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The aging process of sapwood ray parenchyma cells in four woody species Yang, Kung Chi 1990

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THE AGING PROCESS OF SAPWOOD RAY PARENCHYMA CELLS IN FOUR WOODY SPECIES By RUNG CHI YANG B.Sc.F., National Chung-Hsing University, 1965 M.Sc.F., The University of New Brunswick, 1970 M.Sc., The University of Toronto, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Forestry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1990 © Rung chi Yang, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of FORESTRY The University of British Columbia Vancouver, Canada D a t e August 9, 1990 DE-6 (2/88) ABSTRACT A g i n g of r a y parenchyma c e l l s from the young sap-wood to r e c e n t l y formed heartwood was s t u d i e d i n s i n g l e stems of P i n u s  b a n k s i a n a L a m b . , P i c e a mar iana (Mi11 . ) B . S . P . , A b i e s balsamea ( L . ) M i l l , and Populus t r e m u l o i d e s M i c h x . Season , r a d i a l l o c a t i o n of c e l l w i t h i n sapwood, and c e l l l o c a t i o n v e r t i c a l l y w i t h i n a ray at a g i v e n r a d i u s were c o n s i d e r e d as f a c t o r s which might i n f l u e n c e the a g i n g p r o c e s s . A 12 mm increment core was e x t r a c t e d at b r e a s t h e i g h t , from the n o r t h a s p e c t of a t r e e o f each s p e c i e s i n May and J u l y f o r m o i s t u r e c o n t e n t d e t e r m i n a t i o n . Another s e t of c o r e s from the south a s p e c t of the same t r e e s was c o l l e c t e d i n May, J u n e , J u l y , A u g u s t , O c t o b e r , and November or December. These c o r e s were used to i n v e s t i g a t e the p h y s i o l o g i c a l and c y t o l o g i c a l p r o p e r t i e s of l i v i n g sapwood r a y parenchyma c e l l s . Q u a l i t a t i v e and q u a n t i t a t i v e o b s e r v a t i o n s were made of the s t a t u s o f r a y c e l l s b o t h w i t h 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 i n o r d e r to draw i n f e r e n c e s c o n c e r n i n g the sapwood/heartwood t r a n s f o r m a t i o n from the a g i n g of sapwood r a y parenchyma c e l l s . The sapwood m o i s t u r e c o n t e n t of the t h r e e c o n i f e r s s t u d i e d was h i g h e r than t h a t of hear twood , whereas i n Populus i i t r e m u l o i d e s i t was lower than t h a t of heartwood. The sapwood m o i s t u r e content i n May was c o n s i s t e n t l y g r e a t e r than i n J u l y . V i t a l i t y of the sapwood ray parenchyma c e l l s expressed by a new n u c l e a r e l o n g a t i o n index decreased from the outer sapwood towards the heartwood. The s u r v i v a l r a t e of the c e l l s decreased c u r v i l i n e a r l y from the middle sapwood towards the heartwood. At a g i v e n sapwood increment , a g r e a t e r percentage of dead ray parenchyma c e l l s was found among the m a r g i n a l c e l l s than among the c e n t r a l c e l l s of a r a y . No s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e was found between the v i t a l i t y of the m a r g i n a l and c e n t r a l c e l l s , nor between any two cont iguous sampl ing p e r i o d s w i t h e x c e p t i o n s i n P inus b a n k s i a n a and P i c e a  mariana between two cont iguous sampl ing p e r i o d s from J u l y to December. No t y p i c a l p a t t e r n f o r the d i s t r i b u t i o n of l i p i d content was f o u n d . The p a t t e r n of s t a r c h d i s t r i b u t i o n d i s p l a y e d s i g n i f i c a n t s p e c i e s , r a d i a l , v e r t i c a l and s e a s o n a l v a r i a t i o n and showed two g e n e r a l p a t t e r n s a c r o s s the sapwood. P a t t e r n A d e s c r i b e d a d e c r e a s i n g t r e n d from the o u t e r sapwood towards the i n n e r sapwood. P a t t e r n B was c h a r a c t e r i z e d by a r e l a t i v e l y low s t a r c h content both i n the outer as w e l l as the i n n e r sapwood. The s t a r c h c o n t e n t i n Populus t r e m u l o i d e s and the i i i l i p i d content i n P inus banks iana and P i c e a mariana d i s p l a y e d no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between m a r g i n a l and c e n t r a l ray c e l l s . The m a j o r i t y of ray parenchyma c e l l s showed a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between two cont iguous sampl ing p e r i o d s i n s t a r c h and l i p i d c o n t e n t s . There was no i n v e r s e r e l a t i o n s h i p between the s t a r c h and l i p i d content over the growing season s t u d i e d . Young ray parenchyma c e l l s were r i c h i n c h r o m a t i n and c y t o p l a s m which c o n t a i n e d numerous c e l l o r g a n e l l e s . These c e l l s were c h a r a c t e r i z e d by a m y l o p l a s t s which possessed one or more e l o n g a t e d s t a r c h g r a n u l e s w i t h t h y l a k o i d s and o s m i o p h i l i c g l o b u l i , numerous s m a l l l i p i d d r o p l e t s and most ly r o d - l i k e m i t o c h o n d r i a . In c o n t r a s t , aged ray parenchyma c e l l s f e a t u r e d an aggrega ted , dense nuc leus and c y t o p l a s m which c o n t a i n e d few c e l l o r g a n e l l e s . These aged c e l l s possessed e n l a r g e d swo l l en s t a r c h g r a n u l e s , l a r g e l i p i d d r o p l e t s or lumps w i t h two s t a i n i n g d e n s i t i e s , round shaped m i t o c h o n d r i a w i t h i n c o n s p i c -uous c r i s t a e and a r o u g h / b r o k e n plasmalemma. Some heartwood substances o r i g i n a t e d from the l i p i d lumps which appeared f r e q u e n t l y i n d y i n g ray c e l l s . Based on m i c r o s c o p i c o b s e r v a t i o n s and measurements of the l o s s of v i t a l i t y of ray parenchyma c e l l s , a d e c l i n i n g s u r v i v a l i v r a t e , the d i s i n t e g r a t i o n of c e l l o r g a n e l l e s and the o r i g i n of heartwood substances from l i p i d lumps, i t can be conc luded t h a t heartwood f o r m a t i o n i s l a r g e l y a s s o c i a t e d w i t h the death of sapwood ray parenchyma c e l l s . The death of these c e l l s i s due to the passage o f . t i m e . v TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES i x LIST OF FIGURES x i i LIST OF PLATES x i v ACKNOWLEDGEMENTS x v i 1. INTRODUCTION . . 1 2. LITERATURE REVIEW 4 2.1 Chemical Content 4 2.2 P h y s i o l o g y 9 2.3 Nuc lear Morphology 12 2.4 Heartwood Format ion 15 2 .4 .1 S u r v i v a l P a t t e r n s of Ray Parenchyma C e l l s from Cambium to Heartwood 15 2 .4 .2 Timing of Heartwood Format ion 18 2.5 U l t r a s t r u c t u r e 20 2 .5 .1 Seasonal V a r i a t i o n 20 2 .5 .2 Aging 24 3. OBJECTIVES AND NOVELTY 31 3.1 O b j e c t i v e s 31 3.2 Nove l ty 32 4. GENERAL DESCRIPTION OF SPECIMENS 34 5. STUDIES, 40 5.1 Sapwood and Heartwood as Determined by M o i s t u r e Content V a r i a t i o n 40 5 .1 .1 Sampling and Methods 40 5 .1 .2 R e s u l t s 45 5 . 1 . 2 . 1 P inus banks iana 45 5 . 1 . 2 . 2 P i c e a mariana 46 5 . 1 . 2 . 3 Ab ie s balsamea 46 5 . 1 . 2 . 4 Populus t r e m u l o i d e s 47 5 .1 .3 D i s c u s s i o n 47 v i 5.2 S u r v i v a l Rate 50 5 .2 .1 Sampling and Methods 50 5 .2 .2 R e s u l t s 55 5 . 2 . 2 . 1 P inus banks iana 55 5 . 2 . 2 . 2 P i c e a mariana 57 5 . 2 . 2 . 3 Ab ie s balsamea 57 5 . 2 . 2 . 4 Populus t r e m u l o i d e s 58 5 .2 .3 D i s c u s s i o n 59 5 . 3 . N u c l e a r E l o n g a t i o n Index 66 5 .3 .1 Specimen P r e p a r a t i o n and Methods . . 66 5 .3 .2 R e s u l t s 68 5 . 3 . 2 . 1 P inus b a n k s i a n a 69 5 . 3 . 2 . 2 P i c e s mariana 75 5 . 3 . 2 . 3 Ab ie s balsamea 76 5 . 3 . 2 . 4 Populus t r e m u l o i d e s 78 5 .3 .3 D i s c u s s i o n 79 5.4 Relative S t a r c h Content 83 5 .4 .1 Specimen P r e p a r a t i o n and Methods . . 83 5 .4 .2 R e s u l t s 85 5 . 4 . 2 . 1 P inus b a n k s i a n a 85 5 . 4 . 2 . 2 P i c e a mariana 90 5 . 4 . 2 . 3 Ab ie s balsamea 94 5 . 4 . 2 . 4 Populus t r e m u l o i d e s 95 5 .4 .3 D i s c u s s i o n 96 5.5 R e l a t i v e L i p i d Content 106 5 .5 .1 Specimen P r e p a r a t i o n and Methods . . 106 5 .5 .2 R e s u l t s 106 5 . 5 . 2 . 1 P inus b a n k s i a n a 106 5 . 5 . 2 . 2 P i c e a mariana I l l 5 . 5 . 2 . 3 Ab ie s balsamea 114 5 . 5 . 2 . 4 Populus t r e m u l o i d e s 115 5 .5 .3 D i s c u s s i o n 116 5.6 U l t r a s t r u c t u r e of C e l l O r g a n e l l e s . 122 v i i 5 . 6 . 1 Specimen P r e p a r a t i o n 122 5 .6 .2 O b s e r v a t i o n 125 5 . 6 . 3 R e s u l t s and D i s c u s s i o n 129 5 . 6 . 3 . 1 P inus banks iana 129 5 . 6 . 3 . 1 . 1 Seasonal V a r i a t i o n 129 5 . 6 . 3 . 1 . 2 Aging Process 131 5 . 6 . 3 . 2 P i c e a mariana 141 5 . 6 . 3 . 2 . 1 Seasonal V a r i a t i o n 141 5 . 6 . 3 . 2 . 2 Aging Proces s 143 5 . 6 . 3 . 3 Ab ie s balsamea 147 5 . 6 . 3 . 3 . 1 Seasonal V a r i a t i o n 147 5 . 6 . 3 . 3 . 2 Aging Process 150 5 . 6 . 3 . 4 Populus t r e m u l o i d e s 154 5 . 6 . 3 . 4 . 1 Seasonal V a r i a t i o n 154 5 . 6 . 3 . 4 . 2 Aging Proces s 158 5 . 6 . 4 Heartwood Substances 161 5 . 6 . 5 Summary of U l t r a s t r u c t u r e 164 6. CONCLUSIONS AND RECOMMENDATIONS 244 LITERATURE CITED 255 APPENDIX 1. S c i e n t i f i c Name of Spec ie s 283 APPENDIX 2. An example of procedure f o r data d e r i v a t i o n 286 APPENDIX 3. Summary of u l t r a s t r u c t u r a l changes of f i v e c e l l o r g a n e l l e s i n l i v i n g sapwood ray parenchyma c e l l s due to age and seasona l v a r i a t i o n i n P inus b a n k s i a n a , P i c e a m a r i a n a , Ab ie s balsamea and Populus t r e m u l o i d e s 289 ABBREVIATIONS 290 P inus banks iana 291 P i c e a mariana 292 Abie s balsamea 293 Populus t r e m u l o i d e s 294 v i i i LIST OF TABLES Table No. 1. P e r i o d s of i n i t i a t i o n of heartwood f o r m a t i o n i n v a r i o u s s p e c i e s : A summary 2. Some a n a t o m i c a l f e a t u r e s of the four s p e c i e s s t u d i e d 3. S p e c i e s , age, h e i g h t and diameter of sampled t r e e s and the dates of c o l l e c t i o n f o r m i c r o -s c o p i c s t u d i e s 4. A t - t e s t of the mean d i f f e r e n c e i n m o i s t u r e content between May and J u l y i n sapwood and heartwood of four s p e c i e s s t u d i e d 5. A t - t e s t of the mean d i f f e r e n c e i n s u r v i v a l r a t e between the m a r g i n a l and c e n t r a l c e l l s 6. The number of growth r i n g s counted from the cambium marking the f i r s t observance of death and l a s t observance of l i f e i n the ray parenchyma c e l l s 7. The boundary of sapwood and heartwood expressed as the r i n g age counted from the cambium as determined by two d i f f e r e n t methods: d i s t r i b u t i o n of mo i s ture content (MC) and the s u r v i v a l r a t e (SR) of ear lywood c e n t r a l r a y parenchyma c e l l s d u r i n g May 8. C o r r e l a t i o n c o e f f i c i e n t s between the m o i s t u r e content and s u r v i v a l r a t e i n May specimen 9. T - t e s t s of the d i f f e r e n c e s between the means of n u c l e a r e l o n g a t i o n index over two c o n t i g u o u s sampl ing p e r i o d s and t h e i r v a r i a n c e s f o r both m a r g i n a l (M) and c e n t r a l (C) ray parenchyma c e l l s Page 19 35 38 44 54 56 63 65 77 i x 10. C o r r e l a t i o n c o e f f i c i e n t s between the n u c l e a r e l o n g a t i o n index and the moi s ture content and s u r v i v a l r a t e i n May specimen . . 11. T - t e s t s of the d i f f e r e n c e s between the means of r e l a t i v e s t a r c h content i n the m a r g i n a l and c e n t r a l ray parenchyma c e l l s and F - t e s t s of t h e i r v a r i a n c e s 81 91 12. T - t e s t s of the d i f f e r e n c e s between the means of r e l a t i v e s t a r c h content over two cont iguous sampl ing p e r i o d s and F - t e s t s of t h e i r v a r i a n c e s f o r both m a r g i n a l (M) and c e n t r a l (C) ray parenchyma c e l l s 93 13. C o r r e l a t i o n c o e f f i c i e n t s between the r e l a t i v e s t a r c h content and the m o i s t u r e c o n t e n t , s u r v i v a l r a t e and n u c l e a r e l o n g a t i o n index i n May specimen 104 14. T - t e s t s of the d i f f e r e n c e s between the means of r e l a t i v e l i p i d content of the m a r g i n a l and c e n t r a l ray parenchyma c e l l s and F - t e s t s of t h e i r v a r i a n c e s H 2 15. T - t e s t s of the d i f f e r e n c e s between the means of r e l a t i v e l i p i d content over two cont iguous sampl ing p e r i o d s and F - t e s t s of t h e i r v a r i a n c e s f o r both m a r g i n a l (M) and c e n t r a l (C) Ray parenchyma c e l l s 1 1 3 16. C o r r e l a t i o n c o e f f i c i e n t s between the r e l a t i v e l i p i d content and the m o i s t u r e c o n t e n t , s u r v i v a l r a t e , n u c l e a r e l o n g a t i o n index and the r e l a t i v e s t a r c h c o n t e n t i n May specimen . 17. C o r r e l a t i o n c o e f f i c i e n t s between the mean r e l a t i v e s t a r c h and l i p i d c o n t e n t s of ray parenchyma c e l l s over v a r i o u s sampl ing p e r i o d s 120 121 18. The l o c a t i o n of specimens c o l l e c t e d 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 . Numbers r e f e r to the growth r i n g numbers counted from the cambium 123 19. The main c h a r a c t e r i s t i c f e a t u r e s of f i v e c e l l o r g a n e l l e s i n the o u t e r sapwood ray parenchyma c e l l s of P inus banks iana i n J u l y and December 132 x 20. The main c h a r a c t e r i s t i c f e a t u r e s of f i v e c e l l o r g a n e l l e s of ray parenchyma c e l l s i n P inus  b a n k s i a n a i n the outer and i n n e r sapwood 140 21. The main c h a r a c t e r i s t i c f e a t u r e s of f i v e c e l l o r g a n e l l e s i n the o u t e r sapwood ray parenchyma c e l l s of P i c e a mariana i n J u l y and December 144 22. The main c h a r a c t e r i s t i c f e a t u r e s of f i v e c e l l o r g a n e l l e s of ray parenchyma c e l l s i n P i c e a  mariana i n the outer and i n n e r sapwood 148 23. The main c h a r a c t e r i s t i c f e a t u r e s of f i v e c e l l o r g a n e l l e s i n the outer sapwood ray parenchyma c e l l s of Ab ie s balsamea i n J u l y and November . 151 24. The main c h a r a c t e r i s t i c f e a t u r e s of f i v e c e l l o r g a n e l l e s of ray parenchyma c e l l s of A b i e s  balsamea i n the outer and i n n e r sapwood 155 25. The main c h a r a c t e r i s t i c f e a t u r e s of f i v e c e l l o r g a n e l l e s i n the outer sapwood ray parenchyma c e l l s of Populus t r e m u l o i d e s i n J u l y and November 157 26. The main c h a r a c t e r i s t i c f e a t u r e s of f i v e c e l l o r g a n e l l e s of ray parenchyma c e l l s i n Populus t r e m u l o i d e s i n the outer and i n n e r sapwood 162 x i LIST OF FIGURES Figure No. Page 1. The l o c a t i o n and sequence of specimen e x t r a c t i o n s at v a r i o u s sampl ing p e r i o d s i n the f o u r s p e c i e s s t u d i e d 37 2. The r a d i a l d i s t r i b u t i o n of May (A) and J u l y (•) m o i s t u r e content (%) i n sapwoods and heartwoods 42 3. The s u r v i v a l r a t e of r a y parenchyma c e l l s expressed as the percentage of l i v i n g sapwood ray parenchyma c e l l s i n the m a r g i n a l (•) and c e n t r a l (A) p o s i t i o n s i n May 52 4. The n u c l e a r l e n g t h s of the c e n t r a l ray parenchyma c e l l s i n P inus b a n k s i a n a , P i c e a m a r i a n a , A b i e s balsamea and Populus  t r e m u l o i d e s at v a r i o u s l o c a t i o n s w i t h i n the sapwood at d i f f e r e n t sampl ing p e r i o d s 70 5. V i t a l i t y of m a r g i n a l (•) and c e n t r a l (•) ray parenchyma c e l l s expressed as the n u c l e a r e l o n g a t i o n index a c r o s s the sapwood at v a r i o u s sampl ing p e r i o d s . Each p o i n t r e p r e s e n t s the average o f 20 c e l l measurements 71 6. The r e l a t i v e s t a r c h content of m a r g i n a l (•) and c e n t r a l (A) ray parenchyma c e l l s a c r o s s the sapwood at v a r i o u s sampl ing p e r i o d s . Each p o i n t r e p r e s e n t s the average of 20 ray measurements 86 7. The r e l a t i v e l i p i d content of l i v i n g m a r g i n a l (•) and c e n t r a l (A) r a y parenchyma c e l l s a c r o s s the sapwood at v a r i o u s sampl ing p e r i o d s . Each p o i n t r e p r e s e n t s the average of 10 ray measurements 107 8. An e l e c t r o n m i c r o g r a p h of a t r a n s v e r s e s e c t i o n of an A b i e s balsamea outer sapwood ray parenchyma c e l l c o l l e c t e d i n November, i n d i c a t i n g the l o c a t i o n and d i s t r i b u t i o n of the c e l l o r g a n e l l e s 127 x i i A s i m p l i f i e d s t r u c t u r e of f i v e c e l l o r g a n e l l e s i n a young ray parenchyma c e l l (upper) and an aged ray parenchyma c e l l ( lower) i n 1. P inus  b a n k s i a n a , 2. P i c e a m a r i a n a , 3. Ab ie s balsamea and 4. Populus t r e m u l o i d e s  x i i i LIST OF PLATES Page ABBREVIATIONS USED IN PLATE ILLUSTRATIONS 171 Plate No. 1-4 Pinus b a n k s i a n a ray parenchyma c e l l s i n the o u t e r sapwood r i n g 172 5-6 Pinus b a n k s i a n a ray parenchyma c e l l s i n the middle sapwood r i n g 180 7-8 Pinus b a n k s i a n a ray parenchyma c e l l s i n the i n n e r sapwood r i n g 184 9 Pinus b a n k s i a n a ray parenchyma c e l l s i n the heartwood 188 10-12 P i c e a mariana ray parenchyma c e l l s i n the outer sapwood r i n g 190 13-15 P i c e a mariana ray parenchyma c e l l s i n the middle sapwood r i n g 196 16-17 P i c e a mariana ray parenchyma c e l l s i n the i n n e r sapwood r i n g 202 18 P i c e a mariana ray parenchyma c e l l s i n the heartwood 206 19-21 Abies balsamea ray parenchyma c e l l s i n the outer sapwood r i n g 208 22-23 Abies balsamea ray parenchyma c e l l s i n the middle sapwood r i n g 214 24-25 Abie s balsamea ray parenchyma c e l l s i n the i n n e r sapwood r i n g 218 26 Abies balsamea ray parenchyma c e l l s i n the heartwood 222 27-29 Populus t r e m u l o i d e s r a y parenchyma c e l l s i n the outer sapwood r i n g 224 30-31 Populus t r e m u l o i d e s r a y parenchyma c e l l s i n the middle sapwood r i n g 230 x i v 32-33 Populus t r e m u l o i d e s r a y parenchyma c e l l s i n the i n n e r sapwood r i n g . . 34 Populus t r e m u l o i d e s ray parenchyma c e l l s i n the heartwood 35-36 Heartwood Substances . , . . xv ACKNOWLEDGEMENTS I w i sh to express my deep g r a t i t u d e to the members of my a d v i s o r y commit tee , D r . R. W. Kennedy ( C h a i r m a n ) , D r . J . W . W i l s o n , D r . R . M . K e l l o g g ( F o r i n t e k Canada C o r p . ) and D r . I . E . P . T a y l o r f o r t h e i r h e l p and a d v i c e i n p l a n n i n g the r e s e a r c h p r o j e c t at the b e g i n n i n g of t h i s s t u d y . S p e c i a l thanks are due to D r . Kennedy, Dean, F a c u l t y of F o r e s t r y , f o r h i s i n v a l u a b l e comments and s u g g e s t i o n s d u r i n g the whole course of the s t u d y . I am i n d e b t e d to Lakehead U n i v e r s i t y f o r f i n a n c i a l a s s i s t a n c e and equipment to c a r r y cu t t h i s r e s e a r c h p r o j e c t , and am d e e p l y g r a t e f u l to D r . G . H a z e n b e r g , S c h o o l of F o r e s t r y , Lakehead U n i v e r s i t y , who has p r o v i d e d me w i t h encouragement , a d v i c e i n s t a t i s t i c a l a n a l y s e s and a s s i s t a n c e i n the m a n u s c r i p t p r e p a r a t i o n . Thanks are a l s o extended to M r . B a r r y Wong, computer programmer, F a c u l t y of F o r e s t r y , the U n i v e r s i t y of B r i t i s h C o l u m b i a , f o r h i s h e l p i n s t a t i s t i c a l t e s t s and to M r . A l M a c K e n z i e , Lakehead U n i v e r s i t y , f o r h i s t e c h n i c a l a s s i s t a n c e i n e l e c t r o n m i c r o s c o p y . I want to express the deepest t h a n k f u l n e s s to my f a m i l y ; w i f e J a n e t , daughter T e r r i and son G r a n t , f o r t h e i r s u p p o r t and c o - o p e r a t i o n f o r the p e r i o d of t h i s s t u d y . I t would have been i m p o s s i b l e to complete the r e s e a r c h and w r i t i n g o f t h i s d i s s e r t a t i o n w i t h o u t my w i f e ' s u n d e r s t a n d i n g and s u p p o r t . A p p r e c i a t i o n i s a l s o extended to my p a r e n t s , f a t h e r P r o f . Y . P . Yang and mother Y u - L i , f o r t h e i r encouragement and i n s p i r a t i o n . xv i 1. I N T R O D U C T I O N 1 During tree growth, the outer part of the xylem, i.e., sapwood, is converted into heartwood. This change can modify wood quality and affect wood uti l i z a t i o n significantly for processes such as wood preservation, gluing and pulping. It is known that sapwood may be lighter in colour, more permeable, less decay resistant in service and lower in extractive content than heartwood. The differences between sapwood and heartwood are mainly caused by the death of living sapwood parenchyma cells and the formation of extractives, which i n f i l t r a t e and encrust c e l l walls and pit membranes and trigger the formation of aspirated p i t s . The moisture content of sapwood is higher than that of heartwood in conifers, but in angiosperms the sapwood moisture content may or may not be higher than in heartwood. In some species, crystals are formed in the dying parenchyma c e l l s . It is the v i t a l i t y of the parenchyma cells in sapwood which is the main biological distinction between sapwood and heartwood. Transformation of sapwood into heartwood is a complex process and the specific mechanism of heartwood formation is not completely understood. 2 There are s e v e r a l hypotheses c o n c e r n i n g the i n i t i a t i n g events of heartwood f o r m a t i o n , i n c l u d i n g water d e f i c i t (Trende l enburg 1939, H a r r i s 1954, Huber 1956, Stewart 1966, Zimmermann and Brown 1971); a c c u m u l a t i o n of t o x i c subs tances (Stewart 1966, H i g u c h i 1970, Carrodus 1971); f u n g a l a t t a c k (Busgen 1929, Chattaway 1952); a e r a t i o n a n d / o r d e s i c c a t i o n ( P r i e s t l y 1932, Zycha 1948, P a c l t 1953, Jorgensen 1962, Nekrasova 1967); and ag ing proces se s ( F r e y - W y s s l i n g and Bosshard 1959, Kondo 1964, Bosshard 1965, 1966, 1968, Fukazawa and H i g u c h i 1965, Hugentobler 1965, Necesany 1958, 1965, 1966, Nobuchi and Harada 1968, Nobuchi 1975, Nobuchi et aJL. 1976, Nobuchi 1985). One u n i t i n g aspec t of these v a r i o u s p r o p o s a l s i s the t ene t t h a t death of the l i v i n g sapwood parenchyma c e l l s p l a y s a key r o l e i n the sapwood-heartwood t r a n s f o r m a t i o n . One c o n t r a r y view (Bamber 1976) i s tha t heartwood f o r m a t i o n i s a growth-r e g u l a t i n g proces s t h a t i s r e l a t e d to development and the ma in ta inance of an optimum sapwood volume. Bamber (1976) proposed t h a t the death of sapwood parenchyma c e l l s i s the r e s u l t of heartwood f o r m a t i o n r a t h e r than the cause . 3 The importance of l i v i n g sapwood r a y parenchyma c e l l s i n t h i s change suggested t h a t a s tudy of the s e a s o n a l change i n these c e l l s at v a r i o u s l o c a t i o n s i n sapwood would p r o v i d e u s e f u l i n f o r m a t i o n about heartwood f o r m a t i o n . The p r e s e n t s tudy emphasizes m i c r o s c o p i c and u l t r a s t r u c t u r a l changes . The l a t t e r have not been c a r r i e d out s y s t e m a t i c a l l y b e f o r e . I t i s w e l l known ( K l i e n e b e r g e r 1917, DuPraw 1968, Brown and Ber tke 1969) t h a t m e t a b o l i c a c t i v i t y can be r e l a t e d to the m o r p h o l o g i c a l s t r u c t u r e of c e l l o r g a n e l l e s ; f o r i n s t a n c e , a v i g o u r o u s c e l l always has a l a r g e nuc l eus and numerous c r i s t a e i n s i d e the m i t o c h o n d r i o n . The use of bo th 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 microscopy was expected to p r o v i d e more r e l i a b l e i n f o r m a t i o n about these i n t r i c a t e and important changes i n the sapwood c e l l s of l i v i n g t r e e s . 2. LITERATURE REVIEW There i s s u b s t a n t i a l l i t e r a t u r e on c h e m i c a l , p h y s i o l o g i c a l and c y t o l o g i c a l d i f f e r e n c e s between sapwood and heartwood. However, s e v e r a l a spec t s of l i v i n g c e l l s i n sapwood are s t i l l obscure and t h e r e are s e v e r a l c o n t r a d i c t o r y o b s e r v a t i o n s . 2.1 Chemical Content Heartwood has a d i f f e r e n t c h e m i c a l c o m p o s i t i o n from sapwood. In most c a s e s , e x t r a c t i v e s are p r e s e n t i n g r e a t e r amounts i n heartwood than i n sapwood and a l s o are g e n e r i c or s p e c i f i c i n n a t u r e . U s u a l l y the e x t r a c t -i v e content i n c r e a s e s s h a r p l y at the heartwood boundary but D i e t r i c h s and Funke (1967) r e p o r t e d t h a t the t a n n i n or e x t r a c t i v e content of F r a x i n u s e x c e l s i o r , Quercus  r o b u r , Mansonia a l t i s s i m a and Tectona q r a n d i s reaches a maximum v a l u e i n s i d e the outer heartwood and not a t the boundary . The p h e n o l i c e x t r a c t i v e content of the most r e c e n t l y formed heartwood s t e a d i l y i n c r e a s e s w i t h the age of the t r e e (MacLean and Gardner 1956, Sandermann 5 and Dietrichs 1959, Gardner and Barton 1960). Nobuchi et a l . (1987a) found a similar distribution of heartwood phenols in Cryptomeria japonica. However, they found that most heartwood phenols were in the lower 3 m of outer heartwood in the trunk. Above 3 m, there were more in the inner heartwood. However, Swan et al_. (1969) found the highest amount of lignan in Thuja plicata at the eighth ring from the pith, i.e., in the middle of the heartwood, and not at the boundary The origin of heartwood substances is s t i l l unclear. It has been reported that carbohydrates and nitrogenous compounds are translocated from one part of the plant to another (Crafts 1956). Many workers (Roux 1958, King and White 1957, Hergert and Goldschmid 1958, Alston e_t a_l. 1963) consider that polyphenolic compounds are translocated in a similar manner. In contrast, evidence collected by Wardrop and Cronshaw (1962), H i l l i s and Hasegawa (1963), Hasegawa and Shiroya (1965), H i l l i s and Inoue (1966), Parameswaran and Bauch (1975), Higuchi (1976), Kuo and Arganbright (1980), Nobuchi and Harada (1985), Nobuchi (1985) strongly support the view that polyphenolic compounds are not translocated from other parts of a tree, but 6 form in s i t u by c a r b o h y d r a t e metabol ism of the l i v i n g sapwood ray and a x i a l parenchyma c e l l s . The gross c h e m i c a l c o m p o s i t i o n of the sapwood b e f o r e t r a n s f o r m a t i o n i n t o heartwood may p r o v i d e c l u e s f o r i n t e r p r e t i n g the changes i n the sapwood zone. Fukazawa and H i g u c h i (1965, 1966), i n t h e i r s e r i e s of s t u d i e s on the mechanism of heartwood f o r m a t i o n i n C r y p t o m e r i a j a p o n i c a and o t h e r s p e c i e s , r e p o r t e d t h a t DNA was more or l e s s c o n s t a n t from the cambium to the t r a n s i t i o n zone between sapwood and heartwood, and t h e r e were o c c a s i o n a l l y s l i g h t i n c r e a s e s i n the t r a n s i t i o n zone ( H i g u c h i e t a_l. 1964) . The h i g h e s t amount of RNA was found i n the outermost sapwood and l e v e l d e c l i n e d a c r o s s s e v e r a l c e l l l a y e r s towards the i n n e r sapwood. MacDougal and Smith (1927) i n Sequoia semperv irens and D i e t r i c h s (1964) i n Faqus q r a n d i f o l i a found t h a t the d i m i n u t i o n of s t a r c h i n the rays c o i n c i d e d w i t h the t r a n s i t i o n from sapwood to heartwood. H i g u c h i e_b a l . (1964) observed i n C r y p t o m e r i a j a p o n i c a , Fahn and Arnon (1963) i n Tamarix a p h y l l a and Nobuchi e t a_l. (1987b) i n P t e r o c a r y a r h o i f o l i a , A e s c u l u s t u r b i n a t a and M a g n o l i a g r a n d i f l o r a t h a t the s t a r c h content was h i g h -7 e s t c l o s e to the cambium and decreased g r a d u a l l y toward the t r a n s i t i o n zone. In c o n t r a s t , H i l l i s (1962, 1965, 1968, 1987) r e p o r t e d t h a t most s t a r c h i n E u c a l y p t u s  l a t i f o l i a was i n the middle of the sapwood, a l t h o u g h the v e r t i c a l d i s t r i b u t i o n of s t a r c h was i r r e g u l a r and v a r i e d w i t h i n each t r e e . Jones and B r a d l e e (1933) s t u d i e d s e a s o n a l l y r e l a t e d v a r i a t i o n i n Acer rubrum and found t h a t s t a r c h content was h i g h e s t i n the summer and lowest i n w i n t e r and t h a t sugar content v a r i e d i n v e r s e l y . Sauter (1966a) found the h i g h e s t s t a r c h content i n Populus euramer i ca d u r i n g e a r l y autumn and w i t h a lower peak i n e a r l y s p r i n g . Nobuchi e t a_l. (1986) r e p o r t e d a s i m i l a r b imodal p a t t e r n i n f i f t e e n b r o a d - l e a v e d s p e c i e s i n Japan d u r i n g l a t e summer and e a r l y s p r i n g . These f i n d i n g s are s i m i l a r to those i n A n t i a r i s a f r i c a n a ( O l o f i n o b o b a 1969) and s e v e r a l p i n e s , (Fukazawa e_t a l . 1980) , but c o n t r a s t w i t h the work by P a n d a l a i e t a l . (1985), who r e p o r t e d t h a t t h e r e was no v a r i a t i o n i n s t a r c h and l i p i d s of M e l i a azedarach d u r i n g the y e a r . L i p i d s , which i n c l u d e f a t s and o i l s , occur as s t o r a g e substances i n the sapwood of t r e e s of c e r t a i n genera ( Z i e g l e r 1964) and are r e a d i l y i n t e r c o n v e r t i b l e 8 w i t h c a r b o h y d r a t e s (Pomeroy and S i m i n o v i t c h 1971, Zimmermann and Brown 1971) . H i g u c h i e_t a_l. (1964) s t u d i e d C r y p t o m e r i a j a p o n i c a and found t h a t the q u a n t i t y of l i p i d s i n c r e a s e d w i t h i n c r e a s i n g d i s t a n c e from the cambium towards the t r a n s i t i o n zone , but t h a t the c o n c e n t r a t i o n of l i p i d s d i f f e r s among v a r i o u s g e n e r a , e . g . , T i l i a , B e t u l a and R o b i n i a ( H o l l and P o s c h e n r i e d e r 1975). Kramer and K o z l o w s k i (1979) r e p o r t e d t h a t the h i g h e s t l i p i d content i n T i l i a  m i q u e l i a n a , P inus d e n s i f l o r a and Quercus q l a u c a was found d u r i n g the w i n t e r or s p r i n g and the lowest l e v e l s o c c u r r e d d u r i n g the summer. A s i m i l a r r e s u l t was r e p o r t e d by Fukazawa et a_l. (1980) f o r s e v e r a l p i n e s . They a l s o observed a c o r r e l a t i o n i n P inus s t r o b u s i n which low amounts of sapwood l i p i d s i n September corresponded w i t h c e s s a t i o n of w a l l t h i c k e n i n g and l i g n i f i c a t i o n by ray parenchyma c e l l s . They d i d not f i n d l i p i d s at the sapwood/heartwood boundary d u r i n g any season , and suggested t h a t n e c r o s i s of ray parenchyma c e l l s may occur at any t ime . I t can be seen t h a t the g r e a t e s t amount of e x t r a c t i v e substances i s p r e s e n t i n the most r e c e n t l y formed heartwood at b r e a s t h e i g h t of a t r e e . The o r i g i n 9 of heartwood substances may be a s s o c i a t e d w i t h the l i v i n g sapwood parenchyma c e l l s . The c h e m i c a l sub-s tances w i t h i n the sapwood, such as DNA and RNA, d e c l i n e from the o u t e r sapwood towards the i n n e r sapwood. The s torage m a t e r i a l i n the sapwood, such as s t a r c h , decreases towards the boundary of sapwood and heartwood. In c o n t r a s t , the q u a n t i t y of l i p i d s i n c r e a s e s w i t h r a d i a l d i s t a n c e from the o u t e r sapwood towards the i n n e r sapwood. Seasonal and s p e c i e s v a r i a t i o n of these s t o r a g e m a t e r i a l s have been r e g u l a r l y r e p o r t e d , but d i s c r e p a n c i e s e x i s t r e g a r d i n g the seasons i n which maximum and minimum amounts are p r e s e n t , p a r t l y owing to confounding w i t h the r a d i a l p o s i t i o n i n the sapwood. 