v i n g t r a n s f u s i o n parenchymal c e l l s (TP) w h i c h a r e c i r c u l a r t o o v a l i n t r a n s e c t i o n , and have t h i c k p r i m a r y c e l l w a l l s ( f i g u r e 1 ) . 29 I I. The C y t o l o g y o f the Chiorenchyma i n t h e Summer Condi t i o n A. L i g h t M i c r o s c o p y A f r o z e n s e c t i o n o f summer chlo r e n c h y m a p r e p a r e d by t h e method o u t l i n e d i n A ppendix II i s shown i n f i g u r e 10. F r o z e n s e c t i o n s o f summer and w i n t e r m a t e r i a l have a v e r y g r a n u l a r and opaque app e a r - ance but s e v e r a l c y t o p l a s m i c f e a t u r e s can be i d e n t i f i e d . The c y t o - plasm appears as a t h i n p e r i p h e r a l band c o n t a i n i n g c h l o r o p l a s t s ( a r r o w h e a d s ) , and the n u c l e u s (N) i s c e n t r a l l y l o c a t e d w i t h i n the e e l 1 . When c r y o s e c t i o n s a r e a l l o w e d t o thaw, the g r a n u l a r i t y l a r g e l y d i s a p p e a r s and c y t o p l a s m i c o b j e c t s a r e more d i s t i n c t . F i g u r e 11 i s a thawed s e c t i o n a t h i g h e r m a g n i f i c a t i o n . C h l o r o p l a s t s (arrowheads) can be seen t o be r e g u l a r l y spaced a l o n g t h e c e l l w a l l and the s i d e s o f t h e t r a b i c u l u m ( T ) . The s p e r i c a l appearance o f t h e c h l o r o p l a s t s i n f r o z e n and then thawed c r y o s e c t i o n s appears t o be an a r t i f a c t o f c r y o s e c t i o n i n g . The c e l l i n f i g u r e 10 has an unusual shape due t o the U-shaped i n d e n t a t i o n i n i t s upper s u r f a c e w h i c h i s t h e s u b s t o m a t a l space t h a t c o n n e c t s the a i r spaces o f the n e e d l e i n the l o n g i t u d i n a l p l a n e . The guard c e l l s (Gd) o f the stomata a r e a l s o shown i n t h i s f i g u r e . F r e s h c r y o s e c t i o n s t r e a t e d w i t h e i t h e r f e r r o u s s u l f a t e based s t a i n 30 f i x a t i v e f o r t a n n i n ( J o h a n s e n , 1940), the n i t r o u s a c i d " s t a i n i n g p r o - c e d u r e o f Reeve (1951) o r one p e r c e n t osmic a c i d r e s u l t e d i n t h e i n - t e n s e s p e c i f i c s t a i n i n g o f t h e t a n n i n s w i t h i n t h e main v a c u o l e . The i n t e n s i t y o f t h e s t a i n i n g o f t h e main c e l l v a c u o l e when t h e Johansen p r o c e d u r e i s used i s i l l u s t r a t e d i n f i g u r e 12. In o r d e r t o a v o i d c o n f u s i o n w i t h o t h e r v a c u o l a r s t r u c t u r e s t o be d i s c u s s e d l a t e r , t h i s t a n n i n - f i l l e d v a c u o l e w i l l be c a l l e d t he t a n n i n v a c u o l e (TV). The t a n n i n v a c u o l e i n f i g u r e 1.2 o u t l i n e s c l e a r l y t h e t h i n band o f un- s t a i n e d p a r i e t a l c y t o p l a s m , ( c y t o p l a s m between a r r o w h e a d s ) . A nuc- l e u s i s l o c a t e d c l o s e t o t h e t i p o f a l o n g t r a b i c u l u m ; t h i s i s a t y p i c a l l o c a t i o n o f the n u c l e u s i n the summer c e l l . When f o r m a l i n f i x e d c r y o s e c t i o n s i n t h e summer c o n d i t i o n were s t a i n e d w i t h . S u d a n B l a c k B, many s m a l l o i l d r o p l e t s (0) appeared i n t h e c y t o p l a s m . The l a r g e s t o f t h e s e were about two m i c r o n s i n d i a - meter ( a r r o w s , f i g u r e 13) and t h e i r s i z e extended down t o t h e l i m i t o f r e s o l u t i o n ( a r r o w s , f i g u r e 1 4 ) . In t h e endodermis and t h e t r a n s - f u s i o n parenchyma-, numerous and c o n s i d e r a b l y l a r g e r o i l d r o p l e t s ( arrowhead, f i g u r e 13) can be seen. . No c r y o s e c t i o n s were c u t o f o t h e r c o n i f e r s p e c i e s mentioned i n t h i s work. 31 Ul t r a s t r u c t u r a l Changes i n the Chlorenchyma Re1 a t e d t o t h e Ag i n g of t he Need 1e A l t h o u g h t h e e l u c i d a t i o n o f the p r o c e s s o f senesenee i n p i n e c h l o - renchyma was not the o b j e c t o f t h i s i n v e s t i g a t i o n , i t i s e s s e n t i a l t h a t any major changes due t o a g i n g i n t h e summer ne e d l e . b e c h a r a c t e r i z e d so t h a t t h e s e f e a t u r e s w i l l not be c o n f u s e d w i t h changes r e l a t e d t o s e a s o n - a l dependent f a c t o r s . F o r t h i s p u r p o s e , summer and w i n t e r n e e d l e s from c u r r e n t season n e e d l e f l u s h t o f i v e y e a r s o f age were examined. Three g e n e r a l c y t o l o g i c a l c o n d i t i o n s dependent on n e e d l e age were i d e n t i f i e d . In the f i r s t c o n d i t i o n , the chlorenchyma has a c h a r a c t e r - i s t i c s w h i c h r e f l e c t a s t a t e o f h i g h m e t a b o l i c and s y n t h e t i c a c t i v i t y . The ground c y t o p l a s m i s f i l l e d w i t h numerous polysomes and a l a r g e amount o f RER, p a r t i c u l a r l y near the n u c l e u s ( f i g u r e 2 2 ) . Dictyosomes were f r e q u e n t l y seen i n s e c t i o n s and were su r r o u n d e d by numerous v e s i c l e s . In many c a s e s , numerous d i c t y o s o m e v e s i c l e s a r e l o c a t e d i n t h e a r e a between the d i c t y o s o m e s and the plasma membrane ( f i g u r e 2 9 ) . In o t h e r c a s e s , a l a r g e number o f t h e d i c t y o s o m e a s s o c i a t e d v e s i c l e s appear i n r e g i o n s where v a c u o l i z a t i o n o c c u r s ( f i g u r e 2 6 ) . M i t o c h o n d r i a were l a r g e and e x t e n s i v e l y lobed and had numerous c r i s t a e . T a n n i n i s p r e s e n t a t a v e r y e a r l y s t a g e o f n e e d l e e l o n g a t i o n but i n young e x p a n d i n g n e e d l e s , i t i s o f t e n clumped i n t o i r r e g u l a r a g g r e g a t e s and does n o t o c c u p y the f u l l v o l - ume o f t h e main v a c u o l e ( f i g u r e 2 0 ) . It was c o n c l u d e d t h a t t h e f i r s t c o n d i t i o n o b s e r v e d i n young expand- 32 ing c u r r e n t season n e e d l e s i n June and J u l y r e p r e s e n t e d c e l l s c h a r a c t e r - i z e d by f e a t u r e s u s u a l l y a s s o c i a t e d w i t h a c t i v e m e t a b o l i s m . In t h i s work, t h i s c e l l c o n d i t i o n wi 1 1 be r e f e r r e d t o as t h e t y p e I c e l l t y p e . In l a t e J u l y , the young n e e d l e s had completed e l o n g a t i o n and t h e c y t o p l a s m had t r a n s f o r m e d i n t o t he second c y t o p l a s m i c c o n d i t i o n w h i c h w i l J be r e f e r r e d t o as t h e typ e II c e l l . T h i s c o n d i t i o n o f n e e d l e c y t o - l o g y i s o b s e r v e d i n t h e c u r r e n t season n e e d l e s d u r i n g l a t e J u l y and Au- g u s t . When s e c o n d , t b i r d and f i f t h y e a r n e e d l e s a r e examined, the major- i t y o f c e l l s appear t o a l s o b e l o n g t o t h i s c o n d i t i o n . I t appears t o be c h a r a c t e r i z e d by t h e c e l l b e i n g committed t o , and a c t i v e i n p h o t o s y n t h e s i s and r e s p i r a t i o n . The c y t o p l a s m i s dominated by l a r g e numbers o f c h l o r o - p l a s t s and m i t o c h o n d r i a , and c o n t a i n s few o t h e r o r g a n e l l e s . Other f e a - t u r e s o f t h i s c e l l t y p e a r e : a. Dictyosomes were r a r e l y seen and u s u a l l y a s s o c i a t e d w i t h few s u r r o u n d i n g v e s i c l e s . b. Ribosomes o c c u r e d i n t h e ground cytoplasm-. c. Only smal 1 f r a g m e n t s o f RER were seen,i u s u a l l y i n e a r t h e " n u c l e u s . d. The n u c l e u s had e x t e n s i v e a r e a s o f h e t e r o c h r o m a t i r i . The t h i r d c o n d i t i o n o f summer chlorenchyma c e l l s c o n s i s t s o f what appeared t o be o l d o r damaged c e l l s t o a v a r i e t y o f d e g r e e s . Some o f t h e s e a r e p r o b a b l y caused by p r e p a r a t i v e p r e c e d u r e s f o r e l e c t r o n m i c r o s - copy. These c e l l s a r e c h a r a c t e r i z e d by o r g a n e l l e s t h a t a r e damaged and p o o r l y f i x e d , o r by s c a r c i t y , o f o r g a n e l l e s i n d i c a t i n g t h e low l e v e l or!' 33 c e s s a s i o n o f c e l l u l a r f u n c t i o n s . C e l l s o f t h i s t y p e had some o r most o f the f o l l o w i n g c h a r a c t e r i s t i c s : a. L a r g e q u a n t i t i e s o f o s m i o p h i l i c m a t e r i a l w h i c h tend t o o b s c u r e the c y t o p l a s m i c d e t a i l . The o s m i o p h i l i c m a t e r i a l i s u s u a l l y t a n n i n - l i k e o r 1 i p o i d a 1 i n appearance. F i g u r e 70 i l l u s t r a t e s p a r t o f a c e l l . o f t h i s t y p e . The c y t o p l a s m c o n t a i n s a l a r g e o s m i o p h i l i c o i l - l i k e body (0), and p o o r l y d i s e e r n a b l e c h l o r o p l a s t s . D e s p i t e e l e c t r o n d e n s i t y and l a c k o f membranous d e t a i l , the c y - t o p l a s m i s p r o b a b l y s t i l l f u n c t i o n a l as e v i d e n c e d by t h e p r e - sence o f s t a r c h g r a i n s w i t h i n t h e c h l o r o p l a s t s . However, many c e l l s o f t h i s t y p e d i d not c o n t a i n t h e amount o f f i n e s t r u c t u r a l d e t a i l shown i n t h i s f i g u r e and s t a r c h g r a i n s , w e r e a b s e n t . b. T o n o p l a s t and/or plasma membrane fragmented o r a b s e n t . c. Very poor d e t a i l and/or w i t h a p p a r e n t . m e c h a n i c a l damage t o the c e l l , i . e . , s w o l l e n o r g a n e l 1 e s , r u p t u r e d c y t o p l a s m i c membranes. T h i s c o u l d be a p h y s i c a l phenomenon due t o changes i n o s m o t i c p r e s s u r e and membrane p e r m e a b i 1 i t y w i t h i n t h e s e o l d e r c e l l s . Type I I I c e l l s were not o b s e r v e d i n young m a t u r i n g n e e d l e s ( f i r s t summer), but t h e f r e q u e n c y o f t h e i r o c c u r e n c e i n c r e a s e s w i t h n e e d l e age. A p p r o x i m a t e l y t w e n t y - f i v e p e r c e n t o f the chlorenchyma c e l l s i n f i v e y e a r o l d n e e d l e s were o f t h i s t y p e . Most n e e d l e s o f one y e a r o l d o r o l d e r have o c c a s i o n a l c h l o r o t i c p a t c h e s s c a t t e r e d a l o n g the l e n g t h o f the n e e d l e . These c h l o r o t i c p a t - ches i n c r e a s e i n s i z e and f r e q u e n c y w i t h r e s p e c t t o t h e age o f t h e nee- d l e . Small c h l o r o t i c ( l e s s than 1 mm) p a t c h e s do not u s u a l l y s p r e a d a l o n g the n e e d l e a x i s but extend around t h e n e e d l e as a r i n g o r a g i r d l e . L a r g e r c h l o r o t i c p a t c h e s , however, not o n l y e x t e n d c o m p l e t e l y about t h e n e e d l e but a l s o s p r e a d t o a lengh o f a c e n t i m e t e r o f more a l o n g the n e e d l e a x i s . Type 111 e e l 1s usua11y o c c u r r e d i n l a r g e groups and appeared t o be more e x t e n s i v e w i t h i n t h e t r a n s v e r s e p l a n e o f t h e n e e d l e than t h e y a r e i n t h e p l a n e o f t h e l o n g i t u d i n a l n e e d l e a x i s . They show t h e same b a s i c p a t t e r n o f o r i e n t a t i o n as t h e m a c r o s c o p i c c h l o r o t i c a r e a s . Summer f i x a t i on problems i n c r e a s e d i n p r o p o r t i o n w i t h n e e d l e age and adequate f i x a t i o n s o f t h r e e and f i v e y e a r o l d n e e d l e s was o n l y a c h i e v e d w i t h t h e a c r o l e i n - g l u t a r a l d e h y d e p r i m a r y f i x a t i v e s ( f i g u r e s 69,70 and 71 as ex- a m p l e s ) . One problem a s s o c i a t e d w i t h t h e use o f a c r o l e i n based f i x a t i v e on p i n e chlorenchyma i s t h a t t h e c y t o p l a s m becomes c o n s i d e r a b l y more o s - m i o p h i l i c than w i t h t h e f o r m a l i n - g l u t a r a l d e h y d e f i x a t i v e s . P r e d o m i n e n t l y green n e e d l e s were o b s e r v e d on young open grown t r e e s t h a t were as o l d as. t w e l v e y e a r s . A t t e m p t s t o f i x such n e e d l e s were un- s c c e s s f u l . However, s t a r c h g r a i n s and c h l o r o p l a s t s were o b s e r v e d i n c r y o - s e c t i o n s o f p r i m a r y f i x e d ( a c r o l e i n - g l u t a r a l d e h y d e ) m a t e r i a l a t t h e l i g h t m i c r o s c o p y l e v e l . 35 C. O b s e r v a t i o n s on S p e c i f i c O r g a n e l l e s : E l e c t r o n M i c r o s c o p y A l l o r g a n e l l e s i n summer p i n e c hlorenchyma appear t o be v e r y s i m i l a r t o t h o s e d e s c r i b e d f o r t h e chlorenchyma o f o t h e r h i g h e r p l a n t s . Only t y p e s I and II c e l l s w i l l be d e s c r i b e d s i n c e t h e s e a r e the c e l l s w h i c h a r e o f f u n c t i o n a l s i g n i f i c a n c e t o the t r e e . The plasma membrane i s w e l l d e f i n e d i n t y p e s I and II c e l l s . At t h e f i x a t i v e o s m o l a r i t y used f o r e l e c t r o n m i c r o s c o p y , i t was u s u a l l y t i g h t l y a p p r e s s e d t o the c e l l w a l l s and the w a l l s o f t h e t r a b i c u l a e . However, the h i g h o s m o l a r i t y o f t h e f o r m a l i n f i x a t i v e used t o s t a i n t a n n i n s f o r l i g h t m i c r o s c o p y o f t e n caused p l a s m o l y s i s o f t h e p r o t o p l a s t . ( f i g u r e 12). PIasmodesmata were o b s e r v e d f r e q u e n t l y when s e c t i o n s were c u t near the ends o f t h e c e l l ( s u r f a c e C, and t h e c o r n e r areas' near i t s j u n c t i o n o f w a l l s u r f a c e C and B i n f i g u r e 2). The t o n o p l a s t i s a l s o w e l l d e f i n e d and remains i n t a c t i n a l l t y p e s ! and II c e l l s o b s e r v e d . T a n n i n m a t e r i a l appeared t o s h r i n k and s e p a r a t e d somewhat from t h e t o n o p l a s t f o r m i n g a gap. T h i s i s e v i d e n c e f o r membrane c o n t i n u i t y s i n c e s t r a n d s a r e c l e a r l y seen l i n k i n g t h e two s t r u c t u r e s . Such a phenomenon al so appears t o cause t h e c o n v o l u t i o n i n the p r o f i l e o f the t o n o p l a s t (TO, open a r r o w s , f i g u r e 23). The s i z e o f the gap between t o n o p l a s t and t a n n i n appears t o be f i x a t i o n dependent, v a r y i n g c o n s i d e r - a b l y from one f i x a t i o n t o - t h e next (see f i g u r e s 22, 23 and 26). A c r o l e i n f i x a t i v e s d i d not appear t o form a gap between t h e t o n o p l a s t and t h e t a n - n i n m a t e r i a l . In some s e c t i o n s , t h e t o n o p l a s t i s o b s e r v e d t o i n t e r d i g i - 36 t a t e e x t e n s i v e l y i n t o the adjacent cytoplasm (open arrows, f i g u r e 2 7 ) . When the tannin vacuole i s sectioned o b l i q u e l y , the i n t e r d i g i t a t i n g tonoplast forms complex membrane patterns (open arrows, f i g u r e 2 8 ) . The tonoplast i s a l s o most s u s c e p t i b l e to damage caused by needle s u b d i v i s i o n , or due to osmotic changes during f i x a t i o n . When the tono- p l a s t does rupture, the cytoplasm appears f i l l e d with t a n n i n , and c e l l - u l a r f i n e s t r u c t u r e i s obscured. The s e n s i t i v i t y of the tonoplast dur- ing f i x a t i o n s appears to be dependent on the osmo l a r i t y of the f i x a t i v e . F i x a t i v e s w i t h a high osmotic pressure cause the tannin material to ap- pear to con t r a c t and separate from the tonoplast .(forming the gap pre- v i o u s l y mentioned). F i x a t i v e w i t h osmotic pressures below that used fo r t h i s work tended to rupture the tonoplast which r e s u l t e d in the d i s p e r s i o n of tannin throughout the cytoplasm and the d e s t r u c t i o n of cytoplasmic f i n e s t r u c t u r e . In very young (2mm) needles, the main vacuole i s l a r g e l y e l e c t r o n - transparent ( f i g u r e 20) and tannin d e p o s i t s , i f a n y , o c c u r as scattered clumps. However, we l l before needle elongation i s completed, the main vacuole becomes f i l l e d with t a n n i n , and the main vacuole i s then,as stated p r e v i o u s l y , r e f e r r e d to as the tannin vacuole. In the c e l l of the mature summer chlorenchyma, the tannin vacuole i s a dominant feature and occupies most of the c e l l volume. The tannin material has a very uniform, granu1 ar appearance, but . o c c a s i o n a l l y a f i b r i l l a r f i n e s t r u c t u r e was a l s o seen along w i t h the granular t e x t u r e . 37 In t y p e II and t y p e I I I c e l l s p a r t i c u l a r l y , t h e a p p e a r a n c e o f t h e t a n n i n i s m o d i f i e d by m i n u t e f r a c t u r i n g and k n i f e c h a t t e r a r t i f a c t s formed d u r - i n g s e c t i o n i n g . An example o f t h i s t y p e o f a r t i f a c t i s shown i n t h e t a n - n i n v a c u o l e o f f i g u r e 70. The t a n n i n m a t e r i a l i s e x c e p t i o n a l l y d i f f i c u l t t o c u t and t h i s d i f f i c u l t y a p p e a r s t o i n c r e a s e w i t h n e e d l e a g e . O s m i o - p h i l i c m a t e r i a l w i t h t h e t e x t u r e o f t a n n i n was not s e e n o u t s i d e o f t h e main v a c u o l e i n t y p e s I and II c e l l s i n summer f i x a t i o n s . In some o l d e r n e e d l e s ( t h r e e t o f i v e y e a r s ) , t h e h o m o g e n e i t y o f t h e t a n n i n i n t h e t a n n i n v a c u o l e was o f t e r i . i r e p l a c e d by t h e p r e s e n c e o f l a r g e and s m a l l i n c l u s i o n s . The i n c l u s i o n s were c o n s i s t e n t l y v e r y o s m i o p h i l i c , and smooth i n t e x t u r e and a r e t h u s d e s c r i b e d as a 1 i p o i d a l t y p e ( f igure 70 ) . In t h i s c e l l , t h e t o n o p l a s t c o u l d n o t be r e s o l v e d and t h u s t h e l i p i d m a t - e r i a l (0) c o u l d be w i t h i n a c y t o p l a s m i c v a c u o l e o r w i t h i n t h e t a n n i n v a - c u o l e . In t y p e s I and II c e l l s , a v a r i e t y o f c y t o p l a s m i c v e s i c l e s and v a c u - o l e s a r e seen w i t h i n t h e p e r i p h e r a l c y t o p l a s m o f the. c e l l i n a d d i t i o n t o t h e main t a n n i n v a c u o l e . These c y t o p l a s m i c v a c u o l e s may c o n t a i n s m a l l o s m i o p h i l i c b o d i e s (OB) a n d / o r m a t e r i a l t h a t i s p o s s i b l y : : o f membranous o r i g i n ( f i g u r e s 22, 23 and 25). "The o s m i o p h i l i c b o d i e s d i s c u s s e d t h r o u g h - o u t t h i s work a r e s t r u c t u r e s o f unknown c h e m i c a l c o m p o s i t i o n . They a r e i d e n t i f i e d on t h e b a s i s o f : s i z e , ( u s u a l l y l e s s t h a n 1 Aim i n d i a m e t e r ) , t e x t u r e ( v e r y homogeneous and "1 i p i d-1 i k e " ) and s t a i n i n g i n t e n s i t y , ( t h e y a r e t h e most o s m i o p h i l i c s t r u c t u r e s i n most s e c t i o n s ) . Compare t h e a p - 38 pearance o f o s m i o p h i l i c b o d i e s w i t h t a n n i n m a t e r i a l i n f i g u r e s 22, 23, 2k and 25, and w i t h t h e o i l body i n f i g u r e 29. In t h i s work, c y t o p l a s m i c v a - c u o l e s a r e d i f f e r e n t i a t e d from t h e t a n n i n v a c u o l e on t h e b a s i s o f t h e form- e r b e i n g much s m a l l e r than t h e t a n n i n v a c u o l e ( l e s s than 5 >um i n d i a m e t e r ) and c o n t a i n i n g e l e c t r o n t r a n s p a r e n t lumens, o r membranous m a t e r i a l o r mat- e r i a l t h a t s t a i n s s i m i l a r t o t h a t o f t h e o s m i o p h i l i c b o d i e s . The c y t o p l a s m - i c v a c u o l e s were not o b s e r v e d t o c o n t a i n m a t e r i a l t h a t had s t a i n i n g and t e x t u r a l p r o p e r t i e s s i m i l a r t o t a n n i n . The number o f o s m i o p h i l i c b o d i e s t e n d s t o i n c r e a s e i n l a t e summer i n n e e d l e s o f a l l ages. Most o s m i o p h i l i c b o d i e s a r e found w i t h i n c y t o p l a s m i c v a c u o l e s ( f i g u r e 22), but some a r e found i n the c y t o p l a s m w i t h o u t any a p p a r e n t bounding membranes ( f i g u r e 23, c f . upper and lower o s m i o p h i l i c bod i e s ) . As p r e v i o u s l y n o t e d , d i c t y o s o m e s were v e r y numerous i n t y p e I c e l l s . In e l o n g a t i n g n e e d l e s , d i c t y o s o m e s appear a s s o c i a t e d w i t h t h e t r a b i c u l a r r e g i o n s o f t h e c e l l w a l l . These organel1es appear t o o f t e n have t h e i r mat- u r i n g f a c e towards t h e plasma membrane. D i c t y o s o m a l v e s i c l e s a l s o o f t e n oc- c u p i e d t h e space between t h e c e l l w a l l and t h e d i c t y o s o m e s . These v e s i c l e s g e n e r a l l y were the s m a l l e s t i n s i z e when l o c a t e d near t h e m a t u r i n g f a c e , the l a r g e s t i n t h e p r o x i m i t y o f the c e l l w a l l ( f i g u r e 27). Dictyosomes a r e a l s o a s s o c i a t e d w i t h t h e c y t o p l a s m i c v a c u o l e s d e s c r i b e d p r e v i o u s l y ( f i g - u r e 25). A t r a n s i t i o n a l s e r i e s o f v e s i c l e s may o c c u r between d i c t y o s o m a l f a c e s and t h e c y t o p l a s m i c v a c u o l e s ( f i g u r e 25 ). In t y p e II c e l l s o f 39 the mature summer t i s s u e , the number o f d i c t y s o m e s i s g r e a t l y reduced as compared w i t h e x p a n d i n g n e e d l e s . But i n mature n e e d l e s , the c l o s e a s s o - c i a t i o n between d i c t y o s o m e v e s i c l e s and the c y t o p l a s m i c v a c u o l e s (which do not c o n t a i n o s m i o p h i l i c b o d i e s ) i s s t i l l o b s e r v e d ( f i g u r e 26). In t h e summer n e e d l e , t h e c h l o r o p l a s t s o f the chlorenchyma have t h e c h a r a c t e r i s t i c d i s c o i d a l shape seen In h i g h e r p l a n t s ' w i t h one s i d e o f the p l a s t i d f l a t t e n e d a g a i n s t the plasma membrane ( f i g u r e s 19, 20 and 21). The shape and u n i f o r m d i s t r i b u t i o n o f t h e c h l o r o p l a s t s a l o n g t h e c e l l w a l l and the t r a b i c u l a e remain c o n s t a n t r e g a r d l e s s o f n e e d l e age i n t y p e I and t y p e I I e e l 1s. The c h l o r o p l a s t e n v e l o p e i s d i s t i n c t and c o n s i s t s o f two s h a r p l y c o n t r a s t e d membranes ( f i g u r e 21). The grana were made up o f not more t h a n f i v e o r s i x s t a c k e d t h y l a k o i d s i n a l l c e l l t y p e s . T h e - g r a n a l t h y - l a k o i d s a r e o f v e r y d i f f e r e n t s i z e s , thus t h e r e s u l t i n g s t a c k s o f t h y l a - k o i d s have v e r y i r r e g u l a r edges. P a r t i t i o n s a r e o f t e n e l o n g a t e d , r e - s u l t i n g i n v e r y low and broad p r o f i l e s o f granna ( f i g u r e 21). The g r e a t - l y e l o n g a t e d grana shown i n f i g u r e 21 i s made up o f two t o t h r e e s t a c k e d t h y l a k o i d s . T h i s t y p e o f grana i s more common i n c h l o r o p l a s t s from o l d - e r n e e d l e s , however, e x c e p t i o n s were n o t e d . The c h l o r o p l a s t s o f t h e t h r e e y e a r o l d c e l l i n f i g u r e 71 show wel1 d e v e l o p e d g r a n a l s t a c k s s i m - i l a r i n height t o t h o s e seen i n younger c e l l s . S t a r c h g r a i n s were u s u a l l y found i n summer c h l o r o p l a s t s o f t y p e I ko and t y p e II c e l l s and o f t e n i n t y p e I'll c e l l s i n w h i c h t h e c h l o r o p l a s t s remain i n t a c t . The s t a r c h g r a i n s i n the c h l o r o p l a s t s i n t h i s work ( f i g - u re 21 f o r example) a r e u s u a l l y s m a l l because a l l summer m a t e r i a l was f i x - ed a t dawn. A f t e r n o o n f i x a t i o n s o f summer t i s s u e were not con d u c t e d be- cause s t a r c h i n c e l l s f i x e d i n t h i s manner would o b s c u r e much o f t h e i n - t e r n a l s t r u c t u r e s o f the p l a s t i d s and g r e a t l y d i s t o r t e d t h e i r shape and th e s u r r o u n d i n g c y t o p l a s m . Prominent p l a s t o g l o b u l i a r e a f e a t u r e o f the c h l o r e n c h y m a l c h l o r o - p l a s t .of Junius £cjitor_ta. In v e r y young t y p e I c e l l s , t h e p l a s t o g l o b u l i a r e s m a l l ( a r r o w s , f i g u r e 2 0 ) , but i n t y p e II c e l l s , t hey a r e l a r g e r but remain a t about the same r e l a t i v e number per c h l o r o p l a s t p r o f i l e . A f t e r an i n c r e a s e i n p i a s t o g l o b u l i s i z e from t y p e I t o t y p e II c e l l s , p l a s t o - g l o b u l i s i z e t h e n appears t o be independent o f t h e age o f the c e l l i n t y p e s II and I I I e e l 1s. The v a r i a t i o n o f c h l o r o p l a s t s t r u c t u r e from c e l l t o c e l l o r w i t h i n one c e l l i s g r e a t e r i n o l d e r n e e d l e s . The c h l o r o p l a s t i n f i g u r e 71 f o r example has fewer p i a s t o g 1 o b u l i and more e x t e n s i v e t h y l a k o i d s .than i s t y - p i c a l f o r a t h r e e y e a r o l d type II e e l 1 ; The s t r u c t u r e o f c h l o r e n c h y m a l m i t o c h o n d r i a i n summer a l s o v a r i e s w i t h n e e d l e age. In t y p e I c e l l s , m i t o c h o n d r i a appeared t o be cup-shaped o r m u l t i - l o b e d when s e c t i o n e d i n t h e p l a n e p a r a l l e l t o and j u s t below the c e l l w a l l ( f i g u r e s 27 and 2 8 ) . When t r a n s v e r s e s e c t i o n s o f the c e l l a r e t a k e n w i t h r e s p e c t t o the c e l l w a l l , the m i t o c h o n d r i a a r e not so ex- 41 t e n s i v e l y l o b e d i n appearance and a r e u s u a l l y o v o i d i n s e c t i o n a l p r o f i l e ( f i g u r e 19)- The degr e e o f l o b i n g o f t h e m i t o c h o n d r i a i s g r e a t l y d i m i n - i s h e d i n t y p e II c e l l s . In t h i s c e l l t y p e , s e c t i o n a l p r o f i l e s o f m i t o - c h o n d r i a u s u a l l y appear o v o i d ! i n a l l p l a n e s o f s e c t i o n i n g . Summer m i - t o c h o n d r i a i n b o t h t y p e s I and II c e l l s have e x t e n s i v e t u b u l a r c r i s t a e . The n u c l e u s i n t h e summer chlorenchyma i s u s u a l l y l o c a t e d i n t h e c e n t r a l r e g i o n o f the c e l l , t y p i c a l l y near t h e ends o f one o r more o f the l o n g t r a b i c u l a e . In t h i s p o s i t i o n , t h e n u c l e u s i s n e a r l y s u r r o u n d e d by t h e t a n n i n v a c u o l e . Immediately about t h e n u c l e u s i s a r e g i o n o f p e r i n u c l e a r c y t o p l a s m . The p e r i n u c l e a r c y t o p l a s m i s con n e c t e d by s e v e r - a l broad c y t o p l a s m i c s t r a n d s t h r o u g h t h e t a n n i n v a c u o l e t o the p e r i p h e r - a l c y t o p l a s m near t h e t r a b i c u l a e and/or the c e l l w a l l ( f i g u r e 9 shows two such c o n n e c t i o n s ) . In t y p e II c e l l s , t h i s a r e a o f c y t o p l a s m i s the o n l y a r e a where RER and c y t o p l a s m i c ribosomes were c o n s i s t a n t l y n o t e d . S e c t i o n s from t h i s a r e a o f c y t o p l a s m a l s o appeared t o have a h i g h e r p r o - b a b i l i t y o f c o n t a i n i n g p r o f i l e s o f a d i c t y o s o m e than o t h e r p a r t s o f the ee l 1 . Small o i l b o d i e s up t o t h r e e t o four/urn i n d i a m e t e r were a l s o a common o c c u r e n c e i n t h i s and o t h e r a r e a o f c y t o p l a s m o f t y p e I and t y p e s II e e l 1s ( f i g u r e 3 2 ) . In g e n e r a l , t h e n u c l e o l i appear t o be c i r c u l a r i n s e c t i o n a l p r o f i l e . The n u c l e o l u s i s predominent1y composed o f pars g r a n u l o s a e m a t e r i a l s u r - kl rounded by an unusual h e t e r o c h r o m a t i n - f r e e e l e c t r o n t r a n s p a r e n t r i n g o f n u c l e o p l a s m ( f i g u r e 30). In t y p e I c e l l s , as many as t h r e e n u c l e o - l a r p r o f i l e s were o b s e r v e d ; ( f i g u r e 31) i n s e c t i o n s . The number o f nu- c l e o l a r p r o f i l e s appears t o be u s u a l l y one and o c c a s i o n a l l y two per s e c t i o n i n t y p e II c e l l s ( f i g u r e 32). In t h i r d and f i f t h y e a r n e e d l e s , n u c l e o l i were r a r e l y o b s e r v e d . The n u c l e u s o f a l l c e l l t y p e s c o n t a i n s dense a r e a s o f h e t e r o c h r o m a - t i n , between w h i c h a r e a r e a s o f c l e a r n u c l e o p l a s m c o n s i s t i n g o f s c a t t e r - ed g r a n u l a r m a t e r i a l and v e r y f i n e f i b r i l l a r m a t e r i a l . The e x t e n t o f the h e t e r o c h r o m a t i n r e g i o n s i n t h e n u c l e u s a p pears t o s l i g h t l y i n c r e a s e i n o l d e r n e e d l e s ( c f . f i g u r e s 32 and 69). 43 D. E x p e r i m e n t s on Summer Ne e d l e s I t has been a l r e a d y noted t h a t t h e c h l o r o p l a s t s i n c r y o s e c t i o n e d summer n e e d l e s s w e l l i n t o s p h e r i c a l shapes ( f i g u r e 11). When t h e s e nee- d l e s were kept i n a f r e e z e r a t -18°C o r p l a c e d i n t o l i q u i d n i t r o g e n and then f i x e d f o r e l e c t r o n m i c r o s c o p y a t 4°C, images s i m i l a r t o t h a t shown in f i g u r e 72 a r e o b t a i n e d . An a r e a o f the c y t o p l a s m c o n t a i n i n g what appears t o be p l a s t o g l o b u l i and s t a r c h g r a i n s p r o b a b l y r e p r e s e n t s what may have been a c h l o r o p l a s t . No c l e a r c u t membrane b o u n d a r i e s can be seen. The m i x t u r e o f t a n n i n m a t e r i a l i n t h i s c e l l r e g i o n may i n d i c a t e t h e breakdown o f t h e t o n o p l a s t . Summer t i s s u e f r o z e n t o -18°C and then f i x e d i n t h e w i n t e r p r o c e - dure a t -4°C ( w i t h o u t thawing) a l s o was s e v e r e l y d i s r u p t e d but more c y t o - p l a s m i c membranes remain v i s i b l e . F o l l o w i n g t h i s t r e a t m e n t , c h l o r o p l a s t s were g r e a t l y s w o l l e n w i t h the e n v e l o p e o f t e n r u p t u r e d . A l t h o u g h t h e y s t i l l c o n t a i n e d t h y l a k o i d s , most o f them were e x t e n s i v e l y d i l a t e d . In t h e s e c e l l s , t h e t o n o p l a s t had a p p a r e n t l y r u p t u r e d and t a n n i n m a t e r i a l was p r e s e n t w i t h i n t h e c y t o p l a s m . The v a r i o u s o s m o t i c extremes used i n some o f t h e w i n t e r f i x a t i o n s were a l s o c a r r i e d o u t on summer t i s s u e s as c o n t r o l e x p e r i m e n t s . Ex- t e n s i v e p l a s m o l y s i s o f t h e p r o t o p l a s t was noted w i t h t h e f i x a t i v e con- t a i n i n g 10% s u c r o s e . In t h i s f i x a t i o n , t h e c y t o p l a s m appeared c r u s h e d o r compacted a g a i n s t t h e t a n n i n v a c u o l e and was v e r y o s m i o p h i l i c . The major c e l l o r g a n e l l e s however c o u l d be o b s e r v e d and they appeared t o be S t r u c t u r a l l y i n t a c t . C y t o p l a s m i c o r g a n e l l e s were s w o l l e n and com- p l e t e l y d i s r u p t e d by f i x a t i v e made up i n e i t h e r sea water o r d i s t i l l e d w a t e r . In both c a s e s , t a n n i n m a t e r i a l was a g a i n r e l e a s e d t h r o u g h o u t th e c y t o p l a s m and t h e t o n o p l a s t was not o b s e r v e d . I I I . Autumnal Changes i n t h e U l t r a s t f u c t u r e o f the Chlorenchyma F i x a t i o n s o f m a t e r i a l between August 20 and November 26, 1972 were not s a t i s f a c t o r y . M a t e r i a l : f i x e d on August 20th r e v e a l e d b a s i c a l l y a summer c o n d i t i o n w i t h i n t h e c e l l s . There a r e , however., some s l i g h t mod- i f i c a t i o n s compared t o e a r l i e r summer f i x a t i o n s : t he e l e c t r o n d e n s i t y , s i z e and number o f t h e o i l d r o p l e t s i n c r e a s e d above t h a t seen i n t h e e a r l y and mid-summer f i x a t i o n s ; t h e c h l o r o p l a s t s m a i n t a i n e d t h e i r even d i s t r i b u t i o n w i t h i n t h e c e l l but appeared t o be l o c a t e d . s l i g h t l y away from t h e plasma membrane. M a t e r i a l f i x e d i n l a t e November had the g e n e r a l u l t r a s t u r c t u r a l appearance o f t h e w i n t e r c o n d i t i o n . The Chlorenchyma i n t h e W i n t e r Cond i t ion A. L i g h t M i c r o s c o p y At t h e 1ight m i c r o s c o p y l e v e l , changes i n t h e n a t u r e and p o s i - t i o n o f c y t o p l a s m i c s t r u c t u r e s were o b s e r v e d i n the w i n t e r c o n d i t i o n . A photograph o f an unthawed s e c t i o n o f w i n t e r t i s s u e p r e p a r e d by t h e p r o c e d u r e o u t l i n e d i n A p p e n d i x II i s shown i n f i g u r e 33- The band o f p e r i p h e r a l granul.ar c y t o p l a s m (between a r r o w s ) i s s e v e r a l t i m e s g r e a t - e r , i n w i d t h , than o f comparable t i s s u e i n t h e summer c o n d i t i o n . Very l i t t l e d e t a i l i s d i s c e r n a b l e i n s i d e t h e c y t o p l a s m and t h e c h l o r o - p l a s t s l i n i n g t h e c e l l w a l l , (which c h a r a c t e r i z e d . s u m m e r t i s s u e p r e - pared by t h e same p r e p a r a t i v e p r o c e d u r e s ) , cannot be c l e a r l y v i s u a l - i z e d ( e . g . , c f . f i g u r e 7). The o n l y ; > r e c o g n i z a b l e s t r u c t u r e s a r e the. n u c l e i and t h e c l e a r t a n n i n v a c u o l e s w h i c h appear s i m i l a r i n s i z e and l o c a t i o n t o o b s e r v a t i o n s made on t h e s e s t r u c t u r e s i n c h e m i c a l l y f i x e d mater i a l . M e c h a n i c a l l y , t h i s u n f i x e d f r o z e n w i n t e r t i s s u e was more s u s c e p - t i b l e t o damage caused by t h e s t r e s s e s o f c r y o - o r f r e e h a n d s e c t i o n - i n g . S e c t i o n s tended t o c rumble o r t h e main v a c u o l e s o f t e n f e l l o ut as i n t a c t u n i t s when t h e s e c t i o n s were c u t . I f t h e s e c t i o n s a r e a l - lowed t o thaw w h i l e b e i n g o b s e r v e d , t h e t a n n i n v a c u o l e s seem t o r e t - a i n t h e i r shape, w h i l e the c y t o p l a s m i c a r e a s appear t o l i q u i f y w i t h t h e g r a n u l a r m a t e r i a l w i t h i n i t moving about r a p i d l y i n f l u i d con- v e c t i o n c u r r e n t s . 47 F i g u r e 34 shows a s e c t i o n o f s e v e r a l c e l l s from w i n t e r i n g p i n e c h l o - renchyma t h a t have been s t a i n e d by t h e Johansen.'s t a n n i n s t a i n s o l u t i o n as d e s c r i b e d i n A ppendix ,1. The w ide band o f c y t o p l a s m i s i n marked c o n - t r a s t t o t h e d e n s e l y s t a i n e d t a n n i n v a c u o l e s . U s i n g 1 yum s e c t i o n s o f p l a s t i c - e m b e d d e d m a t e r i a l s t a i n e d w i t h t o l - u i d i n e b l u e and s a f r a n i n , i t was p o s s i b l e t o observe- the s t r u c t u r e o f t h e w i n t e r c hlorenchyma c y t o p l a s m i n more d e t a i l ( f i g u r e 35). The most r e m a r k a b l e f e a t u r e i s t h e c l u m p i n g o f t h e c h l o r o p l a s t s . These clumps appeared t o be compartmented.'in a r e a s bounded by t h e t r a b i c u l a e , t h e c e l l w a l l s and by t h e t a n n i n v a c u o l e . C l o s e e x a m i n a t i o n o f each clump o f c h l o r o p l a s t s i n d i c a t e d i t t o be composed o f d e n s l y s t a i n e d i n t e r n a l mem- brane systems w i t h a d i s c o i d a l shape s i m i l a r t o t h a t o f t h e summer c h l o - r o p l a s t . S u r r o u n d i n g each membrane system i s a medium d e n s i t y homogen- eous a r e a w h i c h i s p r o b a b l y c o r r e s p o n d i n g t o the s t r o m a l r e g i o n o f the c h l o r o p l a s t . I t i s t h e s w o l l e n s t r o m a l a r e a s o f t h e c h l o r o p l a s t s w h i c h a r e r e s p o n s i b l e f o r the g e n e r a l l y c i r c u l a r o r i r r e g u l a r p r o f i l e s o f t h e s e clumped c h l o r o p l a s t s . In some c h l o r o p l a s t s , t h e i n t e r n a l t h y l a k o i d s y s - tem appears t o be t w i c e as l a r g e as t h e a d j a c e n t c h l o r o p l a s t s . The n u c l e u s i n t h e e e l 1 o f w i n t e r m a t e r i a l o c c u p i e d a d i f f e r e n t p o s i t i o n . In marked c o n t r a s t t o t h e summer s t a t e , i t does not p r o t r u d e i n t o the t a n n i n v a c u o l e . I n s t e a d , i t i s c o m p l e t e l y l o c a t e d w i t h i n t h e p e r i p h e r a l c y t o p l a s m ( f i g u r e s 33, 34 and 48). The w i n t e r n u c l e u s i s v a r i a b l e i n shape, but i s u s u a l l y o v o i d a l i n s e c t i o n a l p r o f i l e w i t h t h e major a x i s p a r a l l e l t o t h e p r o x i m a l c e l l w a l l . The w i n t e r n u c l e u s i s 48 u s u a l l y found In a s p e c i f i c p a r t o f t h e p e r i p h e r a l c y t o p l a s m . T h i s a r e a i s t h e same c e l l u l a r r e g i o n where the l a r g e r c h l o r o p l a s t clumps a r e f o u n d . The broad band o f p e r i p h e r a l c y t o p l a s m i n f i g u r e 35 appears as a n e t - work o f c l e a r v a c u o l e s and narrow s t r a n d s o f c y t o p l a s m . So e x t e n s i v e i s t h i s c y t o p l a s m i c v a c u o l a r system t h a t i t c o u l d be s u i t a b l y , d e s c r i b e d as 1 sponge-1 i k e ' . Small dense g r a n u l e s can o f t e n be seen i n t h e c y t o p l a s m near t h e c e l l w a l l (open arrowhead, f i g u r e 35). C r y o s e c t i o n s s t a i n e d w i t h Sudan B l a c k B r e v e a l o i l d r o p l e t s u s u a l l y r a n g i n g between 5 and 10 yum i n d i a m e t e r ( f i g u r e 36). They a r e m a i n l y i n th e p e r i n u c l e a r r e g i o n . The volume o f t h e l a r g e s t s u d a n o p h i l i c d r o p l e t s i s a p p r o x i m a t e l y t e n t i m e s t h e ave r a g e s i z e o f t h o s e o b s e r v e d i n t h e sum- mer c o n d i t i o n w h i l e l a r g e r o i l b o d i e s a r e found near the n u c l e u s . S m a l l e r d r o p l e t s , o f t e n t h e s i z e o f t h o s e i n summer c o n d i t i o n a r e u s u a l l y found s c a t t e r e d t h r o u g h o u t t h e c y t o p l a s m . O i l d r o p l e t s a r e a l s o more e x t e n s i v e i n t h e endodermis and t r a n s f u s i o n parenchyma than i n summer. 