2.2 Physiology A p a r t from a r e p o r t t h a t r e s p i r a t o r y gas exchange i n R o b i n i a pseudoacac ia i s g r e a t e s t i n the i n n e r sapwood ( H o l l and L e n d z i a n 1973), r e s p i r a t i o n of the sapwood c e l l s seems to decrease g r a d u a l l y from the c a m b i a l r e g i o n toward the t r a n s i t i o n zone (Kondo 1964, H i g u c h i e t a_l. 1967) . Enzymat ic a c t i v i t i e s , which 10 i n c l u d e h y d r o l a s e s , o x i d a s e s ( H i g u c h i e_b a_l. 1969) , l i p a s e s , amylases (Sameshima 1967) i n C r y p t o m e r i a  j a p o n i c a , and g l u c o s e - 6 - p h o s p h a t e dehydrogenase i n P inus r a d i a t a (Shain and Mackay 1973a,b) , decrease from the cambium inward to the t r a n s i t i o n zone , where i t d i s p l a y s some renewed i n t e n s i t y . Z i e g l e r (1968) found t h a t i n P inus s t r o b u s the amount of w a t e r - s o l u b l e v i t a m i n s i n c r e a s e d i n the sapwood near the heartwood. P e r o x i d a s e a c t i v i t y was h i g h i n the i n n e r sapwood of E u c a l y p t u s e laeophora (Wardrop and Cronshaw 1962), but i n Quercus robur the a c t i v i t y decreased from the outer sapwood to the middle sapwood and then i n c r e a s e d i n the i n n e r sapwood (Ebermann and S t i c h 1985). E t h y l e n e i s c l o s e l y a s s o c i a t e d w i t h heartwood f o r m a t i o n , ( i . e . the f o r m a t i o n of p o l y p h e n o l s ) , i n both gymnosperm and angiosperm s p e c i e s (Shain and H i l l i s 1973, H i l l i s 1975, Ne l son 1978, Ne l son et a l . 1981) . I t was r e p o r t e d by Sha in and H i l l i s (1973) and Nelson (1978) i n P inus r a d i a t a , J u g l a n s n i g r a and Prunus  s e r o t i n a t h a t e t h y l e n e p r o d u c t i o n decreased from the outer sapwood towards the i n n e r sapwood d u r i n g the growing season . E t h y l e n e p r o d u c t i o n at the t r a n s i t i o n zone of E u c a l y p t u s t e r e t i c o r n i s was the h i g h e s t i n l a t e 11 autumn and e a r l y w i n t e r ( H i l l i s 1975) . In P inus  r a d i a t a , a s i g n i f i c a n t q u a n t i t y of e t h y l e n e was produced d u r i n g w i n t e r i n the i n n e r sapwood (Sha in and H i l l i s 1973) . F u r t h e r m o r e , the e t h y l e n e p r o d u c t i o n i n the i n n e r sapwood of J u g l a n s n i g r a reached a maximum e a r l y i n the dormant p e r i o d of t r e e growth and was low when the cambium was a c t i v e (Nelson 1978) . E t h y l e n e p r o d u c t i o n i n the i n n e r sapwood of Prunus s e r o t i n a was g r e a t e r than i n the midd le and the outer sapwood d u r i n g e a r l y dormancy and reached a h i g h l e v e l l a t e i n the growing season . The f o r m a t i o n of e t h y l e n e was g r e a t e s t d u r i n g the l a t e dormancy p e r i o d (Nelson 1978) . Ne l son et a l • (1981) found t h a t a mean e t h y l e n e p r o d u c t i o n d i f f e r e d c o n s i s t e n t l y between two p l a n t a t i o n s of J u g l a n s n i g r a under d i f f e r e n t s o i l c o n d i t i o n s . . A v a i l a b l e ev idence i n d i c a t e s t h a t a l t h o u g h m e t a b o l i c a c t i v i t y ( r e s p i r a t i o n , v a r i o u s enzyme a c t i v i t i e s and e t h y l e n e p r o d u c t i o n ) of sapwood parenchyma c e l l s decreases from the outer towards the i n n e r sapwood, a h i g h l e v e l of a c t i v i t y remains i n the i n n e r sapwood area and the amount of v i t a m i n s i s h i g h . E t h y l e n e p r o d u c t i o n and v a r i o u s enzymat ic a c t i v i t i e s a l s o appear to show s e a s o n a l v a r i a t i o n . E t h y l e n e 12 p r o d u c t i o n assumes a h i g h l e v e l at the end of the growing season and peaks during the dormant p e r i o d i n the inner sapwood. 2.3 Nuclear Morphology The change i n the nucleus of ray parenchyma c e l l s i n softwood and hardwood xylem has been s t u d i e d by many r e s e a r c h e r s . Each newly formed ray parenchyma c e l l at the cambial zone shows a square c r o s s - s e c t i o n and possesses a rounded n u c l e u s . These c e l l s elongate d u r i n g earlywood/latewood formation and t h e i r n u c l e i i n c r e a s e i n volume during c e l l d i f f e r e n t i a t i o n (Hugentobler 1965, Nobuchi 1985). Frey-Wyssling and Bosshard (1959) found th a t the n u c l e i i n the ray parenchyma c e l l s of seven c o n i f e r o u s s p e c i e s and f i v e hardwoods changed shape at the t r a n s i t i o n zone. Higuchi et a_l. (1964) reported t h a t c o n i f e r o u s ray parenchyma n u c l e i were r o d - l i k e or e l l i p s o i d , whereas those from hardwoods were f u s i f o r m . Hugentobler (1965) found that the n u c l e a r shape was more or l e s s s p e c i e s - s p e c i f i c . He a l s o found th a t elongated n u c l e i became rounded, then i r r e g u l a r , p r i o r to d i s i n t e g r a t i o n and eventual 13 d i s a p p e a r a n c e from the sapwood/heartwood b o u n d a r y . The change from round to i r r e g u l a r shape b e f o r e n u c l e a r d i s i n t e g r a t i o n at the l a s t phase of c e l l l i f e was a l s o o b s e r v e d by many o t h e r r e s e a r c h e r s ( F r e y - W y s s l i n g and Bosshard 1959, Bosshard 1965, 1968, Necesany 1968, Fukazawa and H i g u c h i 1965, Nobuchi 1985) . The n u c l e a r volume of r a y parenchyma c e l l s a l s o d e c r e a s e d g r a d u a l l y from the cambium towards the t r a n s i t i o n zone ( F r e y -W y s s l i n g and Bosshard 1959, Fukazawa and H i g u c h i 1965, B o s s h a r d 1965, 1966, 1968, H u g e n t o b l e r 1965) . F r e y - W y s s l i n g and Bosshard (1959) used the n u c l e a r s l e n d e r n e s s r a t i o (NSR) as a measure to r e l a t e c h a n g i n g c e l l a c t i v i t y w i t h n u c l e a r s i z e . The NSR i s d e f i n e d as the l e n g t h of the n u c l e u s d i v i d e d by i t s w i d t h . Many s t u d i e s have used the NSR to e v a l u a t e the a c t i v i t y o f l i v i n g sapwood r a y parenchyma c e l l s ( H i g u c h i e t a_l. 1964, 1967, Bosshard 1965, 1966, Necesany 1966, H u g e n t o b l e r 1965, S t a h e l 1968, Nobuchi et a l , 1976, N a i r et a l . 1981, N a i r and Chavan 1983) . In most c a s e s , the NSR d e c r e a s e d p r o g r e s s i v e l y from the o u t e r sapwood to the i n n e r sapwood. However, Fukazawa and H i g u c h i (1965) r e p o r t e d i n C r y p t o m e r i a  j a p o n i c a t h a t the NSR d e c r e a s e d g r a d u a l l y w i t h i n 1-3 14 growth r i n g s from the cambium, but was c o n s t a n t towards the i n n e r sapwood. Hugentob ler (1965) found t h a t . t h e NSR i n Quercus robur and Prunus avium was g r e a t e r i n the t r a n s i t i o n zone than i n the middle of the sapwood. In the procumbent ray c e l l s of B e i l s c h m i e d i a tawa the r a t i o i n c r e a s e d w i t h i n c r e a s i n g d i s t a n c e from the cambium (Bosshard 1968). A s tudy by N a i r and Chavan (1983) i n L a g e r s t r o e m i a i n d i c a a l s o showed an i n c r e a s i n g NSR from the cambium towards the i n n e r sapwood. I t i s thus not s u r p r i s i n g t h a t the use of NSR as a marker of c e l l a c t i v i t y r e q u i r e s g r e a t c a u t i o n , a l t h o u g h i t i s not c l e a r whether the u n c e r t a i n t y i s due to the d e f i c i e n c i e s i n the e x p e r i m e n t a l method or i n a fundamental f e a t u r e of the r a t i o . A new parameter , the n u c l e a r e l o n g a t i o n index ( N E I ) , was proposed by Yang (1986, 1987) i n an attempt to i n c r e a s e the a c c u r a c y of e x p r e s s i o n of c e l l v i t a l i t y . The NEI i s d e f i n e d as the l e n g t h of the nuc leus i n a ray parenchyma c e l l d i v i d e d by the l e n g t h of the ray parenchyma c e l l , m u l t i p l i e d by 100. The NEI i s d i f f e r e n t from the NSR i n t h a t i t takes i n t o account the l e n g t h of the c e l l as a f u n c t i o n of the i n d e x , whereas the NSR c o n s i d e r s on ly the s i z e and shape of the n u c l e u s . 15 R e g a r d l e s s of q u a n t i t a t i v e d i f f e r e n c e s i n the shape of the n u c l e u s , a g e n e r a l t r e n d of n u c l e a r change e x i s t s from the o u t e r sapwood towards the i n n e r sapwood. T h i s t r e n d shows a t r a n s i t i o n from an e l o n g a t e d nuc l eus i n the o u t e r sapwood to a rounded n u c l e u s i n the i n n e r sapwood. The rounded nuc l eus i n the i n n e r sapwood becomes i r r e g u l a r , and e v e n t u a l l y d i s i n t e g r a t e s and d i s a p p e a r s i n the newly formed heartwood. 2.4 Heartwood Formation 2 . 4 . 1 Survival Patterns of Ray Parenchyma Cells from Cambium to Heartwood Nobuchi e t a l . (1979) , Fukazawa e t a l . (1980) and Nobuchi (1985) r e p o r t e d t h a t i n some c o n i f e r o u s woods not a l l sapwood r a y and a x i a l parenchyma c e l l s are a l i v e . They s t u d i e d twenty c o n i f e r o u s s p e c i e s i n Japan and, based on the p o s i t i o n of the d y i n g ray parenchyma c e l l s , d e s c r i b e d three types of s u r v i v a l c u r v e s . In type I , e . g . , P inus d e n s i f l o r a and Thuj a o r i e n t a l i s , 16 a l l c e l l s s u r v i v e d from the cambium to the t r a n s i t i o n zone between sapwood and heartwood. In type I I , e . g . , C r y p t o m e r i a , Pseudotsuqa or T s u q a , some ray parenchyma c e l l s d i e d i n the middle or i n n e r sapwood and the number of dead c e l l s i n c r e a s e d towards the boundary of sapwood and heartwood. In t h i s t y p e , the m a r g i n a l c e l l s , i . e . , those on the extreme upper and lower edges of the r a y s , such as i n C r y p t o m e r i a j a p o n i c a or those i n the rows a d j a c e n t to the r a y t r a c h e i d s , such as i n Pseudotsuqa j a p o n i c a , and Tsuga s i e b o l d i i , g e n e r a l l y d i e d f i r s t . In type I I I , e . g . , Ab ie s s p . some ray parenchyma c e l l s d i e d e a r l y i n the outer sapwood, so t h a t the number of dead c e l l s i n c r e a s e d from the cambium towards the boundary of sapwood and heartwood. In t h i s t y p e , the death of ray parenchyma c e l l s proceeded from the upper or lower m a r g i n a l p o s i t i o n or from both p o s i t i o n s . In L a r i x l e p t o l e p i s and P i c e a a b i e s , the f i r s t c e l l death o c c u r r e d among those c e l l s i n c o n t a c t w i t h ray t r a c h e i d s . Yamamoto (1982) r e p o r t e d a type I s u r v i v a l curve from P i n u s s t r o b u s and P inus d e n s i f l o r a and a type II s u r v i v a l curve from P inus b a n k s i a n a . Nobuchi et al_. (1987b) r e p o r t e d t h a t most hardwoods 17 showed a type I s u r v i v a l c u r v e . However, they a l s o found i n C e r e i d i p h y H u m japonicum t h a t a l a r g e number of dead c e l l s o c c u r r e d immediate ly a d j a c e n t to the cambium. The s u r v i v a l p a t t e r n s d i s c u s s e d above are c h a r a c t e r i s t i c f e a t u r e s of a g i n g ( Z i e g l e r 1968, Thimann 1980, Bowen and L o c k s h i n 1981, Karp 1984). Nobuchi et al. (1979) and Nobuchi (1985) a l s o observed i n some s p e c i e s , e . g . , hard p i n e s , t h a t death of ray parenchyma s t a r t e d i n the upper and lower m a r g i n a l c e l l s of the r a y s . T h i s was s i m i l a r to both the l o c a t i o n of c r y s t a l s i n the genus A b i e s (Greggus 1955, Kennedy et al. 1968) , and the c h r o n o l o g y of m a t u r a t i o n of ray c e l l s i n some hard p i n e s ( B a l a t i n e c z and Kennedy 1967). Nobuchi e t a l . (1987b) r e p o r t e d a s t r o n g c o r r e l a t i o n between the number of sapwood dead c e l l s and the amount of heartwood p h e n o l s . They found t h a t the i n c r e a s e d amounts of e x t r a c t i v e s i n the i n n e r sapwood corresponded to the decreased percentage of l i v i n g sapwood ray parenchyma c e l l s . In most c o n i f e r o u s s p e c i e s (wi th the e x c e p t i o n of type I ) , i t i s apparent t h a t some ray parenchyma c e l l s d i e i n a l l sapwood r e g i o n s and thus i n c r e a s e the number 18 of dead c e l l s towards the boundary of sapwood and heartwood . In most hardwoods, many ray parenchyma c e l l s d i e i n the sapwood next to the sapwood/heartwood boundary . The p a t t e r n of c e l l death i n sapwood appears to be s p e c i e s - s p e c i f i c . In some s p e c i e s , at a g i v e n growth r i n g , the c e l l s a t the upper or lower p a r t of a r a y , or those a d j a c e n t to the ray t r a c h e i d s , d i e e a r l i e r than c e l l s i n the c e n t r a l p a r t of a r a y . 2.4.2 Timing of Heartwood Formation The t ime when heartwood f o r m a t i o n beg ins can p r o v i d e v i t a l i n f o r m a t i o n f o r u n d e r s t a n d i n g t h i s i n t r i c a t e p r o c e s s . T a b l e 1 i n d i c a t e s t h a t f o r m a t i o n of the secondary w a l l and l i g n i f i c a t i o n o f the ray parenchyma c e l l s c o i n c i d e w i t h the f o r m a t i o n of heartwood. ( B a l a t i n e c z and Kennedy 1967, Yamamoto e_t a1 . 1979b, Fukazawa e t al. 1980) . In P i n u s s t r o b u s Yamamoto ejt a_l. (1978) found t h a t the f o r m a t i o n of the secondary w a l l and l i g n i f i c a t i o n of e p i t h e l i a l c e l l s i n r e s i n c a n a l c o i n c i d e w i t h the f o r m a t i o n of heartwood. However, no s i n g l e c r i t e r i o n a l l o w s us to i d e n t i f y the p r e c i s e t ime of i n i t i a t i o n of heartwood f o r m a t i o n f o r TABLE 1. Periods of initiation summary. of heartwood formation in various species: a t Species Iiocation criterion Used Authors Initiation Month' Cryptomeria 1aponica Japan (Kyoto) Colouration Nobuchi (1985) July-Sept.-Mar. Juqlans niqra U.S.A. Ethylene Nelson (1978) Oct,-Nov. Juqlans niqra U.S.A. Peroxidase Nelson (1978) Aug.-Oct. Larix leptolepsis Japan (Hokkaido) Colouration Hirai (1951) July-Nov. Pinus radiata Australia Ethylene Shain & Hillis (1973 ) Dormant season Pinus radiata Australia Respiration Shain & Mackay Dormant season (1973b) Pinus strobus Japan (Hokkaido) Wall thickening/ Lignif ication Yamamoto et al. 1979b) Sept. (1978, Pinus strobus Japan (Hokkaido) Wall thickening/ Lignification Yamamoto (1982) Aug.-Oct. Pinus densiflora Japan (Hokkaido) Wall thickening/ Lignification Fukazawa et Aug.-Sept. al. (1980) Yamamoto (1982) Pinus banksiana Japan (Hokkaido) Wall thickening/ Lignification Yamamoto et Aug.-Sept. al. (1979a) Yamamoto (1982) Pinus serotina U.S.A. Ethylene Nelson (1978) July-Oct. Robinia pseudoacacia Japan (Kyoto) Colouration/ Tyloses Nobuchi et al. (1984) Nobuchi (1985) July-Sept. Robinia pseudoacacia Japan (Hokkaido) Colouration/ Tyloses Ishida et al. (1976) Aug.-Sept. 20 a l l t r e e s p e c i e s . 2.5 Ultrastructure 2 . 5 . 1 Seasonal Variation Mia (1972) s t u d i e d the u l t r a s t r u c t u r e of ray parenchyma c e l l s i n the o u t e r sapwood of Populus  t r e m u l o i d e s . He found t h a t season has an apparent e f f e c t on the c y t o p l a s m i c c o m p o s i t i o n of ray parenchyma c e l l s . In the s p r i n g , the ray parenchyma c e l l s c o n t a i n e d a dense n u c l e u s , p l a s t i d s , m i t o c h o n d r i a , a rough endoplasmic r e t i c u l u m ( E R ) , abundant r ib osomes , g o l g i b o d i e s , numerous v e s i c l e s , p h e n o l i c s u b s t a n c e s , and abundant f a t s but no s t a r c h g r a n u l e s . The abundant f a t s and p h e n o l i c subs tances remained i n the c e l l s through the second week of May, but d i s a p p e a r e d i n June and e a r l y J u l y . By m i d - J u l y when c a m b i a l a c t i v i t y had dropped s h a r p l y , f a t body a c c u m u l a t i o n r e s t a r t e d and l a s t e d u n t i l the end of O c t o b e r . In the f a l l , the ray parenchyma c e l l s were crowded w i t h g l o b u l a r f a t bod ie s 21 and the v a c u o l e s were f i l l e d w i t h abundant p h e n o l i c s u b s t a n c e s . In the w i n t e r , the ray parenchyma c e l l s r e t a i n e d the nuc leus w i t h an unusua l e l e c t r o n d e n s i t y . In these w i n t e r c e l l s , p l a s t i d s , m i t o c h o n d r i a , and ER were not c l e a r l y d i s c e r n i b l e . Tsuda (1975 a ,b) s t u d i e d the u l t r a s t r u c t u r e of the v a s c u l a r cambium and i t s d e r i v a t i v e s i n t w e n t y - e i g h t c o n i f e r o u s s p e c i e s i n J a p a n . The n u c l e u s of ray parenchyma c e l l s at the growth r i n g next to the cambium c o n t a i n e d grouped chromat in i n most of the s p e c i e s s t u d i e d and showed no seasona l d i f f e r e n c e . In P inus  d e n s i f l o r a grouped chromat in d i d not e x i s t i n the nuc l eus on A p r i l 17. P r o p l a s t i d s were p r e s e n t i n a l l s p e c i e s d u r i n g every month of specimen c o l l e c t i o n . The e x i s t e n c e of s t a r c h g r a n u l e s i n the p r o p l a s t i d s was v e r y s p e c i e s - s p e c i f i c and s u b j e c t to s e a s o n a l i n f l u e n c e Tsuda a l s o found i n some s p e c i e s , such as C r y p t o m e r i a  j a p o n i c a c o l l e c t e d on March 2 and Cepha lo taxus  h a r r i n g t o n i a c o l l e c t e d on A p r i l 15, t h a t s t a r c h g r a n u l e s appeared i n the p r o p l a s t i d s , whereas i n o t h e r s , such as Tsuqa s i e b o l d i i and Chamaecyparis  ob tusa b o t h c o l l e c t e d on A p r i l 16, no s t a r c h g r a n u l e s 22 appeared . Tsuda (1975b) found t h a t t h e r e were more l i p i d d r o p l e t s i n the ray parenchyma c e l l s o f most s p e c i e s d u r i n g the dormant p e r i o d than d u r i n g the a c t i v e p e r i o d . However, he a l s o observed t h a t Podocarpus  m a c r o p h y l l a c o l l e c t e d on March 21 showed no l i p i d d r o p l e t s i n the c e l l s . Round and e longated m i t o c h o n d r i a were p r e s e n t i n a l l s p e c i e s s t u d i e d r e g a r d l e s s of the sample c o l l e c t i o n p e r i o d . Tsuda a l s o found more e l o n g a t e d m i t o c h o n d r i a w i t h s w o l l e n c r i s t a e i n the J u l y c e l l s than i n the c e l l s observed i n o t h e r months. Tsuda (1975a) r e p o r t e d t h a t l a r g e v a c u o l e s c h a r a c t e r i z e d the ray c e l l s of P i c e a q l e h n i i c o l l e c t e d on May 8 and Ta iwan ia c r y p t o m e r i o i d e s c o l l e c t e d on J u l y 24. In c o n t r a s t , numerous s m a l l v a c u o l e s appeared i n these c e l l s d u r i n g the dormant p e r i o d of t r e e growth . The c e l l s i n P inus d e n s i f l o r a c o l l e c t e d on A p r i l 17, d u r i n g a dormant p e r i o d , showed a few l a r g e v a c u o l e s but numerous s m a l l ones . The c o n f l i c t i n g o b s e r v a t i o n s as p r e s e n t e d above might be due to s p e c i e s d i f f e r e n c e s , the p o s i t i o n of c e l l s where the s e c t i o n s were made, and the l i m i t e d number of c e l l s o b s e r v e d . The main 23 c h a r a c t e r i s t i c f e a t u r e s of a c t i v e and dormant ray parenchyma c e l l s i n the o u t e r sapwood can be summarized as f o l l o w s : An a c t i v e ray parenchyma c e l l c o n t a i n s 1 . A l a r g e n u c l e u s ; 2 . P r o p l a s t i d s w i t h or w i thout s m a l l s t a r c h g r a n u l e s ; 3 . Few l i p i d d r o p l e t s ; 4. E l o n g a t e d m i t o c h o n d r i a w i t h swo l l en c r i s t a e ; 5. A r e l a t i v e l y l a r g e v a c u o l e ; and 6. A rough endoplasmic r e t i c u l u m and numerous r ibosomes . A dormant ray parenchyma c e l l c o n t a i n s 1. A n u c l e u s as l a r g e as i n the a c t i v e p e r i o d ; 2 . P r o p l a s t i d s w i t h a few s t a r c h g r a n u l e s ; 3. Numerous l i p i d d r o p l e t s ; 4. Rounded m i t o c h o n d r i a w i t h unswol l en c r i s t a e ; 5. Some s m a l l v a c u o l e s ; and 6 . A rough endoplasmic r e t i c u l u m and numerous r i b o s o m e s . 24 2 . 5 . 2 Aging The terms ' a g i n g ' and ' senescence ' of a l i v i n g c e l l have been confused by r e s e a r c h e r s f o r a l o n g t i m e . Medawar (1957), L e o p o l d (1961) , S t r e h l e r (1977) and Thimann (1980) d e f i n e d senescence as the d e t e r i o r a t i v e p r o c e s s t h a t i s the n a t u r a l p r e l u d e to d e a t h , whereas ag ing r e f e r s to p r o c e s s e s of a c c r u i n g m a t u r i t y w i t h the passage of t i m e . These f o u r authors a l l agreed t h a t f o u r c r i t e r i a , i . e . , u n i v e r s a l i t y , i n t r i n s i c a l i t y , p r o g r e s s i v e n e s s and d e l e t e r i o u s n e s s , have to be met by a l i v i n g organism to e x p e r i e n c e a g i n g . The p r o g r e s s of a p l a n t from a s e e d l i n g to a j u v e n i l e p l a n t and then from a j u v e n i l e p l a n t to a mature p l a n t i s a t y p i c a l example of a g i n g . In c o n t r a s t , the autumnal c o l o u r i n g of l e a v e s and d ieback of s p r i n g bu lbs i n e a r l y summer are two examples of s enescence . Both Mia (1972) and P a n d a l a i e t a l . (1985) used ag ing and senescence i n t e r c h a n g e a b l y . For Populus  t r e m u l o i d e s Mia (1972) r e p o r t e d t h a t young ray parenchyma c e l l s l o c a t e d next to the cambium possessed a dense c y t o p l a s m which c o n t a i n e d s m a l l v a c u o l e s , 25 e n l a r g e d p l a s t i d s w i t h t h y l a k o i d s , l a r g e m i t o c h o n d r i a , l a r g e f a t b o d i e s , a rough endoplasmic r e t i c u l u m , even ly w e l l d i s t r i b u t e d r ibosomes and an e l o n g a t e d n u c l e u s . Old ray parenchyma c e l l s c o n t a i n e d an e l l i p t i c a l n u c l e u s , w i t h l i g h t l y - s t a i n i n g n u c l e o p l a s m , a m y l o p l a s t s w i t h two or more s t a r c h g r a n u l e s , e l o n g a t e d m i t o -c h o n d r i a w i t h l e s s prominant c r i s t a e , a smooth endo-p l a s m i c r e t i c u l u m and numerous membrane-bound v e s i c l e s between the c e l l w a l l and plasmalemma. The ray parenchyma c e l l s i n the sapwood-heartwood t r a n s i t i o n r e g i o n showed a g e n e r a l d e g e n e r a t i o n of the o r g a n e l l e s . The m i t o c h o n d r i a and the p l a s t i d s l o s t t h e i r i n t e r n a l membrane system, w h i l e the p l a s t i d s s t i l l c o n t a i n e d s t a r c h g r a n u l e s . The c y t o p l a s m c o n t a i n e d fewer r i b o s o m e s , and the o r i g i n a l l y rough endoplasmic r e t i c u l u m was smooth. The a c c u m u l a t i o n of f a t bod ie s and p h e n o l i c substances i n the v a c u o l e s c h a r a c t e r i z e d d y i n g c e l l s . P a n d a l a i et a_l. (1985)* r e p o r t e d t h a t i n most of the sapwood r a y parenchyma c e l l s of M e l i a azedarach the n u c l e u s showed even ly d i s t r i b u t e d c h r o m a t i n , whereas the n u c l e u s showed f r a g m e n t a t i o n and l o s s o f c h r o m a t i n 26 i n the d y i n g c e l l s a t the t r a n s i t i o n zone . Mia (1972) r e p o r t e d t h e r e were some p r o p l a s t i d s packed w i t h t h y l a k o i d s i n the ray parenchyma c e l l s of the o u t e r sapwood i n Populus t r e m u l o i d e s . P a n d a l a i e_t a l . (1985) found t h a t many s t a r c h g r a n u l e s i n the ray parenchyma c e l l s of the o u t e r sapwood i n M e l i a  a z e d a r a c h had deve loped c r a c k s . Whether these c r a c k s are the r e s u l t of p e n e t r a t i o n of a m y l o l y t i c enzymes as suggested by Chandorkar and Badenhuizen (1967) , or due to o t h e r causes i s s t i l l unknown. P a n d a l a i et a l . (1985) r e p o r t e d f u r t h e r t h a t no c r a c k s c o u l d be found on the s t a r c h g r a n u l e s of the r a y parenchyma c e l l s i n the i n n e r sapwood r e g i o n . H o l l and L e n d z i a n (1973) used spectrum a b s o r p t i o n to demonstrate t h a t the s t a r c h g r a n u l e s i n the outer sapwood of R o b i n i a p s e u d o a c a c i a c o n s i s t e d of amylose m o l e c u l e s . In c o n t r a s t , the s t a r c h i n the innermost sapwood was main ly composed of a m y l o p e c t i n m o l e c u l e s . P a n d a l a i et a l . (1985) r e p o r t e d i n M e l i a azedarach t h a t t h e r e were few l i p i d d r o p l e t s i n the r a y parenchyma c e l l s of the outer sapwood. On the o ther hand , M i a ' s 1972 s t u d i e s i n Populus t r e m u l o i d e s showed 27 t h a t l a r g e f a t bod ies were found f r e q u e n t l y i n the o u t e r sapwood. S t u d i e s by H o l l and P o s h e n r i e d e r (1975) i n R o b i n i a pseudoacac ia and Nobuchi (1985) i n softwoods showed t h a t more and l a r g e r l i p i d d r o p l e t s were found i n the innermost sapwood than i n the outer sapwood. P a n d a l a i et_ a_l. (1985) observed i n M e l i a  azedarach t h a t the number of l i p i d d r o p l e t s i n the i n n e r sapwood i n c r e a s e d , accompanied by a decrease i n the number of s t a r c h g r a n u l e s . T h i s i n t e r - r e l a t i o n s h i p between the q u a n t i t y of the l i p i d d r o p l e t s and s t a r c h g r a n u l e s was not observed by Mia (1972). In young ray parenchyma c e l l s of C o n v o l v u l u e  a r v e n s , m i t o c h o n d r i a were s m a l l , numerous and r o d l i k e or d u m b - b e l l i n shape , whereas i n o l d c e l l s , the m i t o c h o n d r i a were few i n number and g r a n u l a r i n shape (Andrew 1960) . In g e n e r a l , a p o s i t i v e r e l a t i o n i n p l a n t c e l l s between the number of m i t o c h o n d r i a and the l e v e l of r e s p i r a t i o n and p h o s p h o r y l a t i o n has been r e p o r t e d by many r e s e a r c h e r s (Lund et aJL. 1958, E r n s t e r 1965, S i m i n o v i t c h e t a_l. 1967, Brown and B e r t k e 1969, Buvat 1969, Loewy and S i e h e v i t z 1969, Munn 1969, Bonner 1973, Ducet and Lance 1978). In h i g h e r p l a n t s an abundance of 28 i n t e r n a l membranes was a s s o c i a t e d w i t h a h i g h r a t e of enzymat ic a c t i v i t y . D e R o b e r t i s e t a l ^ (1960) , Esau (1963) , Munn (1974), Douce (1985), P a n d a l a i et. a l . (1985) r e p o r t e d t h a t d u r i n g a u t o l y s i s of the ray parenchyma c e l l s the c r i s t a e became l e s s p r o m i n e n t . The s i z e of p l a n t m i t o c h o n d r i a v a r i e s from s p e c i e s to s p e c i e s . In g e n e r a l , they are 0.5 to 1.0 um i n d iameter and have an average l e n g t h of 3 to 4 um when e l o n g a t e d (Douce 1985) . Due to t h e i r r e l a t i v e l y l a r g e s i z e , u l t r a - s t r u c t u r a l changes of m i t o c h o n d r i a can be observed by e l e c t r o n m i c r o s c o p y . The shape of a m i t o c h o n d r i o n and s t r u c t u r e of the c r i s t a e i n h i g h e r p l a n t s are two of the most u s e f u l i n d i c a t o r s f o r a b i o l o g i s t to e v a l u a t e the s t a t u s of a l i v i n g c e l l . I t has been r e p o r t e d t h a t the plasmalemma has a rough s u r f a c e , w i t h numerous a t t a c h e d v e s i c l e s d u r i n g the phase of c e l l w a l l t h i c k e n i n g (Cronshaw 1965, Esau e_t a l . 1966, Walker and B i s a l p u t r a 1967, N o r t h c o t e 1969, E v e r t and Deshpande 1970, Cates son 1974, Chafe and Chauret 1974, B e r l y n 1979, Rao and Catesson 1987) . These v e s i c l e s e v e n t u a l l y fuse w i t h the plasmalemma and t h e i r c o n t e n t s are r e l e a s e d to 2 9 become p r e c u r s o r s of c e l l w a l l m a t e r i a l . The secondary w a l l t h i c k e n i n g o f some r a y parenchyma c e l l s of the D i p l o x y l o n p i n e s o c c u r s at the t r a n s i t i o n zone d u r i n g the t r a n s f o r m a t i o n of sapwood i n t o heartwood. The f o r m a t i o n of the secondary w a l l w i t h the f u s i o n of v e s i c l e s i n t o the plasmalemma i s one of the b e s t i n d i c a t i o n s of m a t u r i t y and impending death of these r a y parenchyma c e l l s . The o b s e r v a t i o n o f the s u r f a c e o f the plasmalemma shou ld p r o v i d e d i r e c t ev idence o f the t i m i n g of heartwood f o r m a t i o n i n the D i p l o x y l o n p i n e s . The main c h a r a c t e r i s t i c f e a t u r e s of ray parenchyma c e l l s are summarized be low. Young r a y parenchyma c e l l s c o n t a i n : 1 . A rounded nuc leus i n the c a m b i a l zone, and e l l i p t i c a l n u c l e i w i t h even ly d i s t r i b u t e d c h r o m a t i n e l sewhere; 2 . P r o p l a s t i d s packed w i t h t h y l a k o i d s i n the a c t i v e growing season; a few s m a l l s t a r c h g r a n u l e s may be p r e s e n t i n the dormant season; 30 3. Few s m a l l l i p i d d r o p l e t s i n the a c t i v e growing season and numerous i n the dormant season; 4. Numerous and r o d - l i k e or d u m b - b e l l shaped m i t o c h o n d r i a w i t h w e l l deve loped c r i s t a e i n the a c t i v e growing season and rounded o f f i n the dormant season; 5. S m a l l numerous v a c u o l e s i n the dormant s eason , l a r g e v a c u o l e s i n the a c t i v e growing season; and 6. A rough endoplasmic r e t i c u l u m and numerous r ibosomes . Old ray parenchyma c e l l s c o n t a i n : 1 . A rounded or fragmented n u c l e u s wi thout c h r o m a t i n ; 2. No p r o p l a s t i d s ; a m y l o p l a s t s packed w i t h two or more s t a r c h g r a n u l e s ; some s t a r c h g r a n u l e s w i t h c r a c k s ; 3. More and l a r g e r l i p i d d r o p l e t s ; 4. Few and rounded m i t o c h o n d r i a w i t h l e s s prominent c r i s t a e ; 5. V a r i o u s s i z e d v a c u o l e s p o s s e s s i n g p h e n o l i c s u b s t a n c e s ; and 6. A smooth endoplasmic r e t i c u l u m and l e s s r i b o s o m e s . 31 3. OBJECTIVES AND NOVELTY 3.1 OBJECTIVES The c o n f l i c t i n g o b s e r v a t i o n s r e g a r d i n g the ag ing of l i v i n g c e l l s and subsequent heartwood f o r m a t i o n may be a t t r i b u t e d to the d i f f e r e n c e s between s p e c i e s , c e l l t y p e , l o c a t i o n , season and the methodology employed by v a r i o u s i n v e s t i g a t o r s . L i m i t e d sampl ing and d i f f e r e n t c r i t e r i a f o r ag ing have a l s o c o n t r i b u t e d to these c o n f l i c t i n g o b s e r v a t i o n s . A more d e t a i l e d and s y s t e m a t i c e x p e r i m e n t a l approach i s needed to c l a r i f y some of the c o n t r a d i c t i o n s and to p r o v i d e a b e t t e r u n d e r s t a n d i n g of the a g i n g of r a y parenchyma c e l l s and i t s r o l e i n heartwood f o r m a t i o n . T h e r e f o r e , the e x p e r i m e n t a l work i n t h i s t h e s i s has been des igned t o : 1. D e f i n e the boundary of sapwood and heartwood by u s i n g v a r i o u s c r i t e r i a , such as the d i s t r i b u t i o n of m o i s t u r e content i n sapwood and heartwood and the s u r v i v a l of sapwood r a y parenchyma c e l l s ; 32 2. Study ag ing of r a y parenchyma c e l l s from the young sapwood to r e c e n t l y formed heartwood, as a f u n c t i o n of s p e c i e s , s eason , r a d i a l l o c a t i o n of c e l l s w i t h i n sapwood, and c e l l l o c a t i o n w i t h i n a r a y ; 3. E v a l u a t e ag ing of ray parenchyma c e l l s u s i n g a number o f v a r i a b l e s such as an index of n u c l e a r e l o n g a t i o n , s t a r c h and l i p i d c o n t e n t , and o r g a n e l l e u l t r a s t r u c t u r e , and to i n v e s t i g a t e the c o r r e l a t i o n among these v a r i a b l e s ; and 4. Draw i n f e r e n c e s r e g a r d i n g the sapwood/heartwood t r a n s f o r m a t i o n from the v a r i e t y of events observed r e l a t e d to a g i n g . 