49 B. O b servat i o n s on the V/inter Cond i t i o n : E l e c t r o n M i c r o s c o p y The e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s o f t h e w i n t e r c hlorenchyma c o n f i r m t h e p r o t o p l a s m i c s t r u c t u r e o b s e r v e d i n l i g h t m i c r o s c o p y . Fig/- u r e s 37 t o 40 r e p r e s e n t v i e w s o f the p r o t o p l a s m i c s t r u c t u r e i n w i n t e r c hlorenchyma c e l l s o b t a i n e d from v a r i o u s p l a n e s o f s e c t i o n i n g . These f i g u r e s a r e low m a g n i f i c a t i o n m i c r o g r a p h s showing s e c t i o n a l p r o f i l e s o f n e a r l y e n t i r e c e l l s . They r e v e a l how t h e p o s i t i o n o f t h e c h l o r o p l a s t clumps i s r e l a t e d t o t h e n a t u r e o f t h e c e l l w a l l . F i g u r e 37 shows s e v e r a l c e l l s s e c t i o n e d i n a p l a n e s i m i l a r t o t h e i l l u s t r a t i o n i n f i g u r e 5. There a r e s e v e r a l s m a l l c h l o r o p l a s t clumps shown i n t h e c y t o p l a s m a d j a c e n t t o the c e l l w a l I s c o r r e s p o n d i n g t o the c e l l w a l l f a c e : B i n f i g u r e 2. From t h e s e faces' o f .the c e l l w a l l , t h e s m a l l t r a b i c u l a e i n v a g i n a t e . A l s o shown i n t h i s s e c t i o n a r e the c e l l w a l l s c o r r e s p o n d i n g t o f a c e A i n f i g u r e 2. The c y t o p l a s m next t o t h e s e w a l l s i s marked w i t h a s t e r i s k s , i n f i g u r e 37- Note t h a t c h l o r o p l a s t clumps a r e a b s e nt i n the c y t o p l a s m a d j a c e n t t o t h e s e f a c e s " o f t h e c e l l wa 11. F i g u r e 39 r e p r e s e n t s p o r t i o n s o f two c e l l s s e c t i o n e d i n a p l a n e s i m i l a r t o t h e i l l u s t r a t i o n i n f i g u r e 3- A s h o r t t r a b i c u l u m i s shown i n v a g i n a t i n g from t h e w a l l f a c e B i n each c e l l . A d j a c e n t t o the t r a - b i c u l a e a r e s m a l l c h l o r o p l a s t clumps and i n t h e lower c e l l , a l a r g e o i l body i s a l s o a d j a c e n t t o t h e t r a b i c u l u m . To the l e f t o f f i g u r e 39 i s an a r e a c o r r e s p o n d i n g t o t h e end r e g i o n o f t h e lower c e l l ; i . e . , a d j a - 50 c e n t to t h e c e l l face''' l a b e l l e d C i n f i g u r e 2. Note the c h l o r o p l a s t clumps i n t h i s a r e a . N e a r l y an e n t i r e c e l l s e c t i o n i s shown i n f i g u r e 38. T h i s s e c t i o n i s i n t h e s e c t i o n a l p l a n e o f the c e l l s i m i l a r t o t h e i l l u s t r a t i o n i n f i g u r e 6. Note t h e p r e s e n c e o f l a r g e numbers o f c h l o r o p l a s t s near the ends o f the c e l l ( t o t h e l e f t 'and'- r i g h t ) near t h e c e l l w a l l s c o r r e s - ponding t o f a c e C i n f i g u r e 2. Note the r e l a t i v e l y few c h l o r o p l a s t s i n t h e c y t o p l a s m a d j a c e n t t o t h e c e l l w a l l s c o r r e s p o n d i n g t o the c e l l w a l l f a c e s l a b e l l e d A i n f i g u r e 2. F i g u r e hO i s a s e c t i o n o f s e v e r a l c e l l s i n t h e s e c t i o n a l p l a n e o f t h e c e l l s i m i l a r t o t h a t i l l u s t r a t e d i n f i g u r e 4. In t h i s p l a n e , t h e s i z e o f t h e c h l o r o p l a s t clumps l o c a t e d i n t h e c y t o p l a s m a d j a c e n t ;to t h e end w a l l s o f the c e l l becomes a p p a r e n t . S t u d i e s o f t h e w i n t e r m e s o p h y l l chlorenchyma i n v a r i o u s p l a n e s o f s e c t i o n i n g r e v e a l t h a t the d i s t r i b u t i o n o f c h l o r o p l a s t s i s r e l a t e d t o t h e n a t u r e o f the a d j a c e n t c e l l w a l l . Very few c h l o r o p l a s t s a r e found a d j a c e n t t o the c e l l f a c e s l a b e l l e d A i n f i g u r e 2 where no t r a b i c u l a e i n v a g i n a t e . Small c h l o r o p l a s t clumps ( u s u a l l y two t o t e n o r g a n e l l e s ) a r e found i n t h e c y t o p l a s m a d j a c e n t t o ^ t h e e e l 1 wal 1 f a c e s l a b e l l e d B i n f i g u r e 2 where th e s h o r t t r a b i c u l a e o c c u r . Large c h l o r o p l a s t clumps composed o f up t o twenty or t h i r t y c h l o r o p l a s t s a r e found i n t h e c y t o - plasm a d j a c e n t t o t h e c e l l w a l l f a c e s l a b e l l e d C i n f i g u r e 2. Long t r a - 51 b i e u l a e i n v a g i n a t e from t h i s p a r t o f . t h e . c e l l w a l l . In a d d i t i o n t o the c l u m p i n g phenomena, the w i n t e r c h l o r o p l a s t s have s t r u c t u r a l d i f f e r e n c e s from summer c h l o r o p l a s t s . At low m a g n i f i c a t i o n , the c h l o r o p l a s t s appear bounded by a d i s t i n c t , d a r k boundary. At h i g h e r m a g n i f i c a t i o n s , t h e n a t u r e o f t h e c h l o r o p l a s t e n v e l o p e . c a n be somewhat.- r e s o l v e d ( f i g u r e 4 7 , C E ) . The two bounding membranes o f the c h l o r o p l a s t e n v e l o p e , e a s i l y d i s t i n g u i s h e d i n summer c h l o r o p l a s t s , a r e c o n s i s t e n t l y i n d i s t i n c t i n a l l w i n t e r c h l o r o p l a s t s f i x e d by v a r i o u s . m e t h o d s . D i l a - t i o n o r s w e l l i n g o f t h e inter-membrane space o f the c h l o r o p l a s t e n v e l o p e was never o b s e r v e d i n t h e w i n t e r o b s e r v a t i o n s . I t appears t h e r e f o r e , t h a t the two e n v e l o p e membranes a r e c o n s i s t e n t l y p a r a l l e i t o each o t h e r . F u r t h e r m o r e , e n v e l o p e s o f a d j o i n i n g c h l o r o p l a s t s appear t o be e x t e n s i v e l y i n c o n t a c t w i t h one a n o t h e r (see f i g u r e s 4 2 , 45 and 4.6). A t r i p l e - m e m - brane appearance o f t h e c o n t a c t r e g i o n s between the c h l o r o p l a s t s can be seen i n p l a c e s ( f i g u r e 46), c h a r a c t e r i z e d by a t h i c k c e n t r a l membrane and two t h i n n e r o u t e r membranes. T h i s t r i p l e l a y e r e d membrane system p r o - b a b l y r e p r e s e n t s t h e p a r t i a l f u s i o n a r e a between clumped c h l o r o p l a s t s and can o f t e n be seen a t lower m a g n i f i c a t i o n s ( a r r o w s , f i g u r e 4 2 ) . When th e r e g i o n s o f c h l o r o p l a s t adherence a r e c u t t a n g e n t i a l l.y t o the membrane s u r f a c e , the c o n t a c t r e g i o n appears as a homogeneous dense band ( f i g u r e 4 3 ) . As noted a t t h e l i g h t m i c r o s c o p i c l e v e l , t he t h y l a k o i d systems o f the w i n t e r c h l o r o p l a s t s r e t a i n t h e d i s c o i d a l shape o f t h e summer c h l o r o p l a s t . The arrangement and s i z e o f t h e s t r o m a l and g r a n a l t h y l a k o i d membranes 52 a l s o remain d i s t i n c t and s i m i l a r t o the summer c o n d i t i o n ( f i g u r e 47)• However, i n c o n t r a s t t o t h e summer c o n d i t i o n , two and sometimes t h r e e c o m p l e t e t h y l a k o i d systems a r e found w i t h i n a s i n g l e e n v e l o p e o f w i n t e r c h l o r o p l a s t s . O c c a s i o n a l l y , two c h l o r o p l a s t s appeared, t o be j o i n e d by a c o n t r i c t i o n o r isthmus o f stroma ( f i g u r e 44). When two o r more t h y - l a k o i d systems a r e found w i t h i n a common e n v e l o p e , thay may be a r r a n g e d a t v a r i o u s a n g l e s t o each o t h e r ( a s t e r i s k s , f i g u r e 43). In a l l c a s e s , however, the t h y l a k o i d systems a r e not i n c o n t i n u i t y and a r e always s e p a r a t e d from each o t h e r by an a r e a o f stroma ( f i g u r e 43). The stroma i s v e r y e x t e n s i v e i n w i n t e r c h l o r o p l a s t s and, i n g e n e r a l , t h i s expanse o f stroma a c c o u n t s f o r n e a r l y , h a l f t h e s e c t i o n a l a r e a o f the c h l o r o p l a s t ( f i g u r e s 39, 43 and 47). W i n t e r c h l o r o p l a s t s t h a t have o n l y one i n t e r n a l membrane system w i t h i n t h e e n v e l o p e appear t o be about f i f t y p e r c e n t g r e a t e r i n volume than summer c h l o r o p l a s t s . T h i s volume- t r i c i n c r e a s e i s due t o an i n c r e a s e i n t h e w i d t h o f .winter c h l o r o p l a s t s w i t h o u t a c o r r e s p o n d i n g d e c r e a s e i n l e n g t h ( i . e . , t he c h l o r o p l a s t s s w e l l s u b s t a n t i a l l y i n w i n t e r ) . P l a s t o g l o b u l i have, i n g e n e r a l , . b e e i i . n o t e d i n summer/material t o i n - c r e a s e s l i g h t l y i n s i z e and numbers w i t h i n c r e a s i n g n e e d l e age. T h i s r e l a t i o n s h i p a l s o appears t o be o c c u r i n g i n w i n t e r w i t h o l d e r w i n t e r c h l o r o p l a s t s c o n t a i n i n g l a r g e r and s l i g h t l y more p i a s t o g l o b u l ? ( c f . f i g u r e s 43 and 44). T h i s r e l a t i o n s h i p has many s p e c i f i c e x c e p t i o n s however, as was noted i n the summer o b s e r v a t i o n s . In w i n t e r , t h e p l a s t o g l o b u l i a r e randomly d i s t r i b u t e d w i t h i n t h e c h l o r o p l a s t i n t h e s w o l l e n a r e a s o f the 53 stroma as w e l l as i n the stroma i m m e d i a t e l y around the t h y l a k o i d s ( f i g - u r e s 42, 45 and 4 7 ) . The s t r u c t u r a l and p o s i t i o n a l t r a n s f o r m a t i o n s o f t h e c h l o r o p l a s t s r e p r e s e n t the most d r a m a t i c changes i n ' t h e w i n t e r p r o t o p l a s t s , but o t h e r p r o t o p l a s m i c s t r u c t u r e s a l s o undergo s e a s o n a l changes. The m a t r i x o f the t a n n i n v a c u o l e appeared t o c o n t r a c t i n w i n t e r m a t e r i a l . T h i s con- t r a c t i o n i s a s s o c i a t e d w i t h an a p p r o x i m a t e hundred p e r c e n t v o l u m e t r i c i n c r e a s e i n t h e p e r i p h e r a l c y t o p l a s m ( f i g u r e 3 4 , 3 5 , 36 and 45)- The t a n n i n - c y t o p l a s m i c b o r d e r when viewed i n s e c t i o n s o f e n t i r e c e l l s i s more rounded and conforms l e s s t o the c o n t o u r s o f the c e l l w a l l s ( f i g - u r e s 37, 3 8 , 39 and 4 8 ) . The t e x t u r e o f the t a n n i n m a t e r i a l i s s i m i l a r t o t h a t o b s e r v e d i n t h e summer and y i e l d e d s i m i l a r s h a t t e r a r t i f a c t s w h e n ' s e c t i o n e d . O c c a s i o n a l l y , smal1 b l e b s o f t a n n i n m a t e r i a l appear t o be s e p a r a t e d from t h e main d e p o s i t ( a r r o w h e a d ( s ) , f i g u r e s 39 and 4 0 ) . The t o n o p l a s t , presumably about t h e t a n n i n m a t e r i a l , c o u l d not be c l e a r l y v i s u a l i z e d i n w i n t e r f i x a t i o n s . In w i n t e r , the c y t o p l a s m i s c o n f i n e d t o the c e l l p e r i p h e r y between th e t a n n i n v a c u o l e and the c e l l w a l l and no c y t o p l a s m i c s t r a n d s . a s i ^ d e s - c r i b e d f o r summer c o n d i t i o n i W e r e o b s e r v e d . The w i n t e r p e r i p h e r a l c y t o - plasm i s o f i r r e g u l a r w i d t h ; t h e w i d e s t a r e a s a r e u s u a l l y l o c a t e d i n t h e p o c k e t s formed by t h e l o n g t r a b i c u l a e a t the end o f the c e l l ( f i g u r e s 3 8 , 39 and 4 0 ) . The n a r r o w e s t p o r t i o n s o f c y t o p l a s m o c c u r a l o n g t h e c e l l w a l l s c o r r e s p o n d i n g t o f a c e s A o f f i g u r e 2 ( f i g u r e s 37 and 3 8 ) . 54 In t h e narrower c y t o p l a s m i c r e g i o n s , the c y t o p l a s m i s dominated by ma s s i v e c y t o p l a s m i c v a c u o l a t i o n . These y a c u o l i z e d a r e a s can be d e s c r i b - ed as a p p e a r i n g 'foamy' or'sponge-1ike' and c o n t a i n l i t t l e c y t o p l a s m i c m a t r i x and v e r y few o r g a n e l l e s (see a s t e r i s k s , f i g u r e 48). Large numbers o f v a c u o l e s a r e found about and between t h e c h l o r o - p l a s t clumps i n t h e end r e g i o n s o f t h e c e l l ( a s t e r i s k s , f i g u r e s 41 and 50) and between a r e a s where c y t o p l a s m i c o r g a n e l l e s o c c u r and the plasma membrane ( f i g u r e s 49 and 60). V a c u o l e s a r e not common, however, i n t h e c y t o p l a s m i m m e d i a t e l y a d j a c e n t t o the n u c l e u s and the c h l o r o p l a s t clumps. The n o n - v a c u o l a t e d c y t o p l a s m i n t h e s e a r e a s c o n t a i n s the o c c a s i o n a l polysomes a n d ; o t h e r c e l l o r g a n e l l e s (GC, f i g u r e s 41, 42 and 57). A n o t h e r prominent f e a t u r e o f t h e c y t o p l a s m i s t h e p r e s e n c e o f v e r y l a r g e numbers o f o s m i o p h i l i c b o d i e s . The o s m i o p h i l i c b o d i e s a r e u s u a l l y found i n t h e a r e a s t h a t abound w i t h v a c u o l e s and i n some c a s e s , t h e o s - m i o p h i l i c m a t e r i a l c o n s t i t u t e s t h e b u l k o f the v a c u o l a r m a t e r i a l (OB, f i g u r e 49). The o s m i o p h i l i c m a t e r i a l may a l s o form a t h i n l a y e r l i n i n g t h e i n t e r i o r f a c e o f the c y t o p l a s m i c v a c u o l e (CV and arrowheads, f i g u r e 49). In some s e c t i o n s , a l m o s t e v e r y c y t o p l a s m i c v a c u o l e a p p ears t o con- t a i n an o s m i o p h i l i c body ( f i g u r e s 51 and 52), w h i l e i n o t h e r s e c t i o n s , t h e r e a r e few o s m i o p h i l i c b o d i e s a s s o c i a t e d w i t h the c y t o p l a s m i c v a c u o l e s ( f i g u r e s 50 and 51)• In t he r e g i o n s o f t h e c e l l where the m a j o r i t y o f t h e c h l o r o p l a s t c l u m p s 55 o c c u r and where t h e c y t o p l a s m i s not h i g h l y v a c u o l a t e d , the o s m i o p h i l i c b o d i e s a r e found i n v a c u o l e s a r r a n g e d i n a f i l e o r t i e r a d j a c e n t t o t h e plasma membrane ( f i g u r e s 51 and 5 2 ) . I t i s i n t e r e s t i n g t o note t h a t t h e c y t o p l a s m i c , v a c u o l e s between t h e c h l o r o p l a s t s and the t a n n i n m a t e r i a l (not shown i n f i g u r e 51 ) c o n t a i n v e r y few o s m i o p h i l i c b o d i e s ( d o u b l e end- ed a r r o w ) . I t seems t h a t t h e c y t o p l a s m i c v a c u o l e s appear t o c o n s i s t e n t l y have t h e i r l o n g a x i s p a r a l l e l t o each o t h e r ( f i g u r e 51). C o n f i g u r a t i o n s such as t h i s appear t o i n d i c a t e the s h r i n k a g e o f t a n n i n m a t e r i a l c a u s i n g t h e s t r e t c h i n g o f t h e c y t o p l a s m i c v a c u o l e s . L i p i d i n c l u s i o n s appear t o b u i l d up s i g n i f i c a n t l y i n w i n t e r ( f i g r u r e 3 6 ) . F i g u r e 39 shows a l a r g e o i l body (0) about 5 t o 7 Aim i n diame- t e r , many o f t h e o i l b o d i e s a r e l o c a t e d i n t h e p e r i - n u c l e a r c y t o p l a s m ( f i g u r e s 55 and 5 8 ) . The s i z e , number and d i s t r i b u t i o n o f t h e l a r g e . o i l i n c l u s i o n s noted i n w i n t e r t i s s u e a t the u l t r a s t r u c t u r a l l e v e l a g r e e v e r y w e l l w i t h t h e s i z e , number and d i s t r i b u t i o n o f s u d a n o p h i l i c b o d i e s seen a t the l i g h t m i c r o s c o p y level.-. The m a j o r i t y o f m i t o c h o n d r i a i n t h e w i n t e r c h l o r e n c h y m a l c y t o p l a s m a r e l o o s e l y a s s o c i a t e d w i t h t h e c h l o r o p l a s t clumps ( f i g u r e 3 7 ) . M i t o c h o n - d r i a a r e a l s o o c c a s i o n a l l y l o c a l i z e d near the l a r g e o i l i n c l u s i o n s ( f i g - u r e 5 8 ) . o r a r e randomly d i s t r i b u t e d t h r o u g h o u t the v a c u o l a t e d c y t o p l a s m ( f i g u r e 4 8 ) . The m i t o c h o n d r i a a r e s m a l l e r than i n summer n e e d l e c h l o r e n - chyma and a r e p r e d o m i n e n t l y c i r c u l a r i n s e c t i o n a l p r o f i l e s i n d i c a t i n g a s p h e r i c a l , shape. :As w i t h w i n t e r c h l o r o p l a s t s , the two membranes o f t h e 56 m i t o c h o n d r i a l e n v e l o p e a r e v e r y i n d i s t i n c t and u s u a l l y appear as a s i n - g l e dense l i n e a t low m a g n i f i c a t ion ( f i g u r e 53)- There i s , however, no s i g n o f f u s i o n between m i t o c h o n d r i a . When m i t o c h o n d r i a come i n c l o s e p r o x i m i t y t o one a n o t h e r o r t o c h l o r o p l a s t s , a d i s t i n c t gap was v i s i b l e between t h e i r e n v e l o p e s ( e . g . f i g u r e s 43 and 58, a r r o w s ) . A l t h o u g h some m i t o c h o n d r i a s t i l l have c l e a r and e x t e n s i v e t u b u l a r c r i s t a e i n w i n t e r ( f i g u r e 58), the m a j o r i t y o f m i t o c h o n d r i a appear t o have more p o o r l y d e v e l o p e d c r i s t a e than t h o s e found in- t h e o r g a n e l l e s i n summer ( f i g u r e 53). Dictyosomes were not common i n mature m e s o p h y l l , chlorenchyma t y p e II c e l l s i n both summer and w i n t e r f i x a t i o n s , but t h o s e p r e s e n t i n t h e w i n t e r c o n d i t i o n were c h a r a c t e r i z e d as h a v i n g v e r y few d i c t y o s o m a l v e s - i c l e s a s s o c i a t e d w i t h them ( f i g u r e 54). The few w i n t e r d i c t y o s o m e s o b s e r v e d were found c l o s e t o t h e n u c l e u s o r t h e c h l o r o p l a s t clumps. As noted p r e v i o u s l y , the n u c l e u s changes i t s p o s i t i o n w i t h i n t h e c e l l from a c e n t r a l l o c a t i o n p a r t i a l l y s u r r o u n d e d by t h e t a n n i n v a c u o l e ( i n summer) t o a more p e r i p h e r a l l o c a t i o n near t h e c e l l w a l l s d u r i n g w i n t e r . In summer, t h e n u c l e u s appeared c o n s i s t e n t l y c i r c u l a r i n s e c - t i o n a l p r o f i l e w h i l e i n w i n t e r , t h e most common s e c t i o n a l shape was ov- o i d ( f i g u r e 57) w i t h t h e long a x i s p a r a l l e l t o the c e l l w a l l ( f i g u r e 48). The two l a y e r s o f membranes o f the w i n t e r c e l l n u c l e u s a r e d i s t i n c t 57 and p a r a l l e l w i t h each o t h e r and t h e r e i s no e v i d e n c e o f s w e l l i n g ( f i g - u r e 56). Numerous n u c l e a r pores a r e p r e s e n t ( f i g u r e 5$) and c o n t a i n an amorphous dense m a t e r i a l . Comparison between t h e s t r u c t u r e o f t h e w i n - t e r n u c l e u s a t the l i g h t m i c r o s c o p y and e l e c t r o n m i c r o s c o p y l e v e l s i s i 1 1 u s t r a t e d by f i g u r e s 55 and 57. The d a r k l y s t a i n e d h e t e r o c h r o m a t i c r e g i o n s shown a t t h e l i g h t m i c r o s c o p y l e v e l i n f i g u r e 55 can be seen t o be c o n s i d e r a b l y s m a l l e r and e v e n l y s p a c e d - w i t h i n t h e n u c l e o p l a s m i n con- t r a s t t o the c o u n t e r p a r t r e g i o n s i n summer n u c l e i ( f i g u r e 32). At the u l t r a s t r u c t u r a l l e v e l ( f i g u r e 57), the d a r k e r c h r o m a t i n r e g i o n s o f the n u c l e u s as d e s c r i b e d a t t h e l i g h t m i c r o s c o p y l e v e l have a f i n e g r a n u l - ar t e x t u r e and a r e c o n s i d e r a b l y more d i f f u s e than t h e h e t e r o c h r o m a t i n of the s p r i n g o r summer n u c l e u s . In t h e l i g h t l y s t a i n e d n u c l e o p . l a s m i c ; r e g i o n s , f i n e f i b r i l l a r m a t e r i a l can be seen ( f i g u r e 56). One o r two c i r c u l a r n u c l e o l i were u s u a l l y n o t i c e d i n s e c t i o n a l p r o - f i l e s o f w i n t e r n u c l e i . The n u c l e o l i i n w i n t e r c o n d i t i o n were s t r u c t - u r a l l y s i m i l a r t o t h o s e found i n t h e summer t i s s u e ( f i g u r e 56). The un- u s u a l l e s s dense n u c l e o p l a s m i c a r e a about the n u c l e o l u s seen i n t h e sum- mer t i s s u e ( f i g u r e 30) was not o b s e r v e d . i n w i n t e r f i x a t i o n s . 58 C. W i n t e r M a t e r i a l F i x e d Wi t h D i f f e r i n g F i x a t i v e Sol tit i o n s When w i n t e r c hlorenchyma c e l l s were f i r s t examined, i t was thought t h a t the w i n t e r c y t o p l a s m i c c o n f o r m a t i o n was a t l e a s t p a r t l y induced by o s m o t i c p r e s s u r e damage caused d u r i n g f i x a t i v e p r o c e d u r e s . A s e r i e s o f f i x a t i v e s o l u t i o n s o f v a r y i n g o s m o l a r i t i e s was then made and a p p l i e d t o w i n t e r m a t e r i a l . The r e s u l t s o f t h e s e e x p e r i m e n t s i n d i c a t e d t h a t t h e w i n t e r m a t e r i a l f a i l e d t o v a r y s i g n i f i c a n t l y i n s t r u c t u r a l appearance w i t h any o f t h e f i x a t i v e s o l u t i o n s used. These e x p e r i m e n t s were then ex- panded t o i n c l u d e not o n l y extreme v a r i a t i o n s i n f i x a t i v e o s m o l a r i t y , but a l s o t he use o f e l e c t r o l y t e o r n o n e l e c t r o l y t e a d d i t i v e s i n f i x a t i o n med- i a . An u n b u f f e r e d h y p o t o n i c f i x a t i v e s o l u t i o n was made by u s i n g one p e r c e n t g1utara1dehyde and 0.5% f o r m a l i n i n d i s t i l l e d w a t e r . T h i s f i x a - t i v e s o l u t i o n was below 400 mOsm. T i s s u e f i x e d i n t h i s f i x a t i v e c o u l d not be d i s t i n g u i s h e d from w i n t e r t i s s u e p r e p a r e d by the s t a n d a r d w i n t e r f i x a t i o n p r o c e d u r e s . A v e r y h y p e r t o n i c f i x a t i v e s o l u t i o n was a l s o p r e p a r e d u s i n g sea water and 2% g 1 u t a r a l d e h y d e a n d 0.5% f o r m a l i n as a p r i m a r y f i x a t i v e w i t h - out any b u f f e r o r s u c r o s e i n t h e s o l u t i o n . The o s m o l a r i t y o f t h i s hy- p e r t o n i c e l e c t r o l y t e s o l u t i o n was much g r e a t e r than 1, 050 mOsm. T h i s s o l u t i o n caused some n o t i c e a b l e but minor changes i n t h e u l t r a s t r u c t u r e o f the w i n t e r c h l o r e n c h y m a . 59 In f i g u r e 60, i t can be seen t h a t t h e c h l o r o p l a s t s a r e e l o n g a t e d , but the s t r o m a l m a t e r i a l i s somewhat reduced. In g e n e r a l , t h e a r e a s o f c o n t a c t between the e n v e l o p e s o f a d j a c e n t c h l o r o p l a s t s a r e a l s o reduced ( f i g u r e 6 0 ) . The o s m i o p h i l i c b o d i e s i n t h e c y t o p l a s m appear t o change t h e i r shape from c i r c u l a r forms t o a s t e l l a t e p r o f i l e ( f i g u r e 6 1 ) . The major c y t o p l a s m i c f e a t u r e s t h a t a r e c h a r a c t e r i s t i c o f the w i n t e r f i x a - t i o n s were u n a f f e c t e d by sea w a t e r e l e c t r o l y t e s i n t h e f i x a t i v e s o l u t i o n . A t h i r d t y p e o f f i x a t i v e , t h e n o n - e l e c t r o l y t e f i x a t i v e s o l u t i o n was made up o f 10% s u c r o s e i n a d d i t i o n t o the s t a n d a r d f i x a t i v e s o l u - t i o n . No i d e n t i f i a b l e changes i n w i n t e r c h l o r e h c h y m a l u l t r a s t u r c t u r e was n o t e d w i t h t h i s f i x a t i v e . C o n t r o l s t u d i e s w i t h the above f i x a t i v e s o l u t i o n s were co n d u c t e d on summer t i s s u e from the f i e l d c o l l e c t i n g s i t e s o r from mature n e e d l e s of'' s e e d l i n g s grown under o p t i m a l growth c o n d i t i o n s i n a growth chamber. The f i x a t i v e s o l u t i o n w i t h o u t b u f f e r and t h e s o l u t i o n c o n t a i n i n g sea w a t e r as an a d d i t i v e caused c o m p l e t e d i s r u p t i o n o f the summer c y t o p l a s m . The f i x a t i v e s o l u t i o n c o n t a i n i n g ten p e r c e n t s u c r o s e as an a d d i t i v e r e - s u l t e d i n an i n f e r i o r f i x a t i o n t o m a t e r i a l f i x e d w i t h t h e s t a n d a r d sum- mer f i x a t i v e s o l u t i o n l a r g e l y due t o e x t e n s i v e p l a s m o l y s i s o f the p r o t o - p l a s t . 60 D. E x p e r i m e n t s wi t h Wi n t e r N e e d l e s F r o z e " and Thawed Under F i e l d and L a b o r a t o r y Cond i t io n s I t was found t h a t n e e d l e s c o l l e c t e d i n mid w i n t e r m a i n t a i n e d t h e i r v i a b i l i t y f o r a t l e a s t two months when s t o r e d i n a commercial deep f r e e z e a t -18°C. V i a b i l i t y was t e s t e d by t h e c y t o l o g i c a l methods o u t l i n e d i n the m a t e r i a l and methods s e c t i o n . A t t e m p t s t o s t o r e n e e d l e s i n t h e f r e e - z e r s e c t i o n o f a r e f r i g e r a t o r whose f r e e z e r t e m p e r a t u r e v a r i e d a t l e a s t between -3°C and -8°C were not s u c c e s s f u l . A f t e r one month i n t h e f r e e - z e r compartment o f t h e r e f r i g e r a t o r , no n e e d l e s c o u l d be termed v i a b l e . M a t e r i a l c o l l e c t e d i n mid w i n t e r a t -11°C and t r a n s p o r t e d t o Van- c o u v e r a t s l i g h t l y below f r e e z i n g t e m p e r a t u r e s f a i l e d t o s u r v i v e immer- s i o n i n t o l i q u i d n i t r o g e n . When m a t e r i a l s t o r e d i n t h e c h e s t f r e e z e r f o r s e v e r a l weeks t o f o u r months was p l a c e d i n t o l i q u i d n i t r o g e n and then had t h e v i a b i l i t y t e s t c onducted on t h e n e e d l e s , t h e r e s u l t s were p o s i t i v e . Some o f the c h e s t f r e e z e r s t o r e d m a t e r i a l and a l s o t h e c h e s t f r e e - z e r m a t e r i a l w h i c h had been i n l i q u i d n i t r o g e n was warmed t o about -k°C and f i x e d f o r e l e c t r o n m i c r o s c o p y by the s t a n d a r d w i n t e r p r o c e d u r e . The us u a l w i n t e r s t a t e o f t h e w i n t e r c h l o r e n c h y m a l c y t o p l a s m p r e v i o u s l y des- c r i b e d was o b s e r v e d . The o n l y changes were i n m a t e r i a l t h a t had been i n l i q u i d n i t r o g e n . F i g u r e 62 shows t h a t i n t h i s m a t e r i a l t h e o s m i o p h i l i c b o d i e s have two d i s t i n c t r e g i o n s , a l i g h t o u t e r a r e a and a more e l e c t r o n dense i n n e r c o r e . 61 When mid w i n t e r m a t e r i a l c o l l e c t e d i n t h e f i e l d was p l a c e d i n a growth chamber under summer t e m p e r a t u r e and l i g h t regimes and then ex- amined by l i g h t m i c r o s c o p y , s t a r c h appeared i n t h e c h l o r o p l a s t s and t h e c h l o r o p l a s t clumps become l o o s e a g g r e g a t e s w i t h i n f o u r days. A f t e r e i g h t days i n t h e growth chamber, t h e c h l o r o p l a s t s were e v e n l y d i s t r i - buted a l o n g t h e c e l l and t r a b i c u l a r w a l l s when t i s s u e was examined by l i g h t m i c r o s c o p y . A f t e r about two weeks i n the growth chamber, t h e nee- d l e s s t a r t e d t o become c h l o r o t i c a t t h e n e e d l e t i p s and then d i e d i n s e v e r a l days. In w i n t e r , the n e e d l e s o f P i n u s c o n t o r t a g r o w i n g i n t h e c o l l e c t - i n g a r e a s had changed from a summer green c o l o u r t o y e l l o w w i t h o n l y a l i t t l e p a l e green i n t h e n e e d l e ' s c o l o r a t i o n . T h i s c o l o u r change was most pronounced i n n e e d l e s w i t h f u l l sun e x p o s u r e s ( t h e o n l y n e e d l e s , used i n t h i s w o r k ) . A s i m i l a r but l e s s e x t e n s i v e c o l o u r change was n o t - ed i n t h e w i n t e r f o l i a g e o f P i n u s a l b i c a u l i s l a n d Chamecypar?s n o o t k a t e n - . s i s g r o w i n g on W h i s t l e r m ountain. A c h l o r o t i c c o l o r a t i o n was a l s o noted i n specimens o f P. c o n t o r t a sspC' c o n t o r t a g r o w i n g n a t u r a l l y i n t h e "Her- on Swamp" a r e a o f the U n i v e r s i t y o f B r i t i s h Columbia endowment l a n d s i n mid w i n t e r . Marked w i n t e r c o l o u r changes were not n o t i c e d i n t h e f o l i - age o f o t h e r c o n i f e r s p e c i e s g r o w i n g i n t h e c o l l e c t i n g a r e a s . I t i s i n t e r e s t i n g t o note t h a t a f t e r s e v e r a l days i n t h e summer c o n d i t i o n s o f the growth chamber, t h e n e e d l e s o f p r e v i o u s l y f r o z e n P i n u s contorta ^ S P 1 a t i f o l i a f o K i a g e began t o t u r n t o the .summer-green c o l o u r . 62 The above e x p e r i m e n t s and o b s e r v a t i o n s i n d i c a t e t h a t a f t e r summer c o n d i t i o n s i n a growth chamber f o r s e v e r a l d a y s , the arrangement o f t h e c h l o r o p l a s t s a t l e a s t b e g i n s t o r e v e r t t o t h e summer c o n d i t i o n i n p r e - v i o u s l y f r o z e n n e e d l e s . Two f i x a t i o n s c onducted i n t h e f i e l d on March the t h i r d , 1973 d e m o n s t r a t e d t h a t t h e w i n t e r c y t o p l a s m i c s t a t e i s s t a b l e a t above f r e e z i n g t e m p e r a t u r e s f o r a t l e a s t s e v e r a l h o urs. N e e d l e s f i x - ed a t dawn on t h i s d a t e were f i x e d a t t e m p e r a t u r e s o f between ~3°C and -6°C. T h i s f i x a t i o n showed t h e normal w i n t e r u l t r a s t r u c t u r e f o r t h e ch l o r e n c h y m a . L a t e r i n t h e day, a second f i x a t i o n was con d u c t e d between two and f o u r p.m. when t h e a i r t e m p e r a t u r e was +k t o +6°C. Due t o the b r i g h t s u n s h i n e and l a c k o f w i n d , t h e t e m p e r a t u r e o f the m i c r o - e n v i r o n m e n t about the sun exposed n e e d l e s w h i c h were f i x e d was p r o b a b l y c o n s i d e r a b l y h i g h e r than t h e ambient a i r t e m p e r a t u r e . The n e e d l e s were f i x e d i n s o l u t i o n s i n e q u i l i b r i u m w i t h the a i r t e m p e r a t u r e . The a f t e r n o o n f i x e d n e e d l e s de- m o n s t r a t e d a s i m i l a r appearance t o n e e d l e s f i x e d a t dawn e x c e p t f o r t h e appearance o f s m a l l g r a i n s o f s t a r c h i n n e a r l y a l l o f t h e c h l o r o p l a s t s . N o r m a l l y , s t a r c h g r a i n s were seen o n l y v e r y r a r e l y i n w i n t e r f i x a t i o n s c o nducted a t dawn. On J a n u a r y t h e f i f t h , 1973, a n o t h e r a f t e r n o o n f i x a - t i o n was c o n d u c t e d . T h i s was. a sunny day i n w h i c h t h e t e m p e r a t u r e never went above -8°C. I t i s p o s s i b l e t h a t t h e t e m p e r a t u r e i n t h e m i c r o e n v i r - onment o f t h e n e e d l e s was near o r above f r e e z i n g . No'.starch g r a i n s were o b s e r v e d i n t h e c h l o r o p l a s t s o f m a t e r i a l from t h i s f i x a t i o n . 63 W i n t e r f i x a t i o n s o f t h r e e c o n i f e r s p e c i e s at t h e c o l l e c t i n g s i t e s a l l d e m o n s t r a t e d a s i m i l a r u n i q u e w i n t e r c y t o p l a s m i c s t a t e . W i n t e r a t t h e s e c o l l e c t i n g s i t e s i s c h a r a c t e r i z e d by near c o n t i n u o u s below f r e e z - in g t e m p e r a t u r e s . In e a r l y J a n u a r y , 1973, a f i x a t i o n o f P i n u s c o n t o r t a s s p ; c o n t o r t a g r o w i n g n a t u r a l l y i n t h e "Heron Swamp" r e g i o n o f t h e Un- i v e r s i t y o f - B r i t i s h Columbia endowment l a n d s was-'' f i x e d a t +3°C and a g a i n a week l a t e r when t h e a i r t e m p e r a t u r e was -2°C. A i r t e m p e r a t u r e s i n t h e a r e a f o r the w i n t e r t o t h e d a t e o f f i x a t i o n c o n t a i n e d s e v e r a l days o f m i l d f r o s t s and no t e m p e r a t u r e below - 6°C. The r e s u l t s o f t h e s e f i x a t i o n s were poor but t h e c y t o p l a s m o f the n e e d l e c e l l s o f t h e s e t r e e s -resembled t h e summer s t a t e e x c e p t f o r e x t e n s i v e o i l d r o p l e t s and some osmio- p h i N e m b o d i e s . There was no e x t e n s i v e v a c u o l a t i o n o f t h e c y t o p l a s m and t h e c h l o r o p l a s t s were not; :clumped and c o n t a i n e d ' s m a l 1 s t a r c h g r a i n s . ' The c h l o r o p l a s t s were removed from t h e c e l l w a l l by a narrow r e g i o n o f c y t o - plasm but t h e f l a t f a c e o f t h e p l a s t i d s was o r i e n t a t e d p a r a l l e l t o t h e c e l l w a l l . The p o s i t i o n o f t h e p l a s t i d s was v e r y s i m i l a r t o t h a t shown i n f i g u r e 76. 64 V. V e r n a l Changes i n t h e U l t r a s t r u c t u r e o f t h e Chlorenchyma F i x a t i o n s were co n d u c t e d i n March and A p r i l i n an e f f o r t t o dem- o n s t r a t e any p o s s i b l e c y t o l o g i c a l t r a n s i t i o n phase from the w i n t e r t o the summer c o n d i t i o n . T i s s u e s f i x e d on March t h e t h i r d ( t a b l e 1) show- ed the w i n t e r c o n d i t i o n d e s p i t e t h e f a c t t h a t some o f the m a t e r i a l f i x e d on t h i s d a t e was f i x e d a t above f r e e z i n g t e m p e r a t u r e s . M a t e r i a l f i x e d on June t h e s i x t h ( t a b l e 1) showed t h e summer c o n d i t i o n d e s p i t e l i g h t f r o s t i n t h e a r e a t h e n i g h t b e f o r e . The f i x a t i o n s c o n d u c t e d i n th e , ' : f i e l d on m a t e r i a l c o l l e c t e d on Ap- r i l t h e t w e n t y - s e v e n t h , however, r e v e a l e d a unique u l t r a s t r u c t u r a l con- d i t i o n w h i c h can be i n t e r p r e t e d as an i n t e r m e d i a t e s t a g e between t h e summer and w i n t e r c o n d i t i o n s . The c h l o r o p l a s t s were no l o n g e r i n clumps as noted i n t h e w i n t e r f i x a t i o n s . Most c h l o r o p l a s t were d i s p e r s e d about the p e r i p h e r a l c y t o p l a s m . These c h l o r o p l a s t s were l o c a t e d near o r a p p r e s s - ed t o the plasma membrane w i t h the major s e c t i o n a l a x i s u s u a l l y p a r a l l e l t o t h e c e l l w a l l o r t r a b i c u l a e ( f i g u r e s 63 and 67). The r e m a i n i n g c h l o r - o p l a s t s were a s s o c i a t e d i n s m a l l g r o u p s , ( f i g u r e 65). In t h e s p r i n g f i x a t i o n , t h e d i s t r i b u t i o n o f c h l o r o p l a s t s i n t h e p e r i p h e r a l c y t o p l a s m about t h e c e l l w a l l and t r a b i c u l a e was not u n i f o r m as o b s e r v e d i n summer f i x a t i o n s . In t h e c y t o p l a s m near the ends o f t h e c e l l near t h e long t r a b i c u l a e , the c h l o r o p l a s t d e n s i t y was g r e a t e r than o b s e r v e d i n summer. In o t h e r a r e a s o f t h e c e l l s , p a r t i c u l a r l y i n t h e c e n t r a l r e g i o n s o f t h e c e l l l y i n g i n t h e t r a n s v e r s e p l a n e o f t h e n e e d l e 65 ( c e n t r a l p a r t o f f a c e A, f i g u r e 2), c h l o r o p l a s t s were r a r e , In r e g i o n s where t h e c h l o r o p l a s t d e n s i t y was h i g h , c h l o r o p l a s t s u s u a l l y formed a u n i f o r m t i e r a l o n g t h e plasma membrane w i t h s e v e r a l a d d i t i o n a l c h l o r o p l - a s t s l y i n g deeper i n t h e c y t o p l a s m ( f i g u r e 63). Some o f t h e c h l o r o p l a s t s d i s t r i b u t e d near the plasma membrane were d i s c o i d i n shape w i t h t h e s i d e p r o x i m a l t o t h e plasma membrane b e i n g more f l a t t e n e d ( f i g u r e 66). These c h l o r o p l a s t s appeared t o be v e r y sim- i l a r t o summer c h l o r o p l a s t s . Most o f t h e c h l o r o p l a s t s d i s t r i b u t e d f u r t h - e r from t h e plasma membrane and n e a r l y a l l o f t h e c h l o r o p l a s t s a s s o c i a - t e d i n s m a l l groups appeared i r r e g u l a r i n shape ( f i g u r e s 65 and 67). The i r r e g u l a r s e c t i o n a l appearance o f some o f t h e s p r i n g c h l o r o p l - a s t s i s s i m i l a r t o t h e w i n t e r c h l o r o p l a s t s . However, i n t h e s p r i n g f i x - a t i o n , t h e i r r e g u l a r l y shaped c h l o r o p l a s t s do not appear t o have t h e e x t e n s i v e s t r o m a l a r e a s as seen i n t h e w i n t e r c h l o r o p l a s t s ( f i g u r e 63). The i r r e g u l a r shape o f the s p r i n g c h l o r o p l a s t s appears t o be due r a t h e r t o t h e t w i s t i n g o f t h e i n t e r n a l membrane systems. No s p r i n g c h l o r o p l a s t s were o b s e r v e d t o have more than one i n t e r n a l t h l a k o i d membrane system ( f i g u r e 57). Most s p r i n g c h l o r o p l a s t s had p l a s t o g l o b u l i t h a t appeared g e n e r a l l y s m a l l e r and. s l i g h t l y l e s s numerous than : i n t h e w i n t e r c o n d i t i o n . How- e v e r , i n n e a r l y e v e r y s e c t i o n s t u d i e d , t h e r e were c e r t a i n c h l o r o p l a s t s t h a t were noted f o r t h e v e r y l a r g e masses o f p l a s t o g l o b u l ! , w h i c h appear i n dense c l u s t e r s , ( f i g u r e 65). 66 In r e g i o n s o f t h e s p r i n g c hlorenchyma where the c h l o r o p l a s t den- s i t y was h i g h , t h e c h l o r o p l a s t s l i n i n g t h e plasma membrane and t h o s e l y i n g deeper i n t h e c y t o p l a s m were o f t e n i n v e r y c l o s e p r o x i m i t y . High m a g n i f i c a t i o n s t u d i e s o f t h e s e a r e a s r e v e a l e d a c o n s i s t e n t 75nm t o 150 nm wide space between t h e o u t e r membranes o f a d j a c e n t c h l o r o p l a s t ( f i g - u r e s 63, 64 ( h i g h m a g n i f i c a t i o n o f boxed a r e a i n f i g u r e 63), and open arrows i n f i g u r e 65). There i s no a p p a r e n t c o n t a c t between t h e o u t e r membranes o f t h e c h l o r o p l a s t e n v e l o p e s o f a d j a c e n t c h l o r o p l a s t s . Small s t a r c h g r a i n s were n o t i c e d i n many c h l o r o p l a s t s i n t h e s p r i n g f i x a t i o n ( f i g u r e s 62 and 64). The t a n n i n v a c u o l e s remained p a r t i a l l y c o n t r a c t e d and g e n e r a l l y c i r c u l a r i n s e c t i o n a l p r o f i l e and t h e t o n o p l a s t c o u l d not be r e s o l v e d . Thus the t a n n i n v a c u o l e s appeared s i m i l a r t o t h e w i n t e r s t a t e . The p e r i - p h e r a l c y t o p l a s m s t i l l o c c u p i e d a g r e a t e r volume o f t h e t o t a l c e l l . t h a n t h a t u s u a l l y o b s e r v e d i n t h e summer, but not as g r e a t as t h a t o b s e r v e d i n t h e w i n t e r c o n d i t i o n . The c o n d i t i o n o f t h e ground c y t o p l a s m however, appeared more sim- i l a r t o the summer c o n d i t i o n than t h e w i n t e r s t a t e ( f i g u r e 67). C y t o - p l a s m i c v a c u o l e s were p r e s e n t but were w i d e l y s c a t t e r e d and not a s s o - c i a t e d w i t h o s m i o p h i 1 i c b o d i e s . The o c c a s i o n a l o s m i o p h i l i c body was found s c a t t e r e d i n t h e c y t o p l a s m and near t h e c e l l w a l l (open a r r o w s , f i g u r e 67). The m a j o r i t y o f o s m i o p h i l i c b o d i e s p r e s e n t i n t h e s p r i n g c y t o p l a s m had a d i f f e r e n t morphology than t h a t o b s e r v e d i n t h e w i n t e r c o n d i t i o n . The o s m i o p h i l i c b o d i e s were o f t e n v e r y l a r g e ( f i g u r e 64, 67 arrowheads) o r c o n s i s t e d o f l o c a l i z e d a g g r e g a t e s o f s m a l l e r b o d i e s ( f i g - u r e 68 (0 1 ) ) . The m i t o c h o n d r i a were g e n e r a l l y l o c a t e d c l o s e t o t h e c h l o r o p l a s t s i n t h e s p r i n g f i x a t i o n s ( f i g u r e 63 and 67). The m i t o c h o n d r i a had , o v o i d t o i r r e g u l a r s e c t i o n a l p r o f i l e s ( f i g u r e s 63 and 67) and appeared v e r y s i m i l a r t o t h e d e s c r i p t i o n o f summer m i t o c h o n d r i a i n t y p e II c e l l s . D i ctyosomes were not common and r a r e l y seen i n t h e s p r i n g f i x a t i o n . The n u c l e i i n the s p r i n g f i x a t i o n were l o c a t e d e i t h e r near t h e ends o f t h e c e l l s i n the p e r i p h e r a l c y t o p l a s m ( s i m i l a r t o t h e w i n t e r p o s i t i o n ) o r i n a more c e n t r a l l o c a t i o n i n t h e c e l l u s u a l l y a t t h e end o f a long t r a b i c u l u m ( f i g u r e 66). When l o c a t e d i n a more c e n t r a l l o c a t i o n , t h e n u c l e u s was l o c a t e d i n an i n d e n t a t i o n i n t o the t a n n i n v a c u o l e s i m i l a r t o what was o b s e r v e d i n t h e summer f i x a t i o n s . A l l s p r i n g n u c l e i had a g e n e r a l l y c i r c u l a r s e c t i o n a l p r o f i l e . The n u c l e a r membrane and t h e i n - t e r n a l s t r u c t u r e o f t h e n u c l e u s appear v e r y s i m i l a r t o summer n u c l e i ( f i g u r e 66). N u c l e o l i were o b s e r v e d i n the s p r i n g f i x a t i o n but d e t a i l - ed o b s e r v a t i o n s o f t h e i r s t r u c t u r e were not made. The number o f oi 1 b o d i e s w i t h i n t h e c y t o p l a s m was g r e a t e r i n s p r i n g than any o t h e r s e a s o n . O i l b o d i e s were l o c a t e d near the n u c l e u s ( f i g u r e 66) and a l s o e x t e n s i v e l y t h r o u g h o u t t h e r e s t o f t h e c y t o p l a s m ( f i g u r e s 67 and 68). 68 V I . E x p e r i m e n t s on P i n e S e e d l i n g s i n C o n t r o l l e d E n v i ronments A. A r t i f i c i a l F r o s t H a r d e n i n g : U l t r a s t r u c t u r a l O b s e r v a t i o n s Tumanov (1967) and S a i k i (1973) e l a b o r a t e on t h e i r e a r l i e r s t u - d i e s w h i c h e s t a b l i s h e d t h e c o n d i t i o n s n e c e s s a r y f o r the a r t i f i c i a l i n d u c t i o n o f f r o s t h a r d i n e s s i n c e r t a i n hardy woody p l a n t s . S e e d l - i n g s were grown under optimum c o n d i t i o n s u n t i l t h e y reached t h e eq- u i v a l e n t o f two o r t h r e e y e a r s o f f i e l d g r o w t h. The s e e d l i n g s were a r t i f i c i a l l y hardened f o l l o w i n g t h e p r o c e d u r e o u t l i n e d i n m a t e r i a l s and methods. A l l s e e d l i n g s s u r v i v e d f r e e z i n g c o n d i t i o n s when a r t i - f i c i a l l y hardened but s u s t a i n e d c o n s i d e r a b l e damage. U l t r a s t r u c t u r a l o b s e r v a t i o n s on n e e d l e c hlorenchyma r e v e a l e d some f e a t u r e s c h a r a c t e r i s t i c o f t h e w i n t e r c o n d i t i o n . The t a n n i n v a c u o l e had c o n t r a c t e d t o a s i m i l a r e x t e n t . Some o s m i o p h i 1 i c b o d i e s were found but not t o the same e x t e n t as i n w i n t e r c y t o p l a s m . These b o d i e s were a l s o a s s o c i a t e d w i t h t h e e x t e n s i v e c y t o p l a s m i c v a c u o l a r membrane systems w i t h i n the c y t o p l a s m ( f i g u r e 73). The c y t o p l a s m i c v a c u o l e s were u s u a l l y l a r g e r than t h o s e o b s e r v e d i n t h e w i n t e r c o n d i - t i o n . The c h l o r o p l a s t s were not a l i g n e d a l o n g the c e l l w a l l s . They were instead' s c a t t e r e d i n t h e c y t o p l a s m . , They a r e o f t e n i r r e g u l a r i n shape and many grouped i n t o c l u s t e r s o f between two and f i v e c h l o - r o p l a s t s . The"; o u t e r e n v e l o p e membranes i n t h e clumped c h l o r o p l a s t s were i n c l o s e c o n t a c t ( f i g u r e 7*0. These s m a l l c h l o r o p l a s t groups a r e not e q u i v a l e n t t o t h e e x t e n s i v e c l u m p i n g c h l a r a c t e r i s t i c o f t h e 69 w i n t e r c o n d i t i o n . The n u c l e u s appeared t o be s i m i l a r t o t h e summer con- d i t i o n , and no s i g n i f i c a n t b u i l d - u p o f l a r g e p e r i n u c l e a r l i p i d b o d i e s was o b s e r v e d . Long term damage was o b s e r v e d when t h e s e e d l i n g s were r e t u r n e d t o summer c o n d i t i o n s i n t h e growth chamber. The youngest and the o l d e s t n e e d l e s became c h l o r o t i c , p a r t i c u l a r l y a t the t i p s and bases w i t h i n a week a f t e r f r e e z i n g . Many o f t h e a p i c a l buds f a i l e d t o open. F i g u r e 75 shows a s e e d l i n g (A) w h i c h was g i v e n t h e h a r d e n i n g t r e a t m e n t . The s e e d l i n g was s t i l l a l i v e f o u r months l a t e r , a l t h o u g h more than h a l f o f the n e e d l e s on t h e t r e e were c h l o r o t i c and the youngest n e e d l e s had y e l l o w e d (1ight a r e a s shown by arrow) and l a t e r d i e d . The youngest n e e d l e s were shown i n f i g u r e 71. T r e e B i n f i g u r e 75 was not g i v e n any h a r d e n i n g t r e a t m e n t s ( p h o t o - p e r i o d i c and low t e m p e r a t u r e s ) b e f o r e b e i n g p l a c e d i n t h e f r e e z e r . A f t e r b e i n g p l a c e d i n t h e f r e e z e r , t h i s t r e e q u i c k l y d i e d . U l t r a s t r u c t u r a l o b s e r v a t i o n s on t h e n e e d l e c h l o r e n c h y m a o f t h e c o n t r o l s e e d l i n g ( t r e e B) w h i c h d i d not r e c e i v e p r e - f r e e z i n g h a r d e n i n g t r e a t m e n t s r e v e a l e d t h a t t h e chlorenchyma f i n e s t r u c t u r e was d e s t r o y e d . 70 B. Summer Condi t i o n T r e e s Exposed t o Drought: U 1 t r a s t r u c t u r a l O b s e r v a t i o n s The n a t u r e o f t h e c e l l u l a r . u l t r a s t r u c t u r e o f the n e e d l e s was b r i e f - ly i i h v e s t i g a t e d t o see i f t h e r e i s any c o r r e l a t i o n between t h e d r o u g h t e x p o s u r e c o n d i t i o n and f r o s t - h a r d i n e s s s t a t e s . The t a n n i n m a t e r i a l o f d r ought exposed summer n e e d l e s seems t o be reduced i n volume as i n w i n t e r c o n d i t i o n s , c o i n c i d i n g w i t h a c o n s i d e r a b l e i n c r e a s e i n c y t o p l a s m i c v o l - ume ( f i g u r e 76). The c h l o r o p l a s t s however, remained e v e n l y d i s t r i b u t e d a l o n g t h e c e l l w a l l but had moved s l i g h t l y away from i t ( f i g u r e 76). C h l o r o p l a s t s w i t h c o n s t r i c t i o n s s i m i l a r t o t h o s e o b s e r v e d i n some c a s e s i n w i n t e r ( f i g u r e kh) were o c c a s i o n a l l y o b s e r v e d ( f i g u r e 77). A p p r o x i - m a t e l y t h e same number o f o s m i o p h i 1 i c b o d i e s were p r e s e n t as i n t h e l a t e summer f i x a t i o n s under f i e l d c o n d i t i o n s . There was development o f c y t o - p l a s m i c v a c u o l a r s y s t e m s , ( t h e s e were more e x t e n s i v e than i n normal summer f i x a t i o n s but not n e a r l y as e x t e n s i v e as t h o s e seen i n t h e w i n t - e r c o n d i t i o n , f i g u r e 76). The b u l k o f t h e ground c y t o p l a s m i n t h e d rought c o n d i t i o n c o n s i s t e d o f l i g h t l y s t a i n i n g homogeneous m a t e r i a l w i t h v e r y few ribosomes o r o t h e r i n c l u s i o n s . M i t o c h o n d r i a were few and c i r c u l a r i n p r o f i l e , and s m a l l o i l d r o p l e t s were seen. The n u c l e u s appeared s i m - i l a r t o t h a t seen i n t h e summer c o n d i t i o n . One s e e d l i n g t r e e t h a t had been exposed t o l o n g day p h o t o p e r i o d s and warm growth chamber t e m p e r a t u r e s w h i l e not b e i n g w a t e r e d f o r t h i r t y days was then w a t e r e d once and p l a c e d i n t h e a r t i f i c i a l h a r d e n i n g regime, d e s c r i b e d p r e v i o u s l y . T h i s s e e d l i n g was g i v e n t h e same f r e e z e r f r o s t e x p o s u r e as d e s c r i b e d p r e v i o u s l y f o r s e e d l i n g A. The d r o u g h t exposed 71 s e e d l i n g s u r v i v e d t h e f r o s t e x p o s u r e but subsequent o b s e r v a t i o n r e v e a l e d i t had r e c e i v e d about the same amount o f damage as t r e e A. F i x a t i o n s on the n e e d l e s o f t h i s t r e e were not s u c c e s s f u l . 72 DISCUSSION I. D i s c u s s i o n o f T e c h n i c a l Problems and P r o c e d u r e s A major p o r t i o n o f t h i s work was spent on d e v e l o p i n g p r e p a r a t i v e t e c h n i q u e s f o r e l e c t r o n m i c r o s c o p y w h i c h would g e n e r a t e r e p r o d u c i b l e u l t r a s t r u c t u r a l o b s e r v a t i o n s . I t i s hoped t h a t t h e r e p o r t i n g and d i s - c u s s i o n o f t h e t e c h n i c a l problems d e a l t w i t h i n t h i s work w i l l a s s i s t f u t u r e s t u d i e s on c o n i f e r t i s s u e . The f i x a t i o n o f mature c o n i f e r chlorenchyma f o r u l t r a s t r u c t u r a l s t u d i e s i s v e r y d i f f i c u l t d u r i n g t h e growing season. .Testimony t o t h i s i s shown by t h e f a c t t h a t n e a r l y a l l p u b l i s h e d u l t r a s t r u c t u r a l s t u d i e s on mature c o n i f e r chlorenchyma make r e f e r e n c e t o f i x a t i o n problems; ( P a r k e r and P h i l p o t t , 1961 and 1963; H a r r i s 197\; C a m p b e l l , 1972, and Chabot and Chabot, 1975)- Most o f the above a u t h o r s s p e c u l a t e t h a t t h e prolems stem from t h e i n t e r f e r e n c e w i t h f i x a t i o n c h e m i s t r y by t h e r e s i n o u s and t a n n i n i f e r o u s m a t e r i a l i n t h e m e s o p h y l l t i s s u e s o r i n t h e m e s o p h y l l c h l o r e n c h y m a l c e l l s . A major i n i t i a l problem was found t o be r e l a t e d t o t h e method o f c u t t i n g and s u b d i v i d i n g the n e e d l e f o r f i x a t i o n . In t h e e a r l y s t a g e s o f t h i s work, t h e f l a t s u r f a c e o f t h e n e e d l e ( a d a x i a l s i d e ) was p l a c e d on a p l a s t i c p e t r i d i s h , f l o o d e d w i t h f i x a t i v e s o l u t i o n and s u b d i v i d e d i n t o s m a l l t r a n s v e r s e segments. In t h i s p r o c e d u r e , t h e r a z o r b l a d e used t o c u t t h e n e e d l e d i d not i n i t i a l l y s l i c e and p e n e t r a t e the e p i d e r m a l - hypodermal l a y e r e f f e c t i v e l y . T h i s r e s u l t e d i n t h e c r u s h i n g and mechan- i c a l damage t o t h e d e l i c a t e m e s o p h y l l chlorenchyma s i t u a t e d between t h e tough but f l e x i b l e e p i d e r m i s - h y p o d e r m i s , and t h e t i s s u e s o f t h e endoderm i s and the v a s c u l a r t i s s u e . The problem was a l e v i a t e d by t h e use o f h i g h e r q u a l i t y s t a i n l e s s s t e e l r a z o r b l a d e s and c l e a n i n g them i n an u l t r a - s o n i c a t o r i n a c e t o n e r a t h e r than by hand. The l a t t e r t r e a t m e n t removed o i l f i l m s from t h e b l a d e s w i t h o u t damaging t h e k n i f e edge. T u r n i n g t h e n e e d l e on i t s s i d e a t r i g h t a n g l e s t o the f l a t s i d e o f the n e e d l e a l s o h e l p e d t o p r e v e n t m e s o p h y l l damage. The f l a t s u r f a c e o f the n e e d l e ( a d a x i a l s i d e ) was now a t r i g h t a n g l e s t o the p e t r i d i s h s u r f a c e so t h a t t h e s t r o n g and s u p p o r t i v e e p i d e r m a l - h y p o d e r m a l l a y e r s were a t r i g h t a n g l e s t o the r a - z o r b l a d e , t h u s s u p p o r t i n g t h e chlorenchyma from t h e k n i f e p r e s s u r e r a t h e r than c r u s h i n g . The a d d i t i o n o f f o r m a l i n t o t h e p r i m a r y f i x a t i v e s o l u t i o n r e s u l t e d i n a r a p i d and d e t e c t a b l e h a r d e n i n g o f t h e chlorenchyma so t h a t t r a n s - v e r s e s e c t i o n s o f t h e n e e d l e c o u l d be f u r t h e r s u b d i v i d e d a f t e r about one hour o f p r i m a r y f i x a t i o n . The g e n e r a l h a r d e n i n g o f t h e m e s o p h y l l l e s s e n e d the problem o f t h e k n i f e c r u s h i n g t h e m a t e r i a l . The b e s t r e s u l t s were o b t a i n e d when t h e s e c o n d a r y s u b d i v i s i o n was 74 c a r r i e d out by d i s s e c t i o n and removing as much o f t h e endodermis and hypode r m a l - e p i d e r m a l l a y e r s as p o s s i b l e from t h e m e s o p h y l l . The remo- v a l o f t h e s e c o m p a r a t i v e l y impermeable l a y e r s was found t o f a c i l i t a t e t h e i n f i l t r a t i o n o f the f i x a t i v e and embedding media. Esau (1965) noted t h a t the p i n e n e e d l e s u r f a c e i s c h a r a c t e r i z e d by a t h i c k c u t i c l e and t h a t endodermal e e l 1s may be l i g n i f i e d and c o n t a i n s u b e r i n . Thus t h e d i s s e c t i o n o f t h e s e t i s s u e s from t h e m e s o p h y l l p r o b a b l y f r e e d an a d d i - t i o n a l one o r two f a c e t s o f the t i s s u e b l o c k f o r f i x a t i v e , d e h y d r a t i o n , and embedding media p e n e t r a t i o n . The 0.5 mm t o 1.0 mm t i s s u e p i e c e s s t i l l r e q u i r e d s i x t e e n hours o f p o s t - f i x a t i o n i n osmium t e t r o x i d e a t room t e m p e r a t u r e t o a c h i e v e an u n i f o r m o s m i f i c a t i o n o f the t i s s u e b l o c k . H a r r i s (1971) a l s o a d v o c a t e d s i m i l a r long s e c o n d a r y f i x a t i o n p e r i o d s . The removal o f sc i e r e n c h y m o u s c e l l s a l s o a i d e d i n t h e tr i m m i n g and s e c - t i o n i n g o f the p l a s t i c embedded m a t e r i a l . W i t h r e s p e c t t o the q u a l i t y o f u l t r a s t r u c t u r a l p r e s e r v a t i o n , f i x a - t i o n s w i t h g l u t a r a l d e h y d e as the s o l e p r i m a r y f i x a t i v e were found t o be i n f e r i o r t o a m i x t u r e o f g l u t a r a l d e h y d e and f r e s h l y p r e p a r e d f o r m a l i n . H a r r i s (1971) d i d a c h i e v e adequate f i x a t i o n s w i t h g l u t a r a l d e h y d e as t h e o n l y p r i m a r y f i x a t i v e , but Campbell (1972) and Chabot and Chabot (1975) both used a m i x t u r e o f the two a l d e h y d e s as p r i m a r y f i x a t i v e on c o n i f e r t i s s u e . S r i v a s t a v a and O'Brien (1966) suggest t h a t the r e s i n s i n P i n u s c a m b i a l t i s s u e b i n d and p r e c i p i t a t e g 1utaraldehyde w h i c h may d i s r u p t t h e f i x a t i o n o f the t i s s u e . F i x a t i o n s o f o l d e r (one o r more y e a r s o f age) summer n e e d l e s showed 75 t h e b e s t r e s u l t s when a c r o l e i n was added t o t h e p r i m a r y g l u t a r a l d e h y d e f i x a t i v e s o l u t i o n . Campbell (1972) a l s o o b t a i n e d t h e best r e s u l t s w i t h a c r o l e i n / g l u t a r a l d e h y d e p r i m a r y f i x a t i v e s on mature p i n e n e e d l e t i s s u e s . A c r o l e i n d i f f e r e d from g1utara1dehyde/forma1in f i x a t i v e i n t h a t i t t e n d - ed t o make t h e c y t o p l a s m more o s m i o p h i l i c and a l s o o f t e n a l t e r e d t h e t a n - n i n m a t e r i a l s u b s t r u c t u r e when used as a p r i m a r y f i x a t i ve w i t h g l u t a r a l d - ehyde ( f i g u r e s 69, 70 and 68). (More s t u d i e s s h o u l d be cond u c t e d on the use o f t h i s f i x a t i v e f o r c o n i f e r t i s s u e s ) . Campbell (1972) a l s o used p o t a s s i u m permanganate s o l u t i o n s u c c e s s - f u l l y as a f i x a t i v e on p i n e n e e d l e s but o n l y p u b l i s h e d one f i g u r e o f c h l o - renchyma p r e p a r e d w i t h t h i s c h e m i c a l . I t was a h i g h m a g n i f i c a t i o n photo- m i c r o g r a p h showing o n l y p a r t o f a c h l o r o p l a s t . P a r k e r and P h i l p o t t (1961 and 1963) a l s o used permanganate as t h e s o l e f i x a t i v e on P i n u s c h l o r e n c h y - ma w i t h poor r e s u l t s . A t t e m p t s t o use p o t a s s i u m o f barium permanganate as a f i x a t i v e i n t h i s p r o j e c t f ai 1ed because the m a t e r i a l i n t h e p l a s t i c become so hard t h a t t h e b l o c k s c o u l d not be trimmed o r s e c t i o n e d . A pos- i b l e reason f o r t h i s a p p a r e n t c o n t r a d i c t i o n was t h e use o f barium and po- t a s s i u m permanganate as a se c o n d a r y f i x a t i v e i n t h i s work a f t e r i n i t i a l a l d e h y d e f i x a t i o n . The above a u t h o r s used permanganate o n l y as t h e s o l e f i x a t i v e , a p r o c e d u r e not a t t e m p t e d i n t h i s work. The e x p e r i m e n t a l use o f g l y c o l m e t h a c r y l a t e (Feder and O ' B r i e n , 1968) and Epon r e s i n s f o r embedding p i n e n e e d l e t i s s u e r e s u l t e d i n t h e c o n s i s t e n t f a i l u r e o f t h e s e r e s i n s t o be p r o p e r l y p o l y m e r i z e d w i t h i n t h e t i s s u e s d e s p i t e e x t r e m e l y long d e h y d r a t i o n and i n f i l t r a t i o n p r o c e d u r e s . 76 The f a i l u r e i n p o l y m e r i z a t i o n appeared s p e c i f i c a l l y t o be l o c a t e d i n t h e t a n n i n v a c u o l e s . The embedding r e s i n s i n t h e t a n n i n v a c u o l e s remained se- m i - l i q u i d , w h i l e p o l y m e r i z a t i o n was adequate i n t h e c y t o p l a s m and e x t r a c e - l l u l a r s p a c e s . S p u r r ' s r e s i n , ( S p u r r , 1969), gave adequate p o l y m e r i z a t i o n o n l y a f t e r a l o n g d e h y d r a t i o n and i n f i l t r a t i o n s e r i e s . Campbell (1972) was a p p a r e n t - l y s u c c e s s f u l i n u s i n g Epon r e s i n w h i l e H a r r i s (1971) and Chabot and Cha- bot (1975) both used S p u r r ' s r e s i n . Osmium, and t o a l e s s e r e x t e n d , t h e p l a s t i c r e s i n s , r e a c t w i t h the c o n t e n t s o f t h e t a n n i n v a c u o l e s o f the n e e d l e chlorenchyma t o form an ex- t r e m e l y hard s u b s t a n c e w h i c h i s v e r y d i f f i c u l t t o s e c t i o n . T h i s m a t e r i a l o f t e n v i s i b l y s h a t t e r s g l a s s k n i v e s . Good s e c t i o n s f o r l i g h t o r e l e c t r o n m i c r o s c o p y c o u l d o n l y be c u t s i m p l y from newly broken and e x t r e m e l y good p o r t i o n s o f t h e k n i f e edge. The f a c t t h a t osmium i s i n v o l v e d w i t h t h e f o r m a t i o n o f t h i s v e r y hard m a t e r i a l was deduced from t h e f a c t t h a t a l d - ehyde f i x e d m a t e r i a l was c o n s i d e r a b l y e a s i e r t o s e c t i o n . The t y p e s o f e l e c t r o n m i c r o s c o p i c p r e p a r a t i v e p r o c e d u r a l d i f f i c u l t - i e s d e s c r i b e d above g e n e r a l l y i n c r e a s e d w i t h n e e d l e age d u r i n g summer f i x a t i o n s . In w i n t e r f i x a t i o n s , however, v e r y few p r e p a r a t i v e d i f f i c u l t - i e s were e n c o u n t e r e d . For example, long f i x a t i o n and embedding p r o c e d - u r e s c o u l d be g r e a t l y reduced w i t h o u t d e t e c t a b l e changes i n u l t r a s t r u c t - u r a l p r e s e r v a t i o n . S i m i l a r l y , i t was not n e c e s s a r y t o s u b d i v i d e o r d i s - s e c t t i s s u e b l o c k s as e x t e n s i v e l y as i n t h e summer f i x a t i o n s . W i n t e r 77 n e e d l e s from s i x months o f age t o f i v e y e a r s o f age were f i x e d d u r i n g w i n t e r but no r e l a t i o n s h i p between n e e d l e age and u l t r a s t r u c t u r a l p r e - s e r v a t i o n was o b s e r v e d i n marked c o n t r a s t t o t h e o b s e r v a t i o n s on summer t i s s u e . In t h e w i n t e r f i x a t i o n s , p l a s t i c embedding media o t h e r than S p u r r ' s r e s i n and permanganate f i x a t i v e s were not used. In an a t t e m p t t o redu c e t h e p o s s i b i l i t y o f a r t i f a c t s caused by tha w i n g f r o z e n t i s s u e o r by c h e m i c a l f i x a t i o n and embedding p r o c e d u r e s , the t e c h n i q u e s f o r c r y o m i c r o s c o p y were d e v e l o p e d . The t e c h n i q u e s em- p l o y e d a r e based on the p r i n c i p l e t h a t an i n s i t u n e e d l e i n m i d - w i n t e r a t t h e c o l l e c t i n g s i t e s ( w i t h t e m p e r a t u r e s o f -10^C t o -1'5°C) e x i s t s i n a l m o s t a c o m p l e t e l y f r o z e n s t a t e . The b u l k o f f r e e w a t e r i n n e e d l e t i s s u e a t t h e s e t e m p e r a t u r e s w i l l be c r y s t a l i z e d , (Kaku, 1 9 7 5 ) . The ob- j e c t o f the s t u d y o f f r o z e n t i s s u e was t o f i n d an e f f i c i e n t way t o c o l - l e c t , t r a n s p o r t , s t o r e , and p r o c e s s t h e m a t e r i a l f o r l i g h t m i c r o s c o p y w h i l e m a i n t a i n i n g t h e t i s s u e t e m p e r a t u r e between -10°C t o -15°C. An i m p o r t a n t l i m i t a t i o n t o t h e low t e m p e r a t u r e t e c h n i q u e s was t h e d i f f i c u l t y i n m o n i t o r i n g t e m p e r a t u r e a t a l l s t a g e s o f t h e p r e p a r a t i v e p r o c e d u r e : from t h e f i e l d t o c r y o m i c r o s c o p y . In l i e u o f a c c u r a t e temp- e r a t u r e m e asuring and c o n t r o l s y s t e m s , t h e t i s s u e was c o n s i s t e n t l y kept as c o l d a s , o r c o l d e r than t h e f i e l d t e m p e r a t u r e . The use o f l i q - u i d n i t r o g e n as t h e c o o l a n t m a t e r i a l a t s e v e r a l s t a g e s o f t h e p r e p a r a - t i v e p r o c e d u r e r e s u l t e d i n t e m p e r a t u r e d i f f e r e n c e o f as much as from -10°C a t c o l l e c t i o n s i t e s , t o a p o s s i b l e minimum t e m p e r a t u r e o f -196°C. 78 These f l u c t u a t i o n s p r o b a b l y would not have a f f e c t e d t he s t r u c t u r a l and p o s i t i o n a l n a t u r e o f b u l k t i s s u e . i c e . I t i s a l s o d i f f i c u l t t o e n v i s i o n how the s t r u c t u r a l components o f the c e l l c o u l d s i g n i f i c a n t l y r e a r r a n g e a t t h e s e t e m p e r a t u r e s . S a k a i (1966 and 1973) has dem o n s t r a t e d t h a t e x t r e m e l y hardy l i v i n g woody t i s s u e can s u r v i v e t e m p e r a t u r e s as low as -196°C, p r o v i d e d they had been i n i t i a l l y exposed t o t e m p e r a t u r e s o f 0°C t o -kO°C f o r s e v e r a l hours p r e v i o u s t o immersion. He a l s o d e m o n s t r a t e d t h a t a f t e r t h e i n i t - i a l f r o s t e x p o s u r e , v a r i o u s r a t e s o f f r e e z i n g u s u a l l y d i d not cause damage. S i m i l a r s t u d i e s have been c i t e d by t h e R u s s i a n a u t h o r s (Tumanov, 1967). In t h i s s t u d y , f r e e z e r s t o r e d n e e d l e s t h a t had been c o l l e c t e d i n mid w i n t e r were dropped i n t o a l i q u i d n i t r o g e n v a t and then removed and f i x e d a t -4°C. Very l i t t l e change from t h e normal w i n t e r u l t r a s t r u c t u r e was o b s e r v e d ( f i g u r e 62). These o b s e r v a t i o n s a r e the b a s i s f o r the c o n - c u l s i o n t h a t t h e o b s e r v a t i o n s from m a t e r i a l p r o c e s s e d by c r y o - t e c h n i q u e r e p r e s e n t a v e r y c l o s e c o n d i t i o n o f t h e i n s i t u c y t o p l a s m i c arrangement i n t h e f r o z e n e e l 1. I t would have been d e s i r a b l e t o do more d e t a i l e d s t u d i e s on w i n t e r c e l l s i n t h e f r o z e n s t a t e , but the c o n s t r u c t i o n o f a c r y o m i c r o s c o p e t h a t c o u l d g i v e d i r e c t c o n t r o l o f s t a g e t e m p e r a t u r e f o r f r e e z e - t h a w s t u d i e s i s v e r y complex and e x p e n s i v e ( D i l l e r and Cr.avalho,. 1971 )• .However, the c r y o - 79 tome, c r y o m i c r o s c o p e - a n d h a n d l i n g a p p a r a t u s used f o r t h i s s t u d y were ad- a p t e d from s i m p l e m a t e r i a l s and equipment commonly found i n most u l t r a s t r - u c t u r a l r e s e a r c h l a b o r a t o r i e s . The n e e d l e c h l o r e n c h y m a o f P i n u s c o n t o r t a s s p . l a t i f o l i a was chosen as t h e major e x p e r i m e n t a l m a t e r i a l i n t h i s work due t o s e v e r a l p r o p e r t - i e s w h i c h s i m p l i f i e d and a s s i s t e d s e a s o n a l s t u d i e s on f r o s t h a r d i n e s s . One method o f h i g h l i g h t i n g s e a s o n a l l y r e l a t e d c y t o l o g i c a l changes i s t o s t u d y o r g a n i s m s which d e m o n s t r a t e extreme c a s e s o f the s e a s o n a l phenom- ena. P i n u s c o n t o r t a s s p . l a t i f o l i a specimens c o l l e c t e d i n t h e s u b a l p i n e f o r e s t o f Wyoming and Montana were t e s t e d by S a k a i and .Wej.serTl.9Z3) f o r the d egree o f the t r e e ' s f r o s t h a r d i n e s s . These a u t h o r s have p l a c e d P_. c o n t o r t a s s p . l a t ? f o l i a i n t h e most " h a r d y " c a t a g o r y o f N o r t h American woody p l a n t s . T h e i r specimens were a b l e t o w i t h s t a n d a t l e a s t -80°C i n w i n t e r . In t h i s work, P i n u s c o n t o r t a s s p . 1 a t ? f o 1 i a was sampled a t the w e s t e r n edge o f t h e i n t e r i o r p l a t e a u o f s o u t h w e s t e r n B r i t i s h Columbia a t an a l t i t u d e o f f o u r thousand f e e t . A t t h e sample l o c a t i o n s , t h e c o a s t a l i n f l u e n c e s a r e reduced and t h e c l i m a t e tends t o be more o f t h e h i g h l a t i t u d e c o n t i n e n t a l t y p e . At t h e sample l o c a t i o n s , P_. c o n t o r t a i s a major member o f t h e i n t e r i o r p l a t e a u s u b a l p i n e f o r e s t ( C r i t c h - f i e l d , 1957). Tsuga m e r t e n s i a n a i s found i n a more moderate c l i m a t i c zone i n t h e c o a s t a l s u b a l p i n e f o r e s t s o f B r i t i s h C o l u m b i a , (Sudworth, 1908). However, specimens used i n t h i s work were c o l l e c t e d near t i m b e r l i n e a t f i v e t h o u - sand f e e t i n a l t i t u d e on W h i s t l e r mountain w h i c h i s a s i t e on the c o a s t / 80 range, somewhat removed from t h e moderating": i n f l u e n c e s o f c o a s t a l reg i o n s . P i n u s a l b i c a u l i s i s o f t e n t h e t r e e l i n e s p e c i e s o f th e c o a s t a l f o r - e s t s o f s o u t h w e s t e r n B r i t i s h Columbia and grows i n v e r y exposed s e v e r e l o c a t i o n s (Sudworth, 1908). T r e e s sampled i n t h i s work grew a t about 5,700 f e e t on W h i s t l e r mountain and were found i n f u l l t r e e growth h a b i t s where o t h e r c o n i f e r s were absent o r reduced t o low l y i n g k r u m h o l t z grow- t h h a b i t s . S a k a i and Weiser (1973) d i d not t e s t the h a r d i n e s s o f P i n u s a l b i c a u - 1 i s , b u t on t h e b a s i s o f t h e c l i m a t e o f c o l l e c t i o n s i t e s , t he t r e e must be v e r y f r o s t r e s i s t a n t . These a u t h o r s d i d t e s t Tsuga m e r t e n s i a n a and found i t t o be o n l y m o d e r a t e l y f r o s t h a r d y , t o l e r a t i n g o n l y -40°C. However, t h e s e a u t h o r s sampled Tsuga m e r t e n s i a n a c l o s e t o sea l e v e l near S e a t t l e , Washington. When comparing t h e r e s u l t s o b t a i n e d i n f r o s t h a r d i n e s s s t u d i e s w i t h t h e r e s u l t s o f o t h e r s t u d i e s i n the l i t e r a t u r e , c a r e must be taken t o en- s u r e t h a t t he h a r d i n e s s and growth c o n d i t i o n s o f t h e or g a n i s m s b e i n g com- pared a r e w e l l d e f i n e d . Burke e_t a_l_. (1976) c i t e e v i d e n c e t h a t f r o s t r e s i s t a n t mechanisms may be d i f f e r e n t i n s l i g h t l y , m o d e r a t e l y and e x t r e - mely hardy p l a n t s . H a r r i s (1971) s t u d i e d P i n u s s t r o b u s (an e x t r e m e l y hardy t r e e , S a k a i and W e i s e r , 1973) but h i s s e a s o n a l o b s e r v a t i o n s were c o n d u c t - ed on specimens. g r o w i n g w e l l s o u t h o f t h e t r e e ' s n a t u r a l range on th e campus o f th e u n i v e r s i t y o f A r k a n s a s . Thus H a r i s ' s w i n t e r o b s e r v a - 81 t i o n s may have been v e r y d i f f e r e n t i f conducted i n t h e t r e e ' s n a t u r a l d i s t r i b u t i o n range. The d i f f e r e n t p a r t s o f a woody p l a n t may a l s o d i f - f e r i n t h e degre e o f f r o s t h a r d i n e s s o b t a i n e d , w i t h exposed p a r t s such as t h e n e e d l e s o f c o n i f e r s u s u a l l y o b t a i n i n g t h e maximum h a r d i n e s s l e - v e l s ( P a r k e r , 1963). An i m p o r t a n t c o n c e p t t o be c o n s i d e r e d i n i n t e r p r e t i n g and comparing f r o s t h a r d i n e s s o b s e r v a t i o n s between d i f f e r e n t taxonomic p l a n t groups i s the p o s s i b i l i t y o f d i f f e r i n g f r o s t h a r d i n e s s mechanisms b e i n g employed f o r a g i v e n l e v e l o f h a r d i n e s s a c q u i s i t i o n . The genus P i n u s i s b e l i e v e d t o have e v o l v e d i n t h e h i g h e r l a t i t u d e s o f t h e n o r t h e r n hemisphere ( M i r - ov, 1967) and t h e f r o s t h a r d i n e s s o f most members o f t h e genus i s probab- l y a v e r y a n c i e n t t r a i t . The angiospermous p l a n t s t h a t dominate t h e p l a n - e t today a r e b e l i e v e d t o have been p r o g r e s s i v e l y e x p a n d i n g i n t o more hos- t i l e h i g h a l t i t u d e and l a t i t u d e r e g i o n s s i n c e t he C r e t a c e o u s t i m e p e r i o d (Regay, 1977). Thus the a c q u i s i t i o n o f f r o s t h a r d i n e s s has p r o b a b l y o c- c u r r e d many t i m e s i n g e o l o g i c t i m e and i s p r o b a b l y s t i l l o c c u r r i n g i n g e n e t i c a l l y and range e x p a n d i n g taxonomic groups. These f a c t o r s n e c e s s i - t a t e t h e use o f c a u t i o n when comparing f r o s t h a r d i n e s s s t u d i e s between d i f f e r i n g groups o f p l a n t s , and has p r o b a b l y r e s u l t e d i n t h e f r o s t r e - s i s t a n c e mechanisms o f p l a n t s becoming a v e r y complex phenomenon, p a r t i c u - l a r l y on a c o m p a r a t i v e b a s i s . The development, m a i n t e n a n c e , and l o s s o f f r o s t h a r d i n e s s i s a sea - s o n a l p a r t o f the continuum o f g r o w t h , d i f f e r e n t i a t i o n , and senescence i n p l a n t t i s s u e s . The s t u d y o f f r o s t h a r d i n e s s must t a k e t h e s e phenomena 82 (which o v e r l a p s e a s o n a l c y c l e s ) i n t o c o n s i d e r a t i o n . E x p e r i m e n t a l l y , the p i n e n e e d l e i s w e l l s u i t e d f o r s e p a r a t i n g o t h e r c e l l u l a r p r o c e s s e s w h i c h may c o n f u s e f r o s t h a r d i n e s s s t u d i e s because o f a c h a r a c t e r i s t i c r a p i d growth phase f o l l o w e d by s e v e r a l y e a r s o f m a t u r i t y and f u n c t i o n - a l a c t i v i t y , b e f o r e senescence'becomes a dominant f a c t o r . Sudworth (1908) o b s e r v e d green n e e d l e s o f a t l e a s t e i g h t y e a r s o f age on young P i n u s c o n t o r t a t r e e s . N e e d l e s w h i c h were green and h e a l t h y i n appearance up t o t w e l v e y e a r s i n age were o b s e r v e d on young open grown t r e e s o f P. c o n t o r t a a t t h e s a m p l i n g s i t e s . The age o f n e e d l e s on young P i n u s sp. can be s i m p l y d e t e r m i n e d by c o u n t i n g l o n g shoot i n t e r n o d e s from t h e t e r m i n a l buds t o t h e i n t e r n o d e on t h e br a n c h o r a x i a l t r u n k on w h i c h t h e n e e d l e s o c c u r (Dickmann and K l o z l o w s k i , 1970). The o c c u r r e n c e o f lam- mas o r p r o l y p s i s growth forms would i n v a l i d a t e t h i s method o f n e e d l e age d e t e r m i n a t i o n , but no examples o f t h e s e growth forms were o b s e r v e d . Lam- mas forms o f growth have been r e p o r t e d i n P i n u s d i v a r i c a t a ( b a n k s i a n a ) (Rudolph, 1964),(which i s a h y b r i d i z i n g c l o s e r e l a t i v e o f P. c o n t o r t a , C r l t - c h f i e l d , 1957), i n t h e G r e a t L a k e s a r e a o f e a s t e r n N o r t h A m e r i c a . The common o c c u r r e n c e o f v e r y o l d n e e d l e s on young open grown t r e e s a t t h e s a m p l i n g s i t e s appears t o be dependent on c o n t i n u e d h i g h growth r a t e s and i n s o l a t i o n d u r i n g t h e l i f e o f t h e n e e d l e . The open grown, w e l l spaced young t r e e s a t the s a m p l i n g s i t e s had v e r y l a r g e annual l o n g shoot growth i n c r e m e n t s . T h i s r e s u l t e d i n i n t e r n o d e d i s t a n c e s o f us- u a l l y g r e a t e r than 20 cm, w h i c h when combined w i t h a l a c k o f cr o w d i n g 83 and s h a d i n g between i n d i v i d u a l t r e e s , gave t h e erown a v e r y open growth form and l e s s e n e d the s h a d i n g o f o l d e r n e e d l e s by r e c e n t growth r e s u l t - ing i n t h e c o n t i n u e d i n s o l a t i o n o f o l d e r n e e d l e s . The growth o f new n e e d l e s was o b s e r v e d t o s t a r t i n e a r l y June a t the c o l l e c t i n g s i t e s w i t h t h e emergence o f s h o r t shoots: and r a p i d l o n g shoot e x p a n s i o n . By the end o f J u l y , t h a t season's n e e d l e s had r e a c h - ed t h e l e n g t h o f o l d e r mature n e e d l e s . M i r o v (1967) has o b s e r v e d spec- i a l c a s e s i n w h i c h n e e d l e e l o n g a t i o n c o n t i n u e s f o r a y e a r o r more i n t r o p i c a l and s u b - t r o p i c a l p i n e s . T h i s growth appears t o be .due t o ' t h e a c t i v i t y i n the b a s a l m e r i s t e m o f t h e n e e d l e (Esau, 1965). C e l l d i v i - s i o n does not o c c u r i n t h e a p i c a l m e s o p h y l l r e g i o n s o f f u l l y expanded n e e d l e s o b s e r v e d i n t h i s work. R e s i d u a l a c t i v i t y i n v a s c u l a r cambia w i t h i n t h e s t e l e t h r o u g h o u t the l i f e o f the n e e d l e would a l s o not a f - f e c t t he m e s o p h y l l s i n c e i t appears t o s o l e l y t o pr o p a g a t e v a s c u l a r t i s s u e (Huber, 1947). C y t o l o g i c a l l y , t h e m e s o p h y l l c h l o r e n c h y m a o f P_. c o n t o r t a n e e d l e s i n t h e p e r i o d o f n e e d l e e x p a n s i o n were o b s e r v e d t o have t h e c h a r a c t e r - i s t i c s o f c e l l s w i t h h i g h m e t a b o l i c a c t i v i t y r e l a t i v e t o c e l l s o f o l d e r n e e d l e s . T h i s c e l l t y p e was c l a s s i f i e d as t h e t y p e I c e l l form i n t h i s work. A f t e r n e e d l e e x p a n s i o n was c o m p l e t e d , t h e s i z e and c o m p l e x i t y o f m i t o c h o n d r i a were reduced and the o c c u r e n c e o f d i c t y o s o m e s , d i c t y o s o m - a l v e s i c l e s , RER and SER were g r e a t l y r educed. T h i s c e l l t y p e was 84 termed t h e t y p e II c e l l . I t i s c o n c l u d e d t h a t t h e ty p e II c e l l r e p r e s - e n t s t h e t r a n s f o r m a t i o n o f t h e c y t o p l a s m from a s t a t e o f growth ( t y p e I c e l l ) t o a s t a t e o f mature f u n c t i o n a l a c t i v i t y ( t y p e II c e l l ) . The t y p e II c e l l was found t o be t h e dominant c e l l t y p e i n t h e n e e d l e c h l o - renchyma i n c u r r e n t season n e e d l e s f i x e d i n J u l y and August and i n one, two, t h r e e , and f i v e y e a r o l d n e e d l e s f i x e d between e a r l y June and l a t e August (see t a b l e I ) . A t h i r d c e l l t y p e was i d e n t i f i e d and c a l l e d t h e t y p e I I I c e l l . The i n c i d e n c e o f t h i s t y p e o f c e l l i n c r e a s e d i n p r o p o r t i o n t o n e e d l e age. T h i s c e l l t y p e was i n t e r p r e t e d t o be a , s e n e s c e n t o r d e g e n e r a t i n g c e l l t y p e on t h e b a s i s o f c y t o l o g i c a l p r o p e r t i e s ( B u t l e r and Simon, 1970 and was c h a r a c t e r i z e d i n t h i s work o n l y t o i n s u r e t h a t i t would n o t be con- f u s e d w i t h s e a s o n a l changes. The c y t o l o g i c a l s t a b i l i t y o f the t y p e II c e l l and t h e c o r r e s p o n d - ing l o n g n e e d l e l i f e span made the n e e d l e m a t e r i a l v e r y u s e f u l f o r s t u d y i n g s e a s o n a l l y r e l a t e d phenomena s i n c e t h e same'.type o f t i s s u e can be sampled b e f o r e , d u r i n g and a f t e r w i n t e r t h r o u g h s e v e r a l y e a r l y c y c l e s : In c o n t r a s t , the m a j o r i t y o f t h e c y t o l o g i c a l s t u d i e s on t h e f r o s t h a r d i - ness phenomena i n woody p l a n t s have been co n d u c t e d on t h e v a s c u l a r cam- bium (e.g. Murmanis, 1970) o r se c o n d a r y stem parenchyma (SI i m i n o v l t e h "et". aj_. ,1968) i n w h i c h i n d i v i d u a l c e l l s have a l i f e span o f one t o two y e a r s , d u r i n g w h i c h growth d i f f e r e n t i a t i o n and s e n e s c e n t p r o c e s s e s must be s e g r e g a t e d from c y t o p l a s m i c changes s o l e l y dependent on s e a s o n a l f a c - t o r s . 