3.2 NOVELTY The n o v e l t y of t h i s s tudy i s t h a t q u a n t i t a t i v e and q u a l i t a t i v e o b s e r v a t i o n s on the s t a t u s of l i v i n g c e l l s were c a r r i e d out j o i n t l y w i t h 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 . The s tudy was s y s t e m a t i c a l l y des igned to examine ray c e l l s v a r y i n g i n t h e i r r a d i a l p o s i t i o n from cambium to heartwood and a l s o v a r y i n g i n 33 v e r t i c a l p o s i t i o n w i t h i n a g i v e n r a y . In o r d e r to i n v e s t i g a t e the i n f l u e n c e of season and d i f f e r e n c e due to s p e c i e s , four s p e c i e s were sampled at v a r i o u s t imes over a p e r i o d of e i g h t months, embracing both a c t i v e and e a r l y dormant p e r i o d s of t r e e growth . The s tudy a l s o i n c l u d e d the i n t e r r e l a t i o n s h i p s between v a r i o u s parameters to a l l o w i n f e r e n c e s to be made c o n c e r n i n g the phenomena of sapwood/heartwood t r a n s f o r m a t i o n . 34 4 . GENERAL DESCRIPTION OF SPECIMENS Four s p e c i e s , P inus b a n k s i a n a L a m b . , P i c e a mariana ( M i l l . ) B . S . P . , Ab ie s balsamea ( L . ) M i l l , and Populus  t r e m u l o i d e s M i c h x . growing at the Lakehead U n i v e r s i t y wood lo t , 30 km n o r t h of Thunder Bay, O n t a r i o were chosen f o r t h i s s t u d y . The c o l o u r of heartwood zone, ray s t r u c t u r e and a v a i l a b i l i t y o f sample t r e e s were used as c r i t e r i a of s p e c i e s s e l e c t i o n . One t r e e of each s p e c i e s was sampled. Some of the a n a t o m i c a l f e a t u r e s comparing the ray s t r u c t u r e of these s p e c i e s are l i s t e d i n T a b l e 2. For m i c r o s c o p i c s t u d i e s , a 12 mm increment core was e x t r a c t e d at b r e a s t h e i g h t from the south-west aspec t of a t r e e f r e e of h e a r t r o t . S p e c i a l c a r e was taken to a v o i d "wetwood" i n Populus t r e m u l o i d e s . The t r e e s e l e c t i o n was observed to have no d a r k - c o l o u r e d heartwood which i s o f t e n a s s o c i a t e d w i t h wetwood. The core i n c l u d e d sapwood and heartwood. To min imize the i n f l u e n c e o f p r e v i o u s c o r e e x t r a c t i o n s , each s u c c e s s i v e core was taken 15 cm d i a g o n a l l y away from the p r e v i o u s one. In t h i s manner, a comparat ive s tudy of two Table 2. Some anatomical features of the four species studied. Species Feature Pinus banksiana Picea mariana Abies balsamea Populus tremuloides Non-porous wood X X X Diffuse porous wood X Fusiform rays X X Uniseriate rays X X X X Ray tracheids present X X Dentate ray tracheids X Secondary wall thickening and l ign i f i ca t ion of sapwood parenchyma ce l l s d irect ly behind cambium X X X (Jl 36 a d j a c e n t sampl ing p e r i o d s c o u l d be r e a s o n a b l y c a r r i e d out to l i m i t the v a r i a t i o n due to a s p e c t . The l o c a t i o n and sequence of the specimen e x t r a c t i o n s at v a r i o u s sampl ing p e r i o d s i s i l l u s t r a t e d i n F i g u r e 1. The core was f i x e d immediate ly i n a 3% g l u t a r a l d e h y d e s o l u t i o n . The date of c o l l e c t i o n and the s i z e and age of the sampled t r e e of each s p e c i e s are l i s t e d i n T a b l e 3. These g l u t a r a l d e h y d e - f i x e d specimens were used 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 i c s t u d i e s . 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 were measured or observed i n each of the f o u r s p e c i e s s t u d i e d : 1. M o i s t u r e content (MC) of sapwood and heartwood; 2. S u r v i v a l r a t e (SR) of r a y parenchyma c e l l s ; 3. N u c l e a r e l o n g a t i o n index (NEI) ; 4. R e l a t i v e s t a r c h content (RSC); 5. R e l a t i v e l i p i d c o n t e n t (RLC); and 6. U l t r a s t r u c t u r e of c e l l o r g a n e l l e s . The main o b j e c t i v e s of the s tudy were to e v a l u a t e the impact of age and season on the a c t i v i t y and s t r u c t u r e of sapwood ray parenchyma c e l l s and Figure 1. The locations and sequence of specimen extractions at various sampling periods in the four species studied. Note: The number in the centre indicates the age of the tree at breast height. The numbers outside the sapwood zone (shaded area) indicate the sapwood thickness expressed as the number of growth r ings . Sapwood was defined as those rings containing at least some nuclei in ray parenchyma c e l l s . The cores removed from the north aspect were taken for moisture content determinations. 1. Pinus banksiana, 2. Picea mariana , 3. Abies  balsamea and 4. Populus tremuloides. M-May, Ju-June, J - J u l y , A-August, 0-October, N-November and D-December. Table 3. Species, age, height and diameter of sampled trees and the dates of collection for microscopic studies. Age at Breast Tree Diameter Inside Date of Collection Species Height (Years) Height (m) Bark (cm) a t B H M J u J A O N D Year Pinus 59 22.1 21.6 25 13 20 23 2 2 1983 banksiana Picea 64 19.8 20.2 25 13 20 23 2 2 1983 mariana Abies 65 21.7 18.5 28 25 23 27 3 20 1984 balsamea Populus 46 • 24.3 19.4 28 25 23 27 3 20 1984 tremuloides 39 d i f f e r e n c e s due to the l o c a t i o n of ray parenchyma c e l l s w i t h i n sapwood. No at tempts were made to measure or e v a l u a t e the v a r i a t i o n due to d i f f e r e n t t r e e s i n a g i v e n s p e c i e s . The s tudy was c a r r i e d out at one h e i g h t on a s i n g l e t r e e each of f o u r s p e c i e s . A S t u d e n t ' s t - t e s t was employed to e v a l u a t e the d i f f e r e n c e between the v a r i o u s v a r i a b l e mean of two types of c e l l s or two sampl ing p e r i o d s . An F - t e s t which e s t i m a t e s the homogeneity of the v a r i a n c e was c a r r i e d out p r i o r to the t - t e s t . No t - t e s t s were conducted , i f the F - t e s t i n d i c a t e d a s i g n i f i c a n t d i f f e r e n c e between the v a r i a n c e s . 40 5. STUDIES 5.1 Sapwood and Heartwood as Determined by M o i s t u r e Content V a r i a t i o n . 5 . 1 . 1 . Sampling and Methods The moi s ture content (MC) of sapwood and heartwood was determined from the same t r e e s as those sampled 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 i c s t u d i e s . A 12 mm increment core was e x t r a c t e d from the n o r t h aspec t of each t r e e at b r e a s t h e i g h t . Two c o l l e c t i o n s were made, one i n May and the o ther i n J u l y . The specimens f o r P inus b a n k s i a n a and P i c e a mariana were c o l l e c t e d on May 30 and J u l y 25, 1983 r e s p e c t i v e l y , whereas the specimens f o r A b i e s balsamea and Populus t r e m u l o i d e s were c o l l e c t e d on May 31 and J u l y 26, 1984 r e s p e c t i v e l y . Each core was wrapped w i t h Saran wrap (The Dow C h e m i c a l C o . ) to p r e v e n t m o i s t u r e l o s s d u r i n g t r a n s -p o r t a t i o n . The core was d i s s e c t e d i n t o i t s i n d i v i d u a l annual increments i n the l a b o r a t o r y . C a u t i o n was taken w h i l e each wood c h i p was weighed immediate ly r i g h t 41 a f t e r each c u t t i n g to prevent moi s ture l o s s . Meanwhile the c o r e was always c o v e r e d by Saran wrap. The green and o v e n - d r y weight of each r i n g counted from the cambium was determined on an a n a l y t i c a l b a l a n c e w i t h an a c c u r a c y of + / - 0.001 g . The o v e n - d r y weights o f the specimens were o b t a i n e d when the specimens reached c o n s t a n t w e i g h t . U s u a l l y , i t took two days i n an oven at a temperature of 100 + / - 3 ° C to o b t a i n the oven-dry w e i g h t . The MC of each growth r i n g was expres sed as a percentage of oven-dry w e i g h t . The MC of the i n d i v i d u a l r i n g s i n the May and J u l y specimens was p l o t t e d s e p a r a t e l y a g a i n s t the growth r i n g number counted from the cambium ( F i g u r e 2 ) . The boundary between sapwood and heartwood was determined as the p o i n t where the curve changed a b r u p t l y on the g r a p h s . The average MC of the sapwood and heartwood was then c a l c u l a t e d on the b a s i s of t h i s d i s t i n c t i o n (Table 4 ) . A S t u d e n t ' s t - t e s t of the mean d i f f e r e n c e i n MC between the May and J u l y c o l l e c t i o n s was conducted s e p a r a t e l y f o r each s p e c i e s (Tab le 4 ) . The d i s t r i b u t i o n of MC d i c t a t e d the demarcat ion between sapwood and heartwood . T h i s d e l i n e a t i o n was 180 §" Numb«r ol ftlngt CaunUd Irom Uw Cambium 0 S 10 15 20 25 30 35 40 45 50 55 60 65 Numbtr el Rlngi Countad tram the C*mblum Figure 2. The r a d i a l d i s tr ibut ion of May (A) and July (©) moisture content (%) in sapwoods and heartwoods. 1. Pinus banksiana and 2. Picea mariana. S/H - sapwood and heartwood boundary. N u m b « r o l R i n g * C a u n t i d f rom I n * C i m b l u m Figure 2. (continued). 3. Abies balsamea and 4. Populus tremuloides. Table 4. A t-test of the mean difference in moisture content between May and July in sapwood and heartwood of four species studied. Sapwood Heartwood Mean % Mean %  Species May July t May July t Pinus banksiana 127 120 1.168 ns 30 41 2.390** Picea mariana 153 104 3.557 ** 36 39 1.024ns Abies balsamea 147 141 0.984 ns 41 33 7.353** Populus 95 75 5.987 ** 168 140 6.500** tremuloides ns not s ignif icant a t « o 0.05 ** Significant at<*< 0.01 45 used as a benchmark f o r comparison of a n a t o m i c a l o b s e r v a t i o n s which w i l l be d i s c u s s e d l a t e r . 5 .1 .2 Results 5 . 1 . 2 . 1 Pinus banksiana The MC from the cambium inwards i n P inus banks iana i s shown i n F i g u r e 2 -1 . The average MC of sapwood i n May and J u l y was 127% and 120%, r e s p e c t i v e l y . In c o n t r a s t , the average MC of heartwood i n May and J u l y was 30% and 41% r e s p e c t i v e l y (Tab le 4 ) . A sharp change i n MC, i n both the May and J u l y specimens was found between r i n g s t w e n t y - t h r e e and t w e n t y - s e v e n . The number of r i n g s were counted from the cambium. There was no s i g n i f i c a n t d i f f e r e n c e i n sapwood MC between May and J u l y , whereas the heartwood MC of the May specimen was s i g n i f i c a n t l y lower than t h a t of J u l y (Table 4 ) . 46 5 . 1 . 2 . 2 Picea mariana The MC a c r o s s a r a d i u s of P i c e a mariana i s shown i n F i g u r e 2 -2 . The average MC of sapwood i n May and J u l y was 153% and 104% r e s p e c t i v e l y . In c o n t r a s t , the average MC of heartwood i n May and J u l y was 36% and 39% r e s p e c t i v e l y (Table 4 ) . A sharp change i n MC, i n b o t h May and J u l y was found between r i n g s e i g h t and e l e v e n (counted from the cambium). There was a s i g n i f i c a n t l y h i g h e r sapwood MC i n May than i n J u l y , but i n heartwood, t h e r e was no s i g n i f i c a n t d i f f e r e n c e between the two months. 5 . 1 . 2 . 3 Abies balsamea The MC a c r o s s a r a d i u s of Ab ie s balsamea i s shown i n F i g u r e 2 -3 . The average MC of sapwood i n May and J u l y was 147% and 141% r e s p e c t i v e l y . In c o n t r a s t , the average MC of heartwood i n May and J u l y was 41% and 33% r e s p e c t i v e l y (Table 4 ) . A sharp change i n MC was found between r i n g s f i f t e e n and e i g h t e e n . There was no s i g n i f i c a n t 47 d i f f e r e n c e i n sapwood MC between May and J u l y . In c o n t r a s t , the MC of heartwood i n May was s i g n i f i c a n t l y h i g h e r than t h a t of J u l y (Tab le 4 ) . 5 . 1 . 2 . 4 Populus tremuloides The MC a c r o s s a r a d i u s of Populus t r e m u l o i d e s i s shown i n F i g u r e 2-4 . In c o n t r a s t to the c o n i f e r s , the MC of sapwood, both i n May and J u l y , was lower than t h a t of heartwood. The average MC of sapwood i n May and J u l y was 95% and 75% r e s p e c t i v e l y , whereas the average MC of heartwood was 168% i n May and 140% i n J u l y . (Tab le 4 ) . A sharp MC i n c r e a s e was found from r i n g s twenty to t w e n t y - f o u r . Both sapwood and heartwood had a s i g n i f i c a n t l y h i g h e r MC i n May than i n J u l y . 5 . 1 . 3 Discussion The MC of the sapwood zone was h i g h e r than t h a t of the heartwood zone i n the s i n g l e samples o f the t h r e e c o n i f e r o u s s p e c i e s s t u d i e d . The r e v e r s e was t r u e f o r Populus t r e m u l o i d e s ( F i g u r e 2 ) . T h i s p a t t e r n of 48 m o i s t u r e d i s t r i b u t i o n i n these t h r e e c o n i f e r o u s s p e c i e s was s i m i l a r to t h a t found i n o t h e r c o n i f e r s ( F i e l d i n g 1952, I to 1953, Gibbs 1953, 1958, Yerkes 1967, Z o b e l et a l . 1968, Yamamoto 1982, Nobuchi 1985). The h i g h e r MC of heartwood i n Populus t r e m u l o i d e s was a l s o r e p o r t e d p r e v i o u s l y (Gibbs 1935, Jensen and Dav i s 1953, Yazawa et a l . 1965, Sauter 1966b, S t a h e l 1968) . Gibbs (1953) r e p o r t e d t h a t the MC of the outer sapwood i n P inus b a n k s i a n a was h i g h e r i n J u l y than i n September or May. On the o t h e r hand , Yamamoto (1982) r e p o r t e d t h a t the lowest MC of the outer sapwood i n the same s p e c i e s was found i n August and September. She suggested t h a t the s e a s o n a l l y low MC i n August and September was p o s s i b l y connected w i t h the p r o g r e s s i o n of heartwood f o r m a t i o n . Gibbs (1953) r e p o r t e d t h a t the MC of the o u t e r sapwood i n A b i e s balsamea was h i g h e r i n August than i n o t h e r months. Gibbs (1953) , Yamamoto (1982) and Nobuchi and Harada (1983) found t h a t there was no s e a s o n a l v a r i a t i o n of MC i n c o n i f e r heartwood. As p r e s e n t e d i n T a b l e 4, the average MC of sapwood i n a s i n g l e t r e e of the t h r e e c o n i f e r o u s s p e c i e s s t u d i e d was h i g h e r i n May than i n J u l y , but o n l y P i c e a mariana showed a s i g n i f i c a n t d i f f e r e n c e between these two 49 months. The h i g h MC of sapwood i n May i s i n agreement w i t h t h a t r e p o r t e d by Yamamoto (1982), but d i f f e r s from the f i n d i n g s by Gibbs (1953) . Jensen and Dav i s (1953) r e p o r t e d f o r Populus  t r e m u l o i d e s t h a t the whole t r e e MC was low d u r i n g , w i n t e r and h i g h d u r i n g summer, w i t h the months of May and October as t r a n s i t i o n a l p e r i o d s . Gibbs (1935, 1953) i n d i c a t e d t h a t the MC of whole t r e e s of Populus  t r e m u l o i d e s i n May and September was 125% and 66% r e s p e c t i v e l y . A h i g h e r MC i n b o t h sapwood and heartwood of Populus t r e m u l o i d e s was r e c o r d e d i n May than i n J u l y i n t h i s s t u d y . In o t h e r hardwood s p e c i e s , such as Fagus c r e n a t a (Yazawa 1960) , L i r i o d e n d r o n t u l i p i f e r a ( P h i l l i p s and Schroeder 1973) and C a r y a l a c i n i o s a (Smith and Goebel 1952) , s e a s o n a l v a r i a t i o n of MC was d i f f e r e n t from t h a t o f Populus t r e m u l o i d e s . In t h i s s t u d y , the sapwood MC of s i n g l e t r e e s of t h r e e c o n i f e r o u s s p e c i e s was h i g h e r than t h a t of the heartwood, whereas i n Populus t r e m u l o i d e s the h i g h e r MC was i n the heartwood. 50 5.2 Survival Rate 5 . 2 . 1 Sampling and Methods The g l u t a r a l d e h y d e - f i x e d cores were marked and s l i c e d i n t o s u c c e s s i v e wood b l o c k s seven to ten m i l l i m e t r e s wide from the cambium inwards to the heartwood near the p i t h . T h i r t y - s i x or more r a d i a l s e c t i o n s , 20 um t h i c k , were cut from these wood b l o c k s u s i n g a s l edge microtome. O n e - t h i r d of . the s e c t i o n s were s t a i n e d w i t h h e m a t o x y l i n , to emphasize the n u c l e u s of the r a y parenchyma c e l l s (Gray 1964). Dehydrated and permanent s l i d e s were p r e p a r e d f o r use i n subsequent n u c l e a r morphology s t u d i e s , which i n c l u d e d measurements of s u r v i v a l r a t e (SR) and the n u c l e a r e l o n g a t i o n . The u n s t a i n e d r a d i a l s e c t i o n s were used to s tudy s t a r c h and l i p i d c o n t e n t s . The specimens c o l l e c t e d i n May f o r n u c l e a r morphology s t u d i e s were used to measure the SR. The SR of ray parenchyma c e l l s w i t h i n a g i v e n growth r i n g i s d e f i n e d as the number o f l i v i n g ear lywood ray parenchyma c e l l s i n u n i s e r i a t e r a y s , d i v i d e d by the t o t a l number of dead and l i v i n g ray 51 parenchyma c e l l s r e c o r d e d , m u l t i p l i e d by 100. The number of c e l l s observed on the s l i d e s depended on the t o t a l number of r a y s i n the growth r i n g . In g e n e r a l , the number of c e l l s ranged from 150 to 300 per growth r i n g i n a s e c t i o n . The s u r v i v a l of the m a r g i n a l and c e n t r a l c e l l s of a ray was r e c o r d e d s e p a r a t e l y . I f ray t r a c h e i d s were p r e s e n t , the m a r g i n a l c e l l o f a u n i s e r i a t e ray was d e f i n e d as the ray parenchyma c e l l s f a r t h e s t from the c e n t r e of a r a y . The c e n t r a l c e l l s of a u n i s e r i a t e ray more than three c e l l s h i g h , were d e f i n e d as a l l of the ray parenchyma c e l l s except the m a r g i n a l c e l l s i n a r a y . L i v i n g and dead ray parenchyma c e l l s were determined on the b a s i s of the presence or absence of the c e l l nuc l eus and c y t o p l a s m . In P i n u s b a n k s i a n a the f o r m a t i o n of the secondary w a l l i n some ray parenchyma c e l l s was a f u r t h e r a i d i n j u d g i n g dead c e l l s . The percentages of l i v i n g m a r g i n a l and c e n t r a l ray parenchyma c e l l s were p l o t t e d a g a i n s t the sapwood growth r i n g number from the cambium towards the heartwood ( F i g u r e 3 ) . P a i r e d comparison S t u d e n t ' s t - t e s t s between the m a r g i n a l and c e n t r a l c e l l s o f a ray were conducted f o r the f o u r s p e c i e s s t u d i e d ( T a b l e 5 ) . 52 CC a > IE 3 CO 2 4 6 8 10 12 14 16 18 20 22 24 26 N u m b e r of Rings C o u n t e d from the C a m b i u m "3 <r "ra > 3 to S/H. 4 6 8 10 12 14 16 18 20 22 24 26 N u m b e r of Rings C o u n t e d from the C a m b i u m Figure 3. The survival rate of ray parenchyma c e l l s expressed as the percentage of l i v i n g sapwood ray parenchyma c e l l s in the marginal (•) and central ( A ) positions in May. 1. Pinus banksiana, 2. Picea mariana. S/H - sapwood and heartwood boundary. 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Number of Rings Counted from Ihe Cambium (continued) 3. Abies balsamea and 4. Populus tremuloides. Table 5. A t-test of the mean difference in survival rate between the marginal and central cells. Species Mean % Marginal Central t-value Calculated Table Pinus banksiana 82.4 89.5 3.677** 2.492 Picea mariana 66.8 83.8 4.073** 2.624 Abies balsamea 55.0 89.0 5.905** 2.764 Populus tremuloides 77.3 82.9 6.485** 2.519 ** Significant at ± 0.01 55 5 .2 .2 Results 5 . 2 . 2 . 1 Pinus banksiana The death of the m a r g i n a l and c e n t r a l c e l l s began r e s p e c t i v e l y at the seventh and t e n t h growth r i n g counted from the cambium ( F i g u r e 3-1 and T a b l e 6 ) . The SR of bo th c e l l groups decreased c u r v i l i n e a r l y from the middle sapwood towards the sapwood/heartwood boundary and dropped s h a r p l y from about the s i x t e e n t h growth r i n g (counted from the cambium) inward ( F i g u r e 3 - 1 ) . There was a p a r t i c u l a r l y major drop i n SR d u r i n g the f i n a l two y e a r s , which i n d i c a t e d a r e l a t i v e l y abrupt change from sapwood to heartwood . P r i o r to these f i n a l two y e a r s , more than s i x t y per cent o f the r a y parenchyma c e l l s were s t i l l l i v i n g . The r e s u l t s of a S t u d e n t ' s t - t e s t ( T a b l e 5) show t h a t the SR of the m a r g i n a l c e l l s i s s i g n i f i c a n t l y lower than t h a t of c e n t r a l c e l l s . Table 6. The number of growth rings counted from the cambium marking first observance of death and last observance of life in the ray parenchyma cells. Marginal Cells Central Cells  SPECIES Dead Cell Living Cell Dead Cell Living Cell First Seen Last Seen First Seen Last Seen Pinus banksiana 7 25 10 25 Picea mariana 4 15 8 17 Abies balsamea 2 11 3 16 Populus tremuloides 5 23 5 24 57 5 . 2 . 2 . 2 Picea mariana ; The death of the m a r g i n a l and c e n t r a l c e l l s began r e s p e c t i v e l y at the f o u r t h and e i g h t h growth r i n g ^counted from the cambium) (Table 6 ) . The SR of both c e l l groups dropped markedly a f t e r the n i n t h and t w e l f t h growth r i n g ( F i g u r e 3 - 2 ) . T a b l e 5 i n d i c a t e s t h a t the SR of the c e n t r a l c e l l s was s i g n i f i c a n t l y g r e a t e r than t h a t of the m a r g i n a l c e l l s . T h i s d i f f e r e n c e was most s t r i k i n g between the t e n t h and t h i r t e e n t h r i n g ( F i g u r e 3 - 2 ) . In the m a r g i n a l p o s i t i o n , the l a s t l i v i n g ray parenchyma c e l l s were found i n the f i f t e e n t h growth r i n g counted from the cambium, w h i l e i n the c e n t r a l p o s i t i o n the l a s t l i v i n g c e l l s were found i n the seventeenth r i n g . 5 . 2 . 2 . 3 Abies balsamea The death of the m a r g i n a l and c e n t r a l c e l l s began r e s p e c t i v e l y at the second and t h i r d growth r i n g (Table 6 ) . The SR of m a r g i n a l c e l l s was a p p r o x i m a t e l y 80% from the second to the s i x t h growth r i n g , a f t e r which i t 58 dropped s h a r p l y ( F i g u r e 3 - 3 ) . In the c e n t r a l c e l l s the SR was almost 100 per cent u n t i l the s i x t h growth r i n g , a f t e r which there was a sharp d e c l i n e . The SR of the c e n t r a l c e l l s aga in was s i g n i f i c a n t l y g r e a t e r than t h a t of the m a r g i n a l c e l l s (Tab le 5 ) . The l a s t l i v i n g m a r g i n a l c e l l s were found at the e l e v e n t h r i n g , w h i l e i n the c e n t r a l c e l l s t h i s o c c u r r e d at the s i x t e e n t h r i n g . 5 . 2 . 2 . 4 Populus tremuloides The onset of death of the m a r g i n a l and c e n t r a l c e l l s was at the f i f t h growth r i n g counted from the cambium f o r both p o s i t i o n s of ray c e l l s (Tab le 6). The SR of bo th groups of ray c e l l s decreased v e r y s l owly from the f i f t h to the f i f t e e n t h growth r i n g , a f t e r which t h e r e was a r e l a t i v e l y sharp drop ( F i g u r e 3 - 4 ) . As i n P inus b a n k s i a n a , more than h a l f of the c e l l s remained a l i v e u n t i l the f i n a l two y e a r s , i n d i c a t i n g a r e l a t i v e l y abrupt sapwood-heartwood t r a n s i t i o n . As w i t h the o ther s p e c i e s , the SR of the c e n t r a l c e l l s was s i g n i f i c a n t l y g r e a t e r than t h a t on the margin (Table 5 ) . 59 5 .2 .3 Discussion As presented i n T a b l e 6, the onset of death i n ray parenchyma c e l l s v a r i e d w i t h s p e c i e s and the p o s i t i o n of the c e l l i n a r a y . The death of m a r g i n a l c e l l s o c c u r r e d e a r l i e r than t h a t o f c e n t r a l c e l l s . T h i s was e s p e c i a l l y pronounced i n Abies balsamea whose m a r g i n a l c e l l s had the s h o r t e s t l i f e span of any s p e c i e s i n the s t u d y . T h i s s t r i k i n g l y e a r l y death of m a r g i n a l c e l l s might be r e l a t e d to the absence of ray t r a c h e i d s i n Ab ie s ba lsamea, whose r a d i a l c o n d u c t i o n f u n c t i o n c o u l d be assumed by the dead m a r g i n a l c e l l s . The e a r l y death of m a r g i n a l ray parenchyma c e l l s was a l s o r e p o r t e d by B a l a t i n e c z and Kennedy (1967) and Yamamoto e_t a_l. (1979a ,b) i n hard p ines and by Kennedy et a_l. (1968) i n the genus A b i e s . Nobuchi et a_l. (1979) and Nobuchi (1985) r e p o r t e d t h a t t h i r t e e n out of twenty c o n i f e r o u s s p e c i e s showed a s i m i l a r e a r l y death p a t t e r n as mentioned above. Nobuchi (1985) f u r t h e r i n d i c a t e d t h a t t h i s p a t t e r n of death was o n l y observed i n those s p e c i e s i n which some of the ray parenchyma c e l l s d i e i n the middle or outer sapwood. T h i s d i d not occur i n 600 s p e c i e s i n which a l l of the ray parenchyma c e l l s d i e s i m u l t a n e o u s l y at the boundary of sapwood and heartwood, such as i n P inus d e n s i f l o r a , P inus p e n t a p h y l l a , Taxus  c u s p i d a t a , T h u j o p s i s d o l a b r a t a and J u n i p e r u s r i q i d a . Nobuchi (1985) found s e v e r a l o ther p a t t e r n s of death and s u r v i v a l of sapwood r a y parenchyma c e l l s . For example, i n S c i a d o p i t y s v e r t i c i l l a t a the death of ray parenchyma c e l l s commenced at the c e n t r a l p a r t of a r a y . In wide sapwood s p e c i e s , such as Podocarpus  m a c r o p h y l l a , Podocarpus n a q i and Cephalo taxus h a r r i n q t o n i a , a l l ray parenchyma c e l l s were s t i l l a l i v e even a f t e r the t r e e reached a d iameter of 20 cm. As shown i n F i g u r e 3, the SR of l i v i n g sapwood ray parenchyma c e l l s i n the f o u r s p e c i e s s t u d i e d decreased w i t h c h r o n o l o g i c a l age, or w i t h the number of r i n g s counted from the cambium. T h i s p a t t e r n of SR i s one of the c h a r a c t e r i s t i c f e a t u r e s of ag ing (Medawar 1957, L e o p o l d 1961, S t r e h l e r 1977, Thimann 1980). As i l l u s t r a t e d i n F i g u r e s 3-1 and 3-4 , the death of the m a j o r i t y of ray parenchyma c e l l s o c c u r r e d s h a r p l y i n the l a s t few growth r i n g s near the sapwood/heartwood boundary i n Pinus b a n k s i a n a and 61 Populus t r e m u l o i d e s . In c o n t r a s t , the death of ray parenchyma c e l l s i n c r e a s e d g r a d u a l l y from the outer or middle sapwood towards the sapwood/heartwood boundary i n P i c e a mariana and A b i e s balsamea ( F i g u r e s 3-2 and 3-3 ) . In P inus banks iana the death p a t t e r n of ray parenchyma c e l l s i s the same as t h a t r e p o r t e d by Nobuchi et a l . (1979), Fukazawa et a l . (1980) , Yamamoto (1982) and Nobuchi (1985) f o r a type II s p e c i e s . However, m a r g i n a l c e l l s d i e d e a r l i e r than c e n t r a l c e l l s , an o b s e r v a t i o n not r e p o r t e d e i t h e r by Nobuchi et a l . (1979) or Yamamoto (1982) . The death p a t t e r n of P i c e a mariana i s s i m i l a r to t h a t of P i c e a a b i e s , a type I I I s p e c i e s i n which some ray parenchyma c e l l s d i e d e a r l y i n the outer sapwood (Nobuchi 1985) . The death p a t t e r n of Ab ie s balsamea a l s o corresponds to a type I I I s p e c i e s , such as A b i e s s a c h a l i n e n s i s (Nobuchi et a l . 1979) . The e a r l y death and s h o r t l i f e span of m a r g i n a l c e l l s i n A b i e s balsamea c h a r a c t e r i z e t h i s s p e c i e s . These s u r v i v a l p a t t e r n s ( type I , II and I I I ) have been used to d e s c r i b e death of ray parenchyma c e l l s i n c o n i f e r o u s s p e c i e s , but they seem a p p l i c a b l e to 62 ang iosperms . The death p a t t e r n i n Populus t r e m u l o i d e s i s s i m i l a r to t h a t of a type II s p e c i e s i n which most ray parenchyma c e l l s do not d i e u n t i l the outer sapwood /heartwood boundary i s approached . Nobuchi (1985) suggested t h a t the death p a t t e r n of ray parenchyma c e l l s i s r e l a t e d to the c l a r i t y of the sapwood/heartwood boundary . In type I s p e c i e s , the d i s t i n c t i o n i s very c l e a r . In type II s p e c i e s , i t i s somewhat b l u r r e d . In type I I I s p e c i e s , i t i s d i f f i c u l t to d i s c e r n . However, w i t h the e x c e p t i o n of Pinus  banks iana the r e l a t i o n s h i p between the p a t t e r n of death of ray parenchyma c e l l s and c o l o u r c o n t r a s t between sapwood and heartwood was not o b s e r v e d . The r i n g number d e s c r i b i n g the boundary of sapwood and heartwood v a r i e s w i t h s p e c i e s and c r i t e r i a u s e d . As shown i n T a b l e 7, the boundary of sapwood and heartwood i n P i c e a mariana was found at the e l e v e n t h and seventeenth year (counted from the cambium), r e s p e c t i v e l y , u s i n g MC and SR as c r i t e r i a . There was a d i f f e r e n c e of one or two years between the two c r i t e r i a f o r the o ther three s p e c i e s . The d i s c r e p a n c y may a r i s e from: (a) M o i s t u r e v a r i a t i o n due to aspect where the MC Table 7. The boundary of sapwood and heartwood expressed as the ring age counted from the cambium, as determined by two different methods: distribution of moisture content (MC) and the survival rate (SR) of earlywood central ray parenchyma cells during May. Species Sapwood/Heartwood Boundary (Year) MC SR of Central Ray Cells Pinus banksiana 27 25 Picea mariana 11 17 Abies balsamea 15 16 Populus tremuloides 23 24 64 sample was taken from the n o r t h aspec t of the t r e e , w h i l e the specimen f o r the SR measure was o b t a i n e d from the south aspec t of the t r e e ; (b) Sapwood t h i c k n e s s v a r i a t i o n due to aspec t where the boundary between sapwood and heartwood does not conform to the same growth r i n g s around the e n t i r e p e r i p h e r y (Yang et a l . 