85 I I The D i s c u s s i o n o f Seasona1 Changes i n the Cyto p l a s m o f Needle Chl o- renchyma A f t e r e s t a b l i s h i n g what was o c c u r r i n g T i n t h e chlorenchyma c e l l s w i t h r e g a r d s t o growth, m a t u r i t y and s e n e s c e n c e , i t was then p o s s i b l e t o p r o - ceed w i t h t h e main o b j e c t i v e o f t h i s work wh i c h i n v o l v e s t h e s t u d y o f the s e a s o n a l changes i n t h e c y t o p l a s m o f the n e e d l e chlorenchyma o f P. c o n t o r t a . I t has been e s t a b l i s h e d t h a t t h e ty p e II c e l l was t h e predominant c e l l t y p e o b s e r v e d i n a l l f i x a t i o n s c o n d u c t e d between June 6th and August 10th ( t a b l e I ) . The c y t o l o g i c a l c o n s i s t e n c y o f t h i s c e l l t ype has l e d t o the term " t h e mature summer s t a t e " b e i n g a p p l i e d t o d e s c r i b e t y p e II c e l l s . F i x a t i o n s conducted d u r i n g t h e w i n t e r o f 1972 t o 1973 (November 26th t o March 30th) r e v e a l e d a c o m p l e t e l y d i f f e r e n t c y t o p l a s m i c s t a t e . L i k e the summer s t a t e , equal u l t r a s t r u c t u r a l c o n s i s t e n c y i s o b s e r v e d i n a l l f i x a t i o n s o f a l l n e e d l e ages examined. The r e m a r k a b l e d i f f e r e n t c y - t o p l a s m i c s t a t e i n w i n t e r i s termed " t h e w i n t e r c y t o p l a s m i c s t a t e " . The w i n t e r s t a t e i s b e l i e v e d t o be t h e form o f t h e summer t y p e II c e l l d u r - i n g t h e w i n t e r f r o s t hardy s t a t e between November 16th, 1972 and March 30th, 1973. A number o f w i n t e r s t a t e c e l l s , ( p r o p o r t i o n a l t o n e e d l e age) were b e l i e v e d t o be t h e w i n t e r e q u i l v a l e n t o f ty p e I I I summer s t a t e c e l l s . The more i n t e n s e l y o s m i o p h i l i c w i n t e r e q u i l v a l e n t s o f ty p e I I I c e l l s ap- peared i d e n t i c a l t o t h e i r summer c o u n t e r p a r t s . The l e s s o s m i o p h i l i c t y p e 86 II I e e l I s t h a t had some r e c o g n i z a b l e o r g a n e l l e s and had some o f t h e c y t o - l o g i c a l p r o p e r t i e s o f the w i n t e r s t a t e t y p e II c e l l s . These o b s e r v a t i o n s have l e d t o the c o n c l u s i o n t h a t t h e summer and w i n t e r c y t o p l a s m i c s t a t e s a r e c o m p l e t e l y r e v e r s i b l e on a s e a s o n a l b a s i s . Thus t h e summer and w i n t - e r " s t a t e s " a r e s e a s o n a l t r a n s f o r m a t i o n s o f mature f u n c t i o n a l m e s o p h y l l chlorenchyma c e l l s w h i c h have an u n u s u a l l y l o n g l i f e span f o r c e l l s w i t h - i n t he p l a n t kingdom. In t he w i n t e r c y t o p l a s m i c s t a t e , a l m o s t e v e r y c y t o p l a s m i c s t r u c t u r e and o r g a n e l l e has changed s t r u c t u r a l l y o r p o s i t i o n a l l y . So d i f f e r e n t and unusual i s the w i n t e r c y t o p l a s m compared t o summer f i x a t i o n s and p u b l i s h - ed p h o t o m i c r o g r a p h s o f mature p l a n t t i s s u e t h a t t h e a u t h o r b e l i e v e d i n i t - i a l l y t h a t t h e w i n t e r s t a t e was a f i x a t i o n a r t i f a c t . The most d r a m a t i c change i n t h e w i n t e r chlorenchyma i s t h e s t r u c t u r - a l and p o s i t i o n a l changes i n t h e w i n t e r c h l o r o p l a s t s . The a s p e c t o f sea - s o n a l change i n c h l o r o p l a s t s o f w i n t e r e v e r g r e e n s has r e c e i v e d much a t t e n - t i o n i n t h e e a r l y l i t e r a t u r e . Mohl (1837) n o t i c e d t he s e a s o n a l c h l o r o s i s in some w i n t e r c o n i f e r s and sug g e s t e d t h a t o r g a n i c a c i d s d e s t r o y e d •the pigments i n a s s i m i l a t o r y c e l l s but t h e pigment b o d i e s ( c h l o r o p l a s t s ) remained i n t a c t . Kraus (1874), H a b e r l a n d t (1876) and Schimper (1885) no- t i c e d t he cl u m p i n g o f c h l o r o p l a s t s i n t h e n e e d l e s o f s e v e r a l w i n t e r c o n i - f e r s . However, i n h e r b s , n o n - c o n i f e r o u s d e c i d u o u s and e v e r g r e e n woody p l a n t s , t he b e h a v i o r o f c h l o r o p l a s t s o r p l a s t i d s was h i g h l y v a r i a b l e . In some s p e c i e s , t h e s e a u t h o r s r e p o r t e d no change, some s l i g h t movement from summer p o s i t i o n s , c l u m p i n g , o r c h l o r o p l a s t d i s a p p e a r a n c e . They a l s o r e - 87 p o r t e d g r e a t v a r i a t i o n s i n c h l o r o p l a s t changes w i t h i n i n d i v i d u a l l e a v e s . However, Haber1andt (l876) p a r t i c u l a r l y b e l i e v e d t h a t i n most o v e r w i n t e r i n g p l a n t s , c h l o r o p l a s t s were not d e s t r o y e d . Lewis and . T u t t l e (1923) observed c h l o r o p l a s t c l u m p i n g about the n u c l e u s i n P. g l a u c a i n t h e f a l l and by e a r l y w i n t e r , w i t h f r o s t s o f i n c r e a s i n g se- v e r i t y , t h e c h l o r o p l a s t clumps d i s i n t e g r a t e d and d i s a p p e a r e d . They used the term " l a k e d " ( a p p a r e n t l y an o l d m e d i c a l term more o r l e s s synonynous w i t h l y s e d ) t o d e s c r i b e d t h i s d i s i n t e g r a t i o n . Zacharowa (1929) o b s e r v e d the w i n t e r break down o f c h l o r o p l a s t a f t e r an i n i t i a l c l u m p i n g i n both P i n - cea and P i n u s s p e c i e s , and used the term " z e r f a l l e n " (German: f a l l e n a p a r t ) t o d e s c r i b e d t h i s p r o c e s s . H o l z e r (1958) conducted d e t a i l e d o b s e r v a t i o n s o f c h l o r o p l a s t c l u m p i n g i n the c y t o p l a s m between t h e t r a b i c u l a e i n P i n u s and i n the p a r i e t a l c y t o p l a s m i c r e g i o n s o f the c e l l s i n o t h e r c o n i f e r s . H i s o b s e r v a t i o n s appear t o be v e r y s i m i l a r t o t h o s e r e p o r t e d i n t h i s work. U n f o r t u n a t e l y , H o l z e r ' s work i s not known i n the g e n e r a l l i t e r a t u r e . The c o n t r o v e r s y o v e r the f a t e o f w i n t e r c h l o r o p l a s t s c o n t i n u e d when both G e r h o l d (1959) and P e r r y e_t a_l_. (1965) p u b l i s h e d papers w h i c h i n d i c a t - ed a c o n t i n u e d a c c e p t a n c e o f the p o s s i b i l i t y t h a t t h e c h l o r o p l a s t s i n c o n i - f e r s may break down d u r i n g w i n t e r . Harris (1971) f a i l e d t o n o t i c e any w i n - t e r c l u m p i n g i n the c h l o r o p l a s t s o f P. s t r o b u s but as s t a t e d e a r l i e r , h i s o b s e r v a t i o n s were co n d u c t e d i n a s u b - t r o p i c a l l o c a t i o n . P a r k e r and P h i l - p o t t (1961 and 1963) publ i.shed u l t r a s t r u c t u r a l p h o t o m i c r o g r a p h s o f w i n t e r c h l o r o p l a s t s i n Rhododendron and P i n u s w i t h t h e e x p r e s s purpose o f d i s - p r o v i n g the c h l o r o p l a s t d e s t r u c t i o n c o n c e p t . They a l s o r e p o r t e d e x t e n - 88 s i v e c l u m p i n g o f t h e c h l o r o p l a s t s i n w i n t e r f i x a t i o n s o f P i n u s n e e d l e s and bark parenchyma. In t h i s s t u d y , c h l o r o p l a s t c l u m p i n g was r e g u l a r l y o b s e r v e d i n a l l t h e w i n t e r f i x a t i o n s o f the c o n i f e r s examined. I t was a l s o o b s e r v e d l a t e r by Chabot and Chabot (1975) i n A b i e s . No e v i d e n c e f o r c h l o r o p l a s t d e s t r u c t i o n o r a d e c r e a s e i n t h e i r r e l a t i v e numbers was seen i n t h i s work, nor i t was mentioned by Chabot and Chabot (1975) o r b y . P a r k e r and P h i l p o t t (1961 and 1963)- I t ap p e a r s from t h e s e s t u d i e s t h a t t h e d e s t r u c t i o n t h e o r y - c o n c e r n - ing w i n t e r c h l o r o p l a s t s was e i t h e r an o b s e r v a t i o n a l e r r o r and/or t h e r e s u l t o f p l a s t i d d e s t r u c t i o n d u r i n g p r e p a r a t i o n f o r o b s e r v a t i o n . The s t r u c t u r a l n a t u r e o f c l u m p i n g o f w i n t e r c h l o r o p l a s t s ' i s b e s t r e - v e a l e d w i t h e l e c t r o n m i c r o s c o p y . High m a g n i f i c a t i o n s t u d i e s o f t h e r e g i o n s o f c h l o r o p l a s t c o n t a c t r e v e a l s what may be a t r i p a r t i t e l a y e r i n g o f t h e en- v e l o p e membranes ( f i g u r e s 45 and 46). T h i s s t r u c t u r e i s i n t e r p r e t e d t o i n d i c a t e a f u s i o n o f t h e o u t e r membranes o f each a d j a c e n t c h l o r o p l a s t en- v e l o p e s t o form a s t r u c t u r e cons i s t ing o f two a t t a c h e d membranes. , The f u s e d o u t e r e n v e l o p e membranes a r e b e l i e v e d t o make up t h e c e n t r a l t h i c k component o f t h e t r i p a r t i t e s t r u c t u r e . The i n n e r membranes o f t h e e n v e l o p e o f each p l a s t i d c o n s t i t u t e s t h e o u t e r two t h i n n e r membrane components o f the complex. P a r k e r and P h i l p o t t (1961 abd 1963) d i d not d i s c u s s t h e f i n e s t r u c t u r e o f the c h l o r o p l a s t e n v e l o p e s w i t h i n . t h e c h l o r o p l a s t clumps and t h e i r p u b l i s h e d p h o t o m i c r o g r a p h s a r e o f t o o low a m a g n i f i c a t i o n t o d e t e r - mine t h e s t r u c t u r e o f a d j a c e n t e n v e l o p e membranes. 89 Cons iderab le i n t e r e s t i s now focused on the f u s i o n of l i v i n g p r o t o - p l a s t s of d i f f e r e n t c e l l s from s i m i l a r organisms or comple te l y d i f f e r e n t organisms. Besides removal of the c e l l w a l l ( i f p r e s e n t ) , an apparent l y e s s e n t i a l p r e r e q u i s i t e to s u c c e s s f u l f u s i o n i s the treatment of the c e l l by some m i l d l y t o x i c m a t e r i a l to induce the p r o t o p l a s t s to f u s e . Such methods involved treatment w i th sodium ni t rate(Powerand Fleming, H970) r a d i a t i o n , v i - r a l i n o c u l a t i o n , s p e c i f i c a n t i s e r a (to the membrane prote inous components), high pH, p a r t i a l and b r i e f lysozyme d i g e s t i o n , p o l y e t h y l e n e g l y c o l and p o l y - L - l y s i n e , and probably severa l more methods have been found (see Burgess et a l . (1974) f o r re ferences to the above) . Douglas (1974) c i t e s many cases in which c a l c i u m i s invo lved in the process of the membrane f u s i o n in the e x o c y t o s i s of c y t o p l a s m i c v e s i c l e s and the f u s i o n of plasma membranes. Schober et a l . (1977) has observed that the aggregat ion of i s o l a t e d chromaf f in g ranu les occurs in areas where there has been a c a l c i u m induced area on the g ranu le membrane that i s f r e e of s u r f a c e p a r t i c l e s . What these t reatments and o b s e r v a t i o n s appear to have in common i s that the s i t e of a c t i o n by these t reatments i s the plasma membrane. U l t r a s t r - u c t u r a l s t u d i e s of the adherence zones seem to i n d i c a t e the areas of f u s i o n occur where the apposing plasma membranes of adjacent p r o t o p l a s t s come in to very c l o s e contact (Burgess et a l . , 1974). Subsequent to t h i s , the f u s i o n of p r o t o p l a s t s o c c u r . S i m i l a r process may apply to the f u s i o n between Pinus c h l o r o p l a s t s w h i c h lead to the mix ing of the stroma of ne ighbor ing c h l o r o - - p l a s t s . The e x i s t e n c e of separate systems of t h y l a k o i d s w i t h i n a common stroma seems to support t h i s . 90 Two q u e s t i o n s a r i s e out o f t h e o b s e r v a t i o n o f c h l o r o p l a s t c l u m p i n g . The f i r s t i s what b r i n g s t h e c h l o r o p l a s t s i n t o c l o s e c o n t a c t and t h e s e c - ond i s what may be happening a t t h e s u r f a c e o f the o u t e r e n v e l o p e membranes t o a l l o w t n e i r c l o s e a s s o c i a t i o n . The c a u s e o f c l u m p i n g and c l o s e a p p o s i t i o n o f o u t e r e n v e l o p e membranes i s b e l i e v e d t o be t h e r e s u l t o f f r o s t p l a s m o l y s i s and w i l l be d i s c u s s e d l a t e r . The second q u e s t i o n c o n c e r n s what may be happening a t t h e membrane s u r f a c e . Most membranes i n a p p o s i t i o n t o each o t h e r i n E u k a r y o t i c c e l l s a r e c h a r a c t e r i z e d by an a p p r o x i m a t e l y hO nm o r more gap between s u r f a c e s w h i c h r e s u l t i n r e g u l a r p a r a l l e l s p a c i n g o f t h e membranes. Examples o f such s p a c i n g s would be g o l g i c i s t e r n a e , a l g a l t h y l a k o i d s y s t e m s , n u c l e a r and c h l o r o p l a s t e n v e l o p e s e t c . , and the s p a c i n g between animal t i s s u e c e l l s . T h i s gap i s p r o b a b l y a b a l a n c e between a d h e r e n c e p r o m o t i n g f o r c e s and mutual r e p u l s i v e f o r c e s on t h e a p p o s i n g membrane s u r f a c e s t h a t c a u s e t h i s s t r u c t u r a l a rrangement. R e p u l s i v e f o r c e s a r e t h e n e t n e g a t i v e c h a r g e s a s s o c i a t e d w i t h membrane s u r f a c e p r o t e i n s (Power e_t_ aj_. , 1 9 7 0 ) , and/or the s t r o n g b o n d ing due t o t h e h y d r o p h i l i c phase o f t h e p o l a r ends o f t h e membrane l i p i d s and t h e aqueous phase o f t h e c y t o p l a s m . The a d h e r e n c e f o r - c e s t h a t m a i n t a i n a r e g u l a r p a r a l l e l s p a c i n g , and w h i c h p r e v e n t t h e membr- ane s u r f a c e s from d r i f t i n g a p a r t may due t o t h e c r o s s b r i d g e s formed by p r o t e i n a c o u s b r i d g e s between the p a r a l l e l membranes ( F r a n k e et_ aj_. , 1971) - The c h e m i c a l , p h y s i c a l and b i o l o g i c a l t r e a t m e n t s d e s c r i b e d above f o r the i n d u c t i o n o f p r o t o p l a s t f u s i o n appear t o a f f e c t m a i n l y t h e r e p u l s i v e f o r c e s between c e l l s i n o r d e r t o promote c l o s e r membrane a s s o c i a t i o n . Rad- 91 i a t i o n , e n z y m a t i c d i g e s t i o n and p o s i t i v e l y c h arged e l e c t r o l y t e s a r e p r o b a - b l y t a r g e t e d towards membrane o u t e r s u r f a c e p r o t e i n s ; w h i l e the p o l y - a l c o - h o l s may a f f e c t p r o t e i n s and/or h y d r o p h i l i c l i p i d i n t e r a c t i o n s . S i m i l a r l y , a n t i s e r a would be t a r g e t e d t o membrane s u r f a c e p r o t e i n s . C a l c i u m may neu- t r a l i z e s u r f a c e r e p u l s i v e c h a r g e s on membrane p r o t e i n s thus a l l o w i n g them t o clump and open p r o t e i n f r e e a r e a s . Schober (1977) s u p p o r t s a t h e o r y s u g gest by Ahkong e t a l . (1975) t h a t b e f o r e membrane f u s i o n can o c c u r , mem- brane s u r f a c e p r o t e i n s must be d i s p l a c e d so t h a t t h e l i p i d b i l a y e r s o f a d j a c e n t membranes can come i n t o c l o s e c o n t a c t . The common i n f e r e n c e from a l l t h e above p r o c e d u r e s f o r pr o m o t i n g mem- brane f u s i o n i s t h a t t h e t a r g e t s i t e o f t h e a g e n t s used i s t h e membrane s u r f a c e p r o t e i n s . Membrane s u r f a c e p r o t e i n s ( e c t o p r o t e i n s ) a r e now known t o be l a r g e l y g l y c o p r o t e i n s and a r e p r o t e i n s s p e c i f i c t o the e x t e r n a l plasma membrane s u r f a c e s (Rothman a:nd Lena rd, 1977) • I t can be suggested t h a t t h e g l y - c o p r o t e i n s o f the c h l o r o p l a s t o u t e r e n v e l o p e d membranes a r e damaged o r a l t - e red i n some manner d u r i n g w i n t e r . In t h i s work, one o f t h e few s t r u c t u r a l changes o b s e r v e d w i t h t h e sea wate r e l e c t r o l y t e f i x a t i v e on w a t e r t i s s u e was t h e marked r e d u c t i o n o f c h l o - r o p l a s t a d h e r s i o n z o n e s . T h i s may have r e s u l t e d from some i n t e r a c t i o n be- tween t h e s a l t s i n t h e f i x a t i v e s o l u t i o n and the p h y s i c a l p r o p e r t i e s o f t h e membrane s u r f a c e p r o t e i n s . The o b s e r v a t i o n s on t h e f i n e s t r u c t u r e o f t h e e n v e l o p e membranes o f clumped w i n t e r c h l o r o p l a s t s i n t h i s work have not been r e p o r t e d i n t h e l i t - 92 e r a t u r e . P a r k e r and P h i l p o t t (1961 and 1963) d i d not d i s c u s s t h e f i n e s t r - u c t u r e o f c h l o r o p l a s t e n v e l o p e s i n t h e i r o b s e r v a t i o n s on t h e clumped w i n t - e r c h l o r o p l a s t s o f P i n u s s t r o b u s . T h e i r p u b l i s h e d p l a t e s o f clumped c h l o - r o p l a s t s a r e o f q u i t e low m a g n i f i c a t i o n . However , t h e i r photographs i n d - i c a t e t h a t c l o s e a s s o c i a t i o n between o u t e r membranes o f a d j a c e n t c h l o - r o p l a s t may o c c u r . Chabot and Chabot (1975) mentioned c h l o r o p l a s t c l u m p i n g i n A b i e s sp. but d i d not p u b l i s h a p l a t e showing clumped c h l o r o p l a s t s o r d i s c u s s t h e f i n e s t r u c t u r e o f the e n v e l o p e membranes o f clumped c h l o r o p l a s t s . Only t h e c h l o r o p l a s t s e x h i b i t the clump ing and close outer membrane association p r o p e r t i e s . M i t o c h o n d r i a were o f t e n i n l o o s e c l u s t e r s but never were ob- se r v e d t o adhere. S i m i l a r l y , t h e r e was never any adherence between c h l o r o - p l a s t s and o t h e r o r g a n e l l e s i n w i n t e r f i x a t i o n s . I t can be c o n c l u d e d t h a t c l u m p i n g o f w i n t e r c h l o r o p l a s t s and t h e f u s i o n o f t h e i r o u t e r membranes i n - d i c a t e t h a t t h e c h l o r o p l a s t s o u t e r e n v e l o p e membranes have d i f f e r i n g chemi- c a l p r o p e r t i e s than o t h e r o r g a n e l l e membranes d u r i n g t h e w i n t e r . I t was o b s e r v e d t h a t about t e n t o t h i r t y p e r c e n t o f a l l w i n t e r c h l o r o - p l a s t s had two o r more t h y l a k o i d systems w i t h i n a common e n v e l o p e based on o b s e r v a t i o n s on t h i n s e c t i o n s . T h i s i n d i c a t e s t h a t i n t h e r e a l i t y o f t h r e e d i - m e n s i o n a l s p a c e , t h e m a j o r i t y o f t h e w i n t e r p i a s t i d s may be d o u b l e systems. H a r r i s (1971) and Chabot and Chabot (1975) d i d not r e p o r t any d o u b l e c h l o r o - p l a s t s i n w i n t e r c o n i f e r t i s s u e but P a r k e r and P h i l p o t t (1963) d id mention t h a t " d i v i s i o n 1 i k e c o n s t r i c t i o n s " were n o t i c e d i n o b s e r v a t i o n s d u r i n g t h e f a l l on t h e c h l o r o p l a s t s o f P i n u s s t r o b u s but not d u r i n g w i n t e r . From t h e s e 93 a u t h o r s ' d e s c r i p t i o n o f t h e d i v i s i o n a l s t r u c t u r e s , t h e p l a s t i d s t h e y obs- e r v e d may have l o o k e d l i k e t h e c h l o r o p l a s t s i n f i g u r e kk. I t seems r e a s o n - a b l e f o r P a r k e r and P h i l p o t t t o have c o n c l u d e d t h a t t h e c h l o r o p l a s t ' s n a r r - ow c o n s t r i c t i o n s s i m i l a r t o t h o s e o b s e r v e d i n f i g u r e kh c o u l d r e p r e s e n t p r e - d i v i s i o n a l s t r u c t u r e s . However, i n t h i s work, t h e c h l o r o p l a s t s i n f i g - rue hk were a t y p i c a l , and t h e appearance o f most w i n t e r c h l o r o p l a s t s appear t o be more l i k e l y t h e r e s u l t o f f u s i o n f o r t h e f o l l o w i n g r e a s o n s : 1. Membrane adherence i n c l o s e , a s s o c i a t i o n . - may be a p r e - r e q u i s i t e f o r f u s i o n (Ahkong e t a l . , 1975)• 2. For d i v i s i o n by c o n s t r i c t i o n t o o c c u r , one would e x p e c t t o f i n d c o n t r i c t i o n s o f , o r s e p a r a t i o n s i n t h e i n t e r n a l t h y l a k o i d s y s - tem as w e l l as i n t h e e n v e l o p e membranes. But a l t h o u g h t h y l a k o i d s a r e v i s i b l e between p i a s t i d s c o n s t r i c t i o n s i n f i g u r e kh (open a r - row), t h e y were not o b s e r v e d t o be i n t h e p r o c e s s o f s e p a r a t i n g o r p i n c h i n g a p a r t . C h l o r o p l a s t t h y l a k o i d s c o u l d a l s o g r e a t l y e l - o n g a t e b e f o r e c o n s t r i c t i o n d i v i s i o n . I f t h i s were t h e c a s e , one would e x p e c t c h l o r o p l a s t w i t h v e r y lon g t h y l a k o i d systems would have been o b s e r v e d i n t h i s work. No such s t r u c t u r e s were seen. 3. The t h y l a k o i d systems o f d o u b l e c h l o r o p l a s t s a r e a l l o f t h e same morphology and s i z e as i n normal mature c h l o r o p l a s t s . No s m a l l e r systems than would be e x p e c t e d from c o n s t r i c t i o n d i v i s i o n p r o d - u c t s were o b s e r v e d . k. I t would be unusual t o e x p e c t c h l o r o p l a s t d i v i s i o n s t o be o c c u r - ing i n t h e dormant w i n t e r s t a t e . 5. The t h y l a k o i d systems w i t h i n t h e common e n v e l o p e s were o r i e n t e d a t v a r i o u s a n g l e s t o each o t h e r and o f t e n had l a r g e a r e a s o f s t r o - 94 ma s e p a r a t i n g them ( f i g u r e 43). In post d i v i s i o n p r o d u c t s , one would e x p e c t t o f i n d some t h y l a k o i d membrane systems p a r t i a l l y a t t a c h e d o r a t l e a s t a r r a n g e d i n t h e same p l a n e . On t h e b a s i s o f t h i s d i s c u s s i o n , i t seems more l i k e l y t h a t t h e m u l t i - p l e t h y l a k o i d system c h l o r o p l a s t s were t h e r e s u l t o f f u s i o n between mature p l a s t i d s . A p p a r ent f u s i o n s between c h l o r o p l a s t s have been o b s e r v e d i n a n a t u r - a l p o p u l a t i o n o f t h e h y d r o p h y t e Mimosa p u d i c a (Esau, 1972) and i n r a d i a t i o n induced mutants ( A l l e n et_ aj_., 1972). These a u t h o r s a l so c i t e d s e v e r a l o t h e r c a s e s where r a d i a t i o n has induced c h l o r o p l a s t f u s i o n i n d i f f e r i n g p l a n t s . Ra- d i a t i o n has a l s o been used t o f u s e p l a n t p r o t o p l a s t s as d i s c u s s e d above. F u r - t h e r more, c h l o r o p l a s t s i n t h e r a d i a t i o n exposed p r o t h a l i o f a f e r n used by A l l e n e_t aj_. were o b s e r v e d t o clump t o g e t h e r a f t e r r a d i a t i o n e x p o s u r e and be- f o r e t h e f u s i o n p r o c e s s . I n i t i a l l y , a g g r e g a t e c h l o r o p l a s t s i n r a d i a t i o n expo- sed f e r n p r o t h a l i c o n s i s t o f many i n d i v i d u a l , n o r m a l , d i s c o i d a l a p p e a r i n g t h y l a k o i d systems. But t h e i n d i v i d u a l t h y l a k o i d systems appear t o e v e n t u a l l y f u s e i n t o l a r g e membrane masses ( A l l e n e_t aj_. , 1972).. T h i s does not o c c u r i n w i n t e r p i n e c h l o r e n c h y m a . The t h y l a k o i d systems appear i d e n t i c a l t o t h e sum- mer t h y l a k o i d systems both i n shape and i n i n d i v i d u a l membrane s t r u c t u r e . I t i s i n t e r e s t i n g t o n o t e t h a t t h e t h y l a k o i d membrane o f t h e w i n t e r c h l o r o p l a s t a r e t h e o n l y w i n t e r membranes t h a t a r e s t r u c t u r a l l y s i m i l a r t o summer membranes. By t h e v i r t u e o f t h e i r t r a n s f o r m a t i o n from d i s c o i d a l t o a p p r o x i m a t e l y s p h e r i c a l shapes, t h e w i n t e r c h l o r o p l a s t s i n c r e a s e s u b s t a n t i a l l y i n volume. 95 T h i s c o n c l u s i o n was reached by t r a n s p o s i n g s e c t i o n a l a r e a s i n t o volumes w h i c h i n d i c a t e d t h a t w i n t e r c h l o r o p l a s t s (not i n c l u d i n g t h e f u s e d c h l o r o p l a s t s ) a r e about one and one h a l f t o two t i m e s t h e volume o f summer c h l o r o p l a s t s . T h i s v o l u m e t r i c i n c r e a s e was found t o be due t o t h e i n c r e a s e i n t h e s t r o m a l a r e a s o f t h e c h l o r o p l a s t s . A s i m p l e i n c r e a s e i n stroma p r o b a b l y a l s o a c - c o u n t s f o r t h e change i n shape. The s t a i n d e n s i t y o f c h l o r o p l a s t stroma i n w i n t e r remains t h e same as t h o s e o f summer c h l o r o p l a s t s . Thus t h e v o l u m e t r i c i n c r e a s e due t o a d d i t i o n a l stroma may i n d i c a t e a s u b s t a n t i a l a u g m e n t a t i o n o f t h e s t r o m a l phase o f t h e c h l o r o p l a s t s . Heber (1959) has noted a c o n s i d e r a - b l e i n c r e a s e i n s o l u b l e p r o t e i n and s u g a r s i n t h e c h l o r o p l a s t s o f hardy p l a n t s . The s w e l l i n g o f c h l o r o p l a s t s i n t o s p h e r i c a l forms i s o b s e r v e d i n n e a r l y a l l r e p o r t s on t h e c y t o l o g y o f f r o z e n p l a n t c e l l s (Heber, 1959 and C h e i n a n d W u 1 9 6 5 ) . In non hardy s p e c i e s , the s w e l l i n g a l s o o c c u r s even a t s l i g h t l y above f r e e z i n g t e m p e r a t u r e s ( K i m b a l l e_t a_l_. , 1 9 7 3 ) . In c o n c l u s i o n , d r a m a t i c changes i n w i n t e r c h l o r o p l a s t s i n p i n e c h l o r - enchyma appear t o be c y t o l o g i c a l l y due t o : t h e i r t r a n s l o c a t i o n i n t o c lumps, i n p h y s i c a l p r o p e r t i e s o f t h e c h l o r o p l a s t e n v e l o p e membranes, t h e l a r g e i n - c r e a s e i n s t r o m a l volume and the p o s s i b l e a u g m e n t a t i o n o f s t r o m a l c o n t e n t s . The t h y l a k o i d system a p p e a r s t o r e t a i n a s i m i l a r form t o t h a t o f t h e summer c h l o r o p l a s t s . A d e f i n i t e summer-winter-summer c y c l e was a l s o e s t a b l i s h e d f o r t h e m i t - o c h o n d r i a i n t h e p i n e c h l o r e n c h y m a . In summer, th e m i t o c h o n d r i a appear a s - s o c i a t e d w i t h o r g a n e l l e s w i t h w h i c h t h e y may have a f u n c t i o n a l r e l a t i o n s h i p , i . e . , c h l o r o p l a s t s and o i l b o d i e s . In w i n t e r however, the m i t o c h o n d r i a a r e 96 d i s p e r s e d i n t h e expanded c y t o p l a s m and i n l o o s e c l u s t e r s about the c h l o r o - p l a s t clumps. They cannot be s a i d t o be c o n s i s t e n t l y a s s o c i a t e d w i t h any d e f i n i t e s t r u c t u r e s such as o i l b o d i e s , a l t h o u g h t h i s was o c c a s i o n a l l y ob- s e r v e d ( f i g u r e 58). Chabot and.Chabot (1975) a l s o noted a g e n e r a l d i s p e r s i o n o f w i n t e r m i t o c h o n d r i a i n A b i e s . The shape and volume o f w i n t e r m i t o c h o n - d r i a a l s o changes from l a r g e r and i r r e g u l a r forms ( i n summer) t o t h e s m a l l e r and s p h e r i c a l form i n w i n t e r . Cat.esson(1974) a l s o d e s c r i b e d a s e a s o n a l r e d u c t i o n i n t h e m i t o c h o n d r i a o f maple s e c o n d a r y cambia. M i t o c h o n d r i a became much s i m p l e r and more u n i f o r m ( u s u a l l y o v a t e ) i n s e c t i o n a l shape i n w i n t e r as opposed t o l a r g e r and more complex forms i n f a l l . In e a r l y s p r i n g , he noted an i n c r e a s e i n s i z e and c o m p l e x i t y i n shape. But t h e r e was a r e t u r n t o s i m p l e shapes and s m a l l s i z e i n l a t e s p r i n g and e a r l y summer. T h i s second t r a n s f o r m a t i o n was not o b s e r v - ed i n t h i s s t u d y . M i t o c h o n d r i a l membranes become amorphous and i n d i s t i n c t i n w i n t e r ( f i g - u r e 53) i n both t h e e n v e l o p e and t h e c r i s t a e . The o b s c u r i n g o f s t r u c t u r e , the random d i s t r i b u t i o n o f m i t o c h o n d r i a , and the r e d u c t i o n i n volume may be c o r r e l a t e d t o t h e minimal amount o f r e s p i r a t i o n o b s e r v e d i n o v e r - w i n t e r i n g c o n i f e r s even when th e y a r e b r i e f l y r a i s e d t o above f r e e z i n g t e m p e r a t u r e s ( C l a r k , 1961). R e c e n t l y , m i c r o b o d i e s have been r e p o r t e d i n t h e m e s o p h y l l c hlorenchyma o f n e e d l e s up t o s i x months o l d (November o f t h e y e a r o f n e e d l e f l u s h ) i n P. s t r o b u s and P. m a r i a n a (Chabot and Chabot, 1974). No m i c r o b o d i e s were ob s e r v e d 97 i n P. C o n t o r t a . At the low m a g n i f i c a t i o n s , ( a t w h i c h most s e c t i o n s were s t u d i e d ) , t h e y c o u l d have been m i s s e d by i n t e r p r e t i n g them as m i t o c h o n d r i a . No a t t e m p t was made t o i d e n t i f y them by enzymic l o c a l i z a t i o n . As Chabot and Chabot (1974) noted t h e p r e s e n c e o f m i e r o b o d i e s i n f a t s t o r i n g t r e e s i s v e r y i n t e r e s t i n g , but t h e y were o n l y a b l e t o c y t o c h e m i c a l l y i d e n t i f y c a t a l a s e a c t i v i t y . C h i n g (1970) has d e m o n s t r a t e d the p r e s e n c e o f g l y o x y s o - mes i n t h e o i l b o d i e s o f g e r m i n a t i n g megagametophytes i n P. ponderosa seeds. The p r e s e n c e o f glyoxysome m i e r o b o d i e s ( i f p r e s e n t i n mature p i n e c h l o r e n - chyma) s h o u l d be most a p p a r e n t (based on t h e s t u d i e s i n t h i s work) i n the s p r i n g when i t appears t h a t t h e f a t r e s e r v e s o f t h e chlorenchyma a r e m o b i l - i z e d . The c o n i f e r s appear t o have a m a r k e d l y s t a g e by s t a g e spring-summer growth c y c l e , w i t h r o o t g r o w t h , s e c o n d a r y c a m b i a l a c t i v i t y and shoot grow- t h o c c u r i n g a t d i f f e r i n g t i m e s ( K o z l o w s k i and Winget, 1964). The n e e d l e s t h a t s u r v i v e w i n t e r p l a y an i m p o r t a n t r o l e i n t h e s e growth phases. E x t a n t n e e d l e s s u p p l y f i x e d c a r b o n f o r r o o t growth i n i t i a l l y i n e a r l y s p r i n g . La- t e r , t h e o v e r w i n t e r i n g n e e d l e s s u p p l y t h e s e c o n d a r y c a m b i a l growth (Gord- on and L a r s o n , 1970)and r e p r o d u c t i v e s t r u c t u r e s o f t h e i r r e s p e c t i v e i n t e r - nodes w i t h p h o t o s y n t h a t e s (Dickmann e t a 1., 1970). In e a r l y summer o r l a t e s p r i n g , one y e a r o l d n e e d l e s i n p a r t i c u l a r , s u p p l y a s i g n i f i c a n t amount 1 ̂ o f C - l a b e l l e d c a r b o n t o the l a t e s p r i n g c u r r e n t n e e d l e f l u s h (Loach and L i t t l e , 1973). The s u p p l y o f p h o t o s y n t h a t e s t o the e x p a n d i n g n e e d l e s may s u p p l y t h e f i x e d c a r b o n n e c e s s a r y t o compensate f o r the n e g a t i v e p h o t o s y n - t h e s i s t h a t o c c u r s i n e x p a n d i n g buds and n e e d l e s (due t o v e r y h i g h r e s p i r - a t i o n ; C l a r k , 1961) d u r i n g the p e r i o d o f maximum c u r r e n t n e e d l e growth. 98 O l d e r n e e d l e s a r e n o t as p h o t o s y n t h e t i c a l l y e f f i c i e n t as c u r r e n t y e a r n e e d l e s . However, C l a r k (1961) has shown t h a t s i x y e a r o l d A b i e s balsamea n e e d l e s s t i l l produce more than h a l f t h e net p h o t o s y n t h e s i s o f c u r r e n t n e e d l e s and t h a t the one y e a r o l d and o l d e r f o l i a g e o f P i c a e g l a u c a p r o - duce about 75% o f t h e net p h o t o s y n t h e s i s o f t h e t r e e " d u r i n g the g r o w i n g season. I f t h e s h a d i n g e f f e c t s on o l d e r n e e d l e s by younger n e e d l e s were removed, t h e c o n t r i b u t i o n s o f t h e o l d e r n e e d l e s may be even h i g h e r . The f a c t t h a t t h e o l d e r n e e d l e s r e t a i n an a b i l i t y t o expand t h e i r normal pho- t o s y n t h e t i c c a p a c i t e s has been de m o n s t r a t e d by d e l i b e r a t e l y d e s t r o y i n g c u r r e n t season n e e d l e s (Neales e t a l . , 1968). 