1985); (c) E x p e r i m e n t a l e r r o r s ; (d) The ambigu i ty of d e c i d i n g the p r e c i s e year i n which sapwood MC reaches a c o n s t a n t low v a l u e ; or (e) Any combinat ion of the above f a c t o r s . The d i s c r e p a n c y might be min imized by a b e t t e r specimen c o l l e c t i o n method such as the use of the same increment cores f o r both measures . S t i l l , the SR c r i t e r i o n , which i s based on a c t u a l o b s e r v a t i o n of parenchyma c e l l c o n t e n t s , seems to be a more a p p r o p r i a t e means of d e c i d i n g the sapwood/heartwood boundary . Among the c o n i f e r s , a p o s i t i v e r e l a t i o n s h i p (Table 8) between the MC i n May and the SR (average of the m a r g i n a l and c e n t r a l c e l l s ) was found i n P inus  banks iana and P i c e a m a r i a n a . No l i n e a r r e l a t i o n s h i p between MC and SR was observed i n Abies ba l samea . In Populus t r e m u l o i d e s , a n e g a t i v e r e l a t i o n s h i p Table 8 . Correlation coefficients between the moisture content and survival rate in May specimen. Species MC/SR Pinus banksiana 0.825** Picea mariana 0.862** Abies balsamea 0.357ns Populus tremuloides - 0.597* MC - moisture content SR - survival rate ns - not significant at<=<>0.05 * - significant at0.05 ** - significant at<x<0.01 66 between MC and SR was c a l c u l a t e d . A g r e a t number of r e s e a r c h e r s , such as T r e n d e l e n b u r g (1939) , Huber (1956), Zimmermann and Brown (1971) and Nobuchi and Harada (1983) , b e l i e v e t h a t the death of l i v i n g sapwood ray parenchyma c e l l s i s caused by a change of MC i n sapwood. From the data p r e s e n t e d above, t h i s cause and e f f e c t theory between MC and death of ray parenchyma c e l l s may be t r u e f o r c e r t a i n s p e c i e s , but i t does not app ly to a l l s p e c i e s , such as Abies balsamea and Populus t r e m u l o i d e s . The death p a t t e r n of ray parenchyma c e l l s i n sapwood may appear to be s p e c i e s -s p e c i f i c i n n a t u r e , and not always r e l a t e d to c o r r e s p o n d i n g changes i n MC. 5.3 Nuclear Elongation Index 5 .3 .1 Specimen Preparation and Methods The permanent s l i d e s f o r the SR s tudy ( S e c t i o n 5.2) were a l s o used f o r the n u c l e a r e l o n g a t i o n index (NEI) s t u d y . The v i t a l i t y of a l i v i n g sapwood ray parenchyma 67 c e l l may be expressed q u a n t i t a t i v e l y by the NEI as d e f i n e d on page 14. P r e l i m i n a r y measurements were made from m a r g i n a l and c e n t r a l c e l l s of u n i s e r i a t e rays i n the earlywood of the two youngest growth r i n g s next to the cambium. These measurements were c a r r i e d out f o r a l l f o u r s p e c i e s . I t was e s t a b l i s h e d t h a t between 13 and 18 c e l l s were r e q u i r e d i n o r d e r to come w i t h i n ten per cent of the mean v a l u e w i t h a p r o b a b i l i t y =0.05. Based on these r e s u l t s , a c o n s e r v a t i v e number of twenty ear lywood ray c e l l s and t h e i r n u c l e i were measured from each of the m a r g i n a l and c e n t r a l p o s i t i o n s of each growth r i n g . These twenty c e l l s were s c a t t e r e d through at l e a s t seven u n i s e r i a t e rays w i t h i n a s i n g l e growth r i n g . The upper l i m i t of u n i s e r i a t e r a y s chosen was dependent upon the a v a i l a b i l i t y of l i v i n g ray parenchyma c e l l s . One to t h r e e such c e l l s were randomly s e l e c t e d from each ray chosen , u n t i l a t o t a l of twenty measurements were accumula ted . The measurements were c a r r i e d out f o r the s i x sampl ing p e r i o d s (May to December) and from the f i r s t growth r i n g counted from the cambium to the heartwood boundary. T h i s boundary was f i x e d by the absence of l i v i n g ray parenchyma 68 c e l l s . The number of growth r i n g s i n sapwood v a r i e d w i t h specimens c o l l e c t e d at d i f f e r e n t months as shown i n F i g u r e 1. The number of sapwood r i n g s was taken as the l a r g e s t number encountered i n the sapwood i n the s i x sample p e r i o d s . Some new data were d e r i v e d u s i n g the procedure o f data d e r i v a t i o n as shown i n Appendix 2. A F - t e s t to t e s t homogeneity of v a r i a n c e was c a r r i e d out p r i o r to c a r r y i n g out a t - t e s t of the h y p o t h e s i s tha t the mean NEI i n the m a r g i n a l and c e n t r a l ray parenchyma c e l l s would be e q u a l . I f the F - t e s t showed a s i g n i f i c a n t d i f f e r e n c e , no t - t e s t was c o n d u c t e d . I f the F - t e s t showed a n o n - s i g n i f i c a n t d i f f e r e n c e , then a t - t e s t was c a r r i e d o u t . T h i s s t a t i s t i c a l approach was a l s o a p p l i e d to t e s t the d i f f e r e n c e between the means of two cont iguous monthly sampl ing p e r i o d s . 5 .3 .2 Results A summary of the changes i n the nuc l eus of the c e n t r a l ray parenchyma c e l l s at v a r i o u s l o c a t i o n s 6 9 w i t h i n the sapwood at d i f f e r e n t sampl ing p e r i o d s i s shown i n F i g u r e 4. The n u c l e i of ray parenchyma c e l l s i n the t h r e e c o n i f e r o u s s p e c i e s s t u d i e d appeared e l l i p s o i d a l i n the outer sapwood. These e l o n g a t e d n u c l e i became rounded and more i r r e g u l a r i n shape i n the i n n e r sapwood. The n u c l e i i n Populus t r e m u l o i d e s were f u s i f o r m i n the outer sapwood but became rounded and i r r e g u l a r i n the i n n e r sapwood. The n u c l e i of ray parenchyma c e l l s i n the outer sapwood of P i c e a mariana d i s p l a y e d the l o n g e s t average l e n g t h ( c a . 40 um). The l e n g t h s of the n u c l e i of ray parenchyma c e l l s i n the outer sapwood of Pinus banks iana and A b i e s balsamea were i n the range of 20 to 35 um. The n u c l e i i n Populus  t r e m u l o i d e s were the s h o r t e s t , u s u a l l y l e s s than 20 um i n l e n g t h . The v i t a l i t y of ray parenchyma c e l l s expressed as the NEI a c r o s s the sapwood at v a r i o u s sampl ing p e r i o d s i s i l l u s t r a t e d i n F i g u r e 5. 5.3.2.1 Pinus banksiana For a l l s i x sampl ing p e r i o d s , there was a decrease 70 Pinus banksiana Lamb. LOCATION MAY JUNE JULY AUG. | OCT. DEC. Outer—Sap U M ! H M yip Mid-Sap mi .1*1 Inner—Sap h #" f. I i , I US! Picea mariana (Mill.) B. S. P. Outer—Sap Mid — Sap Inner—Sap • T J j „ 1 • Abies balsamea (L.)Mill. NOV. Outer—Sap Mid-Sap m -1 -.'-I 1.4A\ Inner—Sap 3fe JBSJ 1 * , ! Populus tremuloides Michx. NOV. Outer—Sap t-t. Mid-Sap 1(1 I M sf-7 Inner—Sap L*t:U -J T'-t i 13*1" gjfi 2 0 y m ure 4. The nuclear lengths of the central ray parenchyma cells in Pinus banksiana Lamb., Picea mariana (Mill.) B.S.P., Abies balsamea (L.) Mill, and Populus tremuloides Michx. at various locations within the sapwood at different sampling periods. May June July August October December 4 6 8 10 12 14 16 18 20 22 24 26 Number ol Ring* Counted Irom the Cambium Figure 5. Vitality of margianl (©) and central (A) ray parenchyma cells expressed as the nuclear elongation index across the sapwood at various sampling periods. Each point represents the average of 20 cell measurements. 1. Pinus banksiana. S/H - sapwood and heartwood boundary. Figure 5. (continued). 2. Picea mariana. Figure 5. (continued). 3 . Abies balsamea. 74 20 L, 0 &MJLnLBkMkMLHi^JaBJL^a^L^ULUB 2 4 6 8 10 12 14 16 18 20 22 24 26 Number ol Rings Counted from the Cambium Figure 5. (continued). 4. Populus tremuloides. 75 i n the NEI from the cambium towards the boundary of sapwood and heartwood ( F i g u r e 5 -1 ) . In August and O c t o b e r , the NEI i n c r e a s e d both i n the m a r g i n a l and c e n t r a l c e l l s of sapwood. There were no s i g n i f i c a n t d i f f e r e n c e s between e i t h e r the mean NEI of the m a r g i n a l and the c e n t r a l c e l l s ( F i g u r e 5-1) or t h e i r v a r i a n c e s . T a b l e 9 shows the r e s u l t s of the t - t e s t s of the d i f f e r e n c e s between the means of NEI over two cont inguous sampl ing p e r i o d s , and F - t e s t s of t h e i r v a r i a n c e s f o r both the m a r g i n a l and c e n t r a l c e l l s . In b o t h the m a r g i n a l and c e n t r a l c e l l s , the d i f f e r e n c e s i n the mean between the months of J u l y / A u g u s t and A u g u s t / October were s i g n i f i c a n t . 5 . 3 . 2 . 2 Picea mariana F i g u r e 5-2 i l l u s t r a t e s the v a r i a t i o n of the NEI over the sampl ing p e r i o d . A g e n e r a l l y d e c r e a s i n g t r e n d of the NEI a c r o s s the sapwood i n each g i v e n month was noted except f o r Augus t . In Augus t , the NEI was approx-i m a t e l y c o n s t a n t up to age e l e v e n and then decreased 76 s h a r p l y to age twelve or t h i r t e e n , a f t e r which i t l e v e l l e d o f f u n t i l ages s i x t e e n to e i g h t e e n , by which time a l l the l i v i n g sapwood ray parenchyma had d i e d . The death of the m a r g i n a l r a y parenchyma c e l l s i n P i c e a mariana o c c u r r e d at l e a s t two years e a r l i e r than t h a t of the c e n t r a l ray parenchyma c e l l s ( F i g u r e 5 -2 ) . There was no s i g n i f i c a n t d i f f e r e n c e between the NEI means of these two c e l l s . T a b l e 9 i l l u s t r a t e s the r e s u l t s of S t u d e n t ' s t -t e s t s f o r the d i f f e r e n c e i n the NEI means and the F -t e s t f o r NEI v a r i a n c e s between two adjacent months. The mean v a l u e s were s i g n i f i c a n t l y d i f f e r e n t from October to December. In December the average NEI was at i t s minimum. For both the c e n t r a l and m a r g i n a l c e l l s , the r e s u l t s of the F - t e s t i n d i c a t e d t h a t the NEI v a r i a n c e s d u r i n g the months of J u l y / A u g u s t and A u g u s t / O c t o b e r f a i l e d the t e s t of a common v a r i a n c e at =0.05. 5 . 3 . 2 . 3 Abies balsamea F i g u r e 5-3 shows the NEI f o r Abies balsamea d u r i n g v a r i o u s months of the sampl ing p e r i o d . The death of Table 9 T-tests of the differences between the means of nuclear elongation index over two contiguous sampling periods and F-tests of their variances for both marginal (M) and central (C) ray parenchyma cells. Numbers in parentheses represent probabilities that variation was due to chance. Populus Pinus banksiana Picea mariana Abies balsamea tremuloides  Month  M C M c M C M C May/June ns ns ns ns ns ns ns ns June/July ns ns ns ns ns ns ns ns a ITS July/Aug. (0.000) (0.000) na na ns ns ns ns OJ Aug./Oct. (0.021) (0.027) na na ns ns ns ns Oct./Nov.a (Dec.) ns na (0.000) (0.000) ns ns ns ns May/June ns ns ns ns ns ns ns ns iance June/July ns ns ns ns ns ns ns ns iance July/Aug. ns ns (0.003) (0.016) ns ns ns ns Var Aug./Oct. ns ns (0.000) (0.000) ns ns ns ns Oct./Nov. (Dec.) ns (0.006) ns ns ns ns ns ns L _ : na - not applicable, because of significant difference between the variances, ns - not significant atc*>0.05 a - November for Abies balsamea and Populus tremuloides. December for Pinus banksiana and Picea mariana. 78 sapwood m a r g i n a l ray parenchyma c e l l s o c c u r r e d f i v e y e a r s e a r l i e r than t h a t of the c e n t r a l c e l l s , so t h e r e were fewer measurements f o r m a r g i n a l c e l l s . A sharp d e c l i n e from the f i r s t to the second growth r i n g was n o t i c e d except i n J u l y . A f t e r the second growth r i n g a g r a d u a l d e c l i n e was n o t e d . S t a t i s t i c a l l y , t h e r e was no s i g n i f i c a n t d i f f e r e n c e between the NEI of the m a r g i n a l and the c e n t r a l c e l l s and the average NEI d i d not v a r y s i g n i f i c a n t l y from month to month (Table 9 ) . 5 . 3 . 2 . 4 Populus tremuloides F i g u r e 5-4 d i s p l a y s the v a r i a t i o n of the NEI i n Populus t r e m u l o i d e s d u r i n g v a r i o u s months of the sampl ing p e r i o d . A h y p e r b o l i c t r e n d of the NEI from the f i r s t r i n g to mid-sapwood i s f o l l o w e d by a n e a r l y c o n s t a n t v a l u e to the sapwood/heartwood boundary . There was no s i g n i f i c a n t d i f f e r e n c e between the NEI of the m a r g i n a l and c e n t r a l c e l l s and the mean NEI d i d not v a r y s i g n i f i c a n t l y from month to month (Table 9 ) . 79 5 . 3 . 3 D i s c u s s i o n The shape and s i z e of the n u c l e i i n the o u t e r sapwood ray parenchyma c e l l s were d i f f e r e n t i n the f o u r s p e c i e s . These o b s e r v a t i o n s are c o n s i s t e n t w i t h r e p o r t s by Hugentobler (1965) t h a t the shape of t h e . n u c l e u s i s more or l e s s s p e c i e s - s p e c i f i c . However, more sample t r e e s are needed to c o n f i r m t h i s s p e c i e s - s p e c i f i c n a t u r e , due to l a c k of r e p l i c a t i o n i n t h i s s t u d y . The v i t a l i t y of l i v i n g sapwood ray parenchyma c e l l s , expressed as the NEI d e c l i n e d from the outer sapwood towards the i n n e r sapwood, except f o r the two s m a l l i n c r e a s e s noted i n the middle sapwood of P inus  b a n k s i a n a i n August and October and the sharp decrease i n P i c e a mariana i n Augus t . T h i s d e c l i n i n g t r e n d was d i f f e r e n t from tha t r e p o r t e d by o ther i n v e s t i g a t o r s ( F r e y - W y s s l i n g and Bosshard 1959, Fukazawa and H i g u c h i 1965, Hugentobler 1965, Bosshard 1968, N a i r and Chavan 1983), who expressed the v i t a l i t y of ray parenchyma c e l l s by the n u c l e a r s l e n d e r n e s s r a t i o (NSR). The v i t a l i t y of ray parenchyma c e l l s even showed an i n c r e a s e from the cambium towards the boundary of 80 sapwood and heartwood i n c e r t a i n s p e c i e s (Bosshard 1968, N a i r and Chavan 1983) . T h i s apparent t r e n d might be a t t r i b u t e d to v a r y i n g l e n g t h s of ray parenchyma c e l l s , a f a c t o r not c o n s i d e r e d i n a p p l y i n g the NSR. F u r t h e r s tudy of the v a r i a t i o n of the l e n g t h of ray parenchyma c e l l s a c r o s s the sapwood might c l a r i f y i t s confounding i n f l u e n c e on the NSR i n d e x . One might expect t h a t v i t a l i t y of a l i v i n g sapwood ray parenchyma c e l l i n May would be c o r r e l a t e d w i t h MC f o r t h i s same month. T h i s e x p e c t a t i o n was t r u e f o r P inus banks iana and P i c e a m a r i a n a , but not i n Ab ie s  balsamea (Table 10) . A n e g a t i v e r e l a t i o n s h i p was found i n Populus t r e m u l o i d e s , s i n c e MC i n c r e a s e d a c r o s s the sapwood w h i l e NEI d e c r e a s e d . A s t r o n g p o s i t i v e r e l a t i o n s h i p between the v i t a l i t y expresses as NEI and the SR was found i n a l l f o u r s p e c i e s s t u d i e d (Table 10) . T h i s r e l a t i o n s h i p i m p l i e s t h a t the s u r v i v a l c e l l s i n the i n n e r sapwood show a r e l a t i v e l y low v i t a l i t y . In the s i x t e e n t h r i n g of P inus b a n k s i a n a , about n i n e t y per cent of the c e l l s were s t i l l a l i v e i n May, w h i l e the NEI of the c e l l s had dropped from f i f t e e n to Table 10. Correlation coefficients between the nuclear elongation index and the moisture content and survival rate in May specimen. Species NET/MC NEI/SR Pinus banksiana 0.871** 0.848** Picea mariana 0.852** 0.860** Abies balsamea 0.345ns 0.907** Populus tremuloides -0.623** 0.602** ns - not significant at ^ > 0.05 ** - significant at ^ £ . 0 . 0 1 NEI - nuclear elongation index MC - moisture content SR - survival rate 82 t e n . In P i c e a mariana at the t e n t h r i n g and A b i e s  balsamea at the seventh r i n g , the SR was n i n e t y per c e n t , whereas the NEI had decreased from 15 to 10 and 20 to 10, r e s p e c t i v e l y . S i m i l a r l y i n Populus  t r e m u l o i d e s at the f i f t e e n t h r i n g , n i n e t y per cent of the c e l l s were s t i l l a l i v e w h i l e the NEI had d e c l i n e d from 15 to 7. T h i s c o n s i s t e n t p a t t e r n seems to i n d i c a t e t h a t a l t h o u g h s u r v i v a l of parenchyma c e l l s remained h i g h i n the outer to middle sapwood, fundamental changes were o c c u r r i n g i n the nuc leus due to a g i n g . T h i s was p a r t i c u l a r l y e v i d e n t i n Ab ie s balsamea and Populus t r e m u l o i d e s , where the NEI decreased to about 50% by the 3rd to 4th year beh ind the cambium. There was no s i g n i f i c a n t d i f f e r e n c e between the m a r g i n a l and c e n t r a l c e l l s i n terms of v i t a l i t y . The l i f e span of the m a r g i n a l c e l l s i s s h o r t e r than t h a t of c e n t r a l c e l l s . T h i s i s more e v i d e n t i n A b i e s balsamea than i n the o ther t h r e e s p e c i e s . Based on t h i s r e s u l t , i t was conc luded t h a t the v i t a l i t y of the m a r g i n a l and c e n t r a l c e l l s i s independent of t h e i r l i f e span . 83 5.4 R e l a t i v e S t a r c h Content 5 . 4 . 1 Specimen P r e p a r a t i o n and Methods T h i r t y - s i x or more r a d i a l s e c t i o n s , 20 um i n t h i c k n e s s , were cu t from the cambium towards the heartwood f o r v a r i o u s l i g h t m i c r o s c o p i c s t u d i e s , ( S e c t i o n 5 . 2 . 1 ) . One t h i r d of these r a d i a l s e c t i o n s were s t a i n e d w i t h an IKI s o l u t i o n ( l g T_2 + 2g KI i n 300 ml d i s t i l l e d water) and mounted w i t h the same IKI s o l u t i o n on m i c r o s c o p i c s l i d e s . The edges of the cover s l i p were s e a l e d w i t h "Coversbond" to prevent the s e c t i o n s from d r y i n g o u t . The r e l a t i v e s t a r c h content (RSC) of the earlywood ray parenchyma c e l l s was determined by the d e n s i t y of s t a r c h g r a n u l e s . Four s c a l e s were used based on the percentage of c e l l c a v i t y o c c u p i e d by s t a r c h g r a n u l e s and expressed as f o l l o w s : 0 - 0%, 1 - 30%, 2 - 30% -70% and 3 - > 70%. P r e l i m i n a r y work u s i n g computer imaging was c a r r i e d out to v e r i f y the a c c u r a c y . o f o b s e r v a t i o n s through the ten c e l l s t e s t s . A l l t en v i s u a l 84 o b s e r v a t i o n s were i n agreement w i t h t h a t of computer imaging w i t h i n the s c a l e s as proposed as above. Ten rays hav ing a c e n t r a l p o r t i o n at l e a s t t h r e e to f o u r c e l l s h i g h were s e l e c t e d f o r s t a r c h content c l a s s i f i c a t i o n . A f t e r the RSC of 2 c e l l s at the c e n t r a l zone was r e c o r d e d , the m i c r o s c o p i c s l i d e was moved v e r t i c a l l y to observe one of the m a r g i n a l c e l l a r e a s . The RSC of 2 m a r g i n a l c e l l s was then d e t e r m i n e d . The average of 20 c e l l s o b t a i n e d from 10 r a y s was c a l c u l a t e d . RSC was measured over the s i x sampl ing p e r i o d s to determine s easona l v a r i a t i o n . The measure-ment of RSC was c a r r i e d out i n each growth r i n g from the outer sapwood to the sapwood/heartwood boundary . A n a l y s e s of the RSC w i t h the mean data were conducted i n a f a s h i o n s i m i l a r to the NEI a n a l y s e s i n c l u d i n g p l o t t i n g of the RSC i n the l i v i n g ray parenchyma c e l l s a g a i n s t the sapwood growth r i n g number over the v a r i o u s sampl ing p e r i o d s . No weighted NEI was c a l c u l a t e d . The hypotheses of equa l RSC i n the m a r g i n a l and c e n t r a l ray parenchyma c e l l s was t e s t e d by a t - t e s t . T - t e s t s were a l s o c a r r i e d out between the means of two cont iguous sampl ing p e r i o d s , a f t e r f i r s t c o n d u c t i n g an F - t e s t to ensure homogeneity of v a r i a n c e s . 85 5 .4 .2 Results 5 . 4 . 2 . 1 Pinus banksiana In May, the RSC of the l i v i n g ray parenchyma c e l l s from the f i r s t to the t h i r t e e n t h growth r i n g ( F i g u r e 6-1) was h i g h . S t a r c h content i n these l i v i n g c e l l s then decreased g r a d u a l l y from the t h i r t e e n t h r i n g towards the sapwood/heartwood boundary. In June , the youngest r a y parenchyma c e l l s d i s p l a y e d a low s t a r c h c o n t e n t , which subsequent ly i n c r e a s e d w i t h t r e e age to a peak at the e i g h t h growth r i n g , a n d then decreased g r a d u a l l y to the boundary . In J u l y , the p a t t e r n of RSC was s i m i l a r to the June p a t t e r n but the peak v a l u e was s h i f t e d to the t e n t h growth r i n g . By Augus t , no s t a r c h g r a n u l e s were apparent i n the f i r s t e l e v e n growth r i n g s . A h i g h RSC o c c u r r e d on ly from the f i f t e e n t h to the seventeenth r i n g . In O c t o b e r , the o v e r a l l RSC i n c r e a s e d once aga in and v a r i e d g r e a t l y a c r o s s the sapwood, w i t h two peaks: one i n the t h i r d and f o u r t h growth r i n g s ; the o ther i n the f o u r t e e n t h and f i f t e e n t h r i n g s . In December, no s t a r c h g r a n u l e s were f o u n d . No Figure 6. The relat ive starch content of marginal (#) and central (A) ray parenchyma ce l l s across the sapwood at various sampling periods. Each point represents the average of 20 c e l l measurements. 1. Pinus banksiana. S/H - sapwood and heartwood boundary. Figure 6. (continued). 2 . Picea mariana. Y \ 1 _! ! I !_ It May June 2 1-* c 1 July = 2 August I I I I ! ! ! L October I } I I 1 I L Nowember 2 4 6 8 10 12 14 16 18 20 22 24 26 Number ol Rings Counted Irom the Cambium Figure 6. (continued). 3 . Abies balsamea. Figure 6. (continued). 4 . Populus tremuloides. 90 s t a r c h g r a n u l e s were observed i n the heartwood ray parenchyma c e l l s at any t i m e . A t - t e s t of the mean d i f f e r e n c e i n the RSC i n d i c a t e d t h a t m a r g i n a l c e l l s had s i g n i f i c a n t l y g r e a t e r s t a r c h content than c e n t r a l c e l l s d u r i n g May, June and J u l y (Table 11) . For Augus t , the t - t e s t f o r the mean d i f f e r e n c e between m a r g i n a l and c e n t r a l ray c e l l s c o u l d not be c a r r i e d out because of a v i o l a t i o n of the assumption of a common v a r i a n c e . In both the m a r g i n a l and c e n t r a l c e l l s , the mean RSC showed a s i g n i f i c a n t d i f f e r e n c e between August and October (Table 12 ) . There was no s i g n i f i c a n t d i f f e r e n c e i n the v a r i a n c e between any two a d j a c e n t sampl ing p e r i o d s except d u r i n g the months of . J u l y / August and October/December i n the m a r g i n a l c e l l s . 5 . 4 . 2 . 2 P i c e a mariana In May, as i l l u s t r a t e d i n F i g u r e 6-2, the RSC of the l i v i n g r a y parenchyma c e l l s was very low both i n the outer and i n n e r sapwood. In June , t h e r e were r e l a t i v e l y l a r g e numbers of s t a r c h g r a n u l e s i n the Table 11. T-tests of the differences between the means of relative starch content in the marginal and central ray cells, and F-tests of their variances. Numbers in parentheses represent probabilities that variation was due to chance. Populus Pinus banksiana Picea mariana Abies balsamea tremuloides Month May (0.020) ns ns ns June (0.004) ns (0.012) ns July (0.012) a (0.002) (0.004) Mean Aug. na na ns ns Mean October ns ns ns ns Nov./Dec b a na na a May ns ns ns ns June ns ns ns ns cu rO - H >^  July ns a ns ns August (0.022) (0.025) ns ns (0 > October ns ns ns ns Nov./Dec. b a (0.014) (0.038) a na - not applicable, because of significant difference between the variances, ns - not s ignif icant a t * > 0.05 a - absence of starch content b - November for Abies balsamea and Populus tremuloides. December for Pinus banksiana and Picea mariana. 92 f i r s t t en growth r i n g s , f o l l o w e d by a d e c l i n e to the sapwood/heartwood boundary . In J u l y , no s t a r c h g r a n u l e s c o u l d be o b s e r v e d , w h i l e i n August a s m a l l number was f o u n d , p a r t i c u l a r l y i n the middle of the sapwood zone. In O c t o b e r , abundant s t a r c h g r a n u l e s were found i n the outer sapwood, but t h i s decreased g r a d u a l l y toward the sapwood/heartwood boundary . The s t a r c h content of the m a r g i n a l c e l l s showed a p a r t i c u l a r l y low l e v e l from the seventh to the n i n t h growth r i n g . In December, a few s t a r c h g r a n u l e s were observed i n the f i r s t four growth r i n g s , but v i r t u a l l y none t h e r e a f t e r . No s t a r c h g r a n u l e s were found i n heartwood ray parenchyma c e l l s . There was no s i g n i f i c a n t d i f f e r e n c e i n mean RSC between the m a r g i n a l and c e n t r a l c e l l s (Tab le 11) . In terms of the v a r i a n c e r a t i o t e s t , the RSC i n August ( F i g u r e 6 and T a b l e 11) was found to have s i g n i f i c a n t l y d i f f e r e n t v a r i a n c e s between the c e n t r a l c e l l s and the m a r g i n a l c e l l s . There was a h i g h l y s i g n i f i c a n t d i f f e r e n c e i n the v a r i a n c e r a t i o f o r the RSC between two sampl ing p e r i o d s except f o r May/June (Table 12) . Table 12. T-tests of the differences between the means of relative starch content over two contiguous sampling periods and F-tests of their variances for both marginal (M) and central (C) ray parenchyma cells. Numbers in parentheses represent probabilities that variation was due to chance. Populus Pinus banksiana Picea mariana Abies balsamea tremuloides Month ra QJ OJ u c m - H > May/June ns ns ns ns June/July ns ns na na July/Aug. na (0.006) na na Aug./Oct. (0.000) (0.001) na na M C M Oct./Nov.b na na na (Dec.) na M C M C na ns na na ns ns na na na na na na na na ns ns na na na na May/June ns ns ns ns (0.029) ns (0.002)(0.000) June/July ns ns (0.000) (0.000) ns ns (0.041)(0.001) July/Aug. (0.003) ns (0.000) (0.000) (0.026)(0.028)(0.001)(0.000) Aug./Oct. ns ns (0.000) (0.014) (0.001)(0.013) ns ns Oct./Nov. (0.000) (0.000) (0.000) (0.000) (0.000)(0.000)(0.000)(0.000) (Dec.) na - not applicable, because of significant difference between the variances. ns - not significant at°<>0.05 ip b - November for Abies balsamea and Populus tremuloides. December for Pinus banksiana and Picea mariana. 94 5 . 4 . 2 . 3 Abies balsamea F i g u r e 6-3 i l l u s t r a t e s v a r i a t i o n i n the RSC of the l i v i n g ray parenchyma c e l l s over v a r i o u s sampl ing p e r i o d s from May to November. In May, the RSC was h i g h from the o u t s i d e to the i n n e r sapwood and then the s t a r c h d i s a p p e a r e d c l o s e to the sapwood/heartwood boundary . In June , the RSC i n the c e n t r a l c e l l s and was m a i n t a i n e d over a number or r i n g s . I t decreased from the e l e v e n t h or t w e l f t h r i n g towards the sapwood/ heartwood boundary . In June and J u l y , the s t a r c h g r a n u l e s i n the m a r g i n a l c e l l s d e c l i n e d from the f i r s t growth r i n g towards the boundary . In Augus t , no s t a r c h g r a n u l e s were d e t e c t e d i n the c e n t r a l c e l l s of the f i r s t seven growth r i n g s , wh i l e a low RSC was observed i n the m a r g i n a l c e l l s . In O c t o b e r , there was a h i g h RSC i n the o u t e r sapwood of bo th c e l l t y p e s . In the c e n t r a l c e l l s t h i s h i g h s t a r c h content decreased from the middle sapwood towards the sapwood/heartwood boundary , w h i l e i t decreased from the outer sapwood i n the m a r g i n a l c e l l s . In November, o n l y a few s t a r c h g r a n u l e s were observed i n both types of c e l l s . In a l l i n s t a n c e s , 95 s t a r c h g r a n u l e s were l a c k i n g i n the m a r g i n a l c e l l s of the two or t h r e e innermost sapwood r i n g s . There was a s i g n i f i c a n t d i f f e r e n c e i n the mean RSC of the m a r g i n a l and c e n t r a l c e l l s , i n June and J u l y (Tab le 11) . In November, the v a r i a n c e was s i g n i f i c a n t l y d i f f e r e n t between the m a r g i n a l c e l l s and the c e n t r a l c e l l s . In m a r g i n a l c e l l s , there was a s i g n i f i c a n t d i f f e r -ence i n v a r i a n c e between two cont iguous months except i n the months of J u n e / J u l y (Tab le 12) . In the c e n t r a l c e l l s , t h i s exemption i n c l u d e d May/June and J u n e / J u l y . 5 . 4 . 2 . 4 Populus tremuloides F i g u r e 6-4 p r e s e n t s the v a r i a t i o n of the RSC i n the l i v i n g ray parenchyma c e l l s f o r v a r i o u s sampl ing p e r i o d s from May to November. In May, a low RSC was found a c r o s s the sapwood zone w i t h a s l i g h t peak i n the midd le of the sapwood r e g i o n . In June , J u l y and August a h i g h RSC was observed a c r o s s the sapwood except f o r the f i r s t few r i n g s and the l a s t growth r i n g near the 96 heartwood boundary . In O c t o b e r , a h i g h RSC appeared from the f i r s t to the s i x t e e n t h growth r i n g , f o l l o w e d by a d e c l i n e towards the heartwood boundary . T h i s d i s t r i b u t i o n was not d i s s i m i l a r to t h a t d u r i n g June . No s t a r c h g r a n u l e s c o u l d be d e t e c t e d i n the November spec imen. There was no s i g n i f i c a n t d i f f e r e n c e i n the RSC between the m a r g i n a l and c e n t r a l c e l l s except i n J u l y (Tab le 11), when the m a r g i n a l c e l l s exceeded the c e n t r a l c e l l s i n RSC. The RSC i n Populus t r e m u l o i d e s ray c e l l s v a r i e d g r e a t l y i n the e a r l y growing season . A l l months except A u g u s t / O c t o b e r appeared to have s i g n i f i c a n t l y d i f f e r e n t v a r i a n c e s (Table 12). 5 . 4 . 3 Discussion As shown i n F i g u r e 6 the p a t t e r n s of RSC i n the l i v i n g ray parenchyma c e l l s d i f f e r e d from s p e c i e s to s p e c i e s and from month to month. However, a common f e a t u r e noted was t h a t t h e r e were very few or no s t a r c h g r a n u l e s i n l a t e November or e a r l y December. The d i s a p p e a r a n c e of s t a r c h g r a n u l e i n the l a t e f a l l and 97 w i n t e r i s a s i m i l a r i t y of f o u r t r e e s s t u d i e d . The s t a r c h g r a n u l e s p r o b a b l y c o n v e r t e i t h e r i n t o l i p i d d r o p l e t s as suggested by Sauter (1966a) and Nobuchi e_t a l . (1986) or c h e m i c a l s . F u r t h e r m o r e , i n most of the specimens c o l l e c t e d at v a r i o u s sampl ing p e r i o d s , the RSC was low or zero i n the sapwood r i n g s a d j a c e n t to the heartwood. The e x c e p t i o n s were P inus b a n k s i a n a and P i c e a mariana i n O c t o b e r , A b i e s balsamea i n August and e s p e c i a l l y Populus t r e m u l o i d e s i n J u l y . T h i s g e n e r a l l y low s t a r c h content i n . the innermost sapwood was r e p o r t e d by many o ther r e s e a r c h e r s ( H i l l i s e_t al. 1962, Rudman 1966, N a i r e t al. 1981, Shah et al. 1981, Yamamoto 1982, Nobuchi 1985) . I t was suggested (Rudman 1966, Fukazawa e_t a l . 1980, Shah et a l . 1981, N a i r et al. 1981, Yamamoto 1982, Nobuchi 1985) t h a t the d i s a p p e a r a n c e o f s t a r c h g r a n u l e s i n the inner sapwood may be r e l a t e d to the f o r m a t i o n of heartwood s u b s t a n c e s . However, i n Ab ie s  ba l samea, the d i s a p p e a r a n c e of s t a r c h g r a n u l e s was found i n c e l l s of one or two r i n g s b e f o r e those h a v i n g dead ray parenchyma c e l l s . An unusual f i n d i n g was the complete l a c k of s t a r c h 98 g r a n u l e s i n the J u l y specimens of P i c e a m a r i a n a . Whether t h i s absence of s t a r c h g r a n u l e s r e l a t e s to c a m b i a l a c t i v i t y or heartwood substance f o r m a t i o n remains to be s t u d i e d . In the outer sapwood, the RSC of the f o u r s p e c i e s was low or zero i n A u g u s t . T h i s may be a t t r i b u t e d to the young ray parenchyma c e l l s which were newly formed d u r i n g the year of specimen c o l l e c t i o n . In g e n e r a l , young c e l l s l a c k e d s t a r c h g r a n u l e s (Mia 1972, Tsuda 1975b). The d i s t r i b u t i o n of s t a r c h content i n the l i v i n g ray parenchyma c e l l s a c r o s s the sapwood showed two g e n e r a l p a t t e r n s . P a t t e r n A showed a d e c r e a s i n g t r e n d from the outer sapwood towards the i n n e r sapwood, such as found i n the May specimens of P inus b a n k s i a n a , the June specimens of P i c e a m a r i a n a , and the October specimens of Ab ie s balsamea and Populus t r e m u l o i d e s . P a t t e r n B was t y p i f i e d by a r e l a t i v e l y low s t a r c h content both i n the o u t e r and i n n e r sapwood, w i t h a r e l a t i v e l y h i g h s t a r c h content i n the middle of the sapwood. Two t y p i c a l examples of P a t t e r n B were the J u l y specimens of P inus banks iana and the June 99 specimens of Populus t r e m u l o i d e s Both p a t t e r n s A and B have been r e p o r t e d i n o ther c o n i f e r o u s and hardwood s p e c i e s . For example, the May and June specimens of P inus d e n s i f l o r a (Yamamoto 1982), the May and December specimens of C r y p t o m e r i a j a p o n i c a (Nobuchi 1985), the A p r i l and May specimens of R o b i n i a p s e u d o a c a c i a ( H o l l and L e n d z i a n 1973) and the February specimen of B r i d e l i a r e t u s a ( N a i r et a_l. 1981) a l l d i s p l a y e d P a t t e r n A . P a t t e r n B was r e p o r t e d i n the May and June specimens of P inus b a n k s i a n a (Yamamoto 1982) , the J u l y specimen of C r y p t o m e r i a j a p o n i c a (Nobuchi 1985) and the mid-summer specimens of Anqophora c o s t a t a ( H i l l i s e t a l . 