14 The s t u d i e s by Gordon and L a r s o n (1970) i n d i c a t e d t h a t C - l a b e l l e d c a r b o n was r e c o v e r e d i n t h e e x p a n d i n g bud a f t e r b e i n g f e d t o one y e a r o l d n e e d l e s i n t h e s p r i n g b e f o r e bud b u r s t and a l s o when f e d t o t h e one y e a r o l d n e e d l e s (then o n l y s e v e r a l months o l d ) the p r e c e e d ing autumn. These a u t h o r s e s t i m a t e d t h a t t e n t o f i f t e e n p e r c e n t o f p h o t o s y n t h a t e s f e d t o n e e d l e s i n t h e f a l l a r e r e c o v e r e d i n t h e t e r m i n a l buds j u s t b e f o r e f l u s h t h e f o l l o w i n g s p r i n g . S i n c e s t a r c h i s not s t o r e d i n t h e n e e d l e o v e r w i n t e r ( C l e m e n t s , 1938; L i t t l e , 1961 and 1970; Chabot and Chabot, 1975; and o b s e r - v a t i o n s i n t h i s w o r k ) , t h e p h o t o s y n t h a t e must be s t o r e d i n some o t h e r form. In t h i s work, o i l b o d i e s were found t o g r a d u a l l y i n c r e a s e i n s i z e d u r i n g t h e summer i n n e e d l e s o f a l l ages. By mid w i n t e r , t h e o i l b o d i e s were a dominant f e a t u r e o f t h e w i n t e r c h l o r e n c h y m a and;appeared t o be u n a f f e c t e d by t h e s e a s o n a l c y t o p l a s m i c changes about them. The o i l b o d i e s * T h i s d a t a i s from t h e a u t h o r o f t h i s paper's c a l c u l a t i o n s based on d a t a s u p p l i e d i n C l a r k ' s 1961 monograph. 99 d i d not appear t o i n c r e a s e f u r t h e r i n s i z e d u r i n g the w i n t e r but were much l a r g e r and more numerous i n t h e s p r i n g f i x a t i o n w h i c h was t h e s e a - s o n a l maxima f o r o i l i n n e e d l e s o f a l l ages t h a t were examined. H a r r i s (1971) noted an i n c r e a s e i n c y t o p l a s m i c o i l b o d i e s i n November f i x a t i o n s compared t o summer f i x a t i o n s o f P i n u s s t r o b u s . But Campbell (1972) d i d not mention any o i l r e s e r v e s i n h i s s t u d i e s on summer and e a r l y f a l l n e e d l e s o f P i n u s n i g r a . Chabot and Chabot (1975) o b s e r v e d s u b s t a n t i a l i n c r e a s e s i n chlorenchyma l i p i d b o d i e s i n the w i n t e r f i x a t i o n s o f A b i e s balsamea. W i n t e r a c c u m u l a t i o n s o f 1ipid b o d i e s a r e a l s o found i n t h e r e s - t i n g cambia o f c o n i f e r s (Murmanis, 1970; I t o h , 1971) and i n angiospermous cambia ( R o b e r t s e t aj_. , 1968 and M i a , 1972). The common i f not u n i v e r s a l p r e s e n c e o f l a r g e o i l s t o r a g e b o d i e s i n the s e c o n d a r y cambia o f both c o n i f e r s and an g i o s p e r m s , as w e l l as c o n i f e r n e e d l e s d u r i n g t h e w i n t e r , and t h e r a p i d d i s s i p a t i o n o f t h e s e o i l b o d i e s i n t h e s p r i n g i n d i c a t e s l i p i d r e s e r v e s a r e s i g n i f i c a n t s e a s o n a l s t o r a g e r e s e r v o i r s o f f i x e d c a r b o n . The imp o r t a n c e o f t h i s s t o r a g e p r o d u c t i s un- d o u b t e d l y i n c r e a s e d by t h e near c o m p l e t e absence o f s t a r c h i n t h e w i n t e r need 1es. L a b e l l e d t r a c e r s t u d i e s c i t e d above a l s o i n d i c a t e d t h a t t h e one ye a r o l d and o l d e r n e e d l e s m o b i l i z e w i n t e r s t o r a g e r e s e r v e s kept w i t h i n t h e n e e d l e , p l u s c u r r e n t p h o t o s y n t h a t e s t o s u p p l y c u r r e n t season growth o f a l l p a r t s o f the t r e e . These o b s e r v a t i o n s may c o n t a i n a p a r t i a l answer t o th e lo n g s t a n d i n g q u e s t i o n c o n c e r n i n g t h e c o m p e t i t i v e advantage o f e v e r - green t r e e s v e r s u s d e c i d u o u s t r e e s . In t h i s work, need 1 e s c o n t a i n e d s t a r c h 100 r e s e r v e s and l a r g e r o i l r e s e r v e s than seen i n w i n t e r i n t h e s p r i n g f i x a t - i o n conducted on A p r i l t h e t w e n t y - s e v e n t h , 1973- However, c u r r e n t n e e d l e f l u s h o f t h a t season was j u s t b e g i n i n g on June t h e s i x t h , 1973, w h i l e one y e a r o l d needles observed on June the s i x t h had o n l y a few o i l b o d i e s , c h a r - a c t e r i s i c o f the summer s t a t e . T h i s would i n d i c a t e t h a t one y e a r and o l d - e r n e e d l e s had been p h o t o s y n t h e t i c a l l y a c t i v e t o some degree from A p r i l t h e t w e n t y - s e v e n t h and t h a t by June t h e s i x t h , they had l a r g e l y expended t h e i r o i l r e s e r v e s . S i n c e d e c i d u o u s aspens g r o w i n g a t t h e c o l l e c t i n g s i t e s were o n l y p a r t i a l l y i n l e a f on June t h e s i x t h , P i n u s c o n t o r t a t r e e s would have been p h o t o s y n t h e t i c a 1 1 y a c t i v e f o r up t o a month b e f o r e the d e c i d u o u s t r e e s o f t h e a r e a . Chabot and Chabot (1975) made s i m i l a r o b s e r - v a t i o n s on A b i e s balsamea. These a u t h o r s d e t e c t e d s t a r c h a c c u m u l a t i o n i n e a r l y March, two months b e f o r e new n e e d l e growth o c c u r s . The l y s o s o m a l system i n h i g h e r p l a n t s has been d e s c r i b e d as a system i n v o l v i n g c y t o p l a s m i c s t r u c t u r a l e n t i t i e s w h i c h m a i n t a i n an e f f e c t i v e c o m p a r t m e n t a l i z a t i o n o f h y d r o l y t i c enzymes from t h e r e s t o f the c e l l u l a r system ( B e r j a k , 1971)- B e r j a k d e s c r i b e s e a r l i e r work which have s u g g e s t - ed t h a t the d e v e l o p m e n t a l sequence of s t r u c t u r e s t h a t appear t o have l y - sosomal p r o p e r t i e s i n d i f f e r e n t i a t i n g r o o t t i p s l e a d s t o t h e e v e n t u a l f o r - m a t i o n o f t h e main o r c e n t r a l v a c u o l e as t h e c e l l s mature. T h i s has de- v e l o p e d i n t o t he c o n c e p t t h a t t h e v a c u o l e i s an i n t e g r a l but not t h e o n l y component o f t h e l y s o s o m a l compartment i n mature c e l l s . In the mature c o n i f e r c h l o r e n c h y m a , t h e main v a c u o l e c o n t a i n s t a n n i n m a t e r i a l , a s u b s t a n c e c h a r a c t e r i z e d by i t s a b i l i t y t o b i n d t o and dena- 101 t u r e p r o t e i n s . A t a n n i n - f i 1 l e d main v a c u o l e would thus not be incompat- i b l e w i t h t h e o l d e r but s t i l l v a l i d c o n c e p t o f the main v a c u o l e i n mature p l a n t c e l l b e i n g a " s i n k " ( D e v l i n , 1969) f o r f u r t h e r n o n - m e t a b o l i z a b l e by- p r o d u c t s o f c e l l u l a r a c t i v i t y . The p r e s e n c e o f t a n n i n may be an answer t o t h e problem o f b y - p r o d u c t s removal from t h e c e l l s o l u t i o n i n v e r y l o n g l i v e d p l a n t c e l l s by b i n d i n g and p r e c i p i t a t i n g them. In t h i s c o n t e x t , the o b s e r v a t i o n t h a t t h e homogeneity o f the t a n n i n m a t e r i a l b r e a k s down i n f i v e y e a r o l d n e e d l e s may be " r e l e v a n t . In o l d e r n e e d l e s , t h e t a n n i n m a t e r i a l i s c h a r a c t e r i z e d by a more heterogeneous appearance due t o t h e p r e s e n c e o f v a r i o u s o s m i o p h i l i c i n c l u s i o n s ( f i g u r e 67) w h i c h may i n d i c a t e t h a t t h e t a n n i n may a s s i s t i n i n a c t i v a t i n g c e l l u l a r b y - p r o d u c t s . I f t h e t a n n i n m a t e r i a l w i t h i n t h e t a n n i n v a c u o l e can be viewed as an i n e r t and p r o b a b l e end s t a g e o f a c e l l ' s l y s o s o m a l system, then the p e r - s i s t e n c e o f r e s i d u a l ER i n the c y t o p l a s m about t h e n u c l e u s ( i t was not d e t e r m i n e d i f t h i s was SER o r RER i n t y p e II c e l l s ) , d i c t y o s o m e s and c y - t o p l a s m i c v a c u o l e s (some o f w h i c h may c o n t a i n membranous and p o s s i b l e o r - g a n e l l e d e b r i s ; f i g u r e s 22. 23 and 25) s u g g e s t t h e a c t i v e l y s o s o m a l com- partment i s c o n f i n e d t o and o p e r a t e s w i t h i n t h e c y t o p l a s m . However, con- f i r m a t i o n o f t h i s s u g g e s t i o n would r e q u i r e enzyme l o c a l i z a t i o n s t u d i e s . O b s e r v a t i o n s on t y p e II c e l l s i n the summer s t a t e i n d i c a t e t h a t t h e t a n n i n m a t e r i a l may be s t r u c t u r a l l y removed from the t o n o p l a s t . F i g u r e s 22, 23 and 26 i n d i c a t e t h a t the t a n n i n m a t e r i a l c o n t r a c t s as a u n i t from the t o n o p l a s t . The s h r i n k a g e o f the t a n n i n i s b e l i e v e d t o be a f i x a t i o n a r t i f a c t s i n c e t h e amount o f s e p a r a t i o n between t a n n i n and t o n o p l a s t was 102 f i x a t i o n dependent. However, i n many c a s e s , i t was o b s e r v e d t h a t t h e t o - n o p l a s t i n v a g i n a t e d i n t o t h e c y t o p l a s m f o r m i n g an i n t e r d i g i t a t i n g complex s e c t i o n a l p r o f i l e ( f i g u r e 27)- T h i s unusual t o n o p l a s t c o n f o r m a t i o n i s more pronounced i n o b l i q u e s e c t i o n s t o the t a n n i n v a c u o l e ( f i g u r e 2 8 ) . T h i s phenomena i s b e l i e v e d t o be u n r e l a t e d t o t h e t a n n i n s h r i n k a g e and may r e p r e s e n t t h e t y p e o f complex membrane p a t t e r n commonly a s s o c i a t e d w i t h t r a n s p o r t a c t i v i t y (Gunning and P a t e , 1974). The i n t e r d i g i t a t i o n a l s o p r o - v i d e s a r e a s where th e t o n o p l a s t and lumen o f t h e i n f o l d i n g s o f the tono- p l a s t a r e p h y s i c a l l y s e p a r a t e d from the t a n n i n . T h i s t y p e o f s t r u c t u r e may be the s i t e o f most o f the e n z y m a t i c a c t i v i t y t h a t would be e x p e c t e d by t o n o p l a s t and the main c e l l ( t a n n i n ) v a c u o l e . Enzyme l o c a l i z a t i o n s t u - d i e s would i l l u c i d a t e t h i s s u g g e s t i o n . In w i n t e r , t h e t a n n i n m a t e r i a l c o n t r a c t s t o a g r e a t e r e x t e n t than o b s e r v e d i n summer. T h i s c o n t r a c t i o n g r e a t l y i n c r e a s e s t h e volume o f the p r o t o p l a s t between t h e t a n n i n m a t e r i a l and the i n n e r s u r f a c e o f t h e c e l l w a l l . T h i s c o n t r a c t i o n may a l s o be an a r t i f a c t as i s b e l i e v e d t o be the c a s e i n t h e summer t a n n i n c o n t r a c t i o n . But s t u d i e s on f r o z e n f r e s h w i n - t e r m a t e r i a l w i t h t h e c r y o m i c r o s c o p e i n d i c a t e s t h e t a n n i n c o n t r a c t i o n na- t u r a l l y o c c u r s ( f i g u r e 33) a l t h o u g h p o s s i b l y not as e x t e n s i v e l y as o b s e r - ved i n c h e m i c a l l y f i x e d m a t e r i a l . In w i n t e r , t h e t o n o p l a s t was not i d e n t i f i e d p o s s i b l y due t o v e r y c l o s e a d h e s i o n t o t h e t a n n i n m a t e r i a l w h i c h i s so o s m i o p h i l i c t h a t i t may mask any o t h e r s m a l l o s m i o p h i 1 i c s t r u c t u r e such as a membrane. The maintenance o f an i n t a c t main c e l l v a c u o l e ( t a n n i n v a c u o l e ) i n w i n t e r was a l s o o b s e r v - 103 ed i n c o n i f e r n e e d l e s by Lewis and T u t t l e (1923), P a r k e r and P h i l p o t t (1961 and 1963), H a r r i s (1971) and Chabot and Chabot (1975). T h i s i s i n marked c o n t r a s t t o t h e breakdown o f t h e main c e l l v a c u o l e i n t h e o v e r w i n - t e r i n g c a m b i a l t i s s u e s o f t r e e s (Kadwai and R o b e r t s , 1969; Robards e t a l . , 1969; M i a , 1970 and 1972; Murmanis, 1970 and i t o h , 1971). The breakdown o f c e n t r a l v a c u o l e has a l s o been o b s e r v e d i n t h e t i l l e r i n g nodes o f w i n t e r wheat ( C h i e n and Wu, 1965), bark parenchyma c e l l s ( S i m i n o v i t c h e t a l . , 1968 and K r a s v t s e v e t a 1., 1971) and w h e a t g r a s s h y b r i d l e a v e s ( K h r i s t o l y u b o v a and S a nfonova, 1973)- The main d i f f e r e n c e between the p l a n t c e l l s where t h e r e was t h e ob- s e r v a t i o n o f c e n t r a l v a c u o l e breadkown i n t o numerous s m a l l v a c u o l e s , and the p l a n t c e l l s where the p r e s e r v a t i o n o f an i n t a c t c e n t r a l v a c u o l e was o b s e r v e d , was t h e p r e s e n c e o r absence o f a t a n n i n f i l l e d main v a c u o l e . Most a u t h o r s who s t u d i e d s e c o n d a r y c a m b i a l t i s s u e i n w i n t e r r e p o r t e d what th e y b e l i e v e d t o be t a n n i n m a t e r i a l s i n the lumens o f t h e many s m a l l w i n t - e r v a c u o l e s t h a t made up t h e b u l k o f t h e w i n t e r p r o t o p l a s t . However, t h e s e m a t e r i a l s d i d not have the same s t r u c t u r e o f homogeneity o f the t a n n i n found i n t h e c o n i f e r n e e d l e main c e l l v a c u o l e ; nor were t h e s e ma- t e r i a l s i n t h e same q u a n t i t y as t h e t a n n i n o b s e r v e d i n c o n i f e r t a n n i n v a- c u o l e s . F u r t h e r more, no t e s t s were mentioned t o i d e n t i f y t h e " t a n n i n m a t e r i a l " . The g r e a t mass o f c y t o p l a s m i c v a c u o l e s i n t h e w i n t e r c hlorenchyma a r e one o f t h e main f e a t u r e s o f w i n t e r c e l l s . The q u e s t i o n o f where t h e v a - c u o l e s come from i s a q u e s t i o n t h a t cannot be answered i n t h i s work be- 104 cause i n t h e August t h e t w e n t i e t h , 1972 f i x a t i o n , t h e y were l a r g e l y absent and by November the t w e n t y - s i x t h , 1972 f i x a t i o n , they were p r e s e n t . Cy- t o p l a s m i c v a c u o l e s were a common f e a t u r e o f the summer f i x a t i o n s but t h e i r number was g r e a t l y l e s s than i n w i n t e r and t h e y were e v e n l y d i s p e r s e d w i t h - i n the c y t o p l a s m . In summer, t h e c y t o p l a s m i c v a c u o l e s were u s u a l l y s e p a r a t e d by ground c y t o p l a s m and o r g a n e l l e s from each o t h e r . In w i n t e r , however, the c y t o - p l a s m i c v a c u o l e s a r e found i n masses i n s p e c i f i c a r e a s w i t h i n t h e c e l l . The v a c u o l a r r e g i o n appears t o have o c c u p i e d most o f t h e space formed w i t h - i n the w i n t e r c y t o p l a s m . b y c o n t r a c t i o n o f t h e t a n n i n m a t e r i a l . The v a c u o l - a t i o n o f the w i n t e r c y t o p l a s m a p p ears t o be a common f e a t u r e o f o v e r w i n - t e r i n g c e l l s (see r e f e r e n c e s i n the d i s c u s s i o n o f the t o n o p l a s t and t a n n - i n v a c u o l e a b o v e ) . B i o c h e m i c a l l y , t h e g r e a t l y i n c r e a s e d s y n t h e s i s o f p h o s p h o l i p i d s i s one o f t h e main f e a t u r e s o f s t a g e one o f f r o s t h a r d i n e s s a c q u i s i t i o n (Sim- i n o v i t c h e_t a]_. , 1975)- The s y n t h e s i s o f p h o s p h o l i p i d s and p r o t e i n s (Sim- i n o v i t c h , 1968) a t t h i s t i m e may p r o v i d e the m a t e r i a l s f o r t h e g r e a t l y i n - c r e a s e d a u g m e n t a t i o n o f c y t o p l a s m i c v a c u o l a r membranes t h a t must o c c u r sometime i n the f a l l . S i m i l a r l y , some o f the i n c r e a s e d l i p i d b o d i e s i n t h e s p r i n g f i x a t i o n may r e p r e s e n t t h e l i p o i d a l remains o f the w i n t e r mem- brane masses. Murmanis (1970) and C h i e n a n d W u , (1965) and o t h e r s who r e p o r t e d v a c - u o l e s f o r m i n g i n t h e w i n t e r c y t o p l a s m o f hardy p l a n t s , b e l i e v e d t h a t : the.. t o n o p l a s t o f t h e o r i g i n a l main c e l l v a c u o l e broke down t o form t h e numer- 105 ous w i n t e r v a c u o l e s w h i c h i n t h e i r v i e w , r e p l a c e d t h e summer main c e l l v a c u o l e . In t h i s work, t h e o b s e r v a t i o n o f t h e t o n o p l a s t i n v a g i n a t i n g i n - t o t h e c y t o p l a s m i n summer and t h e d i f f i c u l t y o f r e s o l v i n g i t from a d j a - c e n t c y t o p l a s m i c v a c u o l e s i n w i n t e r may i n d i c a t e t h a t t h e c y t o p l a s m i c v a - c u o l e s o b s e r v e d i n t h i s work were a l s o o f t o n o p l a s t d e r i v a t i o n o r e x t e n - s i o n i n t o t he c y t o p l a s m . I f t h i s i s t h e ease, then t h e o n l y d i f f e r e n c e s i n t h e o b s e r v a t i o n s o f Murmanis and o t h e r s , and o f the o b s e r v a t i o n s i n t h i s work i s t h a t on t h e c o n i f e r n e e d l e s , t h e t a n n i n m a t e r i a l r e t a i n s i t s s t r u c t u r a l i n t e g r i t y w h i l e t h e t o n o p l a s t p r o l i f e r a t e s w i t h i n t h e c y t o p l a s m as i t does i n c e l l s w i t h o u t the t a n n i n v a c u o l e . An a l t e r n a t e s u g g e s t i o n f o r wh i c h t h e r e i s r e c e n t e v i d e n c e i n t h e l i t e r a t u r e ( K h r i s t o l y u b o v a and Sanfonova, 1973) i s t h a t t h e w i n t e r p r o - l i f e r a t i o n o f v a c u o l e s i n w h e a t g r a s s h y b r i d s o r i g i n a t e s from t h e ER. Mo l l e n h a u e r et_ a]_. (1960) o b s e r v e d the r e s p o n s e s o f p l a n t t i s s u e t o me- c h a n i c a l i n j u r y ; and i m m e d i a t e l y a f t e r t h e i n j u r y , t h e damaged c y t o p l a s m i s c h a r a c t e r i z e d by r a p i d membrane p r o l i f e r a t i o n and v a c u o l i z a t i o n o f the c y t o p l a s m o r i g i n a t i n g from t h e ER. The e n d o p l a s m i c r e t i c u l u m has been o b s e r v e d t o p r o l i f e r a t e i n t o v a c u o l e s i n t h e r o o t t i p s o f wheat when c o o l e d t o near f r e e z i n g t e m p e r a t u r e s i n wheat ( P e t r o y s k a y a - B o r a n o v a , 1972). The w i n t e r c y t o p l a s m i c v a c u o l e s by reason o f t h e i r e x t e n t and s e a s o n a l dependence, may p l a y a kay r o l e i n t h e f r o s t t o l e r a n c e o f w i n t e r c e l l s . S i m i n o v i t c h e t a l . (1975).have b i o c h e m i c a l l y r e l a t e d t h e e x t e n s i v e augment- a t i o n o f p h o s p h o l i p i d s i n h a r d e n i n g t r e e s d u r i n g s t a g e one t o t h e p r o l i f e r - a t i o n o f c y t o p l a s m i c membranes. T h i s membrane p r o l i f e r a t i o n a p p e ars t o be one o f the few f e a t u r e s i n common between t h e c y t o l o g i c a l e x a m i n a t i o n s o f d i f f e r e n t hardy p l a n t s p e c i e s i n w i n t e r . 106 A n o t h e r f e a t u r e o f t h e w i n t e r c y t o p l a s m was t h e g r e a t numbers o f o s - m i o p h i l i c b o d i e s found i n t h e w i n t e r c y t o p l a s m . These s t r u c t u r e s a r e found i n summer f i x a t i o n s but g r e a t l y reduced i n s i z e and q u a n t i t i e s . The o s m i o p h i l i c b o d i e s a r e c h a r a c t e r i z e d i n both summer and w i n t e r by a c l o s e a s s o c i a t i o n w i t h c y t o p l a s m i c v a c u o l e s . U s u a l l y t h e o s m i o p h i l i c b o d i e s a r e found w i t h i n a c l e a r l y d e f i n a b l e membrane o f t h e c y t o p l a s m i c v a c u o l e ( f i g u r e s 22, 23 and 2 5 ) . However, i n some p h o t o m i c r o g r a p h s ( i n summer f i x a t i o n s ) , no bounding membranes c o u l d be o b s e r v e d about t h e o s m i o p h i l i c body ( l o w e r OB, f i g u r e 2 3 ) . S t r u c t u r a l l y , t h e o s m i o p h i l i c b o d i e s appear t o be c l o s e l y a s s o c i a t e d w i t h membrane systems. In summer, ' m y e l i n - 1 i k e 1 s t r u c t u r e s a r e o c c a s i o n a l l y o b s e r v e d ( f i g u r e 2h) w h i l e i n w i n t e r , t h e o s - m i o p h i l i c b o d i e s f r e q u e n t l y form c o a t i n g s o f v a r y i n g t h i c k n e s s on t h e l u - men s i d e o f c y t o p l a s m i c v a c u o l e s ( f i g u r e kS). The o s m i o p h i l i c b o d i e s a r e randomly s c a t t e r e d i n the summer c y t o - plasm, but i n w i n t e r , t h e y a r e found i n v e r y s p e c i f i c l o c a t i o n s w i t h i n t h e c e l l . In w i n t e r , the o s m i o p h i 1 1 c b o d i e s o f t e n form a s i n g l e l a y e r o f o s m i o p h i l i c b o d i e s between t h e plasma membrane and o r g a n e l l e s deeper w i t h i n t h e c y t o p l a s m ( f i g u r e s 51 and 5 2 ) . In r e g i o n s where t h e c h l o r o - p l a s t s and t h e n u c l e u s a r e not f o u n d , they a r e i n t e r m i x e d w i t h t h e masses o f c y t o p l a s m i c v a c u o l e s found i n t h e s e a r e a s . The o s m i o p h i l i c b o d i e s a r e c h a r a c t e r i z e d i n t h i s work by an i n t e n s e s t a i n i n g w i t h osmium t e t r o x i d e . The o s m i o p h i l i c b o d i e s i n osmium f i x e d m a t e r i a l a r e a l w a y s t h e most e l e c t r o n dense m a t e r i a l w i t h i n t h e c y t o p l a s m , b e i n g even more o s m i o p h i l i c than t a n n i n m a t e r i a l . In m a t e r i a l f i x e d o n l y 107 w i t h a l d e h y d e s , they s t a i n w e a k l y w i t h l e a d o r uranium and a r e d i f f i c u l t t o d i s t inqu i sh. High m a g n i f i c a t i o n l i g h t m i c r o s c o p y s t u d i e s o f f r e s h summer m a t e r i a l s t a i n e d w i t h Sudan B l a c k B i n d i c a t e d t h e p r e s e n c e o f numerous v e r y s m a l l s u d a n o p h i l i c s t r u c t u e s i n t h e c y t o p l a s m ( f i g u r e 14). T h i s o b s e r v a t i o n may have been a p o s i t i v e i n d i c a t i o n o f a l i p i d n a t u r e f o r t h e o s m i o p h i - l i c b o d i e s . However, t h i s t y p e o f s t u d y was not e x t e n s i v e l y c o n d u c t e d , and w i n t e r l i g h t m i c r o s c o p y s t u d i e s a t h i g h m a g n i f i c a t i o n w i t h Sudan s t a i n s were not c o n d u c t e d . In a d d i t i o n , t he s m a l l s i z e o f the s t r u c t u r e s o b s e r v e d made i t d i f f i c u l t t o d i s t i n q u i s h between p o s i t i v e s t a i n i n g and a h i g h r e - f r a c t i l i t y . H a r r i s (1971) and Chabot and Chabot (1975) b e l i e v e d s t r u c t - u r e s r e s e m b l i n g o s m i o p h i l i c b o d i e s were t a n n i n m a t e r i a l . T a n n i n s p e c i f i c s t a i n s used i n t h i s work gave no t r a c e o f any t a n n i n i f e r o u s m a t e r i a l be- ing p r e s e n t w i t h i n t h e c y t o p l a s m ( f i g u r e 3 4 ) . In a d d i t i o n , i n t h i s work, t a n n i n m a t e r i a l a l w a y s had a f i b r i l l a r o r g r a n u l a r f i n e s t u r c t u r e w h i l e o s m i o p h i l i c b o d i e s d i d not c o n t a i n any r e s o l v a b l e f i n e s t r u c t u r e . These o b s e r v a t i o n s a l l o w t h e t e n t a t i v e c o n c l u s i o n t o be made t h a t t h e o s m i o p h i - l i c b o d i e s a r e not composed o f t a n n i n . They may be l i p o i d a l i n n a t u r e and appear t o have some f u n c t i o n w i t h i n t h e w i n t e r t i s s u e . T h i s l a s t con- c l u s i o n i s based on t h e i r g r e a t i n c r e a s e i n numbers d u r i n g w i n t e r and t h e i r s p e c i f i c d i s t r i b u t i o n w i t h i n t h e w i n t e r c e l l . A p o s s i b l e f u n c t i o n o f t h e o s m i o p h i l i c b o d i e s t h a t s h o u l d be i n v e s t - i g a t e d i s t h a t t h e y may p l a y a r o l e i n p r o t e c t i n g t h e w i n t e r p h o t o s y n t h e - t i c pigments from p h o t o - o x i d a t i o n . C h l o r o p h y l l i s r e t a i n e d i n t h e n e e d l e s 108 t h r o u g h o u t the w i n t e r but t h e marked w i n t e r c h l o r o s i s i n e v e r g r e e n c o n i - f e r s , p a r t i c u l a r l y i n P i nus s p e c i e s , has been a p o i n t o f note i n t h e l i t - e r a t u r e . Mohl (1837) s u g g e s t e d o r g a n i c a c i d s may d e s t r o y t h e green p i g - ments d u r i n g w i n t e r . H a b e r l a n d t (1976) s u g g e s t e d h i g h w i n t e r l i g h t i n - t e n s i t y may cause a r e d u c t i o n i n c h l o r o p h y l l c o n t e n t i n w i n t e r c o n i f e r s . H o l z e r (1958) s u p p o r t e d H a b e r l a n d t ' s v i e w and r e p o r t e d the o b s e r v a t i o n s t h a t p i n e boughs b u r i e d under the snow d i d not become c h l o r o t i c i n w i n t - e r . C a r o t i n o i d pigments a r e y e l l o w and have a r o l e i n p r o t e c t i o n a g a i n s t p h o t o - o x i d a t i o n ( D e v l i n , 1969)- They a r e a l s o h i g h l y u n s a t u r a t e d l i p i d s , and as a r e s u l t , would s t a i n i n t e n s e l y w i t h osmium t e t r o x i d e . The osmio- p h i l i c b o d i e s were o b s e r v e d t o o f t e n l y e i n t i e r s near t h e c e l l w a l l be- tween t h e c h l o r o p l a s t clumps and the plasma membrane. These o b s e r v a t i o n s i n d i c a t e d t h a t i t may be w o r t h w h i l e t o i n v e s t i g a t e the u n s a t u r a t e d o i l c o n t e n t o f s e a s o n a l l y c h l o r o t i c n e e d l e s . A l a r g e w i n t e r b u i l d up o f c a r - o t i n o i d - l i k e p i g m e n t s , p o s s i b l y as the o s m i o p h i 1 i c b o d i e s , may mask t h e c h l o r o p h y l l c o n t e n t o f the n e e d l e enough t o ac c o u n t f o r the s e a s o n a l chan- ges. The l a s t major d i f f e r e n c e o b s e r v e d i n com p a r i s o n s between w i n t e r and summer t i s s u e o b s e r v e d i n t h i s work i s t h e change i n membrane p r o p e r t i e s . T h i s was o b s e r v e d on a s t r u c t u r a l b a s i s and on a more subj e c t i ve bas i s i n terms o f t he s e a s o n a l r e s p o n s e s o f p i n e chlorenchyma t o f i x a t i o n p r o c e d u r e s . In t h i s work, p r i m a r y and sec o n d a r y f i x a t i o n t i m e s c o u l d be g r e a t l y reduced i n w i n t e r m a t e r i a l w i t h o u t a f f e c t i n g t h e o b s e r v e d u l t r a s t r u c t u r e . 109 S i m i l a r l y , t h e c o m p o s i t i o n and m a t e r i a l c o n c e n t r a t i o n s o f f i x a t i v e s o l u - t i o n s c o u l d be v a r i e d i n w i n t e r t o such extremes t h a t t h e y d e s t r o y e d s u - mmer c e l l u l a r u l t r a s t r u c t u r e w i t h minimal o r no e f f e c t on w i n t e r t i s s u e . P a r k e r and P h i l p o t t (1963) commented on t h e c o m p a r a t i v e ease i n a c h i e v i n g good w i n t e r u l t r a s t r u c t u r a l p r e s e r v a t i o n , p a r t i c u l a r l y when compared w i t h summer f i x a t i o n s . These f i x a t i o n p r o p e r t i e s i n d i c a t e t h a t t h e membranes o f t h e w i n t e r p r o t o p l a s t may have l o s t t o a c o n s i d e r a b l e degree t h e i r p r o p e r t i e s o f s e m i - p e r m e a b i l i t y . The w i n t e r p r o t o p l a s t a p p ears remarka- b l y r e s i s t a n t t o o s m o t i c shock from f i x a t i v e s t h a t v a r i e d g r e a t l y i n comp- o s i t i o n . S l i g h t changes i n f i x a t i v e c o m p o s t i o n i n summer a t l e a s t a l t e r - ed c e l l u l t r a s t u r c t u r e (and u s u a l l y d i s r u p t e d i t ) and o f t e n p l a s m o l i z e d c e l l s ( f i g u r e 1 2 ) . However, i n w i n t e r , none o f t h e f i x a t i v e s o l u t i o n s used i n t h i s work p l a s m o l i z e d c e l l s . P l a s m o l y s i s would be e x p e c t e d i n h i g h l y o s m o t i c f i x a t i v e c o n t a i n i n g m a t e r i a l s t h a t a r e n o r m a l l y impermeable i f t he c e l l s had not l o s t some o f the p r o p e r t i e s o f membrane semi-permea- b i l i t y . S t r u c t u r a l l y , t h e r e i s a l s o c o n s i d e r a b l e e v i d e n c e f o r membrane changes i n w i n t e r . Changes i n t h e c h l o r o p l a s t e n v e l o p e membranes have a l r e a d y been d i s c u s s e d . M i t o c h o n d r i a e n v e l o p e and c r i s t a e membranes were i n d i s t - i n c t and amorphous, n u c l e a r e n v e l o p e membranes c o n t a i n e d numerous n u c l e a r p ores and t h e t o n o p l a s t was d i f f i c u l t t o d i s t i n g u i s h . Only t h e plasma membrane remained s t r u c t u r a l l y s i m i l a r t o the summer c o n d i t i o n . These membrane s t r u c t u r a l changes a r e p r o b a b l y r e l a t e d t o t h e s e a s o n a l p r o c e s s e s i n t h e chlorenchyma s i n c e t h e changes a r e r e v e r s i b l e i n summer. S p e c i f i c d e t a i l e d s t u d i e s would have t o be u n d e r t a k e n i n each o f t h e s e a s o n a l l y 110 v a r y i n g membranes t o d e t e r m i n e how each o b s e r v e d membrane change may a f f e c t f r o s t h a r d i n e s s . A t t h i s s t a g e , one can o n l y c o n c l u d e t h a t s i g n i f i c a n t s e a - s o n a l membrane changes o c c u r and t h a t t h e s e changes a r e v e r y o r g a n e l l e o r s t r u c t u r e s p e c i f i c , i . e . , t h e c h l o r o p l a s t e n v e l o p e membranesa.re d i f f e r e n t a t l e a s t i n w i n t e r from t h y l a k o i d membranes, n u c l e a r membranes and t o n o p l a s t membranes. 111 I I I . D i s c u s s i o n o f P o s s i b l e F r e e z i n g Mechanisms'in t h e Chlorenchyma The summer-winter t r a n s f o r m a t i o n o f each major o r g a n e l l e has been d i s c u s s e d above. A t t h i s s t a g e , some w h o l i s t i c c o n c e p t s can be d i s c u s s - ed which may r e l a t e what has been o b s e r v e d c y t o l o g i c a l l y i n P i n u s c o n t o r t a w i t h t h e p r e v a i l i n g l i t e r a t u r e c o n c e p t s o f f r o s t h a r d i n e s s . In t h e i n t r o d u c t i o n , i t was s t a t e d t h a t t h e r e i s a: g e n e r a l agreement i n the r e c e n t l i t e r a t u r e (Burke e t a l . , 1976) t h a t e x t r e m e l y hardy p l a n t s pass t h r o u g h two o r more s t a g e s o f f r o s t h a r d i n e s s development, w i t h s t a g e one b e i n g g e n e r a l i z e d i n t o a p e r i o d i n w h i c h t h e p l a n t d e t e c t s t h e a p p r o a c h - ing f r o s t season w e l l b e f o r e s e v e r e f r o s t , c e a s e s a c t i v e d i v i s i o n and en- gages i n t h e a c t i v e b i o s y n t h e s i s o f c e r t a i n compounds and s t r u c t u r e s w h i c h may have an a c t i v e r o l e i n t h e f r o s t r e s i s t a n c e o f the c e l l ; i . e . s t a g e one i n v o l v e s a c t i v e p h y s i o l o g i c a l p r o c e s s e s w i t h i n t h e c e l l . The c y t o l o g i c a l e x a m i n a t i o n o f e x t r e m e l y hardy t i s s u e d u r i n g t h e w i n - t e r months would r e v e a l t h e m a t e r i a l s s y n t h e s i z e d d u r i n g s t a g e one o f f r o s t h a r d i n e s s development. W i n t e r f i x a t i o n s o f P i n u s c o n t o r t a chlorenchyma i n - d i c a t e d the f o l l o w i n g m a t e r i a l s t h a t were d i f f e r e n t f r o m , o r more numerous than t y p e II summer c e l l s : l i p i d b o d i e s , o s m i o p h i l i c b o d i e s , t h e membranes o f c y t o p l a s m i c v a c u o l e s , n u c l e a r p o r e s , c h l o r o p l a s t stroma and s t r u c t u r a l and p h y s i o l o g i c a l changes i n membranes. The l i p i d b o d i e s as p r e v i o u s l y d i s - c u s s e d a r e not b e l i e v e d t o be i n v o l v e d w i t h f r o s t h a r d i n e s s mechanisms. However, t h e r e m a i n i n g m a t e r i a l presumably was a c t i v e l y s y n t h e s i z e d i n t h e f a l l and was p r e s e n t i n what appeared t o be u n i f o r m q u a n t i t i e s t h r o u g h o u t 112 t h e w i n t e r and s u b s e q u e n t l y became ab s e n t o r g r e a t l y reduced i n q u a n t i t y t h e f o l l o w i n g summer. I t t h u s seems r e a s o n a b l e t o assume t h a t t h e s e mat- e r i a l s had some r o l e i n t h e f r o s t h a r d i n e s s mechanism o f t h e c e l l . The i n - c r e a s e d numbers o f n u c l e a r pores and changes i n t h e morphology o f c h l o r o - p l a s t and m i t o c h o n d r i a l membranes may r e f l e c t the s p e c i f i c r e s p o n s e o f each t y p e o f o r g a n e l l e t o t h e problem o f r e l e a s i n g f r e e w a t e r d u r i n g f r e e z - i n g . No e x p l a n a t i o n f o r the a p p a r e n t i n c r e a s e i n c h l o r o p l a s t stroma o r t h e i n c r e a s e and s p e c i f i c d i s t r i b u t i o n o f o s m i o p h i l i c b o d i e s d u r i n g w i n t e r i s p o s s i b l e based on the o b s e r v a t i o n s i n t h i s work. Suggested f u n c t i o n s d u r i n g w i n t e r f o r t h e o s m i o p h i l i c b o d i e s has been d i s c u s s e d p r e v i o u s l y . Some i n s i g h t i n t o what r o l e s t h e o b s e r v e d c y t o p l a s m i c changes may p l a y i n a c h i e v i n g f r o s t h a r d i n e s s may be o b t a i n e d by c o n s i d e r i n g what happens i n s t a g e two o f f r o s t h a r d i n e s s development. In t h i s s t a g e , i t i s b e l i e v - ed t h a t t h e c y t o p l a s m p h y s i c a l l y accomodates i t s e l f t o t h e i n c r e a s i n g s e v e r - i t y o f f r o s t o r more s p e c i f i c a l l y i c e i n t h e e n v i r o n o f t h e c e l l and t h e c o r r e s p o n d i n g l o s s o f l i q u i d w a t e r from t h e c e l l . The e s s e n t i a l s t e p i n - v o l v e d w i t h phase two o f h a r d i n e s s a q u i s i t i o n i s t h a t o v e r w i n t e r i n g t i s s u e must be exposed t o s p e c i f c moderate f r o s t s t h a t a r e s p e c i e s o r v a r i e t a l spe- c i f i c b e f o r e i t can t o l e r a t e v e r y heavy f r o s t s up t o i t s u l t i m a t e h a r d i n e s s l e v e l ( S a k a i , 1966 and 1973)- What i s g e n e r a l l y b e l i e v e d t o be happening d u r i n g phase two i s t h a t the c y t o p l a s m i s d e h y d r a t i n g and r e l e a s i n g w a t e r to e x t r a c e l l u l a r i c e n u c l e a t i n g s i t e s ( W e i s e r , 1970). T h i s f r o s t plasmo- l y s i s phenomenon i s i n e q u i l i b r i u m w i t h whatever the t i s s u e t e m p e r a t u r e i s a t a g i v e n i n s t a n t , and a t moderate f r o s t i n t e n s i t i e s , t h e b u l k c e l l w a t e r w i l l be r e l e a s e d t o i c e n u c l e a t i n g s i t e s . But i f t h i s w a t e r i s not a l l o w e d 113 enough t i m e t o m i g r a t e o u t o f the c e l l g r a d u a l l y t o f r e e z e e x t e r n a l l y , then i n t r a c e l l u l a r f r e e z i n g w i l l o c c u r ( W e i s e r , 1970). I n t r a c e l l u l a r f r e e z i n g i n hardy mature p l a n t t i s s u e i s c o n s i d e r e d f a t a l by most a u t h o r s (Tumanov, 1967 and W e i s e r , 1970). F r o s t p l a s m o l y s i s o f f r o z e n p l a n t c e l l s has been w e l l documented a t t h e l i g h t m i c r o s c o p i c l e v e l . Improtant e a r l y s t u d i e s on t h e s u b j e c t a r e by l l j i n (1934), K e s s l e r and Rhuland (1938), and more r e c e n t l y , Samygin (1966). A key s t e p f o r t he f r o s t hardy c e l l would t h u s be t o get t h e f r e e z a - b l e f r e e w a t e r o u t o f the c y t o p l a s m t o e x t e r n a l i c e n u c l e a t i o n s i t e s . T h i s c o u l d be a c h i e v e d by the c e l l l o o s i n g i t s membrane s e m i - p e r m e a b i l i t y . W i t h - out t h e l o s s o f s e m i - p e r m e a b i 1 i t y , t h e r e would be an o s m o t i c g r a d i e n t f o r w ater o p p o s i t e t o the d i r e c t i o n i n w h i c h i t must f l o w t o f r e e z e e x t e r n a l l y . The o b s e r v a t i o n i n t h i s work o f the r e s i s t a n c e o f the w i n t e r c y t o p l a s m t o extreme changes i n f i x a t i v e o s m o t i c c o n c e n t r a t i o n whether e l e c t r o l y t i c ( s e a - w a t e r ) o r n o n - e l e c t r o l y t i c ( s u c r o s e ) s u p p o r t s t h i s c o n c e p t . The f a c t t h a t t h e w i n t e r chlorenchyma c e l l s were a l s o never p l a s m o l y z e d a l s o i n d i c a t e s a l o s s o f s e m i - p e r m e a b i l i t y s i n c e t h e p l a s m o l y s i s t h a t may o c c u r d u r i n g f r e e - z i n g , " f r o s t p l a s m o l y s i s " , w i l l i n v o l v e t h e c o l l a p s e o f the p r o t o p l a s t due to l o s s o f l i q u i d w a t e r and may have l i t t l e t o do w i t h t h e semi-permeabi1i- t y o f membranes as i n o s m o t i c a l l y induced p l a s m o l y s i s . I f t h i s c o n c e p t i s c o r r e c t , i t w i l l e x p l a i n s t u d i e s such as t h o s e c o n d u c t e d by S c a r t h and S i m i o v i t c h ( c i t e d by S i m i n o v i t c h e t a l . , 1968) whi c h i n d i c a t e d t h a t n e a r l y t w i c e t h e c o n c e n t r a t i o n o f p l a s m o l y z i n g f l u i d was r e q u i r e d t o a c h i e v e t h e same degree o f p l a s m o l y s i s i n hardy c e l l s as i n non-hardy c e l l s . I f t h e p r o t o p l a s t had p a r t i a