1962) . In Augus t , a r e l a t i v e l y h i g h s t a r c h content i n the ray parenchyma c e l l s was found i n the i n n e r sapwood i n P inus b a n k s i a n a . T h i s r e s u l t i s i n agreement w i t h t h a t r e p o r t e d by Fukazawa e_t a_l. (1980) and Yamamoto (1982) i n the same s p e c i e s . They suggested t h a t the peak of s t a r c h i n the i n n e r sapwood i n August to September i s a s s o c i a t e d w i t h w a l l development of r a y parenchyma c e l l s which s i g n i f i e s the death of c e l l and heartwood f o r m a t i o n . 100 In the t h r e e c o n i f e r o u s s p e c i e s s t u d i e d , a maximum RSC appeared i n May and June f o l l o w e d by a d e c r e a s e towards the m i d d l e of the growing s e a s o n , e i t h e r i n J u l y or August and then a second peak i n O c t o b e r . T h i s b i m o d a l d i s t r i b u t i o n of s t a r c h i n c o n i f e r o u s s p e c i e s t h r o u g h o u t the growing season has been r e p o r t e d f o r P i n u s s t r o b u s , P inus d e n s i f l o r a , P i n u s b a n k s i a n a (Fukazawa e_t al. 1980, Yamamoto 1982) , and C r y p t o m e r i a  j a p o n i c a (Nobuchi 1985) . Bamber and Humphreys (1976) showed t h a t the low amount o f s t a r c h c o i n c i d e d w i t h p e r i o d s of a c t i v e p r o d u c t i o n of new l e a v e s . The low amount of s t a r c h i n J u l y or August of t h r e e c o n i f e r o u s s p e c i e s may a l s o r e l a t e to the l e a f g r o w t h . In c o n t r a s t , s t a r c h i n Populus t r e m u l o i d e s was r e l a t i v e l y abundant from June to O c t o b e r , and s c a r c e to n o n - e x i s t e n t at the b e g i n n i n g and end of the growing s e a s o n . T h i s d i s t r i b u t i o n d i f f e r e d from the two maxima r e p o r t e d i n some hardwoods, such as A c e r p l a t a n o i d e s (Jones and B r a d l e e 1933) , R o b i n i a p s e u d o a c a c i a ( S i m i n o v i t c h e_t al. 1953) , B e t u l a p e n d u l a and' A l n u s  g l u t i n o s a (Ess iamah and E s c h r i c h 1985) . T h i s d i s c r e p a n c y may be s p e c i e s or s i t e dependent . 101 As demonstrated by O l o f i n b o b a (1969) , O l o f i n b o b a and K o z l o w s k i (1972) and Kramer and K o z l o w s k i (1979) , the drop i n s t a r c h c o n t e n t f o l l o w i n g the s p r i n g maximum i s a r e s u l t of u t i l i z a t i o n of r e s e r v e s f o r shoot and c a m b i a l growth . In c o n t r a s t , L i t t l e (1974) r e p o r t e d t h a t the c u r r e n t shoot growth i n A b i e s balsamea i s independent of the l e v e l of c a r b o h y d r a t e accumulated i n the s p r i n g . The reason f o r these c o n t r a d i c t i n g o b s e r v a t i o n s i s unknown. The v i r t u a l d i s a p p e a r a n c e of s t a r c h i n the f o u r s p e c i e s s t u d i e d d u r i n g f a l l and w i n t e r may r e f l e c t i t s c o n v e r s i o n to s u c r o s e a n d / o r l i p i d s a t low temperature as suggested by Kramer and K o z l o w s k i (1979) . The s e a s o n a l f l u c t u a t i o n of s t a r c h content, i n the f o u r s p e c i e s s t u d i e d was g r e a t e r i n the o u t e r sapwood zone than i n the i n n e r sapwood zone. T h i s o b s e r v a t i o n i s c l e a r e r i n the t h r e e c o n i f e r o u s s p e c i e s than i n Populus t r e m u l o i d e s . S i m i l a r o b s e r v a t i o n s were a l s o r e p o r t e d f o r P inus d e n s i f l o r a (Tsuda and S h i m a j i 1971) and i n C r y p t o m e r i a j a p o n i c a (Nobuchi 1985) . Nobuchi (1985) suggested t h a t the s e a s o n a l f l u c t u a t i o n i n the o u t e r sapwood i s r e l a t e d to c a m b i a l a c t i v i t y , and the 102 d i s a p p e a r a n c e of s t a r c h g r a n u l e s i n the i n n e r sapwood i s r e l a t e d to the f o r m a t i o n of heartwood s u b s t a n c e s . In P inus b a n k s i a n a , a r e l a t i v e l y h i g h s t a r c h c o n t e n t i n the middle of the sapwood was n o t e d . S t a r c h g r a n u l e s were absent from both the i n n e r and o u t e r sapwood except i n May ( F i g u r e 6 - 1 ) . T h i s o b s e r v a t i o n i s the same as t h a t r e p o r t e d by Yamamoto (1982) i n the same s p e c i e s . However, she r e p o r t e d o n l y a s m a l l amount of s t a r c h i n O c t o b e r . In t h i s s t u d y , a medium amount of s t a r c h was found a c r o s s the sapwood i n O c t o b e r . The reason f o r t h i s d i s c r e p a n c y i s unknown. The r e l a t i v e l y h i g h s t a r c h content i n the middle sapwood i n August and October c o i n c i d e d w i t h a h i g h v i t a l i t y of ray parenchyma c e l l s as i l l u s t r a t e d i n F i g u r e 5 - 1 . Whether or not t h i s c o i n c i d e n c e has p h y s i o l o g i c a l i m p l i c a t i o n m e r i t s f u r t h e r s t u d y . In P inus b a n k s i a n a , the RSC of the m a r g i n a l c e l l s i n May, June and J u l y i s h i g h e r than t h a t of the c e n t r a l c e l l s . In c o n t r a s t , i n August , the RSC of the c e n t r a l c e l l s was h i g h e r than t h a t of the m a r g i n a l c e l l s . The s h i f t of RSC d i s t r i b u t i o n from m a r g i n a l c e l l s to the c e n t r a l c e l l s has never been r e p o r t e d i n 103 the p a s t . S ince a r e l a t i o n s h i p between food r e s e r v e s and c a m b i a l a c t i v i t y e x i s t s (Wi lcox 1962, Z i e g l e r 1964) , the s i g n i f i c a n c e of t h i s s h i f t to the t r e e growth a n d / o r c a m b i a l a c t i v i t y i s worth p u r s u i n g f u r t h e r . In c o n t r a s t , i n A b i e s ba lsamea, the RSC of the c e n t r a l c e l l s i n June and J u l y i s h i g h e r than t h a t of the m a r g i n a l c e l l s . I t was found t h a t the RSC has some degree of c o r r e l a t i o n w i t h the sapwood m o i s t u r e c o n t e n t (Table 13) . In b o t h P inus b a n k s i a n a and A b i e s ba l samea , a p o s i t i v e r e l a t i o n s h i p between the RSC and the sapwood m o i s t u r e content was f o u n d , whereas i n Populus  t r e m u l o i d e s , a n e g a t i v e r e l a t i o n s h i p between these v a r i a b l e s was c a l c u l a t e d . In P i c e a mariana no c o r r e l a t i o n between these two v a r i a b l e s c o u l d be e s t a b l i s h e d . In P inus b a n k s i a n a the ray parenchyma c e l l s i n the o u t e r sapwood of the May specimens c o n t a i n more s t a r c h than those of the i n n e r sapwood. The c o r r e l a t i o n c o e f f i c i e n t between the SR and the RSC i s h i g h (Tab le 13 ) . A s i m i l a r p o s i t i v e c o r r e l a t i o n between the SR and the RSC was a l s o found Table 13. Correlation coefficients between the relative starch content and the moisture content, survival rate and nuclear elongation index in May specimen. Species RSC/MC RSC/SR RSC/NEI Pinus banksiana 0.868** 0.924** 0.898** Picea mariana 0.376ns 0.512ns 0.657* Abies balsamea 0.544* 0.820** 0.640* Populus tremuloides - 0.511* 0.327ns 0.111ns ns - not s ignif icant at > 0.05 * - s ignif icant at<*<0.05 ** - s ignificant at <* 1 0.01 RSC - re lat ive starch content MC - moisture content SR - survival rate NEI - nuclear elongation index 105 i n A b i e s balsamaea, but no such r e l a t i o n s h i p c o u l d be e s t a b l i s h e d i n the o t h e r two s p e c i e s s t u d i e d . As d i s c u s s e d e a r l i e r , the s t a r c h content v a r i e d g r e a t l y w i t h v a r i o u s sampl ing p e r i o d s . The r e l a t i o n s h i p between the SR and the RSC i n P inus banks iana and A b i e s  balsamea s h o u l d be i n t e r p r e t e d w i t h s p e c i a l c a u t i o n due to a s e a s o n a l impact . The r e l a t i o n s h i p between the NEI and the RSC i n May v a r i e s w i t h s p e c i e s . I t was s i g n i f i c a n t i n the c o n i f e r o u s s p e c i e s (Tab le 13 ) , but not i n Populus  t r e m u l o i d e s . T h i s i s due to the near absence of s t a r c h i n the outer sapwood of the May spec imen, at a t ime when the NEI was at i t s maximum. The p a t t e r n and q u a n t i t a t i v e d i s t r i b u t i o n of the RSC v a r i e d w i t h s p e c i e s , sampl ing p e r i o d , r a d i a l l o c a t i o n of ray parenchyma c e l l s i n sapwood and even w i t h d i f f e r e n c e s between r a y c e l l p o s i t i o n s , i . e . , m a r g i n a l and c e n t r a l ray c e l l s . Great c a r e s h o u l d be taken to s t a n d a r d i z e these v a r i a b l e s when comparat ive s t u d i e s of s t a r c h d i s t r i b u t i o n are c a r r i e d o u t . T h i s p r o t o c o l has not always been o b s e r v e d , j u d g i n g from c o n f l i c t i n g l i t e r a t u r e r e p o r t s . 106 5.5 Relative Lipid Content 5 .5 .1 Specimen Preparation and Methods O n e - t h i r d of the r a d i a l s e c t i o n s p r e p a r e d f o r l i g h t m i c r o s c o p i c s t u d i e s ( S e c t i o n 5 . 2 . 1 . ) were s t a i n e d w i t h Sudan b l a c k B, mounted on m i c r o s c o p i c s l i d e s and s e a l e d w i t h "Coversbond" to prevent the s e c t i o n s from d r y i n g o u t . The RLC of an earlywood ray parenchyma c e l l was judged v i s u a l l y by the d e n s i t y of l i p i d d r o p l e t s and c l a s s i f i e d i n t o one of f o u r s c a l e s as i n the s tudy of the RSC. No weighted RLC was c a l c u l a t e d . The measurement and data ana lyse s f o r the RLC were the same as those f o r the RSC ( S e c t i o n 5 . 3 . 1 ) . 5 .5 .2 Results 5 . 5 . 2 . 1 Pinus banksiana In May, the RLC was h i g h i n the l i v i n g ray parenchyma c e l l s from the f i r s t to about the e l e v e n t h growth r i n g ( F i g u r e 7 - 1 ) , a f t e r which i t decreased 107 2 4 6 8 10 M 14 16 1B 20 22 24 26 Number of Rings Counted Irom the Cambium Figure 7. The r e l a t i v e l i p i d content of l i v i n g marginal and cen tra l (A) ray parenchyma c e l l s across the sapwood at v a r i o u s sampling p e r i o d s . 1. Pinus banksiana. S/H - sapwood and heartwood boundary. S / H May Juris A/ August October IIAMJI^I»WLIIJ»JJJU^^ Dacemb 2 4 6 3 10 12 14 18 13 20 22 24 25 Number of Rings Counted from the Cambium 2 . Picea mariana. Figure 7. (continued). 3. Abies balsamea. 110 2 4 6 8 10 12 14 18 18 20 22 24 26 Number ol Rings Counted from Iha Cambium Figure 7. (continued). 4 . Populus tremuloides. I l l g r a d u a l l y towards the sapwood/heartwood boundary . In June , a h i g h RLC was found i n the i n n e r sapwood. In both J u l y and O c t o b e r , the l i p i d content was h i g h a c r o s s the whole sapwood. By August , t h e r e was a g r a d u a l d e c l i n e of RLC from the t w e n t i e t h r i n g towards the boundary . In December, the RLC decreased g r a d u a l l y from the cambium to a minimum at the e i g h t h r i n g , and rose to a maximum i n mid-sapwood. A t - t e s t of the mean RLC i n d i c a t e d t h a t t h e r e was no s i g n i f i c a n t d i f f e r e n c e between the m a r g i n a l and c e n t r a l c e l l s (Table 14 ) . There was a s i g n i f i c a n t d i f f e r e n c e i n v a r i a n c e of the RLC between two adjacent sampl ing p e r i o d s (Tab le 15 ) . No t - t e s t was c a r r i e d out between two adjacent sample p e r i o d s . 5 . 5 . 2 . 2 Picea mariana In b o t h May and . June , t h e r e was a h i g h l i p i d c o n t e n t i n the l i v i n g ray parenchyma c e l l s a c r o s s the whole sapwood ( F i g u r e 7 - 2 ) . A g r a d u a l d e c l i n e of RLC i n these l i v i n g c e l l s from the o u t e r sapwood towards the i n n e r sapwood was observed i n the J u l y , August and Table 14. T-tests of the differences between the mean relative lipid content of the marginal and central ray parenchyma cells and F-tests of their variances. Numbers in the parentheses represent probabilities that variation was due to chance. MONTH Pinus banksiana Picea mariana Populus Abies balsamea tremuloides May ns ns na ns June ns ns na na July ns ns na ns Mean Aug. ns (0.007) ns (0.000) Mean October ns ns (0.028) na Nov./Dec b ns ns ns na May ns ns (0.005) ns June ns ns (0.007) (0.004) Lance July August ns ns ns ns (0,007) ns ns ns Vari October ns ns ns (0.009) Nov./Dec. a ns ns ns (0.045) na - not applicable, because of significant difference between the variances, ns - not significant at o< > 0.05 a - November for Abies balsamea and Populus tremuloides. December for Pinus banksiana and Picea mariana. Table 15. T-tests of the differences between the means of relative lipid content over two contiguous sampling periods and F-tests of their variances for both the marginal (M) and central (C) ray parenchyma cells. Numbers in parentheses represent probabilities that variation was due to chance. Populus Pinus banksiana Picea mariana Abies balsamea tremuloides Month M c M C M C M C May/June na na na na (0.000)(0.000) na na June/July na na na ns na na na na ean July/Aug. na na (0.000) (0.000) (0.000) na na na s Aug./Oct. na na (0.054) (0.039) (0.002) na na na Oct./Nov.b (Dec.) na na (0.000) (0.000) (0.000M0.000) na na May/June (0.006)(0.019) (0.019) (0.000) ns ns (0.000)(0.019) cu June/July (0.000)(0.000) (0.007) ns (0.034)(0.016)(0.029)(0.001) u a (0 July/Aug. (0.000)(0.000) ns ns ns <0.024)(0.000)(0.000) •I-I u nj i— Aug./Oct. (0.000)(0.000) ns ns ns (0.005)(0.000)(0.000) »> Oct./Nov. (Dec.) (0.000)(0.000) ns ns ns ns (0.002)(0.000) na - not applicable, because of significant difference between the variances, ns - not significant ato<>0.05 b - November for Abies balsamea and Populus tremuloides. December for Pinus banksiana and Picea mariana. O J 114 October samples. In December, the RLC in these living cells increased from the outer towards the inner sapwood. There was no significant mean difference between the marginal and central c e l l s , except in August (Table 14), when the average l i p i d content in the marginal cells was higher than that in the central c e l l s . There was a significant difference in mean l i p i d content both in marginal and central c e l l s , between July/August, August/October and October/December (Table 15). 5.5.2.3 Abies balsamea Figure 7-3 shows the monthly variation in the RLC of the living ray parenchyma cells across the sapwood. In May, a medium amount of lipids in these living cells were distributed across the sapwood. In June and August, lipids were less abundant, but a slight increasing trend from the outer sapwood inward was observed. In July, a high RLC in living cells across the sapwood was noticed, whereas in November a high li p i d content of the living cells throughout the 115 sapwood z o n e was o b s e r v e d . In O c t o b e r , t h e R L C i n t h e l i v i n g r a y p a r e n c h y m a c e l l s a c r o s s t h e sapwood was u n i f o r m l y l o w . T h e r e was a s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e mean RLC o f t h e m a r g i n a l and c e n t r a l c e l l s f o r O c t o b e r ( T a b l e 1 4 ) . I n a t l e a s t one o f t h e two c e l l p o s i t i o n s a h i g h l y s i g n i f i c a n t d i f f e r e n c e o f l i p i d c o n t e n t b e t w e e n two a d j a c e n t s a m p l e p e r i o d s was o b s e r v e d ( T a b l e 1 5 ) . 5 . 5 . 2 . 4 Populus tremuloides In g e n e r a l t h e r e was a h i g h l i p i d c o n t e n t i n t h e l i v i n g r a y p a r e n c h y m a c e l l s a c r o s s t h e sapwood o f P o p u l u s t r e m u l o i d e s . H o w e v e r , i n O c t o b e r a d e c r e a s i n g t r e n d f r o m t h e o u t e r sapwood i n w a r d was o b s e r v e d ( F i g u r e 7 - 4 ) . I n M a y , a s l i g h t d i p i n l i p i d c o n t e n t o f t h e l i v i n g r a y p a r e n c h y m a c e l l s i n t h e m i d d l e sapwood was d e t e c t e d . A l t h o u g h m a r g i n a l c e l l s g e n e r a l l y showed a h i g h e r c o n c e n t r a t i o n o f l i p i d s ( F i g u r e 7 - 4 ) , no s t a t i s t i c a l d i f f e r e n c e c o u l d be o b s e r v e d b e c a u s e o f g e n e r a l l y h e t e r o g e n e o u s v a r i a n c e s e x c e p t i n A u g u s t , ( T a b l e 1 4 ) , 116 when the l i p i d content i n m a r g i n a l c e l l s was h i g h e r than t h a t i n c e n t r a l c e l l s . Both i n the m a r g i n a l and c e n t r a l c e l l s , t h e r e was a s i g n i f i c a n t v a r i a n c e d i f f e r e n c e between a l l con t iguous sampl ing p e r i o d s (Table 15) , p r e c l u d i n g a n a l y s i s of means. 5 .5 .3 Discussion The d i s t r i b u t i o n p a t t e r n s of the RLC i n the l i v i n g ray parenchyma c e l l s ( F i g u r e 7) d i f f e r e d from s p e c i e s to s p e c i e s and from month to month. In a l l f o u r s p e c i e s the RLC was h i g h , a l t h o u g h i n A b i e s ba l samea, a reduced l i p i d content was found i n June , August and O c t o b e r . These f o u r s p e c i e s can be c a t e g o r i z e d as "fat" t r e e s due to t h e i r h i g h l i p i d c o n c e n t r a t i o n ( F i s h e r 1891, A r r h e n i u s 1942). Shah e_t a_l. (1981) i n d i c a t e d tha t A c a c i a n i l o t i c a specimens c o l l e c t e d i n the summer showed a d e c r e a s i n g l i p i d content toward the sapwood/heartwood boundary . For specimens c o l l e c t e d i n December, an i n c r e a s i n g t r e n d of l i p i d content was r e p o r t e d i n T i l i a c o r d a t a 117 ( H o l l and P r i e b e 1985) and P inus d e n s i f l o r a (Yamamoto 1982) . However, a r e l a t i v e l y h i g h c o n s t a n t l i p i d content i n the l i v i n g ray parenchyma c e l l s a c r o s s the sapwood was r e p o r t e d i n P inus s t robus and Pinus  b a n k s i a n a (Fukazawa et a_l. 1980, Yamamoto 1982) f o r the w i n t e r p e r i o d . In t h i s s t u d y , a wide v a r i e t y of l i p i d content i n the l i v i n g c e l l s i n the inner sapwood near the boundary of the sapwood and heartwood was o b s e r v e d . No t y p i c a l r a d i a l d i s t r i b u t i o n p a t t e r n c o u l d be seen from the data c o l l e c t e d . L i p i d d r o p l e t s may be p r e s e n t throughout the y e a r , and they may i n c r e a s e i n s i z e and number i n the autumn, as r e p o r t e d by Robards and Kidwai (1969) i n S a l i x  f r a q i l i s , by Murmanis (1971) i n P inus s t r o b u s , and by P a r k e r (1957) i n P inus ponderosa . Fukazawa et a l . (1980) and Yamamoto (1982) s t u d i e d P inus banks iana and r e p o r t e d t h a t the l i p i d content was minimal d u r i n g the summer and at a maximum i n w i n t e r . Mia (1972) s t u d i e d Populus t r e m u l o i d e s w i t h t r a n s m i s s i o n e l e c t r o n microscopy and found t h a t f a t s d i s a p p e a r e d i n June and e a r l y J u l y . The presence of l i p i d d r o p l e t s i n the summer was r e p o r t e d by Nobuchi (1985) i n a g r e a t number 118 of s p e c i e s . In t h i s s t u d y , a h i g h RLC i n the l i v i n g ray parenchyma c e l l s i n the f o u r s p e c i e s s t u d i e d was found i n J u l y . The reason f o r the d i s c r e p a n c y of l i p i d c o n t e n t between r e s u l t s o f the present s tudy and those o b t a i n e d by o ther i n v e s t i g a t o r s i s s t i l l unknown. There was a d e c r e a s i n g RLC i n the l i v i n g ray parenchyma c e l l s from the outer sapwood towards the i n n e r sapwood i n samples from a p p r o x i m a t e l y one q u a r t e r of the sample p e r i o d s . S e c t i o n s from a p p r o x i m a t e l y one s i x t h of the sample p e r i o d s showed i n c r e a s e d RLC i n the l i v i n g c e l l s from the o u t e r sapwood inwards . The r e m a i n i n g samples showed u n i f o r m l y h i g h RLC i n the l i v i n g c e l l s a c r o s s the sapwood. No f i x e d p a t t e r n c o u l d be e s t a b l i s h e d f o r a g i v e n t r e e . Nobuchi (1985) found t h a t l i p i d d r o p l e t s i n c r e a s e d i n aged ray parenchyma c e l l s . He a l s o found some l i p i d d r o p l e t s i n the heartwood r e g i o n . In t h i s s t u d y , some l i p i d - l i k e d r o p l e t s and l i p i d - l i k e lumps were observed i n the newly formed heartwood. These d r o p l e t s and lumps were not s t a i n e d w i t h Sudan b l a c k B . T h i s s tudy demonstrated t h a t the l i p i d d i s t r i b u t i o n was s p e c i e s dependent and 119 r e l a t e d to sampl ing p e r i o d s , l o c a t i o n of sapwood, and ray c e l l t y p e . In P inus banks iana the RLC i n the l i v i n g sapwood ray parenchyma c e l l s i n May c o r r e l a t e d p o s i t i v e l y w i t h MC, SR, NEI and RSC (Table 16) . However, no such c o r r e l a t i o n c o u l d be e s t a b l i s h e d i n the o t h e r three s p e c i e s . Many b o t a n i s t s b e l i e v e tha t an a c c u m u l a t i o n of s t a r c h i n the s p r i n g i s accompanied by a decrease i n l i p i d content (Koz lowski and K e l l e r 1966, A l d e n and Hermann 1971, Kramer and Koz lowsk i 1979) . I f they indeed were c o n v e r t i b l e , one would expect s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n c o e f f i c i e n t s between the two measures . However, as shown i n T a b l e 17, the r e l a t i o n -s h i p between the RLC and the RSC v a r i e d between p o s i t i v e and n e g a t i v e and depended on s p e c i e s and sampl ing p e r i o d . Fukazawa et a_l. (1980) and Yamamoto (1982) r e p o r t e d tha t P inus s t r o b u s and P inus b a n k s i a n a had a low or zero l i p i d content i n the summer which was c o i n c i d e n t w i t h a r e l a t i v e l y h i g h s t a r c h c o n t e n t . Nobuchi (1985) a l s o observed a s t r o n g n e g a t i v e c o r r e l a t i o n between the d i s t r i b u t i o n of s t a r c h and l i p i d a c r o s s the sapwood i n C r y p t o m e r i a j a p o n i c a over a Table 16. Correlation coefficients between the relative lipid content and the moisture content, survival rate, nuclear elongation index and the relative starch content in May specimen. Species RLC/MC KLC/SR RLC/NEI RLS/RSC Pinus banksiana 0.711** 0. 912** 0.874** 0.872** Picea mariana 0.171ns 0. 281ns 0.111ns 0.301ns Abies balsamea -0.034ns -0. 548* - 0.441ns -0.560* Populus -0.028ns -0. 279ns - 0.202ns -0.254ns tremuloides ns - not significant at °< > 0.05 * - s ignif icant at <=< £ 0.05 ** - s ignif icant at cK ^ 0.01 RLC - re lat ive l i p i d content RSC - re lat ive starch content MC - moisture content SR - survival rate NEI - nuclear elongation index Table 17. Correlation coefficients between the mean relative starch and lipid contents of ray parenchyma cells over various sampling periods. Populus Pinus banksiana Picea mariana Abies balsamea tremuloides  Month  May 0.872** 0.301ns - 0.560* - 0.254ns June 0.163ns 0.653** 0.271ns 0.683** July 0.481* b 0.868** 0.560** Aug. 0.343ns 0.378ns 0.175ns 0.666** October 0.052ns 0.645** 0.561** 0.720** Nov./Dec. a b -0.525* 0.147ns b a - November for Abies balsamea and Populus tremuloides - December for Pinus banksiana and Picea mariana b - no starch granules ns - not s ignif icant <* > 0.05 * - s ignificant at <* £ 0.05 ** - s ignif icant at < 0.01 122 twelve-month p e r i o d . In the present s t u d y , s t a r c h was absent or n e a r l y so from a l l s p e c i e s i n November/December and from P i c e a  mariana i n J u l y ( F i g u r e 6 ) . In both cases the l i p i d c o n t e n t was h i g h ( F i g u r e 7 ) . Thus , i t appears t h a t l i p i d s may be presen t w h i l e s t a r c h i s a b s e n t , but t h a t the presence of s t a r c h does not p r e c l u d e l i p i d c o n c e n t r a t i o n . At s e v e r a l o ther t i m e s , a p o s i t i v e r e l a t i o n s h i p between the RSC and the RLC was observed i n s e v e r a l cases (Tab le 1 7 ) . T h i s i s p a r t i c u l a r l y c o n s i s t e n t i n Populus t r e m u l o i d e s . T h i s p o s i t i v e r e l a t i o n s h i p between s t a r c h and l i p i d c o n t e n t s has not been r e p o r t e d p r e v i o u s l y . 5.6 Ultrastructure of Cell Organelles 5 .6 .1 Specimen Preparation Four s m a l l wood s t i c k s , 1 mm x 1 mm i n c r o s s -s e c t i o n and 2 mm i n l e n g t h , were cut from each core f o r c e l l u l t r a s t r u c t u r e s t u d i e s . These s m a l l s t i c k s were taken from the growth r i n g s l i s t e d i n T a b l e 18. Few Table 18. The location of specimens collected for ultrastructural studies. Number refer to the growth ring numbers counted from the cambium. Species Outer Sapwood Middle Sapwood Inner Sapwood Heartwood Pinus banksiana 1 12 23-25 26-59 Picea mariana 1 7 13-17 18-64 Abies balsamea 1 7 13-16 17-65 Populus tremuloides 1 10 17-24 25-46 to U) 124 i n d i v i d u a l s t i c k s were taken at the i n n e r sapwood. One s m a l l s t i c k was taken from the middle of the heartwood. The f i r s t growth r i n g i n the August specimen and i n the specimens of subsequent months r e p r e s e n t e d the ear lywood formed i n t h a t c u r r e n t y e a r . . These s m a l l s t i c k s were p l a c e d i n marked v i a l s w i t h a 3% g l u t a r a l d e h y d e s o l u t i o n , vacuum i n f i l t r a t e d a t 500 mm Hg f o r one h o u r , r i n s e d f i v e t imes w i t h a 0.2 M potas s ium phosphate b u f f e r (pH 7.2) over a ten minute i n t e r v a l , and p o s t - f i x e d w i t h 2% osmium t e t r o x i d e i n a 0.2 M potass ium phosphate b u f f e r (pH 7.2) f o r two h o u r s . The f i x e d s t i c k s were r i n s e d w i t h a phosphate b u f f e r , dehydrated i n an acetone-water s e r i e s f o l l o w e d by p r o p y l e n e o x i d e , and embedded i n an u l t r a - l o w v i s c o s i t y medium VCD/HXSA ( O l i v e i r a e t a_l. 1983) e n s u r i n g s u i t a b l e o r i e n t a t i o n f o r s e c t i o n i n g and o b s e r v a t i o n . B o t h , r a d i a l and t r a n s v e r s e s e c t i o n s , 600-900 A t h i c k , were p r e p a r e d w i t h a diamond k n i f e and mounted on an uncoated 150 mesh s i l v e r g r i d . 125 5.6.2 Observation C h a r a c t e r i s t i c f e a t u r e s of the f i v e c e n t r a l ray c e l l o r g a n e l l e s due to s p e c i e s , age, and seasona l v a r i a t i o n were o b s e r v e d . These were: (a) N u c l e i - c h r o m a t i n and n u c l e a r enve lope ; (b) A m y l o p l a s t s - s i z e and shape of s t a r c h g r a n u l e s ; (c) L i p i d d r o p l e t s - s i z e , shape and d e n s i t y ; (d) M i t o c h o n d r i a - s i z e , shape, d i s t r i b u t i o n and c r i s t a morphology; and (e) Plasmalemma- s u r f a c e morphology and v e s i c l e a t tachment . Other c e l l o r g a n e l l e s , i n c l u d i n g the endoplasmic r e t i c u l u m , d ic tyosomes , p l a s t i d s , v a c u o l e and ribosomes were sometimes observed and have been d i s c u s s e d i n r e l a t i o n to the morphology of the f i v e p r i n c i p a l c e l l o r g a n e l l e s ment ioned. These o b s e r v a t i o n s were made on t h i n r a d i a l s e c t i o n s o b t a i n e d from the c e n t r a l c e l l of a ray at the o u t e r , m i d d l e , and i n n e r sapwood, and heartwood (Table 18) . Some t r a n s v e r s e s e c t i o n s were a l s o examined to determine the d i f f e r e n c e between v a r i o u s sapwood zones . The c h a r a c t e r i s t i c c y t o l o g i c a l f e a t u r e s of ray parenchyma c e l l s were r e c o r d e d and a p p r o p r i a t e micrographs ( c a . 2400x) were taken w i t h a t r a n s m i s s i o n e l e c t r o n microscope (TEM). A t r a n s v e r s e s e c t i o n of an outer sapwood ray parenchyma c e l l from the November specimen of Abies  balsamea i s presented i n F i g u r e 8 to i l l u s t r a t e the d i s t r i b u t i o n of c e l l o r g a n e l l e s . The key f e a t u r e s of the c e l l o r g a n e l l e s from each s p e c i e s i n the t h r e e sapwood zones and the heartwood are d i s p l a y e d i n P l a t e s 1 to 34 and t a b u l a t e d i n Appendix 3. Each p l a t e c o n s i s t s of s i x TEM m i c r o - g r a p h s i l l u s t r a t i n g the u l t r a s t r u c t u r e of the c e l l o r g a n e l l e s at the r a d i a l p o s i t i o n i n d i c a t e d . The J u l y and November/December specimens were s c r u t i n i z e d more i n t e n s e l y than those from the o ther months s i n c e these are the months of the h i g h e s t and lowest m e t a b o l i c a c t i v i t y r e s p e c t i v e l y of a ray parenchyma c e l l i n the Thunder Bay a r e a . Figure 8. An e l e c t r o n micrograph of a t r a n s v e r s e s e c t i o n of an A b i e s balsamea outer sapwood ray parenchyma c e l l c o l l e c t e d i n November, i n d i c a t i n g the l o c a t i o n and d i s t r i b u t i o n of the c e l l o r g a n e l l e s . Two l a r g e v a c u o l e s are l o c a t e d at both s i d e s of a nuc l eus which i s surrounded by an o r g a n e l l e f r e e zone. L i p i d d r o p l e t s are l o c a t e d at the r i g h t s i d e of the photo . Note: Am-amylop las t ; C W - c e l l w a l l ; ER-endoplasmic r e t i c u l u m : H B - h a l f - b o r d e r e d p i t - p a i r ; L - l i p i d d r o p l e t ; M-mi tochondr ion; N - n u c l e u s ; Pd-plasmodesmata; P t - p l a s t i d ; V - v a c u o l e . 1 2 9 5 .6 .3 Results and Discussion 5 . 6 . 3 . 1 Pinus banksiana 5 . 6 . 3 . 1 . 1 Seasonal Variation The most s t r i k i n g f e a t u r e s of c e l l o r g a n e l l e s due to season and ag ing are i l l u s t r a t e d i n P l a t e s 1 to 9. The u l t r a s t r u c t u r e of the n u c l e u s showed no d i s t i n g u i s h i n g f e a t u r e s between J u l y and December ( P l a t e s 1-C and 1 - F ) . T h i s f i n d i n g i s i n agreement w i t h t h a t r e p o r t e d by Tsuda (1975 a ,b) i n P inus d e n s i f l o r a and twenty-seven o ther c o n i f e r s . No s t a r c h g r a n u l e s were observed i n the August and December ray parenchyma c e l l s of the o u t e r sapwood. The absence of a m y l o p l a s t s i n autumn and w i n t e r xylem and phloem ray parenchyma c e l l s was r e p o r t e d p r e v i o u s l y by Tsuda and S h i m a j i (1971) from Pinus d e n s i f l o r a and Tsuda (1975b) from a great number o f c o n i f e r o u s s p e c i e s . As i l l u s t r a t e d i n F i g u r e 6-1 , no s t a r c h was r e c o r d e d i n August and December i n the o u t e r sapwood. In December, the ray parenchyma c e l l s c o n t a i n e d 130 more l a r g e l i p i d d r o p l e t s than i n J u l y . T h i s o b s e r v a t i o n i s s i m i l a r to t h a t of Tsuda (1975b) i n members of P i n a c e a e . However, t h e r e was a r e l a t i v e l y l a r g e amount of l i p i d s i n the o u t e r sapwood d u r i n g both J u l y and December ( F i g u r e 1 ) . T h i s d i s c r e p a n c y might be a t t r i b u t e d to a l i m i t e d number of h i g h l y v a r i a b l e ray parenchyma c e l l s s t u d i e d w i t h the TEM. In e s t i m a t i n g the d i s t r i b u t i o n of l i p i d or s t a r c h g r a n u l e s , o b s e r v a t i o n s from l i g h t microscopy are expected to be more r e l i a b l e s t a t i s t i c a l l y than those from the TEM, because of the g r e a t e r number of c e l l s and l a r g e r area o b s e r v e d . Ray parenchyma c e l l s i n J u l y c o n t a i n e d many r o d - l i k e m i t o c h o n d r i a , which c o n t a i n e d numerous, l o n g , t u b u l a r c r i s t a e ( P l a t e s , 2-C and 4 - E ) . Only a few round m i t o c h o n d r i a were observed i n the December c e l l s . T h i s s e a s o n a l v a r i a t i o n i s s i m i l a r to t h a t r e p o r t e d by K e i t h and Godkin (1976) i n P i n u s r e s i n o s a . The s u r f a c e of plasmalemma appeared rough i n J u l y , but smooth or a l i t t l e rough i n December i n both the middle and i n n e r sapwood. Few dictyosomes were observed 131 i n the J u l y c e l l s and none c o u l d be d e t e c t e d i n the December c e l l s . T h i s o b s e r v a t i o n i n d i c a t e d a h i g h m e t a b o l i c a c t i v i t y i n J u l y , i n which the rough s u r f a c e of plasmalemma and presence of d ic tyosomes are a c t i v e i n c e l l w a l l f o r m a t i o n (Cronshaw 1965, Esau et a l . 1966, E v e r t and Deshpande 1970, Chafe and Chauret 1974, B e r l y n 1979, Rao and Catesson 1987). Summary f e a t u r e s of f i v e c e l l o r g a n e l l e s , i . e . , n u c l e u s , a m y l o p l a s t , l i p i d d r o p l e t s , m i t o c h o n d r i a and plasmalemma i n s i x sampl ing p e r i o d s of a l l s p e c i e s s t u d i e d are presented i n Appendix 3. The main c h a r a c t e r i s t i c f e a t u r e s of P inus b a n k s i a n a ray parenchyma c e l l s i n J u l y and December are summarized i n T a b l e 19. 5 . 6 . 3 . 1 . 2 A g i n g Process The u l t r a s t r u c t u r a l d i f f e r e n c e s between young and aged ray parenchyma c e l l s of P inus b a n k s i a n a are shown i n P l a t e s 1-9. The nuc leus i n a young ray parenchyma c e l l ( P l a t e 1) was c h a r a c t e r i z e d by even ly d i s p e r s e d c h r o m a t i n and Table 19. The main characteristic features of five cell organelles in the outer sapwood ray parenchyma cells of Pinus banksiana in July and December. Cell Organelle July 20, 1983 December 2, 1983 Nucleus Chromatin evenly dispersed, smooth nuclear envelope, r i ch in nucleoplasm Same as July Amyloplast L ip id Droplets Absent or rarely present; i f present, amyloplast contains a starch granule with thylakoid structure and osmiophilic globuli A few small l i p i d droplets Absent Numerous small and large l i p i d droplets Mitochondria Rod-like in shape with numerous long tubular cristae Rarely present; i f present, round in shape with few short tubular cristae Plasmalemma Surface rough Surface smooth or a l i t t l e rough 133 a smooth n u c l e a r e n v e l o p e . In c o n t r a s t , the a g g r e g a t i o n of c h r o m a t i n and a l a c k of nuc l eop lasm were pronounced i n the aged c e l l at the i n n e r sapwood. The i r r e g u l a r i t y or l o b i n g of the n u c l e a r envelope ( P l a t e 7) was a common f e a t u r e of aged ray parenchyma c e l l s . The change i n n u c l e u s due to ag ing i s s i m i l a r to t h a t observed i n the seed of Zea mays by B e r j a k and V i l l i e r s (1970, 1 9 7 2 a , b ) . Most a m y l o p l a s t s i n a young ray parenchyma c e l l were s m a l l . S t a r c h g r a n u l e s were e longated and narrow i n the a m y l o p l a s t . Some of the a m y l o p l a s t s a l s o c o n t a i n e d a few t h y l a k o i d s t r u c t u r e s s u r r o u n d i n g the s t a r c h g r a n u l e s . A few o s m i o p h i l i c g l o b u l i were found i n the a m y l o p l a s t ( P l a t e s 3 and 4 ) . Some young ray parenchyma c e l l s l a c k e d a m y l o p l a s t s d u r i n g August and December sugges t ing some s e a s o n a l l y i n f l u e n c e d v a r i a t i o n . In compar i son , s t a r c h g r a n u l e s i n an aged ray parenchyma c e l l changed from an e l o n g a t e d to a round shape ( P l a t e s 7 and 8 ) . The s t a r c h g r a n u l e i n an aged c e l l showed some c r a c k s which might r e f l e c t c h e m i c a l d i f f e r e n c e s due to a g i n g . T h i s i s c o n s i s t e n t w i t h s p e c t r a l a b s o r p t i o n measurements ( H o l l and 134 L e n d z i a n 1973) which demonstrated t h a t s t a r c h g r a n u l e s i n the outer sapwood c o n s i s t e d l a r g e l y of amylose m o l e c u l e s , w h i l e those i n the i n n e r sapwood (aged) were a m y l o p e c t i n m o l e c u l e s . A young ray parenchyma c e l l has s m a l l (approx . 1 um d iameter ) and numerous l i p i d d r o p l e t s ( P l a t e s 1 -4) . In aged ray parenchyma c e l l s , l i p i d d r o p l e t s were of v a r i o u s s i z e s , sometimes even appear ing as b i g lumps ( P l a t e s 7 and 8 ) . The b i g l i p i d - l i k e lump might move to a d j a c e n t t r a c h e i d s i n the heartwood zone as shown i n P l a t e 9. I t i s b e l i e v e d t h a t t h i s i s p a r t of the heartwood substance (Wardrop and Cronshaw 1962, Hasegawa and S h i r o y a 1965, H i l l i s and Inoue 1968, Nobuchi e t al. 1976, P a n d a l a i e t a l . 1985) . Bhat and P a t e l (1980) and Nobuchi (1985) r e p o r t e d some l i p i d d r o p l e t s i n the heartwood zone next to the boundary of sapwood and heartwood. The presence of l i p i d - l i k e d r o p l e t s i n newly formed heartwood might be due to a d i f f e r e n t c h e m i c a l r e a c t i o n d u r i n g the t r a n s f o r m a t i o n of sapwood i n t o heartwood. The l i p i d - l i k e d r o p l e t s and l i p i d - l i k e lumps i n the newly formed heartwood ray parenchyma c e l l s were v i s i b l e i n l i g h t m i c r o s c o p y . 135 However, u n l i k e those i n the sapwood d r o p l e t s and lumps, they were not s t a i n e d by Sudan b l a c k B. One o f the i n t e r e s t i n g f i n d i n g s of t h i s s tudy i s t h a t t h e r e were two s t a i n i n g d e n s i t i e s observed i n many l i p i d d r o p l e t s . These l i p i d d r o p l e t s were most ly found i n the i n n e r or middle sapwood ( P l a t e s 5 - B , E , F and 6-A , D , E , F ) . I t i s known (Robards 1970, J u n i p e r e_t a l . 1970) t h a t u n s a t u r a t e d f a t s show i n t e n s e b l a c k s t a i n i n g w e l l w i t h Os O4 whereas s a t u r a t e d f a t s show l i g h t gray d r o p l e t s w i t h Os 0 4 s t a i n i n g . T h i s d i f f e r e n t i a l Os O4 s t a i n i n g i s a t t r i b u t e d to the a v a i l a b i l i t y of s i t e s i n u n s a t u r a t e d f a t t y a c i d s which b i n d the s t a i n s t r o n g l y ( J u n i p e r et a_l. 1970) . L i p i d d r o p l e t s w i t h two s t a i n i n g responses appeared most ly i n l i p i d r i c h ray parenchyma c e l l s of the middle and i n n e r sapwood ( F i g u r e 7 ) . The decrease i n s t a i n i n g d e n s i t y and c o a l e s c e n c e of l i p i d d r o p l e t s was seen i n the middle of the sapwood ( P l a t e s 5 and 6 ) . In J u l y , the l i g h t l y s t a i n e d l i p i d was seen on the s u r f a c e , expanding toward the c e n t r e of the l i p i d d r o p l e t s . There was an i n c r e a s i n g amount of l i g h t l y s t a i n e d l i p i d noted between the middle and i n n e r sapwood, where the 136 d r o p l e t s were l a r g e and o n l y l i g h t l y s t a i n e d . The change of l i g h t d r o p l e t s t a i n i n g d e n s i t y and t h e i r s i z e i n c r e a s e was f i r s t n o t i c e a b l e i n J u l y and c o n t i n u e d u n t i l December (Appendix 3 ) . Young ray parenchyma c e l l s c o n t a i n e d many round or r o d - l i k e m i t o c h o n d r i a but round m i t o c h o n d r i a were r a r e l y presen t i n aged ray parenchyma c e l l s ( P l a t e s 2, 7 and 8 ) . The number of m i t o c h o n d r i a l c r i s t a e decreased from young to o l d ray parenchyma c e l l s . T h i s changed m i t o c h o n d r i a and c r i s t a l morphology p r o v i d e d ev idence of d e c r e a s i n g m e t a b o l i c a c t i v i t y from the o u t e r sapwood towards the i n n e r sapwood (Brown and B e r t k e 1969, Bonner 1973, Ducet and Lance 1978). The o b s e r v a t i o n t h a t there were fewer m i t o c h o n d r i a i n o l d ray parenchyma c e l l s than i n young c e l l s has a l s o been r e p o r t e d by many r e s e a r c h e r s ( F r e y - W y s s l i n g and Bosshard 1959, Nobuchi and Harada 1968, Mia 1972, Nobuchi 1985). They b e l i e v e d t h a t few i n the number of m i t o c h o n d r i o n l eads to decreased m e t a b o l i c a c t i v i t y of c e l l s i s due to a g i n g . I t i s w i d e l y b e l i e v e d ( E r n s t e r 1965, S i m i n o v i t c h e t a_l. 1967, Munn 1969, Buvat 1969, Loewy and S i e h e v i t z 1969, Bonner 1973, Ducet and Lance 137 1978) t h a t a s t r o n g p o s i t i v e r e l a t i o n s h i p e x i s t s between the number of m i t o c h o n d r i a and the m e t a b o l i c a c t i v i t y of a c e l l . These TEM o b s e r v a t i o n s are c o n s i s t e n t w i t h the NEI measurement ( F i g u r e 5) where the m e t a b o l i c a c t i v i t y of the c e l l expressed by the NEI decreased from the o u t e r sapwood towards the i n n e r sapwood. P l a t e s 5-c and 6-c show t h a t r o d - l i k e m i t o c h o n d r i a appeared f r e q u e n t l y i n the middle of the sapwood d u r i n g Augus t . F i g u r e 5-1 i n d i c a t e s an i n c r e a s e o f the NEI i n the middle of the sapwood i n August and O c t o b e r . The s u r f a c e of the plasmalemma of young ray parenchyma c e l l s i n J u l y appeared somewhat rough ( P l a t e 2 - C ) . V e s i c l e s were a t t a c h e d to the s u r f a c e of the plasmalemma i n an aged ray parenchyma c e l l and t h i s p r o v i d e d a d i s t i n g u i s h i n g m o r p h o l o g i c a l s t r u c t u r e . These v e s i c l e s were found i n May through O c t o b e r , e s p e c i a l l y i n Augus t , but were absent i n December ( P l a t e 8, Appendix 3 ) . The v e s i c l e attachment to the plasmalemma c o u l d a l s o be found i n the middle of the sapwood ( P l a t e 6) where t h e r e were some dead ray parenchyma c e l l s . The v e s i c l e s e v e n t u a l l y fuse w i t h the 138 plasmalemma and release their contents, which are believed to be c e l l wall precursors (Cronshaw 1965, Esau et a_l. 1966, Walker and Bisalputra 1967, Chafe and Chauret 1974, Berlyn 1979, Rao and Catesson 1987). After the fusion of the vesicle contents, the ray parenchyma cells undergo their maturation through c e l l wall thickening and l i g n i f i c a t i o n . It is believed that the fusion of the vesicles is a sign of secondary wall ini t i a t i o n leading to lignification and the eventual death of ray parenchyma c e l l s . Pinus banksiana is one of the Diploxylon pines where the secondary wall thickening of the ray parenchyma cells is delayed from outer sapwood immediately next to the cambium to the sapwood/heartwood boundary. The absence of vesicles in younger ray parenchyma cells may be the cause of the delayed formation of their secondary walls in Pinus  banksiana. It should be noted that death of ray parenchyma cells was not restricted to the sapwood growth ring next to the heartwood. As illustrated in Figure 3, death of these cells began at the seventh growth ring 139 counted from the cambium. Nobuchi (1985) demonstrated t h a t the degree of c o l o u r a t i o n of a ray parenchyma c e l l c o r r e l a t e d p o s i t i v e l y w i t h c e l l d e g r a d a t i o n and the number of dead c e l l s i n the i n n e r sapwood of C r y p t o m e r i a j a p o n i c a . He f u r t h e r demonstrated t h a t the amount of methanol e x t r a c t i v e s i n c r e a s e d from the i n n e r sapwood to the boundary of sapwood and heartwood. The d i s t r i b u t i o n p a t t e r n o f methanol e x t r a c t i v e s c o i n c i d e d w i t h t h a t of the number of dead c e l l s i n the same sapwood zone. Ev idence c o l l e c t e d from the SR, N E I , and u l t r a - s t r u c t u r e of c e l l o r g a n e l l e s a l s o i n d i c a t e d a g r a d u a l d e t e r i o r a t i o n of l i v i n g c e l l s over t i m e . I t i s b e l i e v e d t h a t heartwood f o r m a t i o n i s ma in ly due to the death of l i v i n g sapwood parenchyma c e l l s through an a g i n g p r o c e s s . In P inus b a n k s i a n a , the g r a d u a l a d d i t i o n of a new heartwood r i n g to the e x i s t i n g heartwood beg ins d u r i n g J u l y / A u g u s t . T h i s process may l a s t from t h r e e or f o u r months to a year (Nelson 1978, Yamamoto et a l . 1978, Fukazawa et a l . 1980, Nobuchi 1985). The main c h a r a c t e r i s t i c ag ing f e a t u r e s of f i v e c e l l o r g a n e l l e s of ray parenchyma c e l l s are summarized i n T a b l e 20. Table 20. The main characteristic features of five cell organelles of ray parenchyma cells of Pinus banksiana in the outer and inner sapwood. Cell Organelle Outer Sapwood Inner Sapwood Nucleus Amyloplast L ip id Droplets Chromatin evenly dispersed, smooth nuclear envelope, r i ch in nucleoplasm One, rarely two elongated starch granules with thylakoid and osmiophilic globuli Small, numerous; single staining density Chromatin aggregates patchy, irregularity or lobing of nuclear envelope, lack of nucleoplasm Swollen starch granule occupying the whole amyloplast, sometimes with crack-scar; no thylakoid Numerous, small, large or in lumps; sometimes with two staining densities Mitochondria Numerous, round, and/or rod-shape, with numerous cristae Absent or rarely present, round in shape; cristae few or absent Plasmalemma Smooth to rough surface Mainly rough or broken surface; sometimes small to large vesicles attached 141 5 . 6 . 3 . 2 Picea mariana 5 . 6 . 3 . 2 . 1 Seasonal Variation The u l t r a s t r u c t u r e of the nuc leus at the o u t e r sapwood ray parenchyma c e l l s d i s p l a y e d no s i g n i f i c a n t d i f f e r e n c e s between the v a r i o u s sampl ing p e r i o d s ( P l a t e 10 and Appendix 3 ) . No a m y l o p l a s t s were observed i n the ray parenchyma c e l l s d u r i n g J u l y and December. However, some s t a r c h g r a n u l e s were observed i n December w i t h l i g h t microscopy ( F i g u r e 6 - 2 ) . As mentioned e a r l i e r , the TEM o b s e r v a t i o n s were done on a l i m i t e d number of spec imens . In o ther months, one or two e l o n g a t e d s t a r c h g r a n u l e s were found i n the a m y l o p l a s t s of the ray parenchyma c e l l s , t o g e t h e r w i t h a few t h y l a k o i d s and o s m i o p h i l i c g l o b u l i ( P l a t e s 10-12) . Many p l a s t i d s w i t h p a r a l l e l t h y l a k o i d s and o s m i o p h i l i c g l o b u l i were found ( P l a t e s 11-D and 1 2 - D ) . Whether these p l a s t i d s are p r e c u r s o r s of the a m y l o p l a s t remains to be s t u d i e d . Many s m a l l , h e a v i l y s t a i n e d l i p i d d r o p l e t s were p r e s e n t i n the J u l y ray parenchyma c e l l s from the outer 142 sapwood. L i p i d d r o p l e t s w i t h two s t a i n i n g d e n s i t i e s were observed i n December c e l l s . Sometimes, l a r g e l i p i d d r o p l e t s were observed i n J u l y c e l l s ( P l a t e 1 0 - C ) . An abundance of l i p i d d r o p l e t s was a l s o observed w i t h the l i g h t microscope ( F i g u r e 7 - 2 ) . Many round and r o d - l i k e m i t o c h o n d r i a w i t h t u b u l a r c r i s t a e were found i n ray parenchyma c e l l s at the o u t e r sapwood d u r i n g J u l y . M i t o c h o n d r i a i n December c e l l s were round and c o n t a i n e d few s a c c u l a r c r i s t a e . As r e p o r t e d by many r e s e a r c h e r s (Lund et a l . 1958, E r n s t e r 1965, Bonner 1973, Munn 1974, Douce 1985) , the number of m i t o c h o n d r i a and c r i s t a e show a d i r e c t p o s i t i v e r e l a t i o n s h i p w i t h c e l l m e t a b o l i c a c t i v i t y . The o c c u r r e n c e of few m i t o c h o n d r i a and c r i s t a e i n the December ray parenchyma c e l l s suggest t h e i r r e l a t i v e l y low m e t a b o l i c a c t i v i t y . T h i s o b s e r v a t i o n i s i n agreement w i t h the l i g h t m i c r o s c o p i c s tudy which i n d i c a t e s a r e l a t i v e l y lower NEI i n December than i n J u l y . The s u r f a c e of plasmalemma was smooth i n the J u l y c e l l s but rough i n the December c e l l s . The rough s u r f a c e of plasmalemma i n w i n t e r was a l s o observed by 143 R i d i n g and L i t t l e (1984) and Wisn iewsk i and Ashworth (1986) . Rough s u r f a c e s of plasmalemma were a l s o observed i n May, June and October ( P l a t e 1 1 ) . The main c h a r a c t e r i s t i c f e a t u r e s of ray parenchyma c e l l s i n J u l y and December are summarized i n T a b l e 21. 5 . 6 . 3 . 2 . 2 A g i n g Process The u l t r a s t r u c t u r a l d i f f e r e n c e s between a young and an aged ray parenchyma c e l l of P i c e a mariana are shown i n P l a t e s 10-18. The nuc l eus i n a young ray parenchyma c e l l ( P l a t e 10) , f e a t u r e d evenly d i s p e r s e d chromat in and was r i c h i n n u c l e o p l a s m . In c o n t r a s t the aged c e l l n u c l e u s c o n t a i n e d chromat in aggregated i n t o h e a v i l y s t a i n e d p a t c h e s , and l a c k e d nuc leop lasm ( P l a t e 16 ) . Some n u c l e i , such as t h a t i n P l a t e 16-B, were l o b e d i n a way s i m i l a r to t h a t r e p o r t e d i n aged seeds ( B e r j a k and V i l l i e r s 1970). In young ray parenchyma c e l l s , the a m y l o p l a s t o c c u r r e n c e seemed to depend upon the s e a s o n . I f i t was p r e s e n t , an a m y l o p l a s t possessed one or two e l o n g a t e d Table 21. The main characteristic features of five cells organelles in the outer sapwood parenchyma cells of Picea mariana in July and December. Cell Organelle July 20, 1983 December 23, 1983 Nucleus Chromatin evenly dispersed, smooth nuclear envelope, r i ch in nucleoplasm Same as in July Amyloplast Absent; some plastids with osmiophilic globuli Absent, no plastids L i p i d Droplets Numerous small, black stained l i p i d droplets. Some large l i p i d droplets also exist Very numerous with two staining densities Mitochondria Round and mainly rod-l ike in shape, very numerous, many tubular cristae Round in shape, numerous, few saccular cristae. Plasmalemma Surface smooth Surface rough 145 s t a r c h g r a n u l e s and some o s m i o p h i l i c g l o b u l i ( P l a t e 11 ) . The l a t t e r have been r e p o r t e d p r e v i o u s l y (Badenhuizen 1962, 1969, Valanne 1971, Tsuda 1975 a , b ) , but no attempts have been made to i n t e r p r e t t h e i r s i g n i f i c a n c e . In aged ray parenchyma c e l l s , a s w o l l e n s t a r c h g r a n u l e o c c u p i e d an e n t i r e a m y l o p l a s t ( P l a t e 16-A) and no t h y l a k o i d s t r u c t u r e s or o s m i o p h i l i c g l o b u l i were observed . In O c t o b e r , some s t a r c h g r a n u l e s were observed w i t h l i g h t microscopy ( F i g u r e 6 -2 ) , but not e a s i l y found w i t h the TEM owing to t h e i r i n f r e q u e n c y . In g e n e r a l , t h e r e were many s m a l l l i p i d d r o p l e t s i n young ray parenchyma c e l l s and a few l a r g e r ones , some of which formed lumps ( P l a t e s 16 and 17) i n aged ray parenchyma c e l l s . Two s t a i n i n g d e n s i t i e s commonly found i n l i p i d d r o p l e t s of h i g h RLC ray parenchyma c e l l s i n the middle and i n n e r sapwood ( F i g u r e 7 ) , were not found i n young ray parenchyma c e l l s . L i p i d - l i k e d r o p l e t s or lumps were a l s o found i n the heartwood samples ( P l a t e 18) from June to O c t o b e r . D u r i n g the same p e r i o d s , l i p i d d r o p l e t s were s t a i n e d w i t h two s t a i n i n g d e n s i t i e s , a c o n d i t i o n t h a t 146 o c c a s i o n a l l y extended to December (Appendix 3 ) . M i t o c h o n d r i a i n young ray parenchyma c e l l s were numerous and l a r g e l y r o d - l i k e . The i n n e r membrane of m i t o c h o n d r i a possessed numerous t u b u l a r or s a c c u l a r c r i s t a e . In c o n t r a s t , m i t o c h o n d r i a i n aged ray parenchyma c e l l s were round and had few or no c r i s t a e . The m o r p h o l o g i c a l s t r u c t u r e of m i t o c h o n d r i a and the f requency of appearance i n d i c a t e d a d e c l i n e i n v i t a l i t y from young to aged ray parenchyma c e l l s . T h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h the NEI o b t a i n e d by l i g h t microscopy ( F i g u r e 5-2) which i n d i c a t e s a d e c l i n e i n c e l l v i t a l i t y from the outer to the i n n e r sapwood. In young ray parenchyma c e l l s the plasmalemma s u r f a c e was rough or smooth depending upon the season ( P l a t e s 10 -12) . In an aged c e l l the plasmalemma e x h i b i t e d a rough or fragmented s u r f a c e . No v e s i c l e s were d e t e c t e d . As d i s c u s s e d p r e v i o u s l y i n s e c t i o n 5 . 6 . 3 . 1 . 2 , the v e s i c l e s a t t a c h e d to the plasmalemma i n P inus banks iana and b e l i e v e d r e s p o n s i b l e f o r the f o r m a t i o n of the secondary w a l l i n some ray parenchyma c e l l s of D i p l o x y l o n p i n e s . The secondary w a l l of ray parenchyma c e l l s i n P i c e a mariana was formed near the 147 cambium. A l a c k of v e s i c l e s i n the i n n e r sapwood of P i c e a mariana i s c o n s i s t e n t w i t h the e a r l y development of secondary w a l l s . The main c h a r a c t e r i s t i c f e a t u r e s of f i v e c e l l o r g a n e l l e s of ray parenchyma c e l l s due to ag ing are l i s t e d i n T a b l e 22. 5 . 6 . 3 . 3 Abies balsamea 5 . 6 . 3 . 3 . 1 Seasonal Variation The nuc l eus of a ray parenchyma c e l l i n the outer sapwood d i s p l a y e d a dense nuc leop lasm w i t h aggregated c h r o m a t i n . There was no d i f f e r e n c e i n the n u c l e a r s t r u c t u r e between J u l y and November ( P l a t e s 19 and 21) . The appearance and d i s t r i b u t i o n of the a m y l o p l a s t showed s e a s o n a l v a r i a t i o n . In J u l y , the a m y l o p l a s t u s u a l l y c o n t a i n e d a s i n g l e l a r g e s t a r c h g r a n u l e . The a m y l o p l a s t was absent i n November but a g r e a t number of p l a s t i d s w i t h long t h y l a k o i d s and o s m i o p h i l i c g l o b u l i were f o u n d . The presence of long t h y l a k o i d s and o s m i o p h i l i c g l o b u l i was a l s o observed by Tsuda (1975 Table 22. The main characteristic features of five cells organelles of ray parenchyma cells of Picea mariana in the outer and inner sapwood. Cell Organelle Outer Sapwood Inner Sapwood Nucleus Amyloplast Lip id Droplets Chromatin evenly dispersed, smooth nuclear envelope, r i ch in nucleoplasm If present, small elongated starch granules with osmiophilic globuli and thylakoids Small, numerous, black stained; some large l i p i d lumps, except in December, with two staining densities Chromatin aggregated and patchy; lobing of nuclear envelope; lack of nucleoplasm Swollen starch granule occupied entire amyloplast. No osmiophilic globuli and thylakoids Very numerous, various size or in lumps;some-times with two staining densities Mitochondria Very numerous, round and rod-l ike in shape; either saccular or tubular cristae Absent or rarely present with round shape; cristae few or absent Plasmalemma Surface smooth or rough Broken or rough surface 149 a ,b) i n o ther c o n i f e r o u s s p e c i e s . The s t a r c h c o n t e n t v a r i e d between sampl ing p e r i o d s ( F i g u r e 6 - 3 ) . In o ther months, t h e r e were o f t e n two e longated s t a r c h g r a n u l e s accompanied by t h y l a k o i d s and o s m i o p h i l i c g l o b u l i . The u l t r a s t r u c t u r e of s t a r c h g r a n u l e s a l s o showed s e a s o n a l d i f f e r e n c e s ( P l a t e s 19 -20 ) . Many s m a l l , d a r k - s t a i n e d , l i p i d d r o p l e t s were s c a t t e r e d u n i f o r m l y w i t h i n the J u l y r a y parenchyma c e l l s , but they were l o c a t e d main ly at the p e r i p h e r y of the November ray parenchyma c e l l s ( P l a t e s 19 and 21 and F i g u r e 8 ) . The reason f o r t h i s p e r i p h e r a l arrangement i s not known. Most m i t o c h o n d r i a i n the J u l y ray parenchyma c e l l s appeared r o d - l i k e and those i n the November ray parenchyma c e l l s were round ( P l a t e s 19 -21) . In the J u l y m i t o c h o n d r i a , the c r i s t a e were longer than those i n November. The number of m i t o c h o n d r i a was v e r y l a r g e i n b o t h months. The plasmalemma s u r f a c e of bo th the J u l y and November ray parenchyma c e l l s was r e l a t i v e l y smooth, a l t h o u g h i n some November c e l l s , some roughness was apparent ( P l a t e 2 1 - F ) . 150 The main c h a r a c t e r i s t i c f e a t u r e s of the J u l y and November ray parenchyma c e l l s are l i s t e d i n T a b l e 23. 5 . 6 . 3 . 3 . 2 A g i n g Process The u l t r a s t r u c t u r a l d i f f e r e n c e s between young and aged ray parenchyma c e l l s o f Abies balsamea are i l l u s t r a t e d i n P l a t e s 19-26. In young ray parenchyma c e l l s , the nuc l eus was c h a r a c t e r i z e d by d i s t i n c t i v e patchy c h r o m a t i n and abundant nuc leop lasm ( P l a t e s 19 and 21) . The nuc leus i n an aged c e l l had d i s t i n c t i v e chromat in but l a c k e d n u c l e o p l a s m . A t y p i c a l example of an aged nuc l eus i s shown i n P l a t e 24-D. In young ray parenchyma c e l l s , a m y l o p l a s t s o c c u r r e n c e depended upon the season . When they were p r e s e n t , most of the a m y l o p l a s t s c o n t a i n e d one or two e l o n g a t e d s t a r c h g r a n u l e s accompanied by o s m i o p h i l i c g l o b u l i ( P l a t e s 20A and 21A) . In aged c e l l s , a s w o l l e n s t a r c h g r a n u l e o c c u p i e d the e n t i r e a m y l o p l a s t ( P l a t e s 24 and 25) . Some of the s t a r c h g r a n u l e s i n aged c e l l s showed a c r a c k . As mentioned by o ther r e s e a r c h e r s Table 23. The main characteristic features of five cell organelles in the outer sapwood ray parenchyma cells of Abies balsamea in July and November. Cell Organelle July 23, 1984 November 20, 1984 Nucleus Amyloplast L i p i d Droplets Chromatin aggregated into distinctive patches with r i c h nucleoplasm; smooth nuclear envelope One or more large starch granules Very numerous,small in size,dark black stain, scattered throughout the c e l l Same as in July Absent, but sometimes numerous plastids with long thylakoids and/or osmiophilic globuli Numerous, small in size mainly located at the periphery of the c e l l ; dark black stain Mitochondria Plasmalemma Round and rod-l ike in shape; very numerous; cristae tubular in shape Surface smooth More round shaped than rod shaped; cristae in short tubular form. Surface smooth to s l ight ly rough 152 (Nobuchi 1985, P a n d a l a i et_ a_l. 1985), the c r a c k was o n l y found i n swo l l en s t a r c h g r a n u l e s ; no c r a c k c o u l d be found i n e longated s t a r c h g r a n u l e s . There was some d i f f e r e n c e between the l i p i d d r o p l e t s i n young and aged ray parenchyma c e l l s . Those i n aged c e l l s were s t a i n e d w i t h two s t a i n i n g d e n s i t i e s i n aged c e l l s i n J u l y and August , whereas those i n young ray parenchyma c e l l s were u n i f o r m l y b l a c k . Some l i p i d lumps were found i n some ray parenchyma c e l l s i n the middle sapwood ( P l a t e s 22 and 23) . Whether c e l l s c o n t a i n i n g l i p i d lumps were d y i n g or not i s u n c l e a r . I t i s b e l i e v e d t h a t l i p i d - l i k e heartwood subs tances ( P l a t e 26) were d e r i v e d from l i p i d d r o p l e t s . There were many, round and r o d - l i k e m i t o c h o n d r i a i n young r a y parenchyma c e l l s . M i t o c h o n d r i a i n aged ray parenchyma c e l l s were main ly round i n shape. Some m i t o c h o n d r i a l c r i s t a e were d i s c e r n i b l e i n aged c e l l s . The s o - c a l l e d "branch ing m i t o c h o n d r i a " (Douce 1985) were observed i n the middle of the sapwood ( P l a t e s 22 and 23) . These unusual m i t o c h o n d r i a r e f l e c t a h i g h m e t a b o l i c c e l l a c t i v i t y (Douce 1985). In young ray parenchyma c e l l s , the s u r f a c e of 153 plasmalemma was r e l a t i v e l y smooth, w h i l e i n aged ray parenchyma c e l l s , i t was somewhat rough ( P l a t e 2 5 - A ) . As i l l u s t r a t e d i n F i g u r e 8 and P l a t e s 1 9 - F , 21-F and 22, a c l e a r zone, f r e e of c e l l o r g a n e l l e s and c y t o p l a s m , surrounded the nuc l eus i n the ray parenchyma c e l l s i n May, June and November. T h i s o r g a n e l l e - f r e e zone was not found i n the o ther two c o n i f e r s and has not been r e p o r t e d p r e v i o u s l y . T h i s o r g a n e l l e - f r e e zone was found i n the f a l l / w i n t e r c e l l s . Whether t h i s zone a c t e d as a p r o t e c t i v e zone from c o l d weather or served some other f u n c t i o n m e r i t s f u r t h e r s t u d y . The n u c l e a r c h r o m a t i n i n young parenchyma c e l l s appeared as d i s t i n g u i s h a b l e patches and d i f f e r e d from t h a t of P inus banks iana and P i c e a m a r i a n a . The n u c l e a r s t r u c t u r e a f t e r ag ing i s the same f o r a l l t h r e e c o n i f e r s . The s t r u c t u r e s of aged a m y l o p l a s t s and m i t o c h o n d r i a appeared s i m i l a r to those i n the o t h e r two c o n i f e r s . L i p i d d r o p l e t s i n Ab ie s balsamea d i d not e x h i b i t two s t a i n i n g d e n s i t i e s due to a g i n g as d i d those of the two o ther c o n i f e r s . L i p i d lumping o c c u r r e d i n the middle of the sapwood (Appendix 3) r a t h e r than i n the i n n e r sapwood as i n the o ther two c o n i f e r 154 s p e c i e s . No v e s i c l e s were a t t a c h e d to the plasmalemma as i n P inus b a n k s i a n a . Based on the NEI and u l t r a s t r u c t u r a l changes of the ray parenchyma c e l l s the v i t a l i t y of l i v i n g sapwood ray parenchyma c e l l s i n A b i e s d e c l i n e d over t i m e . T h i s c o n c l u s i o n was the same as t h a t f o r P inus b a n k s i a n a and P i c e a m a r i a n a . The main c h a r a c t e r i s t i c f e a t u r e s of f i v e c e l l o r g a n e l l e s of ray parenchyma c e l l s due to ag ing are l i s t e d i n T a b l e 24. 5 . 6 . 3 . 4 Populus tremuloides 5 . 6 . 3 . 4 . 