l l y l o s t t h e p r o p e r t y o f s e m i - p e r m e a b i l i t y , then i n - 1 c r e a s e d o s m o t i c s o l u t i o n s would be n e c e s s a r y t o a c h i e v e t he same degree o f o s m o t i c p l a s m o l y s i s . The f a c t t h a t a t l e a s t some hardy t i s s u e s become more permeable i n w i n t e r has been known f o r some ti m e ( S c a r t h and L e v i t t , 1937) and more r e - c e n t l y , s u p p o r t e d by McKenzie et_ aj_. (1974). S t u d i e s have a l s o i n d i c a t e d i n c r e a s e f r o s t r e s i s t a n c e i n non-hardy t i s s u e t r e a t e d w i t h compounds t h a t i n c r e a s e membrane p e r m e a b i l i t y ( K u i p e r , 1967)- In t h i s d i s c u s s i o n , t h e l o s s o f membrane s e m i - p e r m e a b i l i t y has been d i s c u s s e d w i t h r e g a r d t o the p r o t o p l a s t as a whole. In r e a l i t y , t h e v a r - i o u s membrane systems w i t h i n t he p r o t o p l a s t may not u n i f o r m l y g a i n t h e same degree o f p e r m e a b i l i t y . In t h i s work, t h e r e i s e v i d e n c e t h a t t h e c h l o r o p l a s t s a t l e a s t r e t a i n some degr e e o f o s m o t i c r e s p o n s i v e n e s s t o d i f f e r i n g f i x a t i v e s o l u t i o n s ( p a r t i c u l a r l y t h e seawater e l e c t r o l y t e f i x a t - i v e s o l u t i o n , f i g u r e 60). The r e t e n t i o n o f some degree o f t u r g i d i t y by s p e c i f i c o r g a n e l l e s c o u l d a f f e c t t h e d i s t r i b u t i o n o f o r g a n e l l e s i n t h e win t e r c y t o p l a s m by t h e o r a n g e l l e s a c t i n g as s t r u c t u r a l u n i t s i n t h e plasm- o l y z i n g p r o t o p l a s t d u r i n g f r e e z i n g . I f p l a s m o l y s i s i s not u n i f o r m t hrough o u t t he c e l l , t hen t h e s e o r g a n e l l e s c o u l d be pushed o r squeezed i n t o spec- i f i c l o c a t i o n s by t h e c o l l a p s i n g c y t o p l a s m . When c o n s i d e r i n g an h y p o t h e s i s t o e x p l a i n what may o c c u r when t h e win t e r p i n e c h l o r e n c h y m a l c e l l f r e e z e s , t h e a s s y m e t r y o f c y t o p l a s m i c s t r u c t - u r e s o b s e r v e d i n t h i s work s h o u l d be c o n s i d e r e d . I t was noted t h a t t h e clumped c h l o r o p l a s t s , n u c l e u s and most summer-like ground c y t o p l a s m was 115 found i n s p e c i f i c p a r t s o f the c e l l t h a t were d e f i n e d by t h e p r e s e n c e o f t r a b i c u l a e i n v a g i n a t i n g from the c e l l w a l l s . In t h e r e g i o n s where t h e long t r a b i c u l a e i n v a g i n a t e from c e l l w a l l f a c e C, i t was o b s e r v e d t h a t the p l a s - modesmata a l s o o c c u r e d . The piasmodesmata r e p r e s e n t s i t e s o f plasma mem- brane bondage t o the c e l l w a l l . Thus i f t h e c y t o p l a s m p l a s m o l y z e d and r e - t r e a t e d from the c e l l w a l l , p l a s m o l y s i s would have t o o c c u r w i t h the c y t o - plasm s t i l l a t t a c h e d t o the p1asmodesmata1 r e g i o n s o f t h e c e l l w a l l . In w i n t e r , t h e c o l l a p s e d p o r t i o n s o f the c y t o p l a s m i n t h e p i n e c h l o - renchyma would be t h e a r e a s o f e x t e n s i v e c y t o p l a s m i c v a c u o l i z a t i o n w h i l e t h e c h l o r o p l a s t clumps tend t o o c c u r i n t h e a r e a s near t h e plasmodesmata. T h i s h y p o t h e s i s a l s o would o f f e r a m e c h a n i c a l e x p l a n a t i o n o f how t h e summer o r g a n e l l e d i s t r i b u t i o n i s d i s r u p t e d . In t h i s c o n c e p t , the p l a s m o l y s i s o f t h e c y t o p l a s m o c c u r s i n t h e r e g i o n o f e x t e n s i v e v a c u o l i z a t i o n . The v a - c u o l i z a t i o n may be a p r o t e c t i v e d e v i c e t o a l l o w f o r t h e e x p a n s i o n and c o l - l a p s e o f the c y t o p l a s m v i a h i g h l y permeable plasma and v a c u o l a r membranes. The f r e e z e - t h a w c y c l e s t h a t must o c c u r on warm days and s u b - f r e e z i n g n i g h t t e m p e r a t u r e s may squeeze the c h l o r o p l a s t s and t h e n u c l e u s i n t o t h e p a r t s o f t h e c e l l where t h e c y t o p l a s m cannot c o l l a p s e due t o t h e at t a c h m e n t t o plasmodesmata. In t h i s m e c h a n i c a l e x p l a n a t i o n t o ac c o u n t f o r t h e w i n t e r c h l o r o p l a s t and n u c l e a r d i s t r i b u t i o n , plasmodesmata a l o n e cannot be t h e o n l y s i t e s where c o m p l e t e p l a s m o l y s i s o f t h e c y t o p l a s m does not o c c u r because s m a l l clumps o f c h l o r o p l a s t s and o c c a s i o n a l l y t h e n u c l e u s a r e a l s o found i n t h e 116 c y t o p l a s m near where t h e s m a l l t r a b i c u l a e i n v a g i n a t e . The t r a b i c u l a e them- s e l v e s may p r o v i d e i n c r e a s e d a r e a s o f a t t a c h m e n t between t h e plasma memb- rane and t h e c e l l w a l l , thus making p l a s m o l y s i s more d i f f i c u l t i n t h e c y t o - plasm a d j a c e n t t o t r a b i c u l a r a r e a s . One f e a t u r e o f p l a s m o l y s i s caused by f r e e z i n g t h a t has l o n g been ob- s e r v e d i n c a s e s where i c e forms o u t s i d e t h e c e l l w a l l i n p r i m a r y and t h i n w a l l e d t i s s u e i s t h e f a c t t h a t t h e c e l l t e n d s t o p a r t i a l l y c o l l a p s e as c e l l t u r g a r p r e s s u r e i s l o s t (see works c i t e d i n L e v i t t , 1972). I f t h i s i s t h e c a s e , the most l i k e l y s i t e f o r e x t r a c e l l u l a r i c e f o r m a t i o n i n t h e p i n e me- s o p h y l l i s i n t h e t r a n s v e r s e c h l o r e n c h y m a a i r spaces between t h e t r a n s e v e r s e f i l e s o f m e s o p h y l l c e l l s . In t h i s c a s e , i c e would form o p p o s i t e c e l l f a c e A i n f i g u r e 2. The c e n t r a l p a r t s o f t h i s c e l l f a c e would a l s o be t h e weakest c e l l f a c e t s t r u c t u r a l l y because the t r a b i c u l a e would o f f e r a b r a c i n g e f f e c t t o the c e l l w a l l s about the p e r i m e t e r o f t h e c e l l ( i n t h e t r a n s v e r s e p l a n e o f t h e n e e d l e ) . The s u p p o r t g i v e n t o t h e s p e c i f i c a r e a s o f the c e l l w a l l by t h e t r a b i c u l a e may t h u s o f f e r p r o t e c t i o n from c e l l w a l l c o l l a p s e i n t h e t r a - b i c u l a r a r e a s . C h l o r o p l a s t s and o t h e r o r g a n e l l e s would a c c u m u l a t e near t h e t r a b i c u l a r a r e a s by t h e s q u e e z i n g e f f e c t o f w a l l c o l l a p s e i n o t h e r p a r t s o f the c e l l . In t h i s c o n c e p t , t h e t r a b i c u l a r w a l l s a r e assumed t o f u n c t i o n as s t r u c t u r a l s u p p o r t s f o r the c h l o r e n c h y m a l c e l l w a l l . The s t r u c t u r a l a r - rangement o f t h e m e s o p h y l l can be d e s c r i b e d as r a d i a l l y e x t e n d i n g (from en- dodermis t o hypodermis) s i n g l e s h e e t s o f c e l l s i n t h e t r a n s v e r s e p l a n e o f t h e n e e d l e . T h i s c e l l s h e et i s p a r t i c u ' 1 a r l y t h i n due t o t h e minor a x i s o f t h e chlorenchyma c e l l s b e i n g i n t h e l o n g i t u d i n a l p l a n e o f the n e e d l e . Thus t h e chlorenchyma e e l 1 s may requ i r e add i t i o n a l s t r e n g t h and bracing to c o u n t e r a c t 117 f o r c e s normal t o the t r a n s v e r s e p l a n e o f t h e n e e d l e , s i n c e t h i s would be the weakest p l a n e o f t h e chlorenchyma c e l l s h e e t . The t r a b i c u l a e ( a r r a n g - ed about a l l c e l l w a l l s not l y i n g i n t h e t r a n s v e r s e p l a n e ) a r e i d e a l l y po- s i t i o n e d t o g i v e maximal b r a c i n g s u p p o r t t o the c e l l s h e et i n t h i s d i r e c t - i o n . They a r e a l s o p o s i t i o n e d t o p r e v e n t the t r a n s v e r s e f a c e s o f the c h l o r - enchymal c e l l s ( f a c e A) from b a l l o o n i n g out under e x c e s s t u r g o r p r e s s u r e and thus o c c l u d i n g the t r a n s v e r s e a i r spaces w i t h i n t h e n e e d l e . The above d i s c u s s i o n i s based on t h e premise t h a t t h e w i n t e r c y t o p l a s m - i c arrangement p r o v i d e s c l u e s t o t h e d i s t o r t i o n and p h y s i c a l r e a rrangements w h i c h may o c c u r i n t h e c y t o p l a s m as a r e s u l t o f t h e f r e e z i n g p r o c e s s . The r e t e n t i o n o f plasma membrane-cell w a l l c o n n e c t i o n s and t h e p o s s i b l e d i s t - o r t i o n o f s p e c i f i c p a r t s o f t h e c e l l w a l l due t o e x t r a c e l l u l a r i c e f o r m a t - ion and/or a l o s s o f t u r g o r p r e s s u r e a r e proposed mechanisms w h i c h may ac- count f o r the o b s e r v e d arrangement o f t h e w i n t e r P i n u s c y t o p l a s m . T h i s i s o n l y a p r o p o s a l s u g g e s t e d as a b a s i s f o r f u r t h e r i n v e s t i g a t i o n . I t s h o u l d be noted t h a t t h e R u s s i a n a u t h o r s (Genkel and Kurkova, 1971) b e l i e v e t h a t plasmodesmatal s t r a n d s b r e a k s o r s e p a r a t e from p r i m a r y p i t f i e l d s d u r i n g i c e induced p l a s m o l y s i s . These o b s e r v a t i o n s i f c o r r e c t would i n v a l i d a t e the c o n c e p t p r e s e n t e d above,which i s based on the plasma membrane remain- ing a t t a c h e d t o the c e l l w a l l i n p r i m a r y p i t f i e l d a r e a s . L e v i t t (1972) i s s k e p t i c a l o f t h e R u s s i a n c o n c e p t o f plasma membrane at t a c h m e n t t o p r i - mary p i t f i e l d s b r e a k i n g d u r i n g w i n t e r . He c i t e s s e v e r a l e a r l y m i c r o s c o p y s t u d i e s w h i c h c l e a r l y showed c y t o p l a s m i c s t r a n d s from i c e i n d u c e d , p l a s m o l - yzed c e l l s r e m a i n i n g a t t a c h e d t o t h e c e l l w a l l . 118 B o l d (1957) s u g g e s t e d t h a t t h e t r a b i c u l a e s e r v e i n t h e l e a v e s o f P i n u s t o i n c r e a s e t h e gas exchange s u r f a c e o f the c hlorenchyma c e l l s . T h i s con- c e p t i s based on t h e f r e q u e n t o b s e r v a t i o n o f c h l o r o p l a s t s l i n i n g t h e t r a - b i c u l a r w a l l s deep w i t h i n t h e c e l l i n t e r i o r . H a r i s (1971) extended t h i s c o n c ept by o b s e r v i n g t h e s p l i t t i n g o f the m i d d l e l a m e l l a a t the ends o f t r a b i c u l a e forms an e x t r a c e l l u l a r a i r space o r tube e x t e n d i n g t h r o u g h t he ch l o r e n c h y m a l c e l l l i n k i n g t he two c e l l f a c e s l a b e l l e d A i n f i g u r e 2. T h i s a i r space was a l s o o b s e r v e d i n t h i s work and t h e a i r spaces a r e thus i l l u s - t r a t e d a t t h e t i p s o f t h e t r a b i c u l a e i n f i g u r e 3- The o b s e r v a t i o n s i n t h i s work a l s o s u p p o r t the above c o n c e p t s but i t i s suggested t h a t t he s t r u c t - u r a l r o l e o f the t r a b i c u l a e may be the w a l l s t r u c t u r e s ' p r i m a r y f u n c t i o n . T r a b i c u l a e a r e not found i n t h e chlorenchyma o f o t h e r c o n i f e r n e e d l e s (see Esau, 1965, f o r r e f e r e n c e s ) even though a t l e a s t some n e e d l e b e a r i n g c o n i f e r s have a m e s o p h y l l i c arrangement s i m i l a r t o t h a t o b s e r v e d i n P i n u s c o n t o r t a (Gambles et_ aj_. , 1974). A p o s s i b l e e x p l a n a t i o n f o r t h i s i s t he shape o f p a l l i s a d e chlorenchyma c e l l s i n o t h e r c o n i f e r s i s u s u a l l y a cy - l i n d r i c a l s shape w h i c h would be a s t r o n g e r b a s i c s t r u c t u r e t o l a t e r a l f o r - ces t o the c y l i n d e r ' s major a x i s than t o t h e t h i n r e c t a n g u l a r box o r o v a l shape o f the p i n e m e s o p h y l l c h l o r e n c h y m a . On t h e b a s i s o f the above d i s c u s s i o n , i t would be e x p e c t e d t h a t due t o f r o s t p l a s m o l y s i s , t h e most l i k e l y p l a c e f o r t h e c e l l w a l l t o p a r t i a l l y c o l l a p s e i n Tsuga m e r t e n s i a n a would be the c e l l w a l l a r e a w i t h t h e l e a s t m e c h a n i c a l s t r e n g t h and the w a l l a r e a bounded by e x t r a c e l l u l a r a i r spaces where i c e c r y s t a l g r o w t h can o c c u r . In the c y l i n d r i c a l c e l l s o f T. mert- 119 e n s I a n a , t h e a i r spaces b o r d e r t h e c y l i n d r i c a l w a l l s o f t h e c e l l w h i c h c o u l d be e x p e c t e d t o be the weakest p a r t o f the c e l l w a l l . The dome shaped ends o f t h e c e l l f i r m l y a t t a c h e d t o a d j a c e n t c e l l s would be t h e s t r o n g e s t p a r t o f t h e c e l l . I t i s i n t e r e s t i n g t o n o t e t h a t c h l o r o p l a s t clumps and n u c l e i a r e found a t the ends o f t h e T. m e r t e n s i a n a c e l l i n w i n t e r and t h e c y t o p l a s m a l o n g t h e c y l i n d r i c a l w a l l s i s p r e d o m i n e n t l y f i l l e d by c y t o p l a s m i c v a c u o l e s ( f i g u r e 18). The c o n c e p t o f i c e p l a s m o l y s i s o f t h e c y t o p l a s m , p a r t i c u l a r l y i n t h e a r e a s o f the c e n t r a l p a r t s o f t h e c e l l w a l l f a c e A w i t h t h e r e s u l t a n t s q u e e z i n g o f some o r g a n e l l e s i n t o t h e c y t o p l a s m near the t r a b i c u l a r r e g i o n s becomes a more l o g i c a l s u g g e s t i o n i f the o b s e r v a t i o n s o f P a r k e r (1960), w h i c h i n d i c a t e the g e l l a t i o n o f t h e t a n n i n v a c u o l e d u r i n g w i n t e r a r e c o r r - e c t . The c o n c e p t o f p r o t o p l a s t g e l 1 a t i o n d u r i n g w i n t e r i n f r o s t hardy c e l l s has long been a m a t t e r o f d e b a t e . S c a r t h and L e v i t t (1937) b e l i e v e d t h a t the c y t o p l a s m remained f l u i d w h i l e o t h e r a u t h o r s (Lewis e t a l . , 1920 and K e s s l e r e t a 1 . , 1938) m a i n t a i n e d t h a t t h e p r o t o p l a s t became more v i s c o u s o r g e l l a t e d . An e x p l a n a t i o n o f a t l e a s t p a r t o f t h i s c o n t r o v e r s y may be t h e f a i l u r e o f some o f the e a r l y a u t h o r s t o d i s t i n g u i s h between what i s o c c u r i n g i n t h e main v a c u o l e v e r u s t h e c y t o p a l s m , and a l s o t h e use o f p l a s - m o l y z i n g f l u i d s t o measure c y t o p l a s m i c v i s c o s i t y o r g e l l i n g . The l a t e r p r e c e d u r e would g i v e i n c o r r e c t r e s u l t s i f p r o t o p l a s t p e r m e a b i l i t y was i n - c r e a s e d t o a wide range o f s u b s t a n c e s and the d i f f e r e n t i a I" p e r m e a b i l i t y o f the p r o t o p l a s t membranes d e c r e a s d as i s i n d i c a t e d by t h e o b s e r v a t i o n s i n t h i s work. 120 I t Is d i f f i c u l t t o c o n c e i v e o f how a l l p a r t s o f t h e c y t o p l a s m as ob- s e r v e d i n t h e w i n t e r p i n e chlorenchyma c o u l d e x i s t i n a g e l 1 s t a t e . For example, t h e r e i s no e v i d e n c e f o r any e l e c t r o n d e n s i t y w i t h i n t h e c y t o p l a s m - i c v a c u o l e s o t h e r t h a n t h e o s m i o p h i l i c b o d i e s t h a t o f t e n c o a t the i n n e r s u r f a c e o f t h e v a c u o l e membrane. From t h i s o b s e r v a t i o n , t h e c y t o p l a s m i c v a c u o l e s may o n l y c o n t a i n m a t e r i a l t h a t e x h i b i t s no s t a i n i n g p r o p e r t i e s o r e l e c t r o n d e n s i t y when p r e p a r e d by u l t r a s t r u c t u r a l t e c h n i q u e s . Such mater- i a l s c o u l d o n l y be c e l l w a t e r and low m o l e c u l a r w e i g h t o r g a n i c compounds such as amino a c i d s , s u g a r s e t c . Such m a t e r i a l s would not be a b l e t o g e l - l a t e . The t a n n i n m a t e r i a l o f t h e t a n n i n v a c u o l e however c o u l d p o s s i b l y gel-> l a t e . . I t meets many o f t h e c h a r a c t e r i s t i c s r e q u i r e d o f a g e l l o u t l i n e d by L e v i t t (1956) and Tumanov (1967). Some o f t h e s e c h a r a c t e r i s t i c s o f t a n n i n s a r e a t h r e e d i m e n s i o n a l m a c r o m o l e c u l a r p o l y m e r i c c o n f i g u r a t i o n o f v e r y l a r g e and i n d e t e r m i n a b l e m o l e c u l a r w e i g h t compounds and c o n t a i n i n g hydro- phi l i e m o i e t i e s t o i n t e r a c t w i t h t h e g e l l s o l v e n t (water) (Swain, 1965). The g e l l p o l y e r must a l s o be v e r y e v e n l y and u n i f o r m l y d i s p e r s e d w i t h i n t h e s o l v e n t i n o r d e r t o g e l l ( L e v i t t , 1956). U l t r a s t r u c t u r a l o b s e r v a t i o n s on the t a n n i n s i s P i n u s i n t h i s work, and e l s e w h e r e ( e . g . , L e d b e t t e r and P o r t e r , 1970 and H a r i s , 1971) c o n s i s t e n t l y i n d i c a t e t h a t t h e t a n n i n i s ev- e n l y d i s p e r s e d and r e m a r k a b l y homogeneous w i t h i n t h e t a n n i n v a c u o l e . The above o b s e r v a t i o n s i n t h e m s e l v e s would not be s u f f i c i e n t arguments f o r the g e l l a t i o n o f t h e t a n n i n v a c u o l e were i t not f o r the d u p l i c a t i o n i n t h i s work o f the e x p e r i m e n t a l o b s e r v a t i o n on a cryotome s t a g e o f P a r k e r 121 (1960) t h a t t h e t a n n i n v a c u o l e o f P i n u s n e e d l e s i n w i n t e r can be p h y s i c a l - l y d i s l o d g e d from t h e p r o t o p l a s t as an i n t a c t u n i t . T h i s o b s e r v a t i o n i n - d i c a t e s t h a t a t l e a s t a t s u b - f r e e z i n g t e m p e r a t u r e s , t h e t a n n i n v a c u o l e i s in a form t h a t can m a i n t a i n i t s s t r u c t u r e independent o f e x t e r n a l s u p p o r t . I f t h e t a n n i n i s i n a g e l l e d s t a t e d u r i n g w i n t e r , i t i s p r o b a b l y not i n a g e l l e d s t a t e d u r i n g summer. E v i d e n c e f o r t h i s i s t h e o b s e r v e d l y s i s o f summer c e l l s when a l l o w e d t o thaw on a c r y o m i c r o s e o p e s t a g e and a l s o t h e o b s e r v a t i o n o f t a n n i n m a t e r i a l becoming d i s p e r s e d t h r o u g h o u t t h e c y t o p l a s m in- summer chlorenchyma t h a t had been f i x e d a f t e r f r e e z i n g ( f i g u r e 7 2 ) . P a r k e r (1960) a l s o d i d not b e l i e v e t h e t a n n i n v a c u o l e g e l l e d i n summer. There a r e a l s o t h e o r e t i c a l arguments i n f a v o r o f t h e g e l l i n g c o n c e p t f o r a t l e a s t p a r t s o f w i n t e r c e l l s . For example, the c e l l must m a i n t a i n some form o f s t r u c t u r a l i n t e g r i t y a f t e r t h e l o s s o f f r e e z a b l e w a t e r (Tuma- nov, 1967) • E x p e r i m e n t s w i t h a r t i f i c i a l o r g a n i c g e l l s i n d i c a t e t h a t a t slow f r e e z i n g r a t e s , t h e y tend t o - r e l e a s e f r e e water t o e x t e r n a l i c e n u c l e - a t i o n s i t e s r a t h e r than a l l o w i n g i c e n u c l e a t i o n t o o c c u r w i t h i n t h e g e l 1 (Per- s i d s k y and L u y e t , 1975). G e l l i n g o f t a n n i n would reduce t h e r i s k o f i t en- t e r i n g t h e c y t o p l a s m d u r i n g t h e p h y s i c a l s t r e s s e s o f w i n t e r . I f t h e t a n n i n v a c u o l e does g e l l i n w i n t e r , then t h e c o n c e p t o f i c e p l a s m o l y s i s o f the c y t o p l a s m w h i c h l e a d s t o the s q u e e z i n g o f t h e c h l o r o - p l a s t and the n u c l e u s i n t o s p e c i f i c a r e a s o f t h e c e l l where t h e w a l l may not be a b l e t o c o l l a p s e i s c o n s i d e r a b l y more a t t r a c t i v e . W i t h a s e m i - s o l - i d t a n n i n d e p o s i t on t h e i n s i d e o f t h e c e l l and a c o l l a p s i n g c e l l w a l l on t h e e x t e r i o r o f t h e c y t o p l a s m , t h e r e would now be two s o l i d e n t i t i e s b e t - ween wh i c h t h e c y t o p l a s m would be c r u s h e d d u r i n g i c e p l a s m o l y s i s . 122 The above d i s c u s s i o n o f what may o c c u r t o cause some o f t h e c h a r a c t - e r i s t i c f e a t u r e s o f the w i n t e r c y t o p l a s m can o n l y be c o n s i d e r e d a p r e l i m i n - a r y t h e o r y . C o n s i d e r a b l y more e x t e n s i v e s t u d y most be c o n d u c t e d b e f o r e t h e s e c o n c e p t s c o u l d be v e r i f i e d . The above d i s c u s s i o n i s based on the c o n c e p t t h a t t h e w i n t e r c y t o p l a s - mic arrangement i n P i n u s and T. m e r t e n s i a n a ^ i s l a r g e l y induced by p h y s i c a l d e f o r m a t i o n s w i t h i n t h e c y t o p l a s m . There i s the p o s s i b i l i t y t h a t some o r a l l o f t h e o r g a n e l l e arrangements t h a t were o b s e r v e d i n w i n t e r may be due t o p h y s i o l o g i c a l l y based movements o f o r g a n e l l e s f o r p r o t e c t i v e o r o t h e r r e a s o n s . For example H a b e r l a n t (1876) has suggested t h a t c h l o r o p l a s t clum- p i n g may be a p h y s i o l o g i c a l l y induced mechanism f o r c h l o r o p l a s t s t o g a i n added p r o t e c t i o n from t h e p h o t o - o x i d i z i n g e f f e c t s o f l i g h t d u r i n g the long dormant s t a t e o f w i n t e r . P r e s u m a b l y , w i t h i n t h e c h l o r o p l a s t clumps, t h e masking e f f e c t s o f t h e o r g a n e l l e s a l l b e i n g d e n s e l y c l u s t e r e d i n one a r e a c o u l d be an advantage. The p r i n c i p l e o f c h l o r o p l a s t movements i n r e l a - t i o n t o e x t e r n a l s t i m u l i ( u s u a l l y v a r y i n g l i g h t i n t e n s i t i e s ) i s wel1 known, ( e . g . , l n o u e and S h i b a t a , 1973)- Such p h y s i o l o g i c a l l y based p o s s i b i l i t i e s have not been d i s c u s s e d i n t h i s work but s h o u l d be s e r i o u s l y c o n s i d e r e d as a l t e r n a t i v e s t o t h e c o n c e p t s p r e s e n t e d i n t h i s work. S t u d i e s o f the chlorenchyma d u r i n g t h e f a l l w h i l e t h e c e l l s a r e becoming f r o s t hardy but a r e s t i l l p h y s i o l o g i c a l l y a c t i v e s h o u l d i l l u c i d a t e whether o r g a n e l l e move- ments a r e based on p h y s i o l o g i c a l r a t h e r than p h y s i c a l , i c e r e l a t e d f a c t o r s . S i m i l a r l y , t h e s t u d y o f f r o s t hardy c e l l s i m m e d i a t e l y b e f o r e and a f t e r t h e f i r s t s e v e r e s e a s o n a l f r o s t may i n d i c a t e whether p h y s i c a l f o r c e s r e l a t e d t o t h e f r o s t have a l t e r e d the o r g a n e l l e arrangement w i t h i n c e l l s . 123 F u r t h e r s t u d y o f t h e c y t o l o g y o f f r o s t r e s i s t a n t p r o c e s s e s i n v e r y h a r d y p l a n t s s h o u l d be c o n d u c t e d . Such s t u d i e s w o u l d i l l u e i d a t e t h e c y - t o l o g i c a l b a s i s o f t h e f i r s t phase o f f r o s t h a r d e n i n g d u r i n g t h e l a t e sum- mer and e a r l y f a l l (a p e r i o d o f t h e s e a s o n a l c y c l e not c o v e r e d i n t h i s work ) and c o u l d , w i t h p r e s e n t l y a v a i l a b l e t e c h n i q u e s , c l a r i f y t h e p h y s i - c a l p r o c e s s e s o c c u r i n g w i h t i n t h e c e l l a s t h e f r o s t h a r d y t i s s u e i s f r o - z e n . A c r y o m i c r o s c o p e > w i t h mechanisms t o p r e c i s e l y c o n t r o l s t a g e t e m p - e r a t u r e s u c h a s t h e one d e v e l o p e d by Di 1 l e r e_t aj_. (1971) w o u l d a l low t h e s t u d y o f i c e f o r m a t i o n and m e l t i n g w i t h i n s p e c i f i c t e m p e r a t u r e r a n g e s . The use o f t h e f r e e z e s u b s t i t u t i o n f i x a t i o n and embedding t e c h n i q u e s s u c h as t h o s e used by R a p a t z et_ a_j_. (1963) w o u l d a l l o w d i r e c t e l e c t r o n m i c r o - s c o p i c o b s e r v a t i o n o f t h e c y t o p l a s m i n t h e s t a t e o f i c e p l a s m o l y s i s . The p r o d u c t i o n o f f r e e z e - e t c h e d p l a t i n u m - c a r b o n r e p l i c a s o f m a t e r i a l f r o z e n t o p r e - a r r a n g e d t e m p e r a t u r e s a t c o n t r o l l e d r a t e s b e f o r e s u b m e r s i o n i n t o l i q u i d n i t r o g e n may o f f e r t h e g r e a t e s t p o t e n t i a l o f a l l p r e s e n t t e c h n i q u e s i n t h e s t u d y o f f r o z e n t i s s u e s i n c e t h e p r e s e n c e o f i c e c r y t a l s t h e m s e l v e s c a n be seen a t t h e r e s o l u t i o n l e v e l o f t h e e l e c t r o n m i c r o s c o p e ( N e ? , 1 9 7 6 ) . In t h i s w o r k , t h e f r e e z e - e t c h t e c h n i q u e had been a t t e m p t e d , but t h e ma- j o r d i f f i c u l t y e n c o u n t e r e d was t h e f a i l u r e t o s u c c e s s f u l l y remove r e p l i c a s f r o m t i s s u e p i e c e s . 124 IV. D i s c u s s i o n o f Exper iments i n C o n t r o l 1ed Env i ronment Chambers The e x p e r i m e n t s on p i n e s e e d l i n g s i n c o n t r o l l e d e n v i r o n m e n t s were de- s i g n e d t o s t u d y c y t o l o g i c a l l y t h e f r o s t h a r d e n i n g p r o c e s s between the non- hardy growth s t a t e and s t a g e two. I t has been noted t h a t s t a g e one i s be- l i e v e d ( W e i s e r , 1970 and Tumanov, 1967) t o be a f r o s t season d e t e c t i o n and s y n t h e s i s o f c r y o p r o t e c t i v e m a t e r i a l s s t a g e . Thus i t was hoped t h a t a f i x - a t i o n o f n e e d l e s from s e e d l i n g s t h a t had been exposed t o two weeks o f s h o r t photo p e r i o d s and p r o g r e s s i v e l y lower t e m p e r a t u r e s may r e v e a l t h e f o l l o w i n g : any component o r components o f t h e w i n t e r c y t o l o g i c a l s t a t e due t o p r i o r s p e c i f i c c h e m i c a l s y t h e s i s and p r e p a r a t i o n by chlorenchyma c e l l s ; and any component o r components due t o t h e mechanica1 d e f o r m a t i o n o f t h e c y t o p l a s m by deep f r e e z i n g ( s t a g e 2 o f f r o s t h a r d i n e s s a c q u i s i t i o n ) . U n f o r t u n a t e l y , t h e f i x a t i o n o f c o n t r o l t r e e s and t h e f i x a t i o n o f t r e e s f i x e d a t v a r i o u s t i m e s d u r i n g t h e a r t i f i c i a l f r o s t a c c l i m a t i z a t i o n p e r i o d i n s t a g e one were not s u c c e s s f u l . A t t e m p t s t o r e p e a t the e x p e r i m e n t were p r e v e n t e d by r e p e a t e d f a i l u r e s o f t h e growth chambers u n t i l t h e s u p p l y o f s e e d l i n g t r e e s ran o u t . The f i x a t i o n c o n d u c t e d a f t e r the a c c l i m a t i z e d t r e e s were p l a c e d i n t h e f r e e z e r a t -18°C f o r e i g h t hours was s u c c e s s f u l . However, the o b s e r v a t i o n s r e c o r d e d on t h i s m a t e r i a l a r e not i d e n t i c a l w i t h t h o s e d e s c r i b e d f o r t h e w i n t e r c o n d i t i o n i n the f i e l d . The s e p a r a t i o n o f c h l o r o p l a s t s from t h e c e l l w a l l , t h e i r adherence i n 125 s m a l l c l u s t e r s , t h e f o r m a t i o n o f c y t o p l a s m i c v a c u o l e s and the c o n t r a c t i o n o f t h e t a n n i n v a c u o l e i m p l y t h a t some o f t h e f e a t u r e s o f t h e w i n t e r u l t r a - s t r u c t u r e were o b t a i n e d . The f a c t t h a t t h e t r e e s were s e v e r e l y damaged a f t e r f r e e z e r e x p o s u r e i n d i c a t e s t h a t a r t i f i c i a l h a r d e n i n g was a t most o n l y a p a r t i a l s u c c e s s . However, t h a t t h e c o n t r o l t r e e s were k i l l e d i m m i d i a t e - l y d u r i n g o r a f t e r f r e e z e r e x p o s u r e i n d i c a t e s t h a t some degree o f a r t i f i - c i a l l y induced h a r d e n i n g was a c h i e v e d . Two v e r y i n t e r e s t i n g o b s e r v a t i o n s were made on t r e e s a r t i f i c i a l l y h a r d - ened and exposed t o t h e f r e e z e r t e m p e r a t u r e s . The f i r s t i s t h a t t h e n e e d l e s f a i l e d t o y e l l o w u n t i l a f t e r t h e f r o s t e x p o s u r e , and the degree o f y e l l o w i n g was v e r y s l i g h t . The second i s t h a t v e r y few o s m i o p h i l i c b o d i e s were ob- s e r v e d i n t h e n e e d l e s . T h i s may be i n d i r e c t e v i d e n c e t h a t t h e c h l o r o - p l a s t c l u m p i n g and/or t h e o s m i o p h i l i c b o d i e s may be r e s p o n s i b l e f o r t h e w i n - t e r c h l o r o s i s o f P i n u s c o n t o r t a ssp. l a t i f o l i a n e e d l e s . T h i s t y p e o f c o n t - r o l l e d e n v ironment s t u d y would be a most i n t e r e s t i n g a r e a f o r f u r t h e r work. P i n u s c o n t o r t a can be d e s c r i b e d as a moderate x e r o p h y t e ( M i r o v , 1967). Tumanov (l967),and A l d e n and Hermann (1971) c i t e l i t e r a t u r e w h i c h d i s c u s s e s t h e p o s s i b l e c o r r e l a t i o n between drought r e s i s t a n c e and f o r s t h a r d i n e s s . L i and Weiser (1970) succeeded i n s l i g h t l y i n c r e a s i n g t h e f r o s t h a r d i n e s s o f stem t i s s u e p i e c e s by p a r t i a l d e h y d r a t i o n b e f o r e f r o s t e x p o s u r e . A dr o u g h t e x p o s u r e e x p e r i m e n t was con d u c t e d on t r e e s w i t h t h e o b j e c t o f : a. D e t e r m i n i n g i f d r o u g h t exposed t r e e s were more f r o s t r e s i s t a n t . b. D e t e r m i n i n g i f t h e u l t r a s t r u c t u r e o f d r o u g h t exposed n e e d l e s had any resemblance t o w i n t e r f r o s t hardy n e e d l e s . 126 N e i t h e r p a r t o f t h i s e x p e r i m e n t i n d i c a t e d an o b v i o u s c y t o l o g i c a l c o r - r e l a t i o n between d r o u g h t c o n d i t i o n s and f r o s t h a r d i n e s s . The u l t r a s t r u c t - u r e o f drought exposed n e e d l e s d i d i n d i c a t e some s e p a r a t i o n o f c h l o r o p l a s t s from t h e c e l l w a l l . However, t h i s was o c c a s i o n a l l y noted i n summer f i x a t - i o n s and may be f i x a t i o n a r t i f a c t . SUMMARY T h i s work has i d e n t i f i e d two s e a s o n a l l y dependent, c y t o p l a s m - i c s t a t e s i n t h e m e s o p h y l l c hlorenchyma o f P i n u s c o n t o r t a spp. l a t - i f o l i a . In one s t a t e , t h e c y t o p l a s m i c c o n s t i t u e n t s o f most mature chlorenchyma c e l l s ( i n n e e d l e s v a r y i n g i n age from j u s t a f t e r t h e c o m p l e t i o n o f n e e d l e e l o n g a t i o n t o n e e d l e s f i v e y e a r s o f age).app- ear v e r y s i m i l a r t o the s t r u c t u r a l and p o s i t i o n a l o r g a n i z a t i o n o f c e l l o r g a n e l l e s and c o n s t i t u e n t s d e s c r i b e d i n t h e l i t e r a t u r e f o r the a s s i m i l a t o r y t i s s u e o f o t h e r h i g h e r p l a n t s . The c h l o r e n c h y m a l c e l l s found i n t h e s t a t e d e s c r i b e d above were a l l o b s e r v e d d u r i n g t h e grow- ing season (June t h r o u g h A u g u s t ) . T h i s s t a t e i s r e f e r r e d t o here as t h e summer s t a t e . F i x a t i o n s on c h l o r e n c h y m a l c e l l s c o l l e c t e d d u r i n g t h e w i n t e r months,.(Pecember t h r o u g h M a r c h ) , r e v e a l e d a c o n s i s t e n t but unusual c y t o p l a s m i c s t a t e , (termed t h e w i n t e r s t a t e ) , w h i c h was r e m a r k a b l y d i f f e r e n t from the s t r u c t u r a l and p o s i t i o n a l appearance o f a l m o s t a l 1 c y t o p l a s m i c s t r u c t u r e s and o r g a n e l l e s o b s e r v e d i n t h e summer, s t a t e . The w i n t e r c y t o p l a s m can be d e s c r i b e d and compared t o t h e summer s t a t e on t h e b a s i s o f s p e c i f i c i n d i v i d u a l o r g a n e l l e s o r s t r u c - t u r e s . C h l o r o p l a s t s : In summer, t h e c h l o r o p l a s t s were d i s c o i d a ! i n shape, l y - 128 ing near the c e l l w a l l w i t h t h e i r l o n g axes ( i n s e c t i o n a l p r o f i l e ) p a r a l l e l t o t h e c e l l w a l l . The d i s t r i b u t i o n o f c h l o r o p l a s t s was u n i f o r m about t h e c e l l w a l l and t r a b i c u l a e . In w i n t e r , the c h l o r o p l a s t s were a g g r e g a t e d i n t o e i t h e r l a r g e clumps o f a t l e a s t ten , o r s m a l l clumps o f l e s s than t e n . Gener- a l l y , t h e l a r g e clumps were i:n t h e p a r t s o f t h e c e l l where the long t r a b i c u l a e a r e found and the s m a l l e r clumps, i n t h e a r e a s near t h e s m a l l t r a b i c u l a e . No c h l o r o p l a s t s were found i n t h e c y t o p l a s m a d j a c e n t t o t h e c e l l w a l l a r e a s w i t h o u t t r a b i c u l a e . The w i n t e r c h l o r o p l a s t s were a p p r o x i m a t e l y c i r c u l a r i n s e c t - i o n a l p r o f i l e and s l i g h t l y l a r g e r i n volume than summer c h l o r o p l a s t s . The i n c r e a s e i n volume appears t o be due t o a t r a n s f o r m a t i o n o f ' t h e c h l o r o p l a s t ' s shape from d i s c o i d a l i n summer t o the s p h e r i c a l shape. The added volume i s due t o an i n c r e a s e i n stroma.The w i n t e r ' t h y l a k o i d system o f t h e c h l o r o p l a s t u s u a l l y r e t a i n e d the d i s c o i d a l appearance. W i n t e r c h l o r o p l a s t s were a l s o c h a r a c t e r i z e d by t h e i r near c o m p l e t e absence o f s t a r c h . The e n v e l o p e membranes o f w i n t e r c h l o r o p l a s t s were not as d i s t i n c t i n w i n t e r as i n summer, t h e y appeared amorph- ous a t h i g h m a g n i f i c a t i o n . T h y l a k o i d membranes appeared t o be sharp and d i s t i n c t i n both summer and w i n t e r f i x a t i o n s . In w i n t e r , t h e o u t e r membranes o f the c h l o r o p l a s t e n v e l o p e s o f a d j a c e n t c h l o r o p l a s t s w i t h i n clumps may f u s e t o form r e g i o n s of close-membrane a s s o c i a t i o n as t h e r e s u l t o f f u s i o n between n e i g h b o r i n g c h l o r o p l a s t s . Many w i n t e r c h l o r o p l a s t s c o n t a i n e d two 129 o r more t h y l a k o i d systems w i t h i n one membrane e n v e l o p e . Mi t o c h o n d r i a : In summer, M i t o c h o n d r i a were p o l y m o r p h i c i n c r o s s s e c t i o n and were u s u a l l y d i s t r i b u t e d u n i f o r m l y between c h l o r o p l a s t s o r o i l b o d i e s . In w i n t e r , m i t o c h o n d r i a were c o n s i d e r a b l y s m a l l e r i n s e c t i o n a l p r o f i l e , and u s u a l l y s p h e r i c a l i n shape. They a r e u s u a l l y l o o s e l y a s - s o c i a t e d w i t h c h l o r o p l a s t clumps and t h e remainder a r e randomly s c a t t e r - ed i n t h e c y t o p l a s m . No m i t o c h o n d r i a l membrane f u s i o n was o b s e r v e d be- tween m i t o c h o n d r i a o r between m i t o c h o n d r i a and o t h e r o r g a n e l l e s . The c r i s t a e were reduced i n number and t h e m i t o c h o n d r i a l membranes were not as d i s t i n c t as o b s e r v e d i n the o r g a n e l l e s i n summer. RER, SER and Di c t y o s o m e s : These s t r u c t u r e s were r a r e i n both w i n t e r and summer c y t o p l a s m . RER and SER were u s u a l l y found near t h e n u c l e u s i n both summer and w i n - t e r f i x a t i o n s . N u c l e u s : In summer, the n u c l e u s i s s p h e r i c a l and i s u s u a l l y found i n a cen- t r a l l o c a t i o n w i t h i n t h e c e l l . The t o n o p l a s t u s u a l l y n e a r l y e n c i r c l e s t h e n u c l e u s e x c e p t f o r broad c y t o p l a s m i c s t r a n d s c o n n e c t i n g t h e pe- r i n u c l e a r c y t o p l a s m t o the p e r i p h e r a l c y t o p l a s m . The n u c l e a r enve- l o p e i s d i s t i n c t but w i t h few n u c l e a r p o r e s . The n u c l e o p l a s m c o n t a i n s v e r y dense r e g i o n s o f h e t e r o c h r o m a t i n i n t e r s p e r s e d w i t h r e l a t i v e l y 130 c l e a r r e g i o n s o f n u c l e o p l a s m c o n t a i n i n g some g r a n u l a r m a t e r i a l and f i n e f i b r i l l a r m a t e r i a l . In w i n t e r , the, n u c l e u s i s found i n t h e p e r i p h e r a l c y t o p l a s m , u s u a l l y i n t h e ends o f t h e c e l l where t h e l o n g t r a b i c u l a e a r e f o u n d , l t i s u s u a l l y o v o i d i n shape w i t h the long a x i s p a r a l l e l t o the ad- j a c e n t c e l l w a l l . The n u c l e a r e n v e l o p e i s c h a r a c t e r i z e d by a v e r y h i g h d e n s i t y o f n u c l e a r p o r e s . The n u c l e o p l a s m a p pears f i n e l y g r a n - u l a r w i t h the dense h e t e r o c h r o m a t i n a r e a s being more d i s p e r s e d i n w i n t - e r . The n u c l e o l u s appears s i m i l a r i n t h e w i n t e r and summer n u c l e u s . The t a n n i n , main v a c u o l e and t o n o p l a s t : The b u l k o f t h e c e l l volume i n both summer and w i n t e r i s o c - c u p i e d by t h e main c e l l v a c u o l e . However, i n t h e p i n e c h l o r e n c h y m a , t h i s v a c u o l e i s f i l l e d w i t h a homogeneous,highly o s m i o p h i l i c m a t r i x w h i c h was i d e n t i f i e d t h r o u g h s p e c i f i c s t a i n i n g as b e i n g t a n n i n mat- e r i a l . The main v a c u o l e was t h u s r e f e r r e d t o as the t a n n i n v a c u o l e . In summer, t h e t o n o p l a s t was v e r y prominent due t o a s l i g h t s h r i n k a g e o f the t a n n i n m a t r i x d u r i n g most f i x a t i o n s . The summer t o n o p l a s t was p a r t i c u l a r l y s e n s i t i v e t o changes i n f i x a t i v e osmo- l a r i t i e s . The volume o f the t a n n i n v a c u o l e was g r e a t e r and t h e c y - t o p l a s m was c o n f i n e d t o : ; a t h i n p e r i p h e r a l s heet about t h e i n n e r s u r f a c e o f t h e c e l l w a l l , i n s l e n d e r s t r a n d s t h r o u g h t h e main v a c u - o l e t o the n u c l e u s , and a t h i n r e g i o n o f p e r i n u c l e a r c y t o p l a s m . 131 In w i n t e r , the t o n o p l a s t was o b s c u r e d ) ' b y t a n n i n m a t e r i a l and changes i n the o s m o l a r i t y o f f i x a t i v e f a i l e d t o a f f e c t t h e t o n o p l a s t o r t h e t a n n i n m a t e r i a l . The t a n n i n v a c u o l e was reduced i n volume and had r e t r a c t e d t o the c e n t r a l a r e a o f the c e l l . The v a c u o l e was more s p h e r i c a l (not f o l l o w i n g t h e c o n t o u r s o f t h e i n n e r s u r f a c e o f the w a l l as c l o s e l y ) and no c y t o p l a s m i c s t r a n d s were o b s e r v e d t r a n s - v e r s i n g i t . The c o n t r a c t i o n o f t h e t a n n i n v a c u o l e g r e a t l y i n c r e a s e d the volume o f the p e r i p h e r a l c y t o p l a s m about t h e i n n e r s u r f a c e o f t h e c e l l w a l l . T h i s i n c r e a s e i n c y t o p l a s m i c volume was g r e a t e s t near t h e ends o f t h e c e l l where t h e long t r a b i c u l a e a r e found. The shape o f the t a n n i n v a c u o l e i n w i n t e r was c o n f i r m e d by o b s e r v a t i o n s on c r y o - s e c t i o n s o f c h e m i c a l l y u n t r e a t e d t i s s u e . C y t o p l a s m i c v a c u o l e s and o s m i o p h i l i c b o d i e s : In summer, sma11 i n t e n s e l y o s m i o p h i l i c b o d i e s were o b s e r v e d w i t h i n t h e c y t o p l a s m . They were not found i n l a r g e numbers and were r a n - domly d i s t r i b u t e d . In w i n t e r , the o s m i o p h i l i c b o d i e s were p r e s e n t i n g r e a t number and were a v e r y prominent f e a t u r e . They w e r e . l o c a l i z e d i n two gen- e r a l a r e a s w i t h i n t h e c e l l : n e ar t h e plasma membrane about t h e en- t i r e c e l l as a s i n g l e u n i f o r m l a y e r , and randomly d i s t r i b u t e d deep i n t h e c y t o p l a s m about t h e c y t o p l a s m i c v a c u o l e s , p a r t i c u l a r l y i n a r e a s not o c c u p i e d by t h e c h l o r o p l a s t clumps o r t h e n u c l e u s . In summer, s m a l l c y t o p l a s m i c v a c u o l e s a r e found s c a t t e r e d 132 t h r o u g h o u t the c y t o p l a s m . They u s u a l l y c o n t a i n o s m i o p h i l i c b o d i e s . In w i n t e r , t h e o s m i o p h i l i c b o d i e s remained a s s o c i a t e d w i t h masses o f c y t o p l a s m i c v a c u o l e s , m a i n l y i n a r e a s o f c y t o p l a s m not o c c u p i e d by t h e c h l o r o p l a s t clumps o r t h e n u c l e u s . The dense masses o f v a c u o l e s can be d e s c r i b e d as b e i n g "foamy" o r "s p o n g - 1 i k e " i n appearance. O'il b o d i e s : In summer, t h e r e a r e some o i l b o d i e s o b s e r v e d w i t h i n t h e c y - t o p l a s m , u s u a l l y near t h e n u c l e u s . i n w i n t e r , t h e s e o i l b o d i e s a r e g r e a t l y i n c r e a s e d i n s i z e and number and a r e a prominent f e a t u r e o f the w i n t e r c y t o p l a s m . Plasma membrane: The plasma membrane appeared as a c l e a r l y d e f i n e d membrane c l o s e l y a p p r e s s e d t o t h e c e l l w a l l i n most summer and a l l w i n t e r f i x a t i o n s . In some summer f i x a t i o n s , t h e plasma membrane was o c - c a s i o n a l l y s l i g h t l y s e p a r a t e d from t h e c e l l w a l l due t o some deg- ree o f c e l l p l a s m o l y s i s . E x p e r i m e n t s w i t h d i f f e r i n g f i x a t i v e and p r e p a r a t i v e p r o c e d u r e s r e s u l t - ed i n improved methods o f p r e p a r i n g p i n e n e e d l e chlorenchyma f o r e l e c t r o n m i c r o s c o p y . These e x p e r i m e n t s a l s o r e v e a l e d " t h a t the w i n t e r c y t o p l a s m i s re m a r k a b l y i n s e n s i t i v e t o r a d i c a l changes i n the o s m o t i c and i o n i c n a t u r e o f f i x a t i v e s o l u t i o n s when compared t o summer t i s s u e . Chemical '. p r e p a r a t i v e 133 p r o c e d u r e s f o r e l e c t r o n m i c r o s c o p y c o u I d be s u b s t a n t i a l 1 y reduced i n d u r a t - i o n f o r w i n t e r t i s s u e w i t h o u t any d e t e c t a b l e d e t e r i o r a t i o n o f u l t r a s t r u c t - u r a l p r e s e r v a t i o n . These d i f f e r e n e e s in. response t o c h e m i c a l t r e a t m e n t s a r e i n t e r p r e t e d as b e i n g i n d i c a t i v e o f a fundamental change i n the permea- b i l i t y o f c y t o p l a s m i c membrane systems i n w i n t e r . I t i s c o n c l u d e d t h a t t h e d r a m a t i c t r a n s f o r m a t i o n i n chlorenchyma u l t - r a s t r u c t u r e between t h e summer and w i n t e r s t a t e s r e p r e s e n t s t h e c y t o l o g i - c a l r e s p o n s e t o t h e p h y s i o l o g i c a l and p h y s i c a l changes w i t h i n t h e p i n e n e e d l e m e s o p h y l l . The p h y s i o l o g i c a l l y induced u l t r a s t r u c t u r a l changes i n - c l u d e t h e changes i n c y t o p l a s m i c membrane p e r m e a b i l i t y and s t r u c t u r e i n w i n t e r , t h u s a l l o w i n g t h e r a p i d p e n e t r a t i o n o f f i x a t i v e s o l u t i o n s and p r e - v e n t i n g any e x t e n s i v e osmotic response by t h e p r o t o p l a s t o r i t s o r g a n e l l e s t o s i g n i f i c a n t changes i n f i x a t i v e c o n c e n t r a t i o n o r c o m p o s i t i o n . The i n - c r e a s e s o b s e r v e d i n c y t o p l a s m i c v a c u o l a t i o n , o s m i o p h i l i c b o d i e s , o i l bo- d i e s , c h l o r o p l a s t stroma and n u c l e a r e n v e l o p e pores must a l s o be t h e r e s u l t o f p h y s i o l o g i c a l and b i o c h e m i c a l processesuwhieh must have o c c u r r e d whi l e t h e chlorenchyma was s t i l l i n an a c t i v e s t a t e . s o m e time i n e a r l y f a l l . S i m i - l a r l y , t h e r e d u c t i o n i n m i t o c h o n d r i a l volume and the a s s u m p t i o n o f s i m p l i - f i e d s p h e r i c a l shapes, and the h y d r o l y s i s o f c h l o r o p l a s t s t a r c h g r a i n s must a l s o have o c c u r red when t h e t i s s u e was s t i l l a c t i v e m e t a b o l i c a l l y . I t i s c o n c l u d e d i n t h i s work t h a t the above c y t o p l a s m i c changes between summer and w i n t e r s t a t e s may be t h e c y t o l o g i c a l r e s p o n s e o f t h e s t a g e one phase o f f r o s t h a r d i n e s s p r e p a r a t i o n . 134 Some o f t h e s t r u c t u r a l d i f f e r e n c e s between summer and w i n t e r c h l o r e n - chyma u l t r a s t r u c t u r e may be r e l a t e d t o p h y s i c a l r a t h e r than p h y s i o l o g i c a l o r b i o c h e m i c a l changes i n t h e ch l o r e n c h y m a . The p h y s i c a l changes a r e be- l i e v e d t o be due t o t h e d i s t o r t i o n s i n d u c e d v w i t h i n t h e p r o t o p l a s t due t o i c e p l a s m o l y s i s . These p h y s i c a l d i s t o r t i o n s may r e p r e s e n t t h e c y t o l o g i c - a l r e sponse o f s t a g e two o f f r o s t h a r d i n e s s development, i n wh i c h t h e p r o - t o p l a s t i s b e l i e v e d t o p h y s i c a l l y adapt and accomodate i c e p l a s m o l y s i s and~ the p r e s e n c e o f i c e c r y s t a l s . C y t o l o g i c a l . m a n i f e s t a t i o n s o f i c e p l a s m o l y - s i s would l e a d t o the* a g g r e g a t i o n o f - c h l o r o p l a s t s i n c e r t a i n s p e c i f i c c y - t o p l a s m i c a r e a s and f i n a l l y t h e f u s i o n o f t h e s e o r g a n e l l e s . The t r a n s l o - c a t i o n and change i n shape o f the w i n t e r n u c l e u s and t h e d i s t r i b u t i o n o f c y t o p l a s m i c v a c u o l e s a r e a l s o b e l i e v e d t o be the r e s u l t o f i c e induced p l a s m o l y t i c f o r c e s . An h y p o t h e s i s based on p h y s i c a l f o r c e s due t o t h e f r e e z i n g o f the w i n - t e r chlorenchyma i s p r e s e n t e d t o e x p l a i n some o f t h e w i n t e r u 1 t r a s t r u c t u r - a l changes o b s e r v e d . T h i s h y p o t h e s i s i s based on t h e co n c e p t o f d i f f e r e n - t i a l p l a s m o l y s i s i n w h i c h the w i n t e r c e l l p l a s m o l y z e s i n r e g i o n s o f e x t e n - s i v e c y t o p l a s m i c v a c u o l i z a t i o n , but does not p l a s m o l y z e as e x t e n s i v e l y i n c e l l r e g i o n s where the c h l o r o p l a s t clumps and the w i n t e r n u c l e u s a r e found. A p o s s i b l e e x p l a n a t i o n f o r t h i s d i f f e r e n t i a l p l a s m o l y s i s i s based on the con c e p t t h a t t h e plasma membrane may adhere t o t h e e e l 1 w a l l more e x t e n s i v e - l y where t h e p r i m a r y p i t f i e l d s a r e l o c a t e d , w h i l e t h e p r o t o p l a s t c o l l a p s e s r e a d i l y i n t h e p r e s e n c e o f i c e d e h y d r a t i o n i n t h e r e g i o n s o f e x t e n s i v e v a - c u o l i z a t i o n . A n o t h e r i m p o r t a n t a s p e c t o f t h i s h y p o t h e s i s i s t h a t t h e t a n - n i n v a c u o l e e n t e r s a g e l - 1 i k e s t a t e w i t h subsequent s t r u c t u r a l i n t e g r i t y 135 a t low t e m p e r a t u r e s and/or i n a d e h y d r a t e d s t a t e due t o the removal o f c e l l f r e e w a t e r by i c e n u c l e a t i o n s i t e s e x t e r n a l t o t h e p r o t o p l a s t . N u c l e a r and c h l o r o p l a s t m i g r a t i o n s from summer l o c a t i o n s would o c c u r by t h e squeez- ing o f t h e s e o r g a n e l l e s from summer l o c a t i o n s as the c y t o p l a s m c o l l a p s e s a g a i n s t the t a n n i n m a t e r i a l . The fundamental t r a n s f o r m a t i o n o f t h e summer p i n e c hlorenchyma c e l l u l t r a s t r u c t u r e t o the w i n t e r s t a t e i s c o m p l e t e l y r e v e r s i b l e . The summer s t a t e o f n e e d l e s a f t e r w i n t e r i s v e r y s i m i l a r t o the summer b e f o r e expo- s u r e t o f a l l and w i n t e r . What may be an i n t e r m e d i a t e o r s p r i n g t r a n s i t - i o n a l s t a t e has been d e s c r i b e d i n t h i s work f o r chlorenchyma f i x e d i n A p r i l . P i n e c hlorenchyma from n e e d l e s t h a t had been kept i n a f r o z e n s t a t e from w i n t e r c o l l e c t i o n were o b s e r v e d i n t h e f r o z e n s t a t e w i t h o u t any chem- i c a l a d d i t i v e s . These o b s e r v a t i o n s i n d i c a t e d t h a t t h e t a n n i n v a c u o l e was i n t he same p o s i t i o n as o b s e r v e d i n c h e m i c a l f i x a t i o n s o f w i n t e r c e l l s . The p o s i t i o n o f o i l b o d i e s and t h e n u c l e u s were a l s o s i m i l a r t o t h a t ob- se r v e d in:chem:i c a l l y f i x e d m a t e r i a l . No o t h e r c y t o p l a s m i c d e t a i l s c o u l d be o b s e r v e d i n f r o z e n s e c t i o n s . I t i s c o n c l u d e d t h a t t h e o b s e r v a t i o n s on f r o - z e n m a t e r i a l i n d i c a t e s t h a t t h e appearance o f c h e m i c a l l y f i x e d w i n t e r t i s - sue may be v e r y c l o s e t o t h e t r u e p h y s i c a l s t a t e o f the w i n t e r f r o z e n c e l l . S t u d i e s i n t h i s work i n d i c a t e d t h a t a m i n o r i t y o f c h l o r e n c h y m a l c e l l s i n n e e d l e s o f a l l ages were s e n e s c e n t o r dead. The numbers o f c e l l s i n t h e s e two c o n d i t i o n s were p r o p o r t i o n a l t o n e e d l e age, but were independent o f s e a s o n . 136 The chlorenchyma o f P i n u s a l b i c a u l i s and Tsuga m e r t e n s i a n a were a l s o examined a t t h e l i g h t m i c r o s c o p y l e v e l , i n summer and w i n t e r . These o b s e r - v a t i o n s i n d i c a t e d a s e a s o n a l c y c l e between summer and w i n t e r c y t o p l a s m i c s t a t e s w h i c h i s v e r y s i m i l a r t o ' t h a t s t u d i e d i n P. c o n t o r t a . The s t r u c t u r e o f t h e m e s o p h y l l c h l o r e n c h y m a l c e l l s were s t u d i e d i n d e t a i l by s e r i a l s e c t i o n i n g and s c a n n i n g e l e c t r o n m i c r o s c o p y . The shape o f t he m e s o p h y l l c e l l was o b s e r v e d t o be r e l a t e d t o t h e d i s t r i b u t i o n o f c e r t a i n c e l l u l a r components i n t h e w i n t e r s t a t e . E x p e r i m e n t s were co n d u c t e d t o a r t i f i c i a l l y i nduce f r o s t h a r d i n e s s i n l a b o r a t o r y grown P i n u s c o n t o r t a s e e d l i n g s , but t h e s e e x p e r i m e n t s were o n l y p a r t i a l l y s u c c e s s f u l and no d e f i n i t i v e c o n c l u s i o n s can be based on them. 137 APPENDIX I S p e c i f i c S t a i n S o l u t i o n 1. M o d i f i e d Johansen (.1940) S t a i n and F i x a t i o n f o r T a n n i n s A d a r k brown s t a i n i n g o f v a c u o l e s w i l l o c c u r w i t h l o n g i t u d i n a l l y c u t n e e d l e s f i x e d f o r 12 hours i n t h e f o l l o w i n g s o l u t i o n : F e r r o u s s u l p h a t e 0.5 gm 1.5% f r e s h l y made f o r m a l i n , made up i n Sodium c a c o d y l a t e b u f f e r 2. Reeve S t a i n f o r P o l y p h e n o l s (1951) To f r e s h s e c t i o n s , t he f o l l o w i n g s o l u t i o n s a r e added i n s u c c e s s - i o n . 10% sodium n i t r i t e • 20% urea s t a b i l i z e r 10% a c e t i c a c i d ; A f t e r 3 t o 5 m i n u t e s , add 2 N sodium h y d r o x i d e . Slow i n d e v e l o p i n g l i g h t p i n k a r e a s were i n t e r p r e t e d t o i n d i c a t e the p r e s e n c e o f t a n n i n s . The b r i g h t c h e r r y - r e d s t a i n i n g o f t a n n i n s d e s c r i b e d by Reeve was never o b s e r v e d . Only a l i g h t p i n k c o l o r a t i o n o f t h e t a n n i n v a c u o l e s was o b s e r v e d . Sudan B l a c k B S o l u t i o n S t a i n was made up i n a b s o l u t e a l c o h o l and a c l e a r i n g s o l u t i o n o f 90% a l c o h o l - 10% d i s t i l l e d w a t e r was used. S t a i n i n g was done f o r f i v e m i n utes f o l l o w e d by 1 minute i n c l e a r i n g s o l u t i o n , f o l l o w e d by two washes i n d i s t i l l e d w a t e r . Dark s p h e r i c a l d e s p o s i t s i n the p r o - t o p l a s t s were i n t e r p r e t e d as p o s i t i v e t e s t s f o r l i p i d s . 138 APPENDIX I I C r y o s e c t i o n i n g and L i g h t M i c r o s c o p y o f Frozen T i s s u e In o r d e r t o vie w t i s s u e i n the f r o z e n s t a t e and unthawed s t a t e , ( f i e l d c o l l e c t i o n was a t below z e r o t e m p e r a t u r e s ) , a R i c h e r t c r y o m i - crotome and Nikon b i o e u l a r m i c r o s c o p e w i t h camera a t t a c h m e n t were mod- i f i e d f o r t h i s p u r p o s e . The p r o c e d u r e and a p p a r a t u s used i s d e s c r i b - ed as f o l l o w s w i t h r e f e r e n c e t o f i g u r e s ?8, 79 and 80. A. C r y o s e c t i o n i n g N e edles s t o r e d i n a commercial f r e e z e r a t -18°C, were s u b d i v i d e d t r a n s v e r s e l y w i t h i n the f r e e z e r i n t o a p p r o x i m a t e l y 0.5 cm l o n g s e g - ments. They were then p l a c e d i n a s e a l e d v i a l w h i c h was immersed i n a b r i n e - i c e bath (A, f i g u r e 78), and t r a n s f e r r e d i n t h i s b a t h t o t h e a p p a r a t u s i l l u s t r a t e d i n f i g u r e 78. The a p p a r a t u s c o n s i s t s o f a s t a n d - a r d R i c h e r t CO2 e x p a n s i o n c r y o t o m e , ( ( B ) , CO^ b o t t l e ; ( C ) , c u t t i n g arm; (D)-, c o o l i n g s t a g e ) . A l a r g e r a z o r b l a d e wi:th a w e l l made o f p l a s t i c i n e a t t a c h e d t o i t s upper s u r f a c e was used as the s e c t i o n i n g k n i f e . L i q u i d n i t r o g e n was t r a n s f e r r e d from the Dewar l i q u i d n i t r o - gen r e s e r v o i r (K) t o t h e p l a s t i c i n e w e l l by an aluminum f o i l l a d l e ( E ) . When v i g o r o u s b i o l i n g o f the l i q u i d n i t r o g e n i n the w e l l s t o p p e d , the k n i f e was ready f o r s e c t i o n i n g . S e v e r a l drops o f gum a r a b i c a s u p p o r t media was c o o l e d on the cryotome s t a g e (D) u n t i l i c e began t o form. T i s s u e was then removed from the b r i n e - i c e b a t h w i t h i n s u l a t e d f o r c e p s 139 (G), t he t i p s o f wh i c h had been c o o l e d i n l i q u i d n i t r o g e n . The s p e c i - men was d i p p e d i n t o l i q u i d n i t r o g e n and t h r u s t i n t o t h e s u p p o r t media. The extreme low t e m p e r a t u r e o f t h e specimen f r o z e t h e s u p p o r t media i m m e d i a t e l y . The specimen and s u p p o r t media were m a i n t a i n e d i n t h e f r o z e n s t a t e by expanding CO^ gas. S e c t i o n s c o u l d now be c u t s i n c e both specimen and k n i f e were a t v e r y low t e m p e r a t u r e s . A p p r o x i m a t e l y 20"/um s e c t i o n s were o b t a i n e d . P l a s t i c c o v e r s l i p s (H) were c u t i n t o 6 mm s q u a r e s ( a r r o w s ) . Some o f the c o v e r s l i p p i e c e s were then c o a t e d about t h e edges w i t h a t h i n band o f s i l i c o n e vacuum g r e a s e . A c o l d s i n k was used t o m a i n t a i n t h e co- v e r s l i p s a t low t e m p e r a t u r e and i t c o n s i s t e d o f a 650 gm. b l o c k o f i r o n (J) wh i c h had p r e v i o u s l y been immersed i n 1 i q u i d n i t r o g e n u n t i l b o i l i n g s t o p p e d . The m a n i p u l a t i o n , s t o r a g e , and t r a n s p o r t o f c o v e r - s l i p s and s e c t i o n s were conducted on t h e s u r f a c e o f t h i s s i n k w h i c h c o u l d be used f o r up t o twenty minutes b e f o r e r e - c o o l i n g became nece- s s a r y . S e c t i o n s were t r a n s f e r r e d from t h e k n i f e t o t h e g r e a s e d c o v e r - s l i p and a non-greased c o v e r s l i p p l a c e d on t o p . T h i s formed a s e a l e d w a f e r c o n s i s t i n g o f t h e two c o v e r s l i p s , t h e s e c t i o n s i n between, and the edges s e a l e d w i t h vacuum g r e a s e . B. C r y o m i c r o s c o p y The a p p a r a t u s used t o o b s e r v e t h e f r o z e n s e c t i o n s i s i l l u s t r a t - ed i n f i g u r e 79. A Nikon b i n o c u l a r With a camera attachment (P) was used. The s t a g e c o n t r o l s were removed and r e p l a c e d w i t h a handmade c o o l i n g a p p a r a t u s (M). T h i s a p p a r a t u s w i l l be d e s c r i b e d w i t h r e f e r - ence t o t h e c r o s s - s e c t i o n a l d i a g r a m shown i n f i g u r e 80. The s t a g e was c o v e r e d w i t h aluminum f o i l (not shown) and then c o a t e d w i t h a l a y e r o f p l a s t i c i n e ( s t i p p l e d a r e a s ) . A heavy gauge h X 10 cm s t r i p o f copper w i t h a 2 mm a p e r t u r e d r i l l e d near one end was p l a c e d i n t h e p l a s t i c i n e w i t h t h e a p e r t u r e o v e r t h e o p t i c a l a x i s o f t h e m i c r o s c o p e . The o t h e r end o f the copper sheet was bent upwards i n t o a l i q u i d n i - t r o g e n r e s e r v o i r and c o a t e d w i t h vacuum g r e a s e t o p r e v e n t l e a k i n g . The heavy guage copper sheet a c t e d as t h e c o n d u c t i n g medium t o lower t h e s t a g e t e m p e r a t u r e by c o n d u c t i n g heat from t h e s t a g e t o t h e l i q u i d n i t r o g e n r e s e r v o i r . A 1 cm a r e a on t h e upper s u r f a c e of t h e copper sheet above the a p e r t u r e was not c o v e r e d w i t h p l a s t i c i n e . The appar- a t u s was deemed ready f o r use when a. drop o f 50% e t h y l e n e g l y c o l s o l - u t i o n f r o z e d q u i c k l y when p l a c e d on t h e exposed copper above t h e aper t u r e . Only t h e 20X and t h e 40X o b j e c t i v e s were used and t h e conden- s e r l e n s e was t u r n e d down t o p r e v e n t i t from coming t o o c l o s e t o t h e c o o l i n g system. Freon gas (N) was c o o l e d t o l i q u i d i n a l i q u i d n i t r o g e n b a t h (0) 141 The exposed copper on t h e s t a g e was brushed w i t h l i q u i d f r e o n and t h e specimen w a f e r q u i c k l y d i p p e d i n t o the f r e o n j u s t b e f o r e o b s e r v a t i o n , i n o r d e r t o quench t h e m a t e r i a l and remove w a t e r vapour f r o s t from t h e s y s - tem. The sepcimen w a f e r was m a n i p u l a t e d on t h e s t a g e w i t h c o o l e d i n - s u l a t e d f o r c e p s (G). 142 PLATES AND EXPLANTIONS LEGEND C C u t i c l e CC C h l o r o p l a s t clump CE C h l o r o p l a s t e n v e l o p e Ch C h l o r o p l a s t Cr C r i s t a e CV C y t o p l a s m i c V a c u o l e Cy C y t o p l a s m D Dictyosome E E p i d e r m i s ER E n d o p l a s m i c R e t i c u l u m (used when d i s t i n c t i o n between RER and SER cannot be d e t e r m i n e d ) . En Endodermis ES E x t r a c e l l u l a r Space FC F i b e r C e l l s FH F i b e r o u s Hypodermal C e l l s G Grannum Gd Guard C e l l s GC Ground C y t o p l a s m HC H e t e r o c h r o m a t i n M M e s o p h y l l Mt M i t o c h o n d r i a MV Main V a c u o l e N N u c l e u s NE N u c l e a r E n v e l o p e Nu N u c l e o l u s 0 O i l body OB O s miophi 1ic Body P Polysomes Pd Plasmodesmata Pg P l a s t o g l o b u l i PM Plasma Membrane RD R e s i n Duct RER Rough E n d o p l a s m i c R e t i c u l u m S Stroma Sc S t a r c h C e n t e r SER Smooth E n d o p l a s m i c R e t i c u l u m S t S t a r c h G r a i n T T r a b i c u l u m TM T a n n i n M a t e r i a l To T o n o p l a s t TP T r a n s f u s i o n Parenchyma TT T r a n s f u s i o n T r a c h e i d s TV T a n n i n V a c u o l e VB V a s c u l a r Bundle W C e l l W a l l WL "Water L a y e r " o f the Hypodermis 144 PLATE I Needle C e l l s and T i s s u e s F i g u r e 1 One h a l f o f the t r a n s v e r s e a r e a o f f r e s h mature n e e d l e m a t e r i a l from an a p p r o x i m a t e l y 2Qwm c y r o s e c t i o n s e c t i o n i s shown by d a r k f i e l d i l l u m i n a t i o n . C e l l s and t i s s u e s from o u t e r s u r f a c e t o n e e d l e i n t e r i o r a r e : t h i c k c u t i c l e (C, a r - row), t h e o u t e r dermal l a y e r s ; s i n g l e t i e r o f e p i d e r m a l c e l l s (E) w i t h o u t v i s i b l e c e l l l u m i n a ; a s i n g l e t i e r o f t h i n - w a l l e d hypodermal c e l l s ( t h e " w ater l a y e r " , WL) and a one o r two c e l l l a y e r o f f i b r o u s hypodermal c e l l s (FH). The m e s o p h y l l (M) c o n s i s t s o f c r e n u l a t e d w a l l e d c h l o r e n c h y m a , e.g. c e l l l a b e l l e d ( A ) . Note m e d i a l l y ( t o the l e f t o f the p l a t e ) , t h e r e a r e two t i e r s o f r e c t a n g u l a r c hlorenchyma c e l l s and l a t e r a l l y ( t o the r i g h t ) a l o n g the n e e d l e edges, t h e r e a r e more than two c e l l l a y e r s . C e l l s i n t h i s a r e a a r e us- u a l l y o v o i d i n t r a n s e c t i o n . In t h e l a t e r a l m e s o p h y l l , a r e s i n d u c t (RD), w i t h d u c t lumen b o r d e r e d by a t h i n w a l l e d s e c r e t o r y e p i t h e l i u m (SE) and a f i b e r c e l l l a y e r (FC) i s . f o u n d . Note t h e boxed a r e a i n the upper m e d i a l m e s o p h y l l r e p r e s e n t s the a r e a where most s e c t i o n s s t u d i e d i n t h i s work were t a k e n . The endodermis (En) o f one c e l l l a y e r e n c l o s e s t r a n s f u s i o n t i s s u e o f two c e l l t y p e s : t r a n s f u s i o n t r a c h e i d s (TT) and t r a n s f u s i o n parenchymal c e l l s ( T P ) . One o f the two v a s c u l a r b u n d l e s (VB) i s shown. A 100 Aim s c a l e i s shown: 175x.  145 PLATE I I The Shape and S e c t i o n a l I n t e r p r e t a t i o n s o f a G e n e r a l i z e d P i n e Mesophy11 Cel1 F i g u r e s 2 t o 6 show the t h r e e d i m e n s i o n a l shape o f a g e n e r a l i z e d .. medial c h l o r e n c h y m a l c e l l and the i n t e r p r e t a t i o n o f v a r i o u s s e c t i o n i n g p l a n e s t h r o u g h i t . F i g u r e 2 Diagram o f the s u r f a c e o f a g e n e r a l i z e d c h l o r e n c h y m a l c e l l . Three c e l l f a c e s a r e shown w i t h r e s p e c t t o the nee- d l e : t r a n s v e r s e f a c e ( A ) , r a d i a l f a c e (B) and one o f t h e ends o f t h e c e l l ( t a n g e n t i a l f a c e , C ) . Dark s u r f a c e grooves a r e t r a b i c u l a r w a l l i n v a g i n a t i o n s . F i g u r e s 3 t o 6 a l l have the same key t o the c e l l u l a r components shown. N u c l e u s , ( c i r c l e and N ) , l i g h t s t i p p l i n g ( c y t o p l a s m ) , d a r k s t i p - p l i n g ( t a n n i n m a t e r i a l o f the t a n n i n v a c u o l e ) , d a r k l i n e s ( c e l l w a l l s and the t r a b i c u l a r i n v a g i n a t i o n s ) . A l l s e c t i o n p r o f i l e s a r e o f s e c t i o n s w h i c h c o u l d be c u t from f i g u r e 2. F i g u r e 3 The g e n e r a l i z e d appearance o f a t r a n s v e r s e n e e d l e s e c t - i o n , i n a p l a n e p a r a l l e l t o f a c e A f i g u r e 2. F i g u r e 4 / • A t a n g e n t i a l n e e d l e s e c t i o n near the end o f the c e l l ( f a c e C ) . Note t h a t t he end w a l l t r a b i c u l a e cause t h e sec- t i o n t o appear as t h r e e , e e l 1-wal1-bounded compartments. F i g u r e 5 A t a n g e n t i a l n e e d l e s e c t i o n c u t t i n g t he m i d - r e g i o n o f the c e l l below the r e g i o n o f t r a b i c u l a r p e n e t r a t i o n . F i g u r e 6 A r a d i a l n e e d l e s e c t i o n o f t h e narrow s i d e o f t h e c e l l i n a p l a n e p a r a l l e l t o f a c e B arid c l o s e enough t o c u t t h e t r a b i c u l u m . , Note t h e t r a b i c u l u m w h i c h i n v a g i n a t e s i n from f a c e B d i v i d e s t h i s s e c t i o n i n t o two compartments.  146 PLATE I I I S c a n n i n g E l e c t r o n M i c r o s c o p y o f Mesophyl1 Chlorenchyma F i g u r e 7 A t r a n s v e r s e v i e w o f t h e l a t e r a l m e s o p h y l l . (FH) = f i b - rous hypodermal c e l l s . Note the chlorenchyma e e l 1s a r e o v o i d i n o u t l i n e , t r a b i e u l a e o f u n i f o r m l e n g t h , i n v a g i n a t e about each c e l l . C e l l w a l l d e b r i s from a c e l l i n t h e s h e e t o f c e l l s t h a t was above the c e l l s shown can be seen s t i l l a t t a c h e d ( s o l - i d a r r o w ) . Open arrow shows where the c e l l w a l l has broken o f f the e e l 1 s u r f a c e . Approx. 600x. F i g u r e 8 Two c e l l s (A l ! and A) a r e viewed o b l i q u e l y i n the t r a n s - v e r s e p l a n e o f the n e e d l e . These c e l l s a r e more a n g u l a r i n o u t l i n e b e i n g from t h e more medial m e s o p h y l l a r e a s . The c e l l f a c e s l a b e l l e d A and A1 '* c o r r e s p o n d t o f a c e A i n f i g u r e 2. S i m i l a r l y , f a c e C c o r r e s p o n d s t o f a c e C i n f i g u r e 2. Note t h e l o n g t r a b i c u l u m (arrow) and t h e h o l l o w t o the l e f t where the c y t o p l a s m Wh ich has-been d i s l o d g e d . Arrowhead i n d i c a t e s c y t o p l a s m i c a r e a s where t h e c e l l w a l l has broken o f f . Approx. 850x. F i g u r e 9 H i g h e r m a g n i f i c a t i o n o f two a d j a c e n t c e l l s . The a r r o w - head shows t h e i n v a g i n a t i o n o f a s h o r t t r a b i c u l a e . Note how the t r a b i e u l a r f o l d s c o m p l e t e l y back on i t s e l f l e a v i n g what appears t o be a p i t i n t o c e l l i n t e r i o r . Arrows p o i n t t o t r a - b i c u l a e whose w a l l s have not f o l d e d back on t h e m s e l v e s com- p l e t e l y but have formed "U" shaped c l e a v a g e s i n the c e l l w a l l s . Note near t h e t o p a r r o w , a p a t c h o f c e l l w a l l has broken o f f e x p o s i n g the c y t o p l a s m . Approx. 1,000x.  147 PLATE IV L i g h t M i c r o s c o p y - Summer Chlorenchyma F i g u r e 10 The s e c t i o n was p r e p a r e d and o b s e r v e d by t h e p r o c e d u r e i n a p p e n d i x I I . An e n t i r e c h l o r e n c h y m a l c e l l i s v i s i b l e , a "U- shaped" e x t r a c e l l u l a r space e x t e n d s i n t o the c e l l . The guard c e l l s (Gd) o f a stomata can be seen. A n u c l e u s (N) and c h l o r o - p l a s t s t i g h t l y a p p r e s s e d t o the c e l l w a l l (arrow) can be seen. A 10 Aim s c a l e i s shown: 1,400x. F i g u r e 11 F o r m a l i n f i x e d m a t e r i a l . The c e l l w a l l s o f two c e l l s (W), a t r a b i c u l u m (T) and n u c l e u s (N) a r e v i s i b l e . I n d i v i d u a l c h l o - r o p l a s t s (arrowheads) u n i f o r m l y l i n e t h e w a l l and t h e t r a b i c u l u m . A 10 Aim s c a l e i s shown: 1 ,700x. F i g u r e 12 T i s s u e s t a i n e d i n the f o r m a l i n f i x a t i v e and t a n n i n s p e c i - f i c s t a i n s o l u t i o n g i v e n i n a p p e n d i x I. Two t y p e s o f t r a b i c u - l a e a r e shown: t h e l o n g end w a l l t r a b i c u l u m (T) and t h e s h o r t e r l a t e r a l w a l l t r a b i c u l a e ( T 1 ) . The c y t o p l a s m can be seen as a t h i n c l e a r p a r i e t a l band (between a r r o w h e a d s ) . Note t h e h e a v i l y s t a i n e d t a n n i n v a c u o l e (TV) and a n u c l e u s (N) a t t h e t i p o f t h e l o n g t r a b i c u l u m . A 25 Aim s c a l e i s shown: 850x. F i g u r e 13 Two l a r g e c h l o r e n c h y m a l and s e v e r a l endodermal c e l l s (En) a r e shown i n u n f i x e d m a t e r i a l s t a i n e d w i t h Sudan B l a c k B. Small s u d a n o p h i l i c b o d i e s a r e found i n the c y t o p l a s m (open a r - rows) and l a r g e r b o d i e s a r e found i n the endodermis (En, a r r o w ) . A 25 Aim s c a l e i s shown: 750x. F i g u r e 14 High m a g n i f i c a t i o n o f the ty p e o f m a t e r i a l i n f i g u r e 13- Note the s u d a n o p h i l i c b o d i e s ( 0 ) - a r e c l o s e t o the c e l l w a l l . Some dar k s t a i n i n g m a t e r i a l i s near the r e s o l u t i o n o f the m i - c r o s c o p e (White.arrows) . A 10 /jm s c a l e i s shown: 2,000x. 147-a 148 PLATE V The Summer _̂ W i n t e r L i g h t M i c r o s c o p y C y t o l o g y o f Tusga m e r t e n s i a n a Summer f i x a t i o n s . F i g u r e 15 T r a n s v e r s e s e c t i o n o f mountain hemlock n e e d l e . Note t a n - n i n ( T V ) , c i r c u l a r n u c l e i (N) and c h l o r o p l a s t s l i n i n g t h e c e l l w a l l ( a r r o w h e a d s ) . P a r t o f the endodermis (En) i s a l s o v i s i - b l e . 700x. F i g u r e 16 A p o r t i o n o f a m e s o p h y l l c e l l . I n d i v i d u a l c h l o r o p l a s t s (Ch) a r e c l e a r l y seen w i t h o v a t e p r o f i l e s , a c y t o p l a s m i c v a - c o u l e i s shown (CV). S t a r c h g r a i n s ( S t ) a r e p r e s e n t i n the c h l o r o p l a s t s and what may be o s m i o p h i l i c b o d i e s (arrowheads) a r e seen a l o n g the c e l l w a l l . TV = t a n n i n v a c u o l e . 1,600x. W i n t e r f i x a t i o n s . F i g u r e 17 C h l o r o p l a s t s clumps (CC) a r e v i s i b l e i n a l l c e l l s shown. Note the p o s i t i o n o f t h e o v a t e n u c l e u s (N) i n t h e c y t o p l a s m w i t h t h e two prominent n u c l e o l i ( a r r o w s ) . C y t o p l a s m i c v a c - u o l e s (CV) and o s m i o p h i l i c b o d i e s can be o b s e r v e d ( a r r o w h e a d s ) . Open arrow p o i n t s t o a p r i m a r y p i t f i e l d between two c e l l s . TV = t a n n i n v a c u o l e . 1,600x. F i g u r e 18 C h l o r o p l a s t clumps (CC) i n two a d j a c e n t c e l l s , n o t e t h e v a c u o l a t e d c y t o p l a s m between the clumps, the t a n n i n v a c u o l e (TV) and.the c e l l w a l l . The v a c u o l e s a l o n g t h e c e l l w a l l , i n p a r t i c u l a r , c o n t a i n numerous o s m i o p h i l i c b o d i e s ( a r r o w h e a d s ) . 1,500x.  H 9 PLATE VI E l e c t r o n M i c r o s c o p y o f Summer Chlorenchyma. F i g u r e s 19 t o 32 a r e e l e c t r o n photomicrographs o f summer t i s s u e f i x e d a t dawn and f i x e d i n t h e s t a n d a r d f i x a t i v e s o l u t i o n . F i g u r e 19 P a r t s o f f o u r c e l l s s e c t i o n e d i n the r e g i o n i l l u s t r a t e d i n f i g u r e 5 . T h i s i s a t h r e e month o l d n e e d l e (August f i x a t - i o n ) . The c e l l s a r e dominated by the homogeneous o s m i o p h i l i c t a n n i n v a c u o l e s (TV). In the p a r i e t a l c y t o p l a s m i s fo u n d : c h l o r o p l a s t s (Ch) w i t h s t a r c h g r a i n s ( S t ) , p o l y m o r p h i c m i t o - c h o n d r i a ( M t ) , homogeneous and l a r g e l y n o n - v a c u o l a t e d ground c y t o p l a s m , a few c y t o p l a s m i c v a c u o l e s (CV) a s s o c i a t e d w i t h t h e o s m i o p h i l i c b o d i e s (OB), and s m a l l ( u s u a l l y 2 m i c r o n s o r l e s s ) o i 1 b o d i e s (0) . A 5 >um s c a l e i s shown: 5 , 0 0 0 x . F i g u r e 20 A 2 mm n e e d l e . T a n n i n has o n l y j u s t begun t o be formed and none i s v i s i b l e i n t h e main v a c u o l e (MV). The c h l o r o p l a s t has f u l l y d e v e l o p e d w i t h s m a l l grana (G, w h i t e a r r o w ) . P l a s t o - g l o b u l i ( d a r k a r r o w s ) a r e v e r y s m a l l and few i n number. The c e l l w a l l (W) and p a r t o f the e x t r a c e l l u l a r space (ES) i s shown. A 1 Aim s c a l e i s shown: 17 ,000x . F i g u r e 21 A c h l o r o p l a s t and m i t o c h o n d r i a (Mt) from a two y e a r o l d n e e d l e . The membranes shown a r e d i s t i n c t . The plasma mem- brane (PM), d o u b l e membraned c h l o r o p l a s t e n v e l o p e (CE, a r r - ow) and m i t o c h o n d r i a l e n v e l o p e membranes (open arrowhead) can be seen. Grana c o n t a i n few t h y l a k o i d s but p a r t i t i o n r e g i o n s a r e e x t e n s i v e (open a r r o w ) . P l a s t o g l o b u l i (Pg) a r e l a r g e , num- e r o u s , and u s u a l l y a s s o c i a t e d w i t h t he t h y l a k o i d membranes. The ground c y t o p l a s m ,'(GC) i s r e l a t i v e l y empty o f i d e n t i f i a b l e s t r u - c t u r e s , e x c e p t f o r f i n e g r a n u l a r and",f i b r i l a r m a t e r i a l . A s t a r c h g r a i n ( S t ) i s shown w i t h i n t h e c h l o r o p l a s t . A 1 Aim s c a l e i s shown: 2 9 , 0 0 0 x .  150 PLATE VI I E l e c t r o n M i c r o s c o p y o f Summer Chlorenchyma: C o n t i n u e d F i g u r e 22 The o r g a n e l l e r i c h c y t o p l a s m near the n u e l c u s (N) i s - shown i n t h i s s e c t i o n from a two month o l d n e e d l e . A c h l o r - o p l a s t (Ch) c o n t a i n i n g a s t a r c h g r a i n ( S t ) and w i t h some o f i t s t h y l a k o i d s s l i g h t l y s w o l l e n i s shown a l o n g the c e l l w a l l (W), the plasma membrane. (PM), s e v e r a l c y t o p l a s m i c v a c u o l e s (CV) c o n t a i n i n g o s m i o p h i l i c b o d i e s (OB) arid membranous and f i b r i l l a r m a t e r i a l , rough e n d o p l a s m i c (RER), a d i c t y o s o m e s (P, arrowheads) a r e shown. A 0.5 Aim s c a l e i s shown: 38,OOOx. F i g u r e 23 Two o s m i o p h i l i c b o d i e s (OB) a r e shown i n t h r e e month o l d n e e d l e t i s s u e . The upper i s e n c l o s e d w i t h i n a c y t o p l a s m i c v a c u o l e (CV) w h i c h a l s o c o n t a i n s some membranous m a t e r i a l ( s o l i d a r r o w h e a d ) . The lower o s m i o p h i l i c body appears not t o be membrane bound. Note t h e t o n o p l a s t (To^ open a r r o w s ) ap- pear s s e p a r a t e d from t h e t a n n i n m a t e r i a l o f the t a n n i n v a c - u o l e (TV). F i b r i l l a r m a t e r i a l appears t o s t r e t c h between the t a n n i n and the t o n o p l a s t (open a r r o w s ) . W = c e l l w a l l . A 0.5 Aim s c a l e i s shown: 40,000x. F i g u r e 2k An o s m i o p h i l i c body (OB) from f o u r t e e n month o l d n e e d l e t i s s u e i s shown a s s o c i a t e d w i t h s e v e r a l c o n c e n t r i c membranes ( a r r o w h e a d s ) . A l s o shown a r e t h e t a n n i n v a c u o l e ( T V ) , and the c e l l w a l l (W). A 0.5/Jm s c a l e i s shown: 33,OOOx. F i g u r e 25 A s m a l l a r e a o f c y t o p l a s m i s shown from t h r e e month o l d n e e d l e t i s s u e . C y t o p l a s m i c v a c u o l e s (CV) a r e shown c o n t a i n - i n g o s m i o p h i l i c m a t e r i a l ( s o l i d a r r o h e a d ) . Two d i c t y o s o m e s (D) a r e shown s u r r o u n d e d by numerous s m a l l v e s i i d l e s (open ar- rowhead) i TV = t a n n i n v a c u o l e . A 0.5 jum s c a l e i s shown: 35,OOOx.  151 PLATE V I I I E l e c t r o n M i c r o s c o p y o f Summer Chlorenchyma: c o n t i n u e d F i g u r e 26 A s m a l l a r e a o f c y t o p l a s m i s shown from two month o l d nee- d l e t i s s u e . S e v e r a l d i c t y o s o m e s (D) a r e v i s i b l e and surrounded by v e s i c l e s . From what may be p a r t o f a d i c t y o s o m e ( i n t h e up- per c e n t e r ) , a s e r i e s o f c y t o p l a s m i c v a c u o l e s ( s o l i d a r r o w s ) can be seen. The l a r g e s t i s c l o s e s t t o and appears t o have f u s - ed w i t h t h e t o n o p l a s t (open a r r o w h e a d ) . Note t h e f i b r i l s ( a r - rows) w h i c h appear t o connect t h e t a n n i n m a t e r i a l w i t h t h e t o n - o p l a s t ( T o ) . TV = t a n n i n v a c u o l e . A 0 .5 Aim s c a l e i s shown: 28,000x. F i g u r e 27 An a r e a o f c y t o p l a s m from a young n e e d l e (one month o l d ) i s shown. A d i c t y o s o m e (D) w i t h a s e r i e s o f c y t o p l a s m i c v e s i - c l e s between i t and the c e l l w a l l (W) i s shown. An unusual s e c t i o n o f a m i t o c h o n d r i o n (Mt) and p a r t o f the t a n n i n v a c u o l e (TV) i s a l s o shown. A 0 .5 Aim s c a l e i s shown: 35,OOOx. F i g u r e 28 T h i s s e c t i o n i s p a r a l l e l t o , and j u s t under t h e c e l l w a l l i n a young (two month o l d ) n e e d l e showing a m u l t i l o b e d m i t o - c h o n d r i a ( M t ) . Note, t h a t the m a t r i x i s f i l l e d w i t h e x t e n s i v e t u b u l a r c r i s t a e ( C r , a r r o w h e a d s ) . P a r t o f the s u r f a c e o f a c h l o r o p l a s t (Ch) i s a l s o shown. A 0.5 jum s c a l e i s shown: 34,OOOx. F i g u r e 29 An o i l body (0) i s shown w i t h t h e m i t o c h o n d r i a (Mt) n e a r - by i n one y e a r o l d n e e d l e t i s s u e . Two o s m i o p h i l i c b o d i e s (OB) a r e a l s o shown a l o n g w i t h p a r t o f a c h l o r o p l a s t (Ch) and the c e l l w a l l (W). A 0.5 Aim s c a l e i s shown: 38,000x... 151-a 152 PLATE IX E l e c t r o n M i c r o s c o p y o f Summer Chlorenchyma: C o n t i n u e d F i g u r e 30 A n u c l e o l u s i s shown from a two month o l d n e e d l e c o n t a i n - in g l a r g e l y p a r s g ranulosum . I t i s s e p a r a t e d from t h e a d j a - c e n t h e t e r o c h r o m a t i n by a r i n g o f c l e a r n u c l e o p l a s m c o n t a i n i n g o n l y f i n e f i b r i l l a r m a t e r i a l . A 0.5 >um s c a l e i s shown: 30,000x. F i g u r e 31 A r e g i o n between two c e l l s i s shown w i t h the n u c l e u s (N) o f one c e l l near the c e l l w a l l o f a t h r e e month o l d n e e d l e . T h i s i s a 1 Aim p l a s t i c s e c t i o n s t a i n e d w i t h osmium, t o l u i d i n e b l u e and S a f f r a n i n and viewed w i t h phase c o n t r a s t o p t i c s . Three n u c l e o l i (Nu, arrowheads) a r e v i s i b l e , each w i t h a c l e a r r i n g o f n u c l e o p l a s m about them. The dense and i r r e g u l a r h e t e r - o c h r o m a t i n i s v i s i b l e (He). S t a r c h g r a i n s ( S t ) a r e seen i n c h l o r o p l a s t s (Ch) spaced e v e n l y a l o n g the c e l l w a l l . The t a n - n i n v a c u o l e s (TV), e x t r a c e l l u l a r space (ES) , and a t r a b i c u l u m (T) a r e a l s o shown. A 5 jum s c a l e i s shown: 3,100x. F i g u r e 32 Most o f a s e c t i o n o f a n u c l e u s from a t h r e e month o l d nee- d l e i s shown. A c l e a r l y d e f i n e d n u c l e a r e n v e l o p e (NE) c o n s i s t - ing o f two d i s t i n c t r ' . membranes i n w h i c h few n u c l e a r p o re p r o - f i l e s can be seen ( a r r o w s ) . The n u c l e o p l a s m c o n t a i n s two nu- c l e o l i (Nu) and i r r e g u l a r dense a g g r e g a t e s o f ' g r a n u l a r h e t e r o - c h r o m a t i n (He, a r r o w h e a d s ) . In t h e c y t o p l a s m near the n u c l e u s rough e n d o p l a s m i c r e t i e u l u r n (RER), an o i l body ( 0 ) , c h l o r o p l a s t s ( C h ) , and t h e t a n n i n v a c u o l e (TV) a r e shown. A 1 Aim s c a l e i s shown: 12,000x. 152 -a 153 PLATE X L i g h t M i c r o s c o p y o f W i n t e r T i s s u e F i g u r e 33 An u n s t a i n e d , n o n - f i x e d , f r o z e n s e c t i o n o f s e v e r a l c e l l s i s shown p r e p a r e d by t h e p r o c e d u r e o u t l i n e d i n a p p e n d i x I I . Note t h e wide band (between arrowheads) o f g r a n u l a r c y t o p l a s m a l o n g the c e l l w a l l . In most a r e a s t h e c y t o p l a s m i s as wide as the d i s t a n c e t he t r a b i c u l a e (T) p e n e t r a t e i n t o t h e c e l l . The o n l y o t h e r r e c o g n i z a b l e f e a t u r e s a r e t h e t a n n i n v a c u o l e s (TV) and a n u c l e u s (N) i n one c e l l . N o t h i n g r e s e m b l i n g c h l o r o p l a s t s can be seen. M a t e r i a l from e i g h t month o l d n e e d l e . A 25 Aim s c a l e i s shown: 800x. S e c t i o n about 20 jum t h i c k . F i g u r e 34 A c r y o s e c t i o n o f c e l l s s t a i n e d and f i x e d i n t h e s p e c i f i c f o r m a l i n f i x a t i v e f o r t a n n i n g i v e n i n a p p e n d i x I. Note t h a t t h e r e i s an u n i f o r m and d r a m a t i c p o s i t i v e t a n n i n s t a i n i n g f o r the t a n n i n v a c u o l e s ( T V ) , but none i n t h e c y t o p l a s m , endoder- mis (En) o r t r a n s f u s i o n t r a c h e i d (TT). The s e v e r a l n u c l e i (N) v i s i b l e a r e l o c a t e d a l o n g t h e c e l l w a l l . ' No s u b s t r u c t u r e can be seen w i t h i n t h e c y t o p l a s m e x c e p t f o r numerous l a r g e , h i g h l y r e f r a c t i l e s p h e r e s ( a r r o w h e a d s ) . These s t a i n p o s i t i v e l y w i t h Sudan B l a c k B. A 50 Aim s c a l e i s shown: 380x. F i g u r e 35 A 1 pm p l a s t i c s e c t i o n o f two c e l l s w i t h osmium, t o l u d i n e b l u e and S a f r a n i n s t a i n i n g i s shown. The i r r e g u l a r n a t u r e o f the t a n n i n v a c u o l e (TV) i s an a r t i f a c t due t o t e a r i n g as a r e - s u l t o f s e c t i o n i n g . Note t h e e x t e n s i v e r e g i o n s o f v a c u o l a t e d c y t o p l a s m (CV). Some o s m i o p h i l i c b o d i e s (arrowheads) a r e l a r g e enough t o be seen amongst the c y t o p l a s m i c v a c u o l e s . Note th e l a r g e number o f c h l o r o p l a s t s w h i c h a r e found i n t h e c h l o - r o p l a s t clumps (CC). Note t h a t t h e d a r k t h y l a k o i d system o f each c h l o r o p l a s t i s c o n t r a s t e d a g a i n s t a s u r r o u n d i n g l i g h t l y s t a i n e d r e g i o n o f stroma. FH = p a r t o f a f i b r o u s hypodermal c e l l . A 10 pm s c a l e i s shown: 1",700x. F i g u r e 36 One c e l l and p a r t s o f s e v e r a l o t h e r s a r e shown i n t h i s f i g - u r e o f an u n f i x e d and a p p r o x i m a t e l y 20 >um c r y o s e c t i o n e d mater- i a l s t a i n e d w i t h Sudan B l a c k B. Note t h e s e v e r a l v e r y l a r g e o i l d r o p l e t s (0) found w i t h i n t h e c e l l . T = long t r a b i c u l a e o f the e e l 1. A 10/jm s c a l e i s shown: 1,400x. 153-a 154 PLATE XI E l e c t r o n M i c r o s c o p y o f W i n t e r Chlorenchyma F i g u r e 37 A n e e d l e t a n g e n t i a l s e c t i o n showing s e v e r a l c e l l s i n the p l a n e o f f i g u r e 5- Note how t h e t a n n i n v a c u o l e s (TV) a r e rounded and do n o t f o l l o w t h e c o n t o u r s o f t h e c e l l w a l l (W) w i t h t h e r e s u l t a n t i r r e g u l a r s e c t i o n a l w i d t h o f the c y t o p l a s m . Small c h l o r o p l a s t clumps (CC) a r e found a l o n g the w a l l s c o r - r e s p o n d i n g t o f a c e B ( f i g u r e 2) w h i l e few o r g a n e l l e s a r e found a l o n g t h e w a l l s c o r r e s p o n d i n g t o f a c e A ( f i g u r e 2 ) , a s t e r i s k . C i r c u l a r m i t o c h o n d r i a a r e found about t h e c h l o r o p l a s t clumps (Mt, a r r o w h e a d s ) . The open a r r o w p o i n t s t o b l e b s o f t a n n i n a l o n g t h e edge o f t h e t a n n i n v a c u o l e . OB = o s m i o p h i l i c b o d i e s , ES = e x t r a c e l l u l a r s p a c e , n = n u c l e u s , seen i n t h e lower l e f t hand c o r n e r . N e e d l e , t w e n t y months o l d . A 5 >um s c a l e i s shown: 4,400x.  155 PLATE XI I E l e c t r o n M i c r o s c o p y o f W i n t e r Chlorenchyma: C o n t i n u e d F i g u r e 38 C e l l s e c t i o n i n t h e p l a n e o f f i g u r e 6 showing n e a r l y an e n t i r e c e l l from a n i n e month o l d n e e d l e . Note t h a t t h e l a r - g e s t c h l o r o p l a s t clumps a r e i n t h e ends o f the c e l l ( l e f t and r i g h t p a r t s o f t h e f i g u r e ) . C y t o p l a s m i c v a c u o l e s tend t o be o r i e n t a t e d normal t o the t a n n i n v a c u o l e (TV) s u r f a c e ( d o u b l e ended a r r o w s ) . The c y t o p l a s m i c v a c u o l e s a r e a l s o a s s o c i a t e d w i t h t h e o s m i o p h i l i c b o d i e s . An o b l i q u e s e c t i o n o f a s h o r t t r a b i c u l u m (T) i s a l s o shown. A 5 yum s c a l e i s shown: k,k00x. F i g u r e 39 C e l l s e c t i o n i n a p l a n e s i m i l a r t o f i g u r e 3 showing p a r t o f a e i g h t month o l d n e e d l e . The c h l o r o p l a s t clumps (CC) a r e l o c a t e d i n the ends o f the c e l l ( t o the l e f t o f the f i g u r e ) and a l o n g t h e w a l l c o r r e s p o n d i n g t o f a c e B, f i g u r e 2 (clumps i n t h e c e n t e r and t o t h e r i g h t , near the s h o r t t r a b i c u l a e ( T ) ) . A l a r g e o i l body i s seen ( 0 ) . Note how c l o s e t h e t a n n i n va- c u o l e (TV) comes t o the ends o f the t r a b i c u l a e . The edge o f t h e t a n n i n v a c u o l e has fragmented i n t o some s m a l l e r p i e c e s o f t a n n i n m a t e r i a l ( a r r o w h e a d ) . OB = o s m i o p h i l i c b o d i e s . ES = e x t r a c e l l u l a r space f i l l e d w i t h what appears t o be c e l l u l a r d e b r i s. A 5 /Jm s c a l e i s shown: 4 6 , 0 0 0 x . ,  156 PLATE X I I I E l e c t r o n M i c r o s c o p y o f W i n t e r Chlorenchyma: C o n t i n u e d F i g u r e 40 A t a n g e n t i a l s e c t i o n o f an e i g h t month o l d n e e d l e (appro- x i m a t e l y t h e p l a n e o f f i g u r e 4). Note the l a r g e c h l o r o p l a s t clumps (CC) c o n s i s t e n t l y f o u n d near t h e end e e l 1 wa11s of' t h e s e c e l l s . T h i s s e c t i o n has j u s t c u t i n t o the s u r f a c e o f a t a n n i n v a c u o l e (TV). Note the i r r e g u l a r s u r f a c e and what a p p e a r s t o be t a n n i n blebs* ( s o l i d arrowheads) i n t h e i m m e d i a t e l y a d j a c e n t c y t o p l a s m . Plasmodesmata (Pd) a r e seen between a d j a c e n t c h l o - renchymal c e l l s . Note t h a t t h e plasma membrane i s c o n s i s t e n t - l y a p p r e s s e d t o t h e c e l l w a l l (open a r r o w h e a d ) . A 5 /Jm s c a l e i s shown: 5,000x. F i g u r e 41! One o f the r e g i o n s o f e x t e n s i v e l y v a c u o l a t e d c y t o p l a s m des- c r i b e d as "foamy" o r ' s p o n g y " i s shown by t h e a s t e r i s k , s e p a r a t - i n g two c h l o r o p l a s t clumps (CC) i n an n i n e month o l d n e e d l e . Note the v e r y s m a l l amounts o f ground c y t o p l a s m (arrowheads) found i n the r e g i o n s o f the most e x t e n s i v e v a c u o l a t i o n . A 2.5 Aim s c a l e i s shown: 75,000x. F i g u r e 42 P a r t o f a c h l o r o p l a s t clump o f an e i g h t month o l d n e e d l e i s shown. Note how t h e c h l o r o p l a s t s a r e i n t e r l o c k i n g w i t h each o t h e r w i t h no open spaces between a d j a c e n t p l a s t i d s . The j u n c t - i o n a l r e g i o n s between t h e c h l o r o p l a s t s appear t o c o n s i s t o f se- v e r a l p a r a l l e l membranes ( s o l i d a r r o w h e a d s ) . Between th e c h l o - r o p l a s t s and t h e m o t o c h o n d r i a , t h e r e i s a u n i f o r m gap o f c y t o - plasm (open a r r o w h e a d s ) . Most o f t h e i r r e g u l a r shaped p o r t i o n s o f t h e c h l o r o p l a s t s c o n s i s t o f s t r o m a l r e g i o n s (S) c o n t a i n i n g no t h y l a k o i d s . Note two t h y l a k o i d systems (each l a b e l l e d w i t h an a s t e r i s k ) l i e a t r i g h t a n g l e s t o each o t h e r w i t h i n one c h l o - r o p l a s t e n v e l o p e . A 1 jam s c a l e i s shown: 13,000x. 156-a 157 PLATE XIV E l e c t r o n M i c r o s c o p y o f W i n t e r Chlorenchyma: C o n t i n u e d F i g u r e 43 Two c h l o r o p l a s t s from an e i g h t month o l d n e e d l e a r e shown. The o b i i q u e s e c t i o n o f the c h l o r o p l a s t e n v e l o p e (CE, s o l i d a r r - owhead) appears as a u n i f o r m g r a n u l a r a r e a . C r o s s s e c t i o n s o f the e n v e l o p e a t low m a g n i f i c a t i o n appear as s i n g l e l i n e s (open a r r o w s ) . Note the two i n t e r n a l t h y l a k o i d systems ( a s t e r i s k s ) l i e w i t h i n one c h l o r o p l a s t e n v e l o p e , s e p a r a t e d by a l a r g e a r e a o f stroma ( S ) . A 2 jum s c a l e i s shown: 15,000x. F i g u r e 44 A clump o f c h l o r o p l a s t s from a f i f t y - j f i v e month o l d n e e d l e i s shown. The c h l o r o p l a s t s have l i t t l e e x c l u s i v e l y s t r o m a l r e - g i o n s , l o n g g r a n a l a r e a s o f no more than two o r t h r e e t h y l a k o i d s and numerous l a r g e r p i a s t o g l o b u l i (Pg, s o l i d arrowheads) a r e shown. The c h l o r o p l a s t s o c c a s i o n a l l y have c o n t r i c t i o n s o r n a r - row i s t h m u s e s between them (open a r r o w ) . A l s o shown i s the ground c y t o p l a s m (GC) c o n t a i n i n g l i t t l e e x c e p t s c a t t e r e d f i b u - l a r m a t e r i a l and a v e r y l a r g e o i l body ( 0 ) . W = c e l l w a l l . A 1 jum s c a l e i s shown: 10,000x. F i g u r e 45 P a r t o f t h e a r e a s o f c o n t a c t between t h r e e c h l o r o p l a s t s i n an e i g h t month o l d n e e d l e . The c h l o r o p l a s t s e n v e l o p e (CE, a r r o w - head) i s v e r y d i f f i c u l t t o r e s o l v e i n t o i t s two membrane compo- n e n t s , and appears as a s e r i e s o f v e s i c l e s a p p r o x i m a t e l y 25 nm t o 40 nm i n d i a m e t e r . In t h e r e g i o n s o f c h l o r o p l a s t c o n t a c t , p a r t s o f t h r e e membranes can sometimes be seen ( w h i t e a r r o w s ) . A 2 jum s c a l e i s shown: 27,000x. F i g u r e 46 An o p t i c a l m a g n i f i c a t i o n o f t h e boxed r e g i o n o f f i g u r e 45. The t r i p a r t i t e n a t u r e o f t h e membranes i n the r e g i o n between a d j a c e n t c h l o r o p . l a s t s i s v i s i b l e (between a r r o w h e a d s ) . Two t h i n outer^membranes.'and a t h i c k e r c e n t r a l membrane can be seen. A 0.5 >um s c a l e i s shown: 54,000x. F i g u r e 47 A c h l o r o p l a s t and m i t o c h o n d r i o n (Mt) o f an e i g h t month o l d n e e d l e a r e shown. Note the i n d i s t i n c t c h l o r o p l a s t e n v e l o p e (CE, a r r o w h e a d ) . In c o n t r a s t , t h e t h y l a k o i d membranes i n t h e u n u s u a l l y l a r g e g r a n n a l a r e a (G) a r e v e r y d i s t i n c t . P l a s t o g l o b u l i (Pg, arrow) a r e e v e n l y d i s t r i b u t e d t h r o u g h o u t the p l a s t i d , even i n the l a r g e s t r o m a l (S) a r e a s . The c l e a r r e g i o n ( s c ) may be t h e s t a r c h f o r m a t i o n c e n t e r . Mt = m i t o c h o n d r i a . A 1 Aim s c a l e i s shown: 27,000x. 157-a 158 PLATE XV E l e c t r o n M i c r o s c o p y o f W i n t e r c h l o r e n c h y m a : c o n t i n u e d F i g u r e 48 The appearance o f p a r t s o f two c e l l s f rom t e n month o l d (March) n e e d l e t i s s u e . The s i n g l e a s t e r i s k (upper r i g h t ) i n - d i c a t e s a r e g i o n o f e x t e n s i v e c y t o p l a s m i c v a c u o l a t i o n a s s o - c i a t e d w i t h the o s m i o p h i l i c b o d i e s (OB) . In the d o u b l e a s t - e r i sk a r e a ( c e n t e r r i g h t ) , l i t t l e o s m i o p h i l i c m a t e r i a l i s seen w i t h t h e c y t o p l a s m i c v a c u o l e s . M i t o c h o n d r i a (Mt, arrowheads) a r e found i n the s i n g l e a s t e r i s k r e g i o n but not i n t h e d o u b l e a s t e r i s k r e g i o n . M i t o c h o n d r i a a r e a l s o l o c a l i z e d i n the non- v a c u o l a t e d c y t o p l a s m i c r e g i o n s about the n u c l e u s (N) and t h e c h l o r o p l a s t clumps (CC). The homogeneity i n t e x t u r e and s t a i n - i n g o f the t a n n i n v a c u o l e s (TV) can be seen from t h e l a r g e a r e a shown. T r a b i c u l a e ( T ) , c e l l w a l l s (W), and e x t r a c e l l u l a r s pace (ES) a r e a l s o shown. A 5 Aim s c a l e i s shown: 6,000x.  159 PLATE XVI E l e c t r o n M i c r o s c o p y o f W i n t e r Chlorenchyma: C o n t i n u e d F i g u r e hS A c y t o p l a s m i c v a c u o l e (CV) i s shown, l i n e d w i t h the mat- e r i a l (arrowheads) from t h e o s m i o p h i l i c b o d i e s , w h i c h i n t u r n l i n e t h e c y t o p l a s m a d j a c e n t t o the c e l l w a l l (W) i n an e i g h t month o l d n e e d l e . P a r t o f a c h a r a c t e r i s t i c w i n t e r n u c l e u s (N) and c h l o r o p l a s t s a r e a l s o shown. A 1 Aim s c a l e i s shown: 10,500x. F i g u r e 50 A s e r i e s o f c y t o p l a s m i c v a c u o l e s s t a r t i n g from the c e l l w a l l (W) and e x t e n d i n g t o the t a n n i n v a c u o l e (TV) and s e p a r a - t i n g two c h l o r o p l a s t s clumps (CC) i n an e i g h t month o l d n e e d l e a r e shown. Note the v a c u o l e s a r e a s s o c i a t e d w i t h an o s m i o p h i - l i c body (OB, a r r o w h e a d ) . There i s dense ground c y t o p l a s m (GC) im m e d i a t e l y about the c h l o r o p l a s t s . A 1 Aim s c a l e i s shown: 12,000x. F i g u r e 51 A s e c t i o n o f a s i x month o l d n e e d l e a t r i g h t a n g l e s t o the c e l l w a l l . Note the o s m i o p h i l i c b o d i e s (OB, arrowhead) predom- i n e n t l y d i s t r i b u t e d i n a t i e r i n the c y t o p l a s m .adjacent t o t h e c e l l w a l l (W). The c y t o p l a s m i c v a c u o l e s between t h e c h l o r o - p l a s t clumps (CC) and t h e t a n n i n v a c u o l e ( j u s t o u t o f th e low- e r l e f t o f the f i g u r e ) appear s t r e t c h e d i n the d i r e c t i o n o f the d o u b l e ended arrow. A 2.5 /Jm s c a l e i s shown: 6,000x. F i g u r e 52 A s e c t i o n t a n g e n t i a l t o the dome-shaped end w a l l ( f a c e C, f i g u r e 2) o f an ten month o l d c e l l . I t can be seen t h a t t h e o s m i o p h i l i c b o d i e s (OB, and arrowheads) a r e found p r e d o m i n e n t l y near the c e l l w a l l (W). C y t o p l a s m i c v a c u o l e s (CV) a r e a s s o c i a t - ed w i t h the o s m i o p h i l i c b o d i e s and a l s o deeper i n the c e l l where t h e r e i s e x t e n s i v e c y t o p l a s m i c membranes, many o f whi c h appear l i k e smooth e n d o p l a s m i c r e t i c u l u m (SER, a r r o w h e a d ) . Two c h l o r o p l a s t s ( C h ) a r e a l s o shown. A 2.5 yum s c a l e i s shown: 6,000x. 159-a. 160 PLATE XVI I E l e c t r o n M i c r o s c o p y o f the W i n t e r Chlorenchyma: C o n t i n u e d F i g u r e 53 A m i t o c h o n d r i o n (Mt) o f an e i g h t month n e e d l e i s shown. The m i t o c h o n d r i a l e n v e l o p e (arrowhead) appears as a s i n g l e i r r - e g u l a r d a r k l i n e . T u b u l a r c r i s t a e ( C r , arrow) a r e i n d i s t i n c t and not as numerous as i n summer. A 0.5 yum s c a l e i s shown: 38,OOOx. F i g u r e 54 A d i c t y o s o m e (D ) . f r o m a n i n e month o l d n e e d l e i s shown. Note t h a t t he budding v i s c l e s a r e a l l a t t a c h e d t o the o r g a n e l - l e andrthere a r e , f e w d i c t y o s o m e a s s o c i a t e d v e s i c l e s i n t h e s u r t r o u n d i n g c y t o p l a s m . A 1 pm s c a l e i s shown: 12,500x. F i g u r e 55 T h i s i s a 1 pm p l a s t i c s e c t i o n s t a i n e d w i t h osmium, t o l u - d i n e b l u e and S a f r a n i n , and photographed w i t h phase c o n t r a s t o p t i c s . A w i n t e r n u c l e u s (N) i s shown w i t h an o i l body (0) and the t a n n i n v a c u o l e (TV) ne a r b y , i n an e i g h t month o l d nee- d l e . Note the e v e n l y d i s p e r s e d g r a n u l a r i t y o f t h e d a r k l y s t a i n - ed h e t e r o c h r o m a t i n and a l s o a s i n g l e n u c l e o l u s (Nu) i s shown. A 5 /Jm s c a l e i s shown: 5,500x. F i g u r e 56 The n u c l e o l u s (Nu) and p a r t o f t h e n u c l e a r e n v e l o p e {HE, a r r o w h e a d ) , c o n t a i n i n g numerous dense n u c l e a r pores a r e shown. T i s s u e i s from an e i g h t month o l d n e e d l e . The h e t e r o c h r o m a t i n (HC) a p p ears t o have a g r a n u l a r s u b s t r u c t u r e . A 1 yum s c a l e i s shown: 28,000x. F i g u r e 57 An e n t i r e w i n t e r n u c l e u s (N) i s shown between two c h l o r o - p l a s t clumps (CC) i n an e i g h t month o l d n e e d l e . A s m a l l n u c l e - o l u s (Nu, arrowhead) appears i n t h i s s e c t i o n and t h e s m a l l clumps o f g r a n u l a r h e t e r o c h r o m a t i c (HC) appear e v e n l y d i s t r i b u t - ed i n t h e n u c l e o p l a s m . S e v e r a l c y t o p l a s m i c v a c u o l e s (CV) a r e shown. A 2.5>um s c a l e i s shown: 9,000x. 160-a 161 PLATE XVI I I E l e e t r o n M i c r o s c o p y o f t h e W i n t e r Chlorenchyma: Cont?nued and Wi n t e r V a r i a t i o n s i n P r e p a r a t i v e P r o c e d u r e s F i g u r e 58 A l a r g e o i l body (0) i s shown next t o t h e n u c l e u s (N) i n a. n i n e month o l d n e e d l e . S e v e r a l m i t o c h o n d r i a (Mt) a r e u s u a l - l y found near the o i l b o d i e s i n s p r i n g . But i n w i n t e r , t h i s o b s e r v a t i o n i s u n u s u a l . Note t h e more e x t e n s i v e t u b u l a r c r i s - t a e than u s u a l . TV = t a n n i n v a c u o l e . A 2 /jm s c a l e i s shown: 12,500x. F i g u r e 59 The v a r i a t i o n s i n w i n t e r f i x a t i o n s between c e l l s a r e shown i n t h i s e l e c t r o n m i c r o g r a p h o f a t h i r t y -two month o l d n e e d l e . C e l l A shows a clump o f c h l o r o p l a s t s i n the normal w i n t e r f i x - a t i o n . In c e l l s B and C ( t h e y may be t h e same c e l l ) , t h e c y t o - plasm i s f i l l e d W i t h t a n n i n m a t e r i a l p o s s i b l e due t o l y s i s o f the t o n o p l a s t . In c e l l D, a l l p r o t o p l a s t s t r u c t u r e s a r e brok- en down. C e l l s B, C and D may be t h e w i n t e r c o r r e l a t e s o f ty p e II I e e l 1s. A 5 yum s c a l e i s shown: 4,000x. F i g u r e 60 t o 62 a r e from m a t e r i a l s t o r e d w i t h i n a f r e e z e r f o r s e v e r a l months at t e m p e r a t u r e -18 C a f t e r c o l l e c t i o n a t below f r e e z i n g t e m p e r a t u r e s i n mid- w i n t e r . F i g u r e 60 A clump o f c h l o r o p l a s t s (CC) from a n i n e month o l d n e e d l e f i x e d i n sea w a t e r b u f f e r i s shown. Note t h e c o n s i s t e n t l y e l o n g a t e d form o f t h e c h l o r o p l a s t s and t h e reduced a r e a s o f c l o s e c o n t - a c t (arrowhead) between a d j a c e n t c h l o r o p l a s t s . The t a n n i n v a c u o l e (TV) and two c e l l w a l l s (W) a r e a l s o shown. A 2.5/Jm s c a l e i s shown: 7,000x. F i g u r e 61 P a r t o f a c e l l f i x e d i n t h e sea water b u f f e r from a n i n e month o l d n e e d l e showing t h e change i n t h e shape o f o s m i o p h i l i c b o d i e s (OB, arrowhead) t o a s t e l l a t e form. Two c h l o r o p l a s t s (Ch) a r e shown, one w i t h a s m a l l s t a r c h g r a i n ( S t ) w h i c h i s unusual i n w i n t e r m a t e r i a l . W = c e l l w a l l . A 1 /im s c a l e i s shown: 12,000x. F i g u r e 62 The appearance o f s u b s t r u c t u r e i n t h e form o f d a r k e r c o r e s (arrowheads) i n t h e o s m i o p h i l i c b o d i e s (OB) a f t e r t h i s n i n e month o l d w i n t e r t i s s u e had been immersed i n t o l i q u i d n i t r o g e n . Some c y t o p l a s m i c v a c u o l e s (CV) a r e a s s o c i a t e d w i t h t h e osmio- p h i l i c b o d i e s and d i c t y o s o m e s (D), a c h l o r o p l a s t ' ( C h ) and a t r a b i c u l u m (T) a r e a l s o shown. A 2/jm s c a l e i s shown: 15,000x.  162 PLATE XIX E l e c t r o n M i c r o s c o p y o f t h e Apr?1 27th 1973 F i x a t i o n F i g u r e 63 S e v e r a l c h l o r o p l a s t s (Ch) a r e shown a l o n g t h e c e l l w a l l (W). One c h l o r o p l a s t ( w i t h t h e s t a r c h g r a i n ( S t ) ) i s not near th e w a l l . Note o c c a s i o n a l d i l a t i o n s o f t h e t h y l a k o i d s and the even d i s t r i b u t i o n o f stroma about the t h y l a k o i d s . A 25 t o 50 nm gap i s a l w a y s o b s e r v e d i n t h i s t i s s u e ( s o l i d arrowheads) be- tween c h l o r o p l a s t s . Note the lobed m i t o c h o n d r i a (Mt, arrow- head) and the c y t o p l a s m i c v a c u o l e (CV) w i t h o u t any a s s o c i a t e d o s m i o p h i l i c m a t e r i a l i n t h e dense ground c y t o p l a s m (GC). (open a r r o w h e a d ) . PM = plasma membrane. TV = t a n n i n v a c u o l e . A 1 pm s c a l e i s shown: 10,000x F i g u r e 64 T h i s e l e c t r o n m i c r o g r a p h i s o f t h e boxed a r e a i n f i g u r e 63- Note t h e c o n s i s t e n t gap o f c y t o p l a s m (Cy, and between arrow- h e a d s ) , between the two c h l o r o p l a s t s . Grana i n t h e s e c h l o r o - p l a s t s c o n s i s t s o f o n l y two o r t h r e e t h y l a k o i d s ( a r r o w s ) . A 0.5 jum s c a l e i s shown: 30,000x. F i g u r e 65 When t h e s p r i n g c h l o r o p l a s t s (Ch) a r e s e c t i o n e d i n t h e p l a n e o f t h e t h y l a k o i d s , l a r g e numbers o f p l a s t o g l o b u l i (Pg) a r e o c c a s i o n a l l y seen t o be a s s o c i a t e d w i t h t h e t h y l a k o i d s . Note t h e i r r e g u l a r shape o f t h e c h l o r o p l a s t s and t h e c o n s i s t - ent gap between a d j a c e n t p l a s t i d s (open a r r o w h e a d s ) . A 5 Aim s c a l e i s shown: 20,000x. F i g u r e 66 A n u c l e u s (N) i n s p r i n g t i s s u e i s shown. .The h e t erochrom- a t i n (He) i s i n an i r r e g u l a r dense f o r m a t i o n t y p i c a l o f t h e sum- mer c o n d i t i o n . The t a n n i n v a c u o l e ( T V ) , an o i l body (0) and a t r a b i c u l u m (T) w i t h c h l o r o p l a s t s (Ch) a l o n g i t a r e a l s o shown. A 5 Aim s c a l e i s shown: 6,000x. F i g u r e 67 A low m a g n i f i c a t i o n o f p a r t o f a c e l l showing t h e e x t e n s i v e a r e a s ground c y t o p l a s m (GC) and o c c a s i o n a l c y t o p l a s m i c v a c u o l e s (CV). Note t h a t the t a n n i n v a c u o l e (TV) b a r e l y e n t e r s t h i s s e c t i o n . The c y t o p l a s m c o n t a i n s , l a r g e amounts o f o s m i o p h i l i c m a t e r i a l r a n g i n g i n s i z e from l e s s than 0.5 Aim b o d i e s ( u s u a l l y found a l o n g t h e c e l l w a l l , open arrowheads) t o l a r g e r i r r e g u l a r b o d i e s ( s o l i d arrowheads) t o s t i l l l a r g e r o i 1 - 1 i k e b o d i e s g r e a t - e r than 2 Aim i n d i a m e t e r . C h l o r o p l a s t s (Ch) a r e o f t e n i r r e g u l a r i n shape but do not have l a r g e s t r o m a l a r e a s . They a r e g e n e r a l - l y found c l o s e t o t h e c e l l wal I s l a n d many have s . t a r c h g r a i n s ( S t ) . M i t o c h o n d r i a (Mt) f r e q u e n t l y have lobed and i r r e g u l a r s e c t i o n a l p r o f i 1 e s . A 5 Aim s c a l e i s shown: 4,000x. 162-o 163 PLATE XX E l e c t r o n M i c r o s c o p y o f Chlorenchyma: S p r i n g F i x a t i o n and M a t e r i a l P r e p a r e d by V a r i o u s P r o c e d u r e s F i g u r e 68 Two t y p e s o f o s m i o p h i l i c c y t o p l a s m i c i n c l u s i o n s seen i n t h e A p r i l 17th, 1973 f i x a t i o n a r e shown. A t the r i g h t , a l a r g e o i l - l i k e i n c l u s i o n (0), and c l u s t e r o f even more o s m i o p h i l i c mater- i a l t o th e l e f t (0). The l a t t e r m a t e r i a l has an appearance sim- i l a r t o th e w i n t e r o s m i o p h i l i c b o d i e s . C y t o p l a s m i c v a c u o l e s (arrowhead) were o c c a s i o n a l l y seen a s s o c i a t e d w i t h t h e l a t t e r s t r u c t u r e s . W = c e l l w a l l . A 5/Jm s c a l e i s shown: 6,000x. F i g u r e 69 The n u c l e u s ( N ) , c h l o r o p l a s t ( C h ) , and t a n n i n v a c u o l e (TV) a r e shown i n a t h r e e y e a r o l d summer n e e d l e . The n u c l e i i n o l d - e r n e e d l e s a r e c h a r a c t e r i z e d by t h e r a r i t y o f n u c l e o l i i n s e c t - i o n s , and e x t e n s i v e , dense h e t e r o c h r o m a t i n (HC). A c r o l e i n f i x a - t i o n . A 5/Jm s c a l e i s shwon: k,500x. F i g u r e 70 P a r t o f a chlorenchyma c e l l from a f i v e y e a r o l d n e e d l e f i x e d i n the a c r o l e i n f i x a t i v e s o l u t i o n . The t a n n i n v a c u o l e (TV) and a l a r g e o s m i o p h i l i c o i l - l i k e body (0) a r e shown. The c y t o - plasm i s p o o r l y s t a i n e d but c h l o r o p l a s t s ( C h ) , c o n t a i n i n g numer- ous p l a s t o g l o b u l i and s t a r c h g r a i n s ( S t ) can be seen. W = c e l l wal 1. A 5>um s c a l e i s shown: 5,700x. F i g u r e 71 P a r t o f a c h l o r o p l a s t ( C h ) , and m i t o c h o n d r i a (Mt) a r e shown from an a c r o l e i n f i x e d t h r e e y e a r o l d summer n e e d l e . The c h l o - r o p l a s t i s unusual i n h a v i n g a few p l a s t o g l o b u l i and some g r a - na (G, arrowhead) c o n t a i n i n g more than t h r e e t h y l a k o i d s . The c y t o p l a s m above t h e o r g a n e l l e s c o n t a i n s e x t e n s i v e o s m i o p h i l i c m a t e r i a l . W = c e l l w a l l . F i g u r e 72 A 1 jam s c a l e i s shown: 22,800x. Mature ( -18°C). summer n e e d l e t i s s u e i s shown a f t e r s e v e r e f r e e z i n g A l l p r o t o p l a s t s t r u c t u r a l o r g a n i z a t i o n i s l o s t i n t h i s t i s s u e . T a n n i n m a t e r i a l (TM) i s found r i g h t up t o th e c e l l w a l l (W). The m a t e r i a l shown may have been a c h l o r o p l a s t s i n c e s t r u c - t u r e s r e s e m b l i n g a s t a r c h g r a i n ( S t ) and p o s s i b l e p l a s t o b l o b u l i (Pg?) a r e shown. A^1 /jm s c a l e i s shown 13,800x. 163 -a 164 PLATE XXI S t u d i e s on P?nus c o n t o r t a S e e d l i n g s S u b j e c t e d t o V a r i o u s T r e a t m e n t s i n C o n t r o l l e d Environment Chambers F i g u r e s 73 and 74 a r e both o f summer n e e d l e s from a t r e e i n w h i c h f r o s t h a r d i n e s s was p a r t i a l l y i n d u c e d . T h i s t i s s u e was f i x e d by t h e w i n t e r p r o c e d u r e a t -k C. F i g u r e 73 P a r t o f t h e c y t o p l a s m i s shown. C y t o p l a s m i c v a c u o l e s (CV) a r e v e r y numerous, and a r e a s s o c i a t e d w i t h t h e o s m i o p h i l i c b o d i e s (OB, and a r r o w h e a d s ) . T a n n i n m a t e r i a l (TM) o f t h e same d e n s i t y and t e x t u r e as t h e t a n n i n v a c u o l e m a t e r i a l i s o f t e n s c a t t e r e d i n t h e c y t o p l a s m i n s m a l l v a c u o l e s . Mt = m i t o c h o n d r i a . A 1 Aim s c a l e i s shown: 26,000x. F i g u r e 74 Two c h l o r o p l a s t s (Ch, A and B) a r e shown w h i c h may be f u s e d a l o n g t h e o u t e r e n v e l o p e membranes. Note t h e absence o f s t a r c h i n t h i s m a t e r i a l . The c y t o p l a s m c o n t a i n s many v a c u o l e s w i t h no d i s c e r n a b l e c o n t e n t s ( a s t e r i s k ) Mt = m i t o c h o n d r i a . W = c e l l - w a l l . A 1 jum s c a l e i s shown: 18,000X. F i g u r e 75 Two o f the t r e e s (pot A) i n w h i c h f r o s t h a r d i n e s s was a r t - i f i c i a l l y induced a r e shown i n p o t s one week a f t e r e x p o s u r e t o -18 C. V i s i b l e damage t o the t r e e s c o n s i s t e d o f the y e l l o w i n g o f t h e y oungest n e e d l e s (open arrowhead, shows l i g h t e r shaded need 1es), most o f w h i c h e v e n t u a l l y d i e d . Second y e a r n e e d l e s ( s o l i d arrowhead) remained green and were used as t h e t i s s u e shown i n f i g u r e s 73 and 7 4. Pot B c o n t a i n s a c o n t r o l t r e e w h i c h had no h a r d i n e s s inducement t r e a t m e n t s . The t r e e i s dead and t h e n e e d l e s a r e d r i e d o u t and f a l l i n g o f f . T h i s t r e e was expo- sed t o the -18 C f r o s t a t the same time as pot A. A 10 cm s c a l e i s shown: t r e e s a r e one f i f t h n a t u r a l s i z e . F i g u r e 76 and 77 a r e o f summer t r e e s which.have been exposed t o warm temp- e r a t u r e s and no w a t e r f o r 30.days. F i g u r e 76 Note t h a t the c h l o r o p l a s t s (Ch) have d e t a c h e d from t h e c e l l w a l l (W). The plasma membrane (PM, arrowhead) i s s t i l l a l o n g t h e c e l l w a l l . O s m i o p h i l i c b o d i e s (OB, arrowheads) a r e more numerous th a n i n normal summer t i s s u e . A 1 >um s c a l e i s shown: 10,000x. F i g u r e 77 The p e r i n u c l e a r c y t o p l a s m c o n t a i n s e n d o p l a s m i c r e t i c u l u m (ER), c i r e u l a r m i t o c h o n d r i a ( M t ) , an unusual c o n s t r i c t e d (open arrowhead) c h l o r o p l a s t (Ch) c o n t a i n i n g a s m a l l s t a r c h g r a i n ( S t ) . The n u c l e u s (N) c o n t a i n s t h e same t y p e o f c h r o m a t i n arrangement seen i n s p r i n g and summer. A s m a l l o i l body (0) i s a l s o seen. W':= c e l l w a l l . A 1 >im s c a l e i s shown: 11,000x. 164-a 165 PLATE XXI I A p p a r a t u s f o r F r o z e n Sect i o n O b s e r v a t i o n F i g u r e 78 The a p p a r a t u s f o r the s e c t i o n i n g o f m a t e r i a l kept c o h t i n - o u s l y f r o z e n . L a b e l l e d items a r e : ( A ) - b r i n e - i c e c o o l i n g b a t h ; (B) - CO ' - b o t t l e ; (C) - s e c t i o n i n g arm o f cryotome w i t h l i q u i d n i t r o g e n r e s e r v o i r on the k n i f e ; (D) - f o i l l a d e l ; (F) - mount- ing media (gum a r a b i c a ) ; (G) - i n s u l a t e d f o r c e p t s ; (H) - p l a s t - i c c o v e r s l i p s c u t i n t o s m a l l s q u a r e s ; ( I ) - vacuum g r e a s e ; (J) - s t e e l , c o l d s i n k ; (K) - Dewar f o r l i q u i d n i t r o g e n s t o r a g e . A 10 cm s c a l e i s shown, a p p a r a t u s i s one f i f t h a c t u a l s i z e . F i g u r e 79 The a p p a r t u s f o r the o b s e r v a t i o n o f f r o z e n s e c t i o n s . Ob- j e c t s not d e s c r i b e d above a r e : (L) - t h e e r y o m i c r o s c o p e s t a g e (which i s d i a g r a m m a t i c a l l y shown i n f i g u r e 80).; (M) - l i q u i d n i t r o g e n r e s e r v o i r ; (N) - f r e o n b o t t l e ; (0) - Dewar o f l i q u i d n i t r o g e n t o l i q u i f y f r e o n gas; (P) - m i c r o s c o p e camera. A 15 cm s c a l e i s shown: a p p a r a t u s i s shown a t one s i x t h a c t u a l s i z e .  166 F i g u r e 80 Diagram o f the c r y o m i c r o s c o p e s t a g e a p p a r a t u s . A c r o s s - s e c t i o n i s shown. FIGURE 8CV- DIAGRAM OF THE C RYO MICRO SCOPE STAGE LENSE 167 BIBLIOGRAPHY Ahkong, Q.F., D. F i s h e r , W. Tampion and J.A. Lucy, 1975- Mechanism o f c e l l f u s i o n . N a t u r e . 253: 194-195- A l d e n , J . and R.K. Hermann, 1971. C o l d h a r d i n e s s mechanisms i n p l a n t s . Bot. Rev. 3]j 37-142. A l l e n , T.D., M.V. 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