1 Seasonal Variation The u l t r a s t r u c u t r e of the nuc leus i n the outer sapwood ray parenchyma c e l l s i n J u l y e x h i b i t e d no c l e a r d i f f e r e n c e from November. The chromat in i n the nuc leus was even ly d i s p e r s e d . Some n u c l e i c o n t a i n e d a h e a v i l y s t a i n e d , r o u n d , n u c l e o l u s . In g e n e r a l , the d iameter of the nuc l eus was l e s s than 2 um which i s f a r s m a l l e r than those i n c o n i f e r o u s s p e c i e s , which measured about Table 24. The main characteristic features of five cell organelles of ray parenchyma cells of Abies balsamea in the outer and inner sapwood. (Jell Organelle Outer Sapwood Inner Sapwood Nucleus Chromatin aggregated into dist inctive patches with r i c h nucleoplasm Chromatin aggregated into distinguishable patches without nucleoplasm Amyloplast L i p i d Droplets If present, elongated or round starch granules with osmiophilic globuli; some plastids with thylakoid and osmiophilic globuli Few to very many, mainly small in size, stained black If present, mainly a single swollen starch granule with cracks in a single membrane amyloplast Very many, small to medium in s ize, stained dark black Mitochondria Round and mainly rod-like in shape; tubular cristae sometimes with branch-mitochondria Mainly round in shape, few to numerous; either short tubular or saccular cristae Plasmalemma Surface smooth Surface smooth to s l ight ly rough 156 5 um. The d i s t r i b u t i o n of the a m y l o p l a s t showed s e a s o n a l v a r i a t i o n . Numerous a m y l o p l a s t s were found i n the J u l y ray parenchyma c e l l s , but none were d e t e c t e d i n November. In J u l y , the a m y l o p l a s t s posses sed s e v e r a l s m a l l round s t a r c h g r a n u l e s . The shape of the a m y l o p l a s t appeared i r r e g u l a r due to the e x i s t e n c e of s e v e r a l s t a r c h g r a n u l e s ( P l a t e s 27-C and 2 8 - C ) . No t h y l a k o i d s or o s m i o p h i l i c g l o b u l i were l o c a t e d i n the a m y l o p l a s t . Many s m a l l and l a r g e , h e a v i l y s t a i n e d l i p i d d r o p l e t s were found i n both J u l y and November. In the J u l y ray parenchyma c e l l s , t h e r e were on ly a few round or r o d - l i k e m i t o c h o n d r i a . In November most of the m i t o c h o n d r i a were r o u n d . The c r i s t a e i n the November m i t o c h o n d r i a were o f t e n s a c c u l a r or not observed i n the i n n e r membrane s t r u c t u r e . The s u r f a c e of the plasmalemma i n b o t h J u l y and November c e l l s was smooth and t i g h t l y p r e s s e d to the c e l l w a l l . The main c h a r a c t e r i s t i c f e a t u r e s of J u l y and November ray parenchyma c e l l s are l i s t e d i n T a b l e 25. Table 25. The main characteristic features of five cell organelles in the outer sapwood ray parenchyma cells of Populus tremuloides in July and November. Cell Orqanelle July 23, 1984 November 20, 1984 Nucleus Chromatin evenly dispersed Same as in July Amyloplast Very numerous/ several small round starch granules within an amyloplast Absent L ip id Droplets Very numerous, small black stained l i p i d droplets; some large droplets also present Same as in July Mitochondria Round and rod-l ike in shape, few in number, tubular cristae Mainly round in shape, few, no cristae or saccular cristae Plasmalemma Surface smooth Surface smooth 158 5 . 6 . 3 . 4 . 2 Ag ing Process The u l t r a s t r u c t u r a l d i f f e r e n c e s between young and aged ray parenchyma c e l l s of Populus t r e m u l o i d e s are shown i n P l a t e s 27-34. In b o t h young and aged r a y parenchyma c e l l s , the n u c l e a r chromat in was even ly d i s t r i b u t e d . However, i n a d y i n g c e l l , c h a r a c t e r i z e d by d i s i n t e g r a t i n g s t a r c h g r a n u l e s and a l a c k of c y t o p l a s m ( P l a t e 30 -D) , the n u c l e a r chromat in appeared l a r g e - g r a i n e d . U n f o r t u n a t e l y , a d y i n g c e l l w i t h an even f u r t h e r d i s i n t e g r a t e d nuc l eus c o u l d not be o b t a i n e d owing to the l i m i t e d number of specimens and the s m a l l s i z e of the n u c l e u s . The a g g r e g a t i o n of chromat in i n the nuc leus c o u l d be t r a c e d i n P l a t e 32-A. In young ray parenchyma c e l l s , the presence of the a m y l o p l a s t depended upon the s eason . I f i t was p r e s e n t , i t c o n t a i n e d s e v e r a l round s t a r c h g r a n u l e s . In an aged ray parenchyma c e l l , the membrane of the a m y l o p l a s t was broken and the s t a r c h g r a n u l e s were d i s p e r s e d . Some of the s t a r c h g r a n u l e s had l o s t t h e i r s p h e r i c a l shape and the c e n t r e s of the s t a r c h g r a n u l e s were empty ( P l a t e s 159 30 to 33 ) . No c r a c k s on the s t a r c h g r a n u l e s c o u l d be d i s c e r n e d . Many d a r k l y s t a i n e d l i p i d d r o p l e t s of v a r i o u s s i z e s were found i n young ray parenchyma c e l l s . With a g i n g , l i p i d d r o p l e t s tended to i n c r e a s e i n s i z e . Some of the d r o p l e t s c o a l e s c e d i n t o l a r g e lumps ( P l a t e s 32 and 33) . F u r t h e r m o r e , the l i p i d d r o p l e t s i n aged ray parenchyma c e l l s or i n d y i n g c e l l s had two s t a i n i n g d e n s i t i e s . U s u a l l y , these ray parenchyma c e l l s possessed a r e l a t i v e l y h i g h l i p i d content ( F i g u r e 7 ) . The change of l i p i d s t r u c t u r e from s m a l l d r o p l e t s to l a r g e lumps, and from a s i n g l e s t a i n i n g d e n s i t y to two s t a i n i n g d e n s i t i e s appeared to be a s i g n of c e l l a g i n g . I t shou l d be noted here t h a t Mia (1972) found an abundance of f a t s d u r i n g the second week of May which d i s a p p e a r e d i n June and e a r l y J u l y . In the present s t u d y , t h e r e was a r e l a t i v e l y h i g h l i p i d content i n the o u t e r sapwood d u r i n g May, June and J u l y ( F i g u r e 7 -4 ) . An abundance of l i p i d d r o p l e t s i n these t h r e e months was a l s o observed w i t h TEM ( P l a t e s 27 to 29) . In young ray parenchyma c e l l s , the m i t o c h o n d r i a were e i t h e r round or r o d - l i k e . In the few m i t o c h o n d r i a 160 p r e s e n t i n aged ray parenchyma c e l l s , the c r i s t a e were not prominent or absent a l t o g e t h e r . Many s c i e n t i s t s (Esau 1963, E r n s t e r 1965, Bonner 1973, Munn 1974, Douce 1985) b e l i e v e tha t the number and shape of the m i t o c h o n d r i a and t h e i r c r i s t a e s t r u c t u r e r e f l e c t m e t a b o l i c a c t i v i t y of a l i v i n g c e l l . The f r e q u e n t rod shaped m i t o c h o n d r i a i n young ray parenchyma c e l l s are c o n s i s t e n t w i t h the h i g h e r m e t a b o l i c a c t i v i t y of those younger c e l l s , which i s a l s o expressed by the NEI ( F i g u r e 5 -5 ) . The s t r u c t u r e of the c e l l s i n J u l y and November ( T a b l e 25) d i d not show s i g n i f i c a n t d i f f e r e n c e s . However, the c e l l o r g a n e l l e s , such as a m y l o p l a s t s , l i p i d d r o p l e t s and m i t o c h o n d r i a look d i f f e r e n t at the d i f f e r e n t sampl ing t imes (Appendix 3; P l a t e s 28 -34) . In young parenchyma c e l l s of Populus t r e m u l o i d e s , the s t a r c h g r a n u l e s were packed w i t h i n an a m y l o p l a s t , w h i l e the s t a r c h g r a n u l e s i n aged c e l l s were s c a t t e r e d . The a m y l o p l a s t membrane was broken i n aged c e l l s . The lumping of l i p i d d r o p l e t s and m i t o c h o n d r i a l changes a s s o c i a t e d w i t h ag ing were s i m i l a r to those i n the t h r e e c o n i f e r s . Based on the ev idence and data 161 c o l l e c t e d w i t h l i g h t m i c r o s c o p y , i t was c o n c l u d e d tha t a l i v i n g sapwood ray parenchyma c e l l undergoes u l t r a s t r u c t u r a l changes due to a g i n g . The main c h a r a c t e r i s t i c f e a t u r e s of f i v e c e l l o r g a n e l l e s of ray parenchyma c e l l s due to ag ing are summarized i n T a b l e 26. 5.6.4 Heartwood Substances P l a t e s 35 and 36 show the v a r i o u s forms of heartwood s u b s t a n c e s , the l o c a t i o n s of these substances i n the ray parenchyma c e l l and t h e i r a d j a c e n t t r a c h e a r y e l ements . These substances were s t a i n e d b l a c k w i t h Os j u s t as the l i p i d d r o p l e t s i n the l i v i n g sapwood ray parenchyma c e l l s . However, these same subs tances were not observed by l i g h t microscopy to have been s t a i n e d w i t h Sudan b l a c k . Thus they f a i l to r e a c t as t y p i c a l l i p i d s , i n t h a t they f a i l e d the Sudan c o l o u r t e s t . L i p i d - l i k e d r o p l e t s or lumps are s e c r e t e d by an unknown mechanism through a h a l f - b o r d e r e d p i t - p a i r from a ray parenchyma c e l l to i t s a d j a c e n t t r a c h e a r y e lements . These l i p i d - l i k e d r o p l e t s or lumps a l s o move Table 26. The main characteristic features of five cell organelles of ray parenchyma cells of Populus tremuloides in the outer and inner sapwood. Cell Organelle Outer Sapwood Inner Sapwood Nucleus Amyloplast L ip id Droplets Chromatin evenly dispersed Present or absent depending upon the season; i f present several small, round starch granules were packed within an amyloplast Very numerous; various sizes of droplets; stained black Chromatin evenly dispersed and aggregated If present, round starch granules were scattered, the membrane of amylo-plast broken Very numerous; small to large in size; some l i p i d lumps; two-density stained l i p i d droplets very common Mitochondria Mostly round in shape, few rod-l ike ; either tubular or saccular cristae; some even lack dist inct ive cristae Absent or rarely present; i f present, round in shape with or without cristae Plasmalemma Surface smooth Surface smooth to rough 163 through a margo of a b o r d e r e d p i t - p a i r on the t r a c h e a r y e l ement . The margo i s the outer r e g i o n of the p i t membrane c o n s i s t i n g of f a s i c u l a t e d m i c r o f i b r i l l a r s t r a n d s of the s u p p o r t i n g membrance l y i n g between the t o r u s and the p i t b o r d e r . T h i s porous r e g i o n p r o v i d e s the channe l s f o r gas and l i q u i d movement between two a d j a c e n t c e l l s as i n d i c a t e d i n P l a t e 35-B and 3 5 - C . The t o r u s i s the c e n t r a l t h i c k e n e d p o r t i o n of the p i t membrane of a p i t - p a i r . I t i s b e l i e v e d t h a t t h i s t o r u s i s impermeable to l i p i d d r o p l e t s , as i n d i c a t e d i n P l a t e 3 5 - E . The m i g r a t i o n of l i p i d - l i k e substance between ray parenchyma c e l l s and a d j a c e n t t r a c h e a r y e lements i n the t r a n s i t i o n zone was a l s o r e p o r t e d by F e n g e l (1970), i n P i n u s s y l v e s t r i s . P l a t e 35-D i l l u s t r a t e s an i n t e r e s t i n g f i n d i n g . A secondary w a l l of a r a y parenchyma c e l l i n P inus  banks iana was d e p o s i t e d at the h a l f - b o r d e r e d p i t - p a i r a r e a . Heartwood substances appeared i n the p i t chamber of t h i s h a l f - b o r d e r e d p i t - p a i r . The f o l l o w i n g q u e s t i o n remains to be answered. "Do the heartwood subs tances i n the ray parenchyma c e l l move through the p i t b e f o r e the secondary w a l l i s d e p o s i t e d or i s t h i s secondary w a l l 164 permeable to the l i p i d substances?" 5 . 6 . 5 Summary of Ultrastructure The s t r u c t u r e of l i v i n g sapwood ray parenchyma c e l l s of a l l s p e c i e s s t u d i e d v a r i e d w i t h season . The changes due to season were not as pronounced as those due to a g i n g . A g e n e r a l i z e d s t r u c t u r e of f i v e c e l l o r g a n e l l e s i n young and aged ray parenchyma c e l l s of f o u r s p e c i e s i s shown i n F i g u r e 9. The morphology of the nuc leus i n young ray parenchyma c e l l s appeared i n d i s t i n c t i v e patches w i t h r i c h n u c l e o p l a s m , or e v e n l y d i s p e r s e d . The c h r o m a t i n i n P inus banks iana and P i c e a mariana i s i n the form of webbing. The nuc leus i n aged ray parenchyma c e l l s was shor tened and formed an i r r e g u l a r n u c l e a r e n v e l o p e . The t h r e e c o n i f e r s a m y l o p l a s t s i n young ray parenchyma c e l l s were c h a r a c t e r i z e d by one or two e l o n g a t e d s t a r c h g r a n u l e s accompanied by a few o s m i o p h i l i c g l o b u l i and t h y l a k o i d s . In Populus  t r e m u l o i d e s , s e v e r a l s m a l l and round s t a r c h g r a n u l e s were packed w i t h i n an a m y l o p l a s t . Swol l en s t a r c h 165 Figure 9. A simplified structure of five c e l l organelles i n a young ray parenchyma c e l l (upper) and an aged ray parenchyma c e l l (lover) in 1. Pinus banksiana, 2. Picea mariana, 3. Abies balsamea and 4. Populus tremuloides. Note: Am-amyloplast; Cw-cell wall; L - l i p i d droplets; M-mitochondrion; N-nucleus; Nu-nucleolus; S-starch granule; PI-plasmalemma; V i - v e s i c l e . Figure 9. (continued). 2. Picea mariana. 167 Figure 9. (continued). 3 . Abies balsamea. 169 g r a n u l e s were observed i n aged ray parenchyma c e l l s of the t h r e e c o n i f e r s p e c i e s . In the aged c e l l s of Populus  t r e m u l o i d e s , the round s t a r c h g r a n u l e s were s c a t t e r e d and the membrane of a m y l o p l a s t s was b r o k e n . L i p i d d r o p l e t s i n young ray parenchyma c e l l s were s m a l l and s t a i n e d b l a c k w i t h a s i n g l e s t a i n i n g d e n s i t y . L i p i d d r o p l e t s i n aged c e l l s were numerous and appeared i n v a r i o u s s i z e s and forms. Many s m a l l to medium s i z e d l i p i d d r o p l e t s were found i n aged c e l l s of A b i e s balsamea which were l a r g e l y b l a c k from a s i n g l e s t a i n i n g d e n s i t y , w h i l e i n the o ther t h r e e s p e c i e s d r o p l e t s were s t a i n e d w i t h two d e n s i t i e s and appeared i n l a r g e d r o p l e t s and lumps. L i p i d d r o p l e t s and l i p i d lumps w i t h two s t a i n i n g d e n s i t i e s were f r e q u e n t l y found i n the middle and i n n e r sapwood ray parenchyma c e l l s which g e n e r a l l y c o n t a i n e d a r e l a t i v e l y h i g h l i p i d c o n t e n t . In g e n e r a l , m i t o c h o n d r i a were very numerous i n young ray parenchyma c e l l s and u s u a l l y absent i n aged c e l l s . M i t o c h o n d r i a i n young ray parenchyma c e l l s were round and r o d - l i k e w i t h d i s t i n c t i v e c r i s t a e . In aged c e l l s they were main ly round and wi thout d i s c e r n i b l e 170 c r i s t a e . Plasmalemma i n young ray parenchyma c e l l s appeared to have a smooth or rough s u r f a c e depending upon the season . Numerous v e s i c l e s a t t a c h e d to the plasmalemma were a s i g n i f i c a n t f e a t u r e i n aged c e l l s of P inus  b a n k s i a n a , but they were absent from the o ther t h r e e s p e c i e s . I t i s conc luded t h a t the u l t r a s t r u c t u r e of sapwood ray parenchyma c e l l s changed from the o u t e r sapwood towards the i n n e r sapwood. The d e t e r i o r a t i o n of c e l l s and c e l l o r g a n e l l e s i n d i c a t e d the l o s s of c e l l v i t a l i t y which i s ma in ly a t t r i b u t e d to the passage of t i m e . 171 ABBREVIATIONS USED IN PLATE ILLUSTRATIONS Am - A m y l o p l a s t CW - C e l l W a l l DW - Dentate W a l l ER - Endoplasmic R e t i c u l u m F - F i b e r HB - H a l f - B o r d e r e d P i t - P a i r L - L i p i d D r o p l e t M - M i t o c h o n d r i o n N - Nucleus Nu - N u c l e o l u s Pd - Plasmodesmata PI - Plasmalemma Pt - P l a s t i d R - Ray Parenchyma Rt - Ray T r a c h e i d S - S t a r c h Granule T - T r a c h e i d V - Vacuo le A l l b l a c k bar s c a l e s on the p i c t u r e s are 1 pm i n l e n g t h u n l e s s o therwise s p e c i f i e d . 172 PLATE 1 Pinus banksiana ray parenchyma cel l s in the outer sapwood ring. A . The s t r u c t u r e of the n u c l e u s , a m y l o p l a s t , m i t o c h o n d r i a and numerous l i p i d d r o p l e t s . May. R a d i a l - s e c t i o n B. The s t r u c t u r e of the nuc l eus and numerous l i p i d d r o p l e t s . June . C r o s s - s e c t i o n . C . A nuc l eus w i t h a d i s t i n c t i v e n u c l e o l u s i n a n u c l e u s , p l a s t i d and m i t o c h o n d r i a . Few l i p i d d r o p l e t s and the absence of the a m y l o p l a s t c h a r a c t e r i z e the c e l l . J u l y . C r o s s - s e c t i o n . D. The s t r u c t u r e of the l i p i d d r o p l e t s and v a c u o l e . Smal l o s m i o p h i l i c g l o b u l i i n a p l a s t i d are n o t i c e d . Augus t . R a d i a l - s e c t i o n . E . The s t r u c t u r e o f the n u c l e u s , e s p e c i a l l y the n u c l e a r envelope (arrow) and the l i g h t s t a i n e d l i p i d d r o p l e t s . A s t a r c h g r a n u l e and two s m a l l o s m i o p h i l i c g l o b u l i i n the a m y l o p l a s t w i t h the c r i s t a - l i k e s t r u c t u r e i n i t are e v i d e n t . A l s o note the t u b u l a r c r i s t a e i n the m i t o c h o n d r i a . O c t o b e r . C r o s s - s e c t i o n . F . The s t r u c t u r e o f the n u c l e u s , n u c l e o l u s and n u c l e a r enve lope . Note numerous l i p i d d r o p l e t s . December. C r o s s - s e c t i o n . 173 P L A T E 1 174 PLATE 2 Pinus banksiana ray parenchyma cells i n the outer sapwood ring. A . A v a c u o l e , l i p i d d r o p l e t s and few e longated m i t o c h o n d r i a . A s a c c u l a r c r i s t a c h a r a c t e r i z e s the m i t o c h o n d r i a . May. C r o s s - s e c t i o n . B . Two l a r g e l i p i d d r o p l e t s and numerous s m a l l l i p i d d r o p l e t s . June . C r o s s - s e c t i o n . C . The e longated shape m i t o c h o n d r i a and s m a l l v e s i c l e s (arrows) a t t a c h e d on the plasmalemma. J u l y . C r o s s - s e c t i o n . D. Few e longated p l a s t i d s and a l i p i d d r o p l e t . Absence of the a m y l o p l a s t i n t h i s m i c r o g r a p h i s a l s o n o t i c e d . A u g u s t . R a d i a l - s e c t i o n . E . The s t r u c t u r e of the m i t o c h o n d r i a and the n u c l e a r envelope of the n u c l e u s . A l i p i d d r o p l e t w i t h v a r i a b l e d e n s i t y i s a l s o n o t i c e d . Arrow i n d i c a t e s the s t r u c t u r e of n u c l e a r enve lope . O c t o b e r . C r o s s -s e c t i o n . F . Numerous heavy s t a i n e d l i p i d d r o p l e t s and a l a r g e v a c u o l e . December. R a d i a l - s e c t i o n . 175 P L A T E 2 176 PLATE 3 Pinus banksiana ray parenchyma cells in the outer sapwood ring . A . The o s m i o p h i l i c g l o b u l i and the t h y l a k o i d s (arrows) i n the p l a s t i d and a m y l o p l a s t . Note the few s m a l l l i p i d d r o p l e t s present b o t h i n the p l a s t i d and a m y l o p l a s t . May. C r o s s - s e c t i o n . B . A l a r g e v a c u o l e and numerous l i p i d d r o p l e t s . Note the absence of the secondary w a l l . June . C r o s s -s e c t i o n . C . The s t r u c t u r e of the l i p i d d r o p l e t s and m i t o c h o n d r i a . Two dictyosomes w i t h 4-6 c i s t e r n a e and numerous s m a l l v e s i c l e s a t b o t h ends of the c i s t e r n a e are n o t e d . J u l y . C r o s s - s e c t i o n . D. The s m a l l v e s i c l e s and p l a s t i d s at the e a r l y stage of development. J u l y . C r o s s - s e c t i o n . E . A s t a r c h g r a n u l e accompanying two t h y l a k o i d s (arrows) i n s i d e an a m y l o p l a s t . The m i t o c h o n d r i a w i t h s a c c u l a r c r i s t a are n o t e d . O c t o b e r . C r o s s -s e c t i o n . F . A l a r g e l i p i d d r o p l e t and numerous s m a l l e r l i p i d d r o p l e t s . No a m y l o p l a s t i s o b s e r v e d . December. R a d i a l - s e c t i o n . 177 P L A T E 3 178 PLATE 4 Pinus banksiana ray parenchyma cells in the outer sapwood ring. A . The s t r u c t u r e of the l i p i d d r o p l e t s and m i t o c h o n d r i a . Note the absence of the secondary w a l l i n t h i s p h o t o . May. C r o s s - s e c t i o n . B . The plasmalemma (arrow) and plasmodesmata at the end w a l l a r e a . May. R a d i a l - s e c t i o n . C . The shape of v a c u o l e s and d i s t r i b u t i o n of the m i t o c h o n d r i a . May. C r o s s - s e c t i o n . D. The s t r u c t u r e o f the plasmodesmata and the numerous m i t o c h o n d r i a c o n c e n t r a t i n g at one s i d e of the ray parenchyma c e l l . J u l y . R a d i a l - s e c t i o n . E . Numerous m i t o c h o n d r i a and two a m y l o p l a s t s w i t h few o s m i o p h i l i c g l o b u l i and s t a r c h g r a n u l e s . J u l y . C r o s s - s e c t i o n . F . A l a r g e v a c u o l e and the plasmodesmata at the s i d e w a l l of the ray parenchyma c e l l . December. R a d i a l -s e c t i o n . 179 180 PLATE 5 Pinus banksiana ray parenchyma cells in the middle sapwood ring. A . A d y i n g c e l l w i t h numerous l a r g e l i p i d d r o p l e t s and t h r e e a m y l o p l a s t s . The c r a c k s (arrows) i n the l a r g e s t a r c h g r a n u l e and the absence of the ground substance c h a r a c t e r i z e the c e l l . June . C r o s s - s e c t i o n . B . A n u c l e u s , a m y l o p l a s t and numerous l i p i d d r o p l e t s w i t h two s t a i n i n g d e n s i t i e s . J u l y . R a d i a l - s e c t i o n . C. A v a c u o l e and the numerous m i t o c h o n d r i a . Augus t . C r o s s - s e c t i o n . D. A m i t o c h o n d r i o n and an a m y l o p l a s t w i t h o s m i o p h i l i c g l o b u l i and t h y l a k o i d ( a r r o w ) . Augus t . C r o s s - s e c t i o n . E . Numerous l i p i d d r o p l e t s w i t h two s t a i n i n g d e n s i t i e s . O c t o b e r . C r o s s - s e c t i o n . F . Numerous l i p i d d r o p l e t s crowded i n t o a n g u l a r shapes . Note the heavy s t a i n e d zone i n the l i p i d d r o p l e t s e x t e n d i n g to the a d j a c e n t d r o p l e t s . December. R a d i a l - s e c t i o n . 181 PLATE 5 182 PLATE 6 Pinus banksiana ray parenchyma cells in the middle sapwood ring. A . Numerous l i p i d d r o p l e t s w i t h two s t a i n i n g d e n s i t i e s . J u l y . R a d i a l - s e c t i o n . B. The s t r u c t u r e of the plasmodesmata and m i t o c h o n d r i a . Note the rough s u r f a c e of the plasmalemma w i t h some s m a l l v e s i c l e s a t t a c h e d to i t ( a r r o w s ) . August . C r o s s - s e c t i o n . C . The s o - c a l l e d "dumb-bel l" shaped m i t o c h o n d r i a which c h a r a c t e r i z e h i g h m e t a b o l i c a c t i v i t y of a c e l l and the rough s u r f a c e of the plasmalemma ( a r r o w ) . August . C r o s s - s e c t i o n . D. The s t r u c t u r e of the n u c l e u s , a m y l o p l a s t and m i t o c h o n d r i a . The l i g h t s t a i n i n g d e n s i t y on the p e r i p h e r a l s u r f a c e of the l i p i d d r o p l e t s c h a r a c t e r i z e the c e l l . O c t o b e r . C r o s s - s e c t i o n . E . The l i g h t s t a i n i n g d e n s i t y l i p i d d r o p l e t s . O c t o b e r . R a d i a l - s e c t i o n . F . The s t r u c t u r e of the plasmalemma (arrow) and two l i p i d d r o p l e t s fused t o g e t h e r w i t h the l i g h t s t a i n i n g d e n s i t y zone on the p e r i p h e r a l s u r f a c e of the d r o p l e t . December R a d i a l - s e c t i o n . 183 PLATE 6 184 PLATE 7 Pinus banksiana ray parenchyma cells in the inner sapwood ring. A . The s t r u c t u r e of the l i p i d d r o p l e t s and a m y l o p l a s t . Note the c r a c k s on the s t a r c h g r a n u l e . May. R a d i a l - s e c t i o n . B . The s t r u c t u r e of the n u c l e u s , l i p i d d r o p l e t s and a m y l o p l a s t . Note the g r o u p i n g of the chromat in (arrow) i n the n u c l e u s . June . R a d i a l - s e c t i o n . C . The s t r u c t u r e of the n u c l e u s , m i t o c h o n d r i a and l i g h t s t a i n e d l i p i d d r o p l e t s . Note the l a c k of c e l l ground s u b s t a n c e . J u l y . R a d i a l - s e c t i o n . D. The h e a v i l y s t a i n e d l i p i d d r o p l e t s , a m y l o p l a s t and n u c l e u s . Note the g r o u p i n g of the chromat in (arrow) i n the n u c l e u s . Augus t . C r o s s - s e c t i o n . E . The s t r u c t u r e of the n u c l e u s , a m y l o p l a s t and l i p i d d r o p l e t s . Note the two s t a i n i n g - d e n s i t y l i p i d d r o p l e t s , w i t h the l i g h t s t a i n i n g zone on the p e r i p h e r y of the d r o p l e t s . O c t o b e r . R a d i a l - s e c t i o n . F . A l a r g e v a c u o l e and the lumping of l i p i d d r o p l e t s . Note the dentate w a l l of the r a y t r a c h e i d . December. R a d i a l - s e c t i o n . 185 P L A T E 7 186 PLATE 8 Pinus banksiana ray parenchyma cells in the inner sapwood ring. A . The attachment of s m a l l v e s i c l e s (arrow) on the plasmalemma. May. R a d i a l - s e c t i o n . B. The s t r u c t u r e of the l i p i d d r o p l e t s and amylop las tThe c r a c k s on the s t a r c h g r a n u l e c h a r a c t e r i z e s the c e l l . June . R a d i a l - s e c t i o n . C . The plasmodesmata on the end w a l l and the attachment of s m a l l v e s i c l e s (arrows) on the plasmalemma. J u l y . R a d i a l - s e c t i o n . D. The s t r u c t u r e of the v a c u o l e , a m y l o p l a s t and l i p i d d r o p l e t s . J u l y . C r o s s - s e c t i o n . E . The s t r u c t u r e of the v a c u o l e , plasmodesmata and l i p i d d r o p l e t s . Note the absence o f v e s i c l e attachment to the plasmalemma. O c t o b e r . R a d i a l - s e c t i o n . F . The l i p i d d r o p l e t s w i t h two s t a i n i n g d e n s i t i e s . December. R a d i a l - s e c t i o n . 187 P L A T E 8 188 PLATE 9 Pinus banksiana ray parenchyma cells in the heartwood. A. The absence of the secondary wall thickening. Cross-section. B. The absence of the secondary wall thickening in two ray parenchyma cells and an adjacent ray parenchyma c e l l with the secondary wall (arrow). Radial-section. C. A ray parenchyma c e l l with the secondary wall thickening (arrows). Note a recess of the secondary wall of the ray parenchyma c e l l at the half-bordered pit-pair area. Cross-section. D. The secondary wall thickening at the end wall (arrow) of a ray parenchyma c e l l but the absence of the secondary wall thickening at the end wall of the adjacent ray parenchyma c e l l . Cross-section . E. The absence of the secondary wall thickening in the ray parenchyma c e l l . Note the heartwood substance in the longitudinal tracheid. Cross-section . F. The secondary wall thickening (arrow) at both sides of the end wall. Note the heartwood substance at the end wall area. Cross-section. 189 P L A T E 9 190 PLATE 10 Picea mariana ray parenchyma cel l s in the outer sapwood ring. A . The s t r u c t u r e of the n u c l e u s , l i p i d d r o p l e t s and a m y l o p l a s t . Note the s u r f a c e s t r u c t u r e of the plasmalemma ( a r r o w ) . May. C r o s s - s e c t i o n . B . The s t r u c t u r e of the n u c l e u s , l i p i d d r o p l e t s and m i t o c h o n d r i a . Note the s u r f a c e s t r u c t u r e of plasmalemma ( a r r o w ) . June . C r o s s - s e c t i o n . C. The s t r u c t u r e of the n u c l e u s , n u c l e o l u s , m i t o c h o n d r i a and l i p i d d r o p l e t s . J u l y . R a d i a l -s e c t i o n . D. Numerous l i p i d d r o p l e t s and m i t o c h o n d r i a . Note the secondary w a l l t h i c k e n i n g (arrow) i n the ray parenchyma c e l l . Augus t . C r o s s - s e c t i o n . E . The s t r u c t u r e of the m i t o c h o n d r i a , l i p i d d r o p l e t s and a m y l o p l a s t . O c t o b e r . C r o s s - s e c t i o n . F . The s t r u c t u r e of the l i p i d d r o p l e t s and v a c u o l e s . December. C r o s s - s e c t i o n . 191 P L A T E 10 192 PLATE 11 Picea mariana ray parenchyma cells in the outer sapwood ring. A . The s t r u c t u r e of the e longated m i t o c h o n d r i a , a m y l o p l a s t s and the rough s u r f a c e of the plasmalemma ( a r r o w ) . Note the t h y l a k o i d s and o s m i o p h i l i c g l o b u l i i n s i d e the a m y l o p l a s t . May. C r o s s - s e c t i o n . B . The s t a r c h g r a n u l e s w i t h the t h y l a k o i d s and o s m i o p h i l i c g l o b u l i i n s i d e the a m y l o p l a s t . June . C r o s s - s e c t i o n . C . The s t r u c t u r e of the a m y l o p l a s t and m i t o c h o n d r i a . Two s t a r c h g r a n u l e s w i t h o s m i o p h i l i c g l o b u l i i n s i d e the a m y l o p l a s t . Note the rough s u r f a c e of the plasmalemma ( a r r o w s ) . June . C r o s s - s e c t i o n . D. Numerous m i t o c h o n d r i a and p l a s t i d s w i t h numerous t h y l a k o i d s . J u l y . R a d i a l - s e c t i o n . E . The s t r u c t u r e o f the a m y l o p l a s t , l i p i d d r o p l e t s and the rough s u r f a c e of the plasmalemma ( a r r o w ) . O c t o b e r . C r o s s - s e c t i o n . F . The s t r u c t u r e of the m i t o c h o n d r i a , l i p i d d r o p l e t s , v a c u o l e and the rough s u r f a c e of the plasmalemma ( a r r o w ) . December. C r o s s - s e c t i o n . 193 PLATE 11 194 PLATE 12 Picea mariana ray parenchyma cells in the outer sapwood ring. A . The s t r u c t u r e of the l i p i d d r o p l e t s and many endoplasmic r e t i c u l a ( E R ) . May. C r o s s - s e c t i o n . B . The s t r u c t u r e of the m i t o c h o n d r i a , v a c u o l e and a m y l o p l a s t . Note the c r a c k s on the s t a r c h g r a n u l e . May. R a d i a l - s e c t i o n . C . The s t r u c t u r e of the m i t o c h o n d r i a , l i p i d d r o p l e t s and many endoplasmic r e t i c u l a . June . C r o s s - s e c t i o n . D. The s t r u c t u r e of the m i t o c h o n d r i a , endoplasmic r e t i c u l u m and p l a s t i d w i t h o s m i o p h i l i c g l o b u l i ( a r r o w ) . Note the absence of the a m y l o p l a s t . J u l y . R a d i a l - s e c t i o n . E . An a m y l o p l a s t and numerous l i p i d d r o p l e t s . O c t o b e r . C r o s s - s e c t i o n . F . The s t r u c t u r e of the endoplasmic r e t i c u l u m , l i p i d d r o p l e t s and m i t o c h o n d r i a w i t h the s a c c u l a r c r i s t a e . Note the o s m i o p h i l i c g l o b u l i (arrows) i n s i d e the m i t o c h o n d r i a . December. C r o s s - s e c t i o n . 195 P L A T E 12 196 PLATE 13 Picea mariana ray parenchyma cells in the middle sapvood ring. A . The s t r u c t u r e of the n u c l e u s , v a c u o l e and l i p i d d r o p l e t s . May. R a d i a l - s e c t i o n . B. The s t r u c t u r e of the n u c l e u s , m i t o c h o n d r i a and a m y l o p l a s t . Note the g r o u p i n g of the chromat in ( a r r o w ) . June . R a d i a l - s e c t i o n . C . The s t r u c t u r e of the nuc l eus and numerous v e s i c l e s ( a r r o w s ) . J u l y . R a d i a l - s e c t i o n . D. The s t r u c t u r e of the l i p i d d r o p l e t s and v a c u o l e . A u g u s t . R a d i a l - s e c t i o n . E . The s t r u c t u r e of the nuc leus and l i p i d d r o p l e t s . Note two s t a i n i n g d e n s i t i e s i n the l i p i d d r o p l e t s . O c t o b e r . R a d i a l - s e c t i o n . F . The s t r u c t u r e of the nuc l eus and l i g h t s t a i n e d l i p i d d r o p l e t s . December. R a d i a l - s e c t i o n . 197 P L A T E 1 3 198 PLATE 14 Picea mariana ray parenchyma cells in the middle sapwood ring. A . The s t r u c t u r e of the plasmodesmata at the end w a l l and l i p i d d r o p l e t s . May. R a d i a l - s e c t i o n . B. The s t r u c t u r e of the plasmmodesmata at the end w a l l . Note the a r c h i n g of the p i t b o r d e r (arrows) of a h a l f - b o r d e r e d p i t - p a i r . June . R a d i a l - s e c t i o n . C . The s t r u c t u r e of the plasmodesmata a t the s i d e w a l l . Note the s t r u c t u r e of the v a c u o l e and the lumping of l i p i d d r o p l e t s w i t h two s t a i n i n g d e n s i t i e s . J u l y . R a d i a l - s e c t i o n . D. The s t r u c t u r e of the plasmodesmata a t the end w a l l . Augus t . R a d i a l - s e c t i o n . E . Two s t a i n i n g d e n s i t i e s of the l i p i d d r o p l e t s . O c t o b e r . R a d i a l - s e c t i o n . F . The s t r u c t u r e of the n u c l e u s , a m y l o p l a s t and l i p i d d r o p l e t s . December. R a d i a l - s e c t i o n . 199 PLATE 14 200 PLATE 15 Picea mariana ray parenchyma cells in the middle sapwood ring. A . The p a r a l l e l arrangement of the endoplasmic r e t i c u l u m between two l i p i d d r o p l e t s . May. R a d i a l -s e c t i o n . B . Numerous heavy s t a i n e d l i p i d d r o p l e t s c l u s t e r i n g at one end of the ray parenchyma c e l l . June . R a d i a l - s e c t i o n . C . The shape of the l i p i d d r o p l e t s and v a c u o l e . Note the absence of the plasmodesma at the s imple p i t - p a i r of the s i d e w a l l . J u l y . R a d i a l - s e c t i o n . D. The s t r u c t u r e of the m i t o c h o n d r i a and l i p i d d r o p l e t s . The heavy s t a i n e d zone extends from one l i p i d d r o p l e t to i t s ad jacent d r o p l e t . J u l y . R a d i a l - s e c t i o n . E . The s t r u c t u r e of the l i p i d d r o p l e t s and v a c u o l e . Two s t a i n i n g d e n s i t i e s of the l i p i d d r o p l e t s . O c t o b e r . R a d i a l - s e c t i o n . F . A l a r g e v a c u o l e i n a r a y parenchyma c e l l . I t s a d j a c e n t ray parenchyma c e l l possesses a few l a r g e l i p i d d r o p l e t s w i t h two s t a i n i n g d e n s i t i e s . December. R a d i a l - s e c t i o n . 201 PLATE 15 202 PLATE 16 Picea mariana ray parenchyma cells in the inner sapwood ring. A. A dying c e l l with the nucleus, l i p i d lump and starch. Note the grouping of the chromatin in the nucleus. May. Cross-section. B. The structure of the nucleus and l i p i d droplets. June. Radial-section. C. The plasmodesmata at the end wall. July. Radial-section. D. A dying c e l l with the nucleus and l i p i d lump. The grouping of the chromatin lacking the nucleoplasm characterizes the c e l l . August. Cross-section. E. The cytoplasm organelles enclosed with the plasmalemma (arrow) in a dying c e l l . October. Radial-section. F. The structure of the nucleus and l i p i d droplets. December. Cross-section. 203 PLATE 16 204 PLATE 17 Picea mariana ray parenchyma cells in the inner sapwood rings. A. The s t r u c t u r e of the m i t o c h o n d r i a , vacuole and l i p i d d r o p l e t s . Note the t u b u l a r c r i s t a (arrow) i n the mitochondrion. May. R a d i a l - s e c t i o n . B. Numerous l i p i d d r o p l e t s and a m y l o p l a s t . The h e a v i l y s t a i n e d zone extends to the adjacent d r o p l e t s . June. R a d i a l - s e c t i o n . C. A l a r g e vacuole and l i p i d lump i n a dying c e l l . J u l y . R a d i a l - s e c t i o n . D. Two l a r g e l i p i d lumps with small l i p i d d r o p l e t s i n a dying c e l l . August. C r o s s - s e c t i o n . E. A dead c e l l w ith a l i p i d - l i k e lump. October. C r o s s - s e c t i o n . F. Two s t a i n i n g d e n s i t i e s of the l i p i d d r o p l e t s . The h e a v i l y s t a i n e d zone extends to the adjacent d r o p l e t s . December. R a d i a l - s e c t i o n . 205 P L A T E 17 206 PLATE 18 Picea mariana ray parenchyma cel l s in the heartwood. A . Some s m a l l d r o p l e t - l i k e heartwood s u b s t a n c e s . C r o s s - s e c t i o n . B . Heartwood substance l i n i n g the w a l l of the ray parenchyma. C r o s s - s e c t i o n . C . Heartwood substance w i t h two s t a i n i n g d e n s i t i e s l i n i n g the w a l l of the ray parenchyma c e l l . C r o s s -s e c t i o n . D. Some L i p i d - l i k e heartwood substance i n a dead ray parenchyma c e l l . C r o s s - s e c t i o n . E . A l a r g e d r o p l e t of heartwood substance w i t h some c y t o p l a s m i c r e s i d u e i n a dead ray parenchyma c e l l C r o s s - s e c t i o n . F . A l a r g e lump of heartwood substance i n a dead ray parenchyma c e l l . C r o s s - s e c t i o n . 207 P L A T E 18 208 PLATE 19 Abies balsamea ray parenchyma cells in the outer sapwood ring. A . Abundance of a m y l o p l a s t s , l i p i d d r o p l e t s and e l o n g a t e d m i t o c h o n d r i a . Note a n u c l e u s w i t h g r o u p i n g of the c h r o m a t i n . May. R a d i a l - s e c t i o n . B . The s t r u c t u r e of the n u c l e u s , a m y l o p l a s t s , e l o n g a t e d m i t o c h o n d r i a and p l a s t i d s w i t h a few o s m i o p h i l i c g l o b u l i ( a r r o w ) . June . C r o s s - s e c t i o n . C . Many e longated m i t o c h o n d r i a , l i p i d d r o p l e t s and a m y l o p l a s t s . J u l y . C r o s s - s e c t i o n . D. The s t r u c t u r e of the nuc leus and l i p i d d r o p l e t s . Note the absence of the a m y l o p l a s t . Augus t . C r o s s - s e c t i o n . E . The s t r u c t u r e of the n u c l e u s , l i p i d d r o p l e t s and a m y l o p l a s t . O c t o b e r . R a d i a l - s e c t i o n . F . The s t r u c t u r e of the n u c l e u s , p l a s t i d , s p h e r i c a l m i t o c h o n d r i a and l i p i d d r o p l e t s . The d i s t i n c t i v e l y grouped c h r o m a t i n and an o r g a n e l l e - f r e e zone (arrow) s u r r o u n d i n g the nuc l eus c h a r a c t e r i z e the c e l l . November. R a d i a l - s e c t i o n . 209 PLATE 19 210 PLATE 20 Abies balsamea ray parenchyma cells in the outer sapwood ring. A . The e longated shape of s t a r c h g r a n u l e s i n an a m y l o p l a s t and numerous l i p i d d r o p l e t s . Note the s t r u c t u r e of the r o d - l i k e m i t o c h o n d r i a and p l a s t i d s . May. C r o s s - s e c t i o n . B . A rounded s t a r c h g r a n u l e i n an a m y l o p l a s t and a few l i p i d d r o p l e t s . Note the e longated m i t o c h o n d r i a . June . C r o s s - s e c t i o n . C . A rounded s t a r c h g r a n u l e i n an a m y l o p l a s t , l i p i d d r o p l e t s and an e l o n g a t e d m i t o c h o n d r i o n . Most of the m i t o c h o n d r i a are s p h e r i c a l i n shape . J u l y . C r o s s - s e c t i o n . D. The s t r u c t u r e of the endoplasmic r e t i c u l u m , f i l a m e n t o u s m i t o c h o n d r i a which c h a r a c t e r i z e h i g h m e t a b o l i c a c t i v i t y ; p l a s t i d s and a few l i p i d d r o p l e t s . Note the absence of the a m y l o p l a s t . Augus t . C r o s s - s e c t i o n . E . The s t r u c t u r e of the m i t o c h o n d r i a and the e l o n g a t e d s t a r c h g r a n u l e i n the a m y l o p l a s t . O c t o b e r . C r o s s - s e c t i o n . F . Numerous p l a s t i d s and s p h e r i c a l m i t o c h o n d r i a . November. R a d i a l - s e c t i o n . 211 PLATE 20 212 PLATE 21 Abies balsamea ray parenchyma cells in the outer sapwood ring. A. The s t r u c t u r e of the m i t o c h o n d r i a and a m y l o p l a s t . Note the o s m i o p h i l i c g l o b u l i (arrow) and e l o n g a t e d s t a r c h g r a n u l e i n s i d e the a m y l o p l a s t . May. R a d i a l -s e c t i o n . B. The s t r u c t u r e of the plasmodesmata a t the end w a l l . Note the i n d e n t u r e (arrows) of the end w a l l . June . C r o s s - s e c t i o n . C . A dictyosome w i t h four c i s t e r n a e and v e s i c l e s ( a r r o w s ) . J u l y . C r o s s - s e c t i o n . D. The s t r u c t u r e of dumb-be l l shaped m i t o c h o n d r i a , the endoplasmic r e t i c u l u m and a few l i p i d d r o p l e t s . Note an unusua l c e l l w a l l i r r e g u l a r i t y at the h a l f - b o r d e r e d p i t - p a i r area ( a r r o w ) . Augus t . C r o s s - s e c t i o n . E . A dictyosome w i t h two c i s t e r n a e and numerous v e s i c l e s ( a r r o w ) . November. C r o s s - s e c t i o n . F . The s t r u c t u r e of the n u c l e u s , endoplasmic r e t i c u l u m , m i t o c h o n d r i a , l i p i d d r o p l e t s , v a c u o l e and plasmalemma (double a r r o w s ) . Note an o r g a n e l l e - f r e e zone (arrow) s u r r o u n d i n g the n u c l e u s . November. C r o s s - s e c t i o n . 213 PLATE 21 PLATE 22 Abies balsamea ray parenchyma cells in the middle sapwood ring. A . The s t r u c t u r e of the n u c l e u s , l i p i d d r o p l e t s and a m y l o p l a s t . Note an o r g a n e l l e f r e e zone ( a r r o w ) . June . R a d i a l - s e c t i o n . B . The s t r u c t u r e of the n u c l e u s , l i p i d d r o p l e t s and a m y l o p l a s t . J u l y . R a d i a l r s e c t i o n . C . The s t r u c t u r e of the n u c l e u s , m i t o c h o n d r i a , l i p i d d r o p l e t s and a m y l o p l a s t . The b r a n c h i n g m i t o c h o n d r i a c h a r a c t e r i z e the h i g h m e t a b o l i c a c t i v i t y of the c e l l . A u g u s t . R a d i a l - s e c t i o n . D. The s t r u c t u r e of the plasmodesmata at the end w a l l . O c t o b e r . R a d i a l - s e c t i o n . E . The s t r u c t u r e of the n u c l e u s , p l a s t i d , m i t o c h o n d r i a and l i p i d d r o p l e t s . The g r o u p i n g of the chromat in i n the nuc leus c h a r a c t e r i z e s the c e l l . November. R a d i a l - s e c t i o n . F . The s t r u c t u r e of the m i t o c h o n d r i a , p l a s t i d and numerous l i p i d d r o p l e t s and two l i p i d lumps. November. R a d i a l - s e c t i o n . 215 PLATE 22 216 PLATE 23 Abies balsamea ray parenchyma cells in the middle sapwood ring. A . The e longated m i t o c h o n d r i o n and l i p i d d r o p l e t s . June . R a d i a l - s e c t i o n . B. A b r a n c h i n g m i t o c h o n d r i o n , l i p i d d r o p l e t s and a m y l o p l a s t . J u l y . R a d i a l - s e c t i o n . C. A b r a n c h i n g m i t o c h o n d r i o n , l i p i d d r o p l e t s and an a m y l o p l a s t w i t h a c r a c k s on the s t a r c h g r a n u l e . A u g u s t . R a d i a l - s e c t i o n . D. Numerous l i p i d d r o p l e t s and an a m y l o p l a s t . O c t o b e r . R a d i a l - s e c t i o n . E . The lumping of l i p i d d r o p l e t s . O c t o b e r . R a d i a l - s e c t i o n . F . The s t r u c t u r e of the p l a s t i d , s p h e r i c a l m i t o c h o n d r i a and l i p i d d r o p l e t s . November. R a d i a l - s e c t i o n . 217 PLATE 23 218 PLATE 24 Abies balsamea ray parenchyma cells in the inner sapwood ring A . The s t r u c t u r e of the n u c l e u s , n u c l e o l u s , a m y l o p l a s t and l i p i d d r o p l e t s . May. C r o s s - s e c t i o n . B . The s t r u c t u r e of the a m y l o p l a s t , s p h e r i c a l m i t o c h o n d r i a and l i p i d d r o p l e t s . J u l y . R a d i a l - s e c t i o n . C . The s t r u c t u r e of the n u c l e u s , a m y l o p l a s t and l i p i d d r o p l e t s . A rounded s t a r c h g r a n u l e i n the a m y l o p l a s t c h a r a c t e r i z e s the c e l l . A u g u s t . R a d i a l - s e c t i o n . D. The s t r u c t u r e of the nuc l eus i n a d y i n g c e l l . The l a c k of ground substance and g r o u p i n g of the chromat in c h a r a c t e r i z e the c e l l . A u g u s t . C r o s s - s e c t i o n . E . Numerous l i p i d d r o p l e t s , e l onga ted m i t o c h o n d r i a and n u c l e u s . November. R a d i a l - s e c t i o n . F . Two p l a s t i d s w i t h many o s m i o p h i l i c g l o b u l i ( a r r o w s ) . November. R a d i a l - s e c t i o n . 219 PLATE 24 2 2 0 PLATE 25 Abies balsamea ray parenchyma cells in the inner sapwood ring A . The s t r u c t u r e of the plasmalemma ( a r r o w ) , a m y l o p l a s t and l i p i d d r o p l e t s . The r o u n d e d - o f f s t a r c h g r a n u l e s c h a r a c t e r i z e the c e l l . May. R a d i a l - s e c t i o n . B . The s t r u c t u r e of the a m y l o p l a s t and l i p i d d r o p l e t s . J u l y . R a d i a l - s e c t i o n . C . The s t r u c t u r e of the a m y l o p l a s t and l i p i d d r o p l e t s . J u l y . R a d i a l - s e c t i o n . D. Numerous l i p i d d r o p l e t s and a m y l o p l a s t s . The r o u n d e d - o f f s t a r c h g r a n u l e s c h a r a c t e r i z e the c e l l . A u g u s t . R a d i a l - s e c t i o n . E . The s t r u c t u r e of the m i t o c h o n d r i a , a m y l o p l a s t and l i p i d d r o p l e t s . The r o u n d e d - o f f s t a r c h g r a n u l e s w i t h c r a c k s and d i s i n t e g r a t i n g membranes of the a m y l o p l a s t c h a r a c t e r i z e the c e l l . A u g u s t . R a d i a l -s e c t i o n . F . The s t r u c t u r e of the n u c l e u s , p l a s t i d s , m i t o c h o n d r i a and l i p i d d r o p l e t s . Numerous o s m i o p h i l i c g l o b u l i (arrow) are i n the p l a s t i d . November. R a d i a l - s e c t i o n . 221 PLATE 25 PLATE 26 Abies balsamea ray parenchyma cells in the heartwood. A . A dark g l o b u l a r - l i k e form of heartwood s u b s t a n c e . R a d i a l - s e c t i o n . B . A g r a n u l a r form of heartwood s u b s t a n c e . R a d i a l -s e c t i o n . C . A lump form of the heartwood subs tance l i n i n g the c e l l w a l l . R a d i a l - s e c t i o n . D. V a r i o u s forms of heartwood s u b s t a n c e . R a d i a l -s e c t i o n . E . A lump form of heartwood substance at the end w a l l a r e a . R a d i a l - s e c t i o n . F . V a r i o u s forms of heartwood s u b s t a n c e . R a d i a l -s e c t i o n . 223 224 PLATE 27 Populus tremuloides ray parenchyma cel l s in the outer sapwood ring. A . Numerous l i p i d d r o p l e t s . May. R a d i a l - s e c t i o n . B . A l a r g e v a c u o l e , l i p i d d r o p l e t s and l i p i d lump, s t a r c h g r a n u l e s and an i s o t r o p i c l a y e r ( a r r o w ) . June . R a d i a l - s e c t i o n . C . The s t r u c t u r e of the l i p i d d r o p l e t s , a m y l o p l a s t s , s p h e r i c a l m i t o c h o n d r i a , plasmodesmata and an i s o t r o p i c l a y e r (arrow) a t the end w a l l a r e a . S e v e r a l round s t a r c h g r a n u l e s i n an a m y l o p l a s t . June . R a d i a l - s e c t i o n . D. The s t r u c t u r e of the m i t o c h o n d r i a , n u c l e u s d r o p l e t s . J u l y . C r o s s -s t a r c h g r a n u l e s , e l onga ted and v a r i o u s s i z e s of l i p i d s e c t i o n . l i p i d d r o p l e t s and - s e c t i o n . E . V a r i o u s forms and s i z e s of m i t o c h o n d r i a . A u g u s t . R a d i a l F . V a r i o u s forms and s i z e s of l i p i d d r o p l e t s and e l o n g a t e d m i t o c h o n d r i a . November. R a d i a l - s e c t i o n . 225 PLATE 27 PLATE 28 226 Populus tremuloides ray parenchyma cel l s in the outer sapwood ring A . The s t r u c t u r e of the nuc l eus and l i p i d d r o p l e t s . May. R a d i a l - s e c t i o n . B . Lumping of l i p i d d r o p l e t s and c l u s t e r i n g of s t a r c h g r a n u l e s . June . R a d i a l - s e c t i o n . C . The s t r u c t u r e of the a m y l o p l a s t and l i p i d d r o p l e t s . S e v e r a l round s t a r c h g r a n u l e s w i t h i n an a m y l o p l a s t . J u l y . C r o s s - s e c t i o n . D. The s t r u c t u r e of the l i p i d d r o p l e t s and d u m b - b e l l shaped m i t o c h o n d r i a . Augus t . R a d i a l - s e c t i o n . E . The s t r u c t u r e of the l i p i d d r o p l e t s and s t a r c h g r a n u l e s . O c t o b e r . R a d i a l - s e c t i o n . F . The s t r u c t u r e of the l i p i d d r o p l e t s and ribosome c l u s t e r ( a r r o w ) . November. R a d i a l - s e c t i o n . 227 PLATE 28 228 PLATE 29 Populus tremuloides ray parenchyma cells in the outer sapwood ring A . The s t r u c t u r e of the l i p i d d r o p l e t s , endoplasmic r e t i c u l u m and m i t o c h o n d r i a . May. R a d i a l - s e c t i o n . B . Four l a r g e l i p i d d r o p l e t s and an a m y l o p l a s t w i t h s t a r c h g r a n u l e s a r r a n g e d i n a s p i r a l f o r m . Note the i s o t r o p i c l a y e r s ( a r r o w s ) . June . C r o s s - s e c t i o n . C . An unusua l form of the endoplasmic r e t i c u l u m . Note the i s o t r o p i c l a y e r s ( a r r o w s ) . J u l y . C r o s s - s e c t i o n . D. The s t r u c t u r e of the m i t o c h o n d r i a and endoplasmic r e t i c u l u m . A b l i n d p i t (arrow) i s to be n o t e d . A u g u s t . C r o s s - s e c t i o n . E . V a r i o u s s i z e s of l i p i d d r o p l e t s and s t a r c h g r a n u l e s . O c t o b e r . R a d i a l - s e c t i o n . F . The p l a s t i d w i t h a few o s m i o p h i l i c g l o b u l i ( a r r o w ) . November. R a d i a l - s e c t i o n . 229 PLATE 29 2 3 0 PLATE 30 Populus tremuloides ray parenchyma cel l s in the middle sapwood ring A . The s t r u c t u r e of the nuc l eus and l i p i d d r o p l e t s . Note the i s o t r o p i c l a y e r ( a r r o w ) . May. R a d i a l -s e c t i o n . B . The abundance of the l i p i d d r o p l e t s . Note the s t r u c t u r e of the endoplasmic r e t i c u l u m . June . R a d i a l - s e c t i o n . C . The s t r u c t u r e of the a m y l o p l a s t , l i p i d d r o p l e t s and m i t o c h o n d r i a . J u l y . R a d i a l - s e c t i o n . D. The s t r u c t u r e of the nuc l eus and l i p i d d r o p l e t s . Some s t a r c h - l i k e g r a n u l e s (double arrows) are empty at the c e n t r e . A l s o note the i s o t r o p i c l a y e r ( a r r o w ) . Augus t . R a d i a l - s e c t i o n . E . Numerous l i p i d d r o p l e t s of v a r i o u s forms and s i z e s . O c t o b e r . R a d i a l - s e c t i o n . F . The s t r u c t u r e of the n u c l e u s , l i p i d d r o p l e t s and the i s o t r o p i c l a y e r ( a r r o w ) . November. R a d i a l -s e c t i o n . 231 PLATE 30 2 3 2 PLATE 31 Populus tremuloides ray parenchyma cells in the middle sapwood ring A . The s t r u c t u r e of the l i p i d d r o p l e t s and some v e s i c l e s (two a r r o w s ) . Note the i s o t r o p i c l a y e r ( a r r o w ) . May. R a d i a l - s e c t i o n . B . The c l u s t e r of the s t a r c h g r a n u l e s and l i p i d d r o p l e t s . June . R a d i a l - s e c t i o n . C . The lumping of the l i p i d d r o p l e t s and d i s i n t e g r a t i n g s t a r c h g r a n u l e s . Some s t a r c h g r a n u l e s are empty at the c e n t r e . Note the i s o t r o p i c l a y e r ( a r r o w ) . J u l y . R a d i a l - s e c t i o n . D. The s t r u c t u r e of the l i p i d d r o p l e t s , v a c u o l e and d i s i n t e g r a t i n g s t a r c h g r a n u l e s . Some s t a r c h g r a n u l e s are empty at the c e n t r e . A u g u s t . R a d i a l -s e c t i o n . E . V a r i o u s s i z e s of l i p i d d r o p l e t s and s t a r c h g r a n u l e s . Note the i s o t r o p i c l a y e r ( a r r o w ) . O c t o b e r . R a d i a l - s e c t i o n . F . Numerous l i p i d d r o p l e t s of v a r i o u s s i z e s and forms . Note the i s o t r o p i c l a y e r (arrow) and the absence of s t a r c h g r a n u l e s . November. R a d i a l -s e c t i o n . 233 234 PLATE 32 Populus tremuloides ray parenchyma cells in the inner sapwood ring A . The s t r u c t u r e of the nuc leus and l i p i d d r o p l e t s w i t h two s t a i n i n g d e n s i t i e s . The h e a v i l y s t a i n e d zone extends from a d r o p l e t to the a d j a c e n t d r o p l e t ( a r r o w ) . J u n e . R a d i a l - s e c t i o n . B . The s t r u c t u r e of the nuc leus and l i p i d d r o p l e t s . A l i p i d - l i k e d r o p l e t (arrow) i s i n s i d e the n u c l e u s . J u l y . R a d i a l - s e c t i o n . C . The s t r u c t u r e of the nuc leus and v a c u o l e . A l i p i d - l i k e d r o p l e t (arrow) i s i n s i d e the n u c l e u s . J u l y . R a d i a l - s e c t i o n . D. The s t r u c t u r e of the l i p i d d r o p l e t s and s t a r c h g r a n u l e s i n a d y i n g c e l l . The l a c k of ground substance c h a r a c t e r i z e s the c e l l . A u g u s t . R a d i a l - s e c t i o n . E . A l a r g e l i p i d d r o p l e t w i t h many s m a l l l i p i d d r o p l e t s of v a r i o u s s i z e s . O c t o b e r . R a d i a l - s e c t i o n . F . The s t r u c t u r e of the nuc leus and l i p i d d r o p l e t s . A l i p i d - l i k e d r o p l e t (arrow) i s i n s i d e the n u c l e u s . November. R a d i a l - s e c t i o n . 235 PLATE 32 236 PLATE 33 Populus tremuloides ray parenchyma cells in the inner sapwood rings A . A l a r g e l i p i d d r o p l e t w i t h two s t a i n i n g d e n s i t i e s . Note the c l u s t e r of s t a r c h g r a n u l e s . June . R a d i a l - s e c t i o n . B . A c l u s t e r of s t a r c h g r a n u l e s between two l a r g e l i p i d lumps which are s t a i n e d to two d e n s i t i e s . The h e a v i l y s t a i n e d zone extends to i t s a d j a c e n t l i p i d d r o p l e t . J u n e . R a d i a l - s e c t i o n . C . Four l a r g e l i p i d d r o p l e t s and many s m a l l l i p i d d r o p l e t s . Note the i s o t r o p i c l a y e r ( a r r o w ) . J u l y . R a d i a l - s e c t i o n . D. Two l a r g e l i p i d lumps and many s t a r c h g r a n u l e s . A u g u s t . R a d i a l - s e c t i o n . E . The s t r u c t u r e of the l i p i d d r o p l e t s , v a c u o l e and c l u s t e r s of s t a r c h g r a n u l e s . O c t o b e r . R a d i a l - s e c t i o n . F . The s t r u c t u r e of the l i p i d d r o p l e t s , p l a s t i d s and the endoplasmic r e t i c u l u m . Note the i s o t r o p i c l a y e r ( a r r o w ) . November. R a d i a l - s e c t i o n . 237 PLATE 33 238 PLATE 34 Populus tremuloides ray parenchyma c e l l s in the heartwood. The heartwood substance i n a dead ray parenchyma c e l l . Note the i s o t r o p i c l a y e r ( a r r o w ) . R a d i a l -s e c t i o n . B . The s t r u c t u r e of the plasmodesmata and the i s o t r o p i c l a y e r and the p i t membrane of the s imple p i t - p a i r s . R a d i a l - s e c t i o n . C . The heartwood substance i n a dead ray parenchyma c e l l . R a d i a l - s e c t i o n . D. Two forms of the heartwood s u b s t a n c e , i . e . , d r o p l e t - l i k e and lump form l i n i n g the c e l l w a l l . R a d i a l - s e c t i o n . E . The c y t o p l a s m i c r e s i d u e i n a dead r a y parenchyma c e l l . R a d i a l - s e c t i o n . F . A l a r g e lump form heartwood s u b s t a n c e . Note the i s o t r o p i c l a y e r ( a r r o w ) . Double -arrows i n d i c a t e the c o n t i n u a t i o n of the i s o t r o p i c l a y e r and the p i t membrane of the s i m p l e p i t - p a i r s . R a d i a l - s e c t i o n . 239 PLATE 34 240 PLATE 35 Heartwood Substances A . Swol l en s t a r c h g r a n u l e s and l i p i d d r o p l e t s i n a r a y parenchyma c e l l and a l o n g i t u d i n a l t r a c h e i d i n the i n n e r sapwood near the heartwood . P inus  b a n k s i a n a . C r o s s - s e c t i o n . B . B l a c k l i p i d d r o p l e t s i n a ray parenchyma c e l l and a ray t r a c h e i d i n the i n n e r sapwood near the heartwood. P inus b a n k s i a n a . C r o s s - s e c t i o n . C . D r o p l e t - l i k e heartwood substances i n margo area of a bordered p i t - p a i r between a ray t r a c h e i d and a l o n g i t u d i n a l t r a c h e i d i n the heartwood . P inus  b a n k s i a n a . C r o s s - s e c t i o n . D. Heartwood substances at the p i t chamber of a h a l f -b o r d e r e d p i t - p a i r i n the heartwood . Arrow i n d i c a t e s the secondary w a l l t h i c k e n i n g of a ray parenchyma c e l l . P inus b a n k s i a n a . C r o s s - s e c t i o n . E . An a s p i r a t e d p i t and a l i p i d - l i k e d r o p l e t a t t a c h e d on the t o r u s i n a heartwood t r a c h e i d . P inus  b a n k s i a n a . C r o s s - s e c t i o n . F . Heartwood subs tances i n a heartwood t r a c h e i d . P inus b a n k s i a n a . C r o s s - s e c t i o n . 241 PLATE 35 242 PLATE 36 Heartwood Substances A . L i p i d d r o p l e t s i n a ray parenchyma c e l l and a l o n g i t u d i n a l t r a c h e i d i n the i n n e r sapwood near the heartwood. P i c e a m a r i a n a . C r o s s - s e c t i o n . B . Heartwood substances at the p i t chamber of a h a l f -b o r d e r e d p i t - p a i r i n the heartwood. P i c e a m a r i a n a . C r o s s - s e c t i o n . C . L i p i d - l i k e d r o p l e t s i n a ray parenchyma c e l l and a l o n g i t u d i n a l t r a c h e i d i n the hear twood . Ab ie s  ba l samea . C r o s s - s e c t i o n . D. Heartwood subs tances i n the c e l l lumen of a l o n g i t u d i n a l t r a c h e i d near the p i t areas i n the heartwood. P i c e a m a r i a n a . C r o s s - s e c t i o n . E . G r a n u l a r l i k e heartwood substances i n the p i t chamber of a h a l f - b o r d e r e d p i t - p a i r i n the heartwood. A b i e s ba l samea . C r o s s - s e c t i o n . F . Heartwood substance i n a ray parenchyma c e l l and the p i t chamber of a s imple p i t - p a i r i n the heartwood. Populus t r e m u l o i d e s . R a d i a l - s e c t i o n . PLATE 36 243 244 6. C O N C L U S I O N S A N D R E C O M M E N D A T I O N S The c y t o l o g i c a l changes i n sapwood r a y parenchyma c e l l s as they age from the cambium to the heartwood have been presented i n d e t a i l . The r e s u l t s i n d i c a t e d t h a t the c e l l s t r u c t u r e o f ray parenchyma c e l l s v a r i e d s i g n i f i c a n t l y w i t h season , r a d i a l l o c a t i o n of c e l l s w i t h i n sapwood and v e r t i c a l p o s i t i o n of c e l l s w i t h i n a r a y . I t should be p o i n t e d out t h a t the d i f f e r e n c e between s p e c i e s i n t h i s s tudy may not be due to s p e c i e s a l o n e , owing to a l a c k of r e p l i c a t i o n . The main f i n d i n g s of t h i s s tudy are summarized below. 1. R a d i a l d i s t r i b u t i o n of m o i s t u r e content i n sapwood and heartwood showed seasona l and s p e c i e s d i f f e r e n c e s . For the t h r e e c o n i f e r s , the m o i s t u r e content i n sapwood was h i g h e r than t h a t of h e a r t -wood, whereas the sapwood mois ture content of Populus t r e m u l o i d e s was lower than t h a t of heartwood. 245 The s u r v i v a l of r a y parenchyma c e l l s decreased c u r v i l i n e a r l y from the outer sapwood towards the i n n e r sapwood. The decreased s u r v i v a l r a t e of the ray parenchyma c e l l s over t ime f o l l o w e d a t y p i c a l p a t t e r n of a g i n g , where the r a t e o f c e l l death a c c e l e r a t e d from the middle sapwood to the sapwood/ heartwood boundary. T h i s boundary was i n d i c a t e d both by an abrupt moi s ture content change and the absence of s u r v i v i n g c e l l s . These two i n d i c a t o r s showed g e n e r a l agreement i n e s t a b l i s h i n g the sapwood/ heartwood boundary, except i n P i c e a mariana i n which a p p r o x i m a t e l y f i f t y per cent of the c e l l s were s t i l l l i v i n g near the apparent boundary d i c t a t e d by m o i s t u r e c o n t e n t . For a g i v e n sapwood increment , a g r e a t e r percentage of dead ray parenchyma c e l l s was found among the m a r g i n a l c e l l s than among the c e n t r a l c e l l s of a r a y . T h i s phenomenon was e s p e c i a l l y pronounced i n Ab ie s balsamea, where a l l m a r g i n a l c e l l s were dead about f i v e years p r i o r to the c e n t r a l c e l l s , and at a t ime when f u l l y h a l f of the c e n t r a l c e l l s were s t i l l l i v i n g . 246 The v i t a l i t y of l i v i n g sapwood ray parenchyma c e l l s expressed by the n u c l e a r e l o n g a t i o n index decreased from the outer sapwood towards the heartwood. The r a t e at which c e l l v i t a l i t y decreased was more pronounced i n the o u t e r sapwood than i n the middle and i n n e r sapwood. In g e n e r a l , no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e was found between the v i t a l i t y of , the m a r g i n a l and c e n t r a l c e l l s , nor between two cont iguous sampl ing p e r i o d s , a l t h o u g h e x c e p t i o n s o c c u r r e d i n J u l y to December i n P inus banks iana and P i c e a m a r i a n a . The p a t t e r n of s t a r c h d i s t r i b u t i o n v a r i e s w i t h s p e c i e s , r a d i a l and v e r t i c a l p o s i t i o n and season . T h i s d i s t r i b u t i o n showed two g e n e r a l p a t t e r n s a c r o s s the sapwood. P a t t e r n A d e s c r i b e d a d e c r e a s i n g t r e n d from the outer sapwood towards the i n n e r sapwood. P a t t e r n B was c h a r a c t e r i z e d by a r e l a t i v e l y low s t a r c h content i n the o u t e r , as w e l l as the i n n e r , sapwood. Both p a t t e r n s d i s p l a y e d a d e c l i n i n g t r e n d from the middle sapwood towards the i n n e r sapwood. Whether t h i s d e c l i n i n g t r e n d i n s t a r c h c o r r e l a t e s w i t h heartwood f o r m a t i o n remains to be answered. 247 In P inus banks iana the s t a r c h content i n the m a r g i n a l c e l l s was s i g n i f i c a n t l y h i g h e r than t h a t of the c e n t r a l c e l l s i n May, June and J u l y , whereas no s i g n i f i c a n t d i f f e r e n c e between these two groups of ray c e l l s i n August , October and December was found . In Ab ie s balsamea the s t a r c h content i n the m a r g i n a l c e l l s was s i g n i f i c a n t l y lower than t h a t of the c e n t r a l c e l l s i n June and J u l y , but no d i f f e r e n c e s i n the r e s t of the sampl ing p e r i o d s were o b s e r v e d . The s t a r c h content i n P i c e a mariana and Populus  t r e m u l o i d e s d i s p l a y e d no s i g n i f i c a n t d i f f e r e n c e between these two groups of ray c e l l s , w i t h one e x c e p t i o n i n J u l y . Most of the t - t e s t s i n a l l s p e c i e s s t u d i e d showed a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e i n s t a r c h content between two cont iguous sampl ing p e r i o d s . L i t t l e or no s t a r c h was found i n November/December i n any s p e c i e s , and i n the t h r e e c o n i f e r s s t u d i e d , a r e l a t i v e l y low s t a r c h content was a l s o observed i n J u l y or August depending upon the s p e c i e s . In Populus t r e m u l o i d e s a r e l a t i v e l y h i g h s t a r c h content was measured from June to O c t o b e r . 248 A relatively high l i p i d content existed in a l l species except Abies balsamea. No typical pattern of l i p i d distribution across the sapwood was observed. In general, there is no s t a t i s t i c a l l y significant difference between l i p i d content in marginal cells and central cells with a few exceptions in August and October. In a l l species studied, significant differences in l i p i d content between two contiguous sampling periods were found. In November/December a high l i p i d content for a l l species studied was found across the sapwood, whereas an absence or minor presence of starch was recorded. However, there was no regular inverse relationship between starch and l i p i d content over the growing season, in contrast to speculation in the literature such a relationship exists. The ultrastructure of living sapwood ray parenchyma cells displayed differences due to season, species and age. In general, the nucleus in young ray 249 parenchyma cells showed an abundance of chromatin and cytoplasm, which contained numerous c e l l organelles. These cells were characterized by amyloplasts which contained one or more elongated starch granules with thylakoids and osmiophilic globuli, numerous small l i p i d droplets and rod-like mitochondria with distinctive and numerous cristae. In contrast, aged ray parenchyma cells featured an aggregated, dense nucleus and contained few c e l l organelles. These cells possessed enlarged, swollen and occasionally cracked starch granules, and large l i p i d droplets or l i p i d lumps with two staining densities. If mitochondria were present, they were round, with or without cristae. In Pinus banksiana, the transformation of sapwood into heartwood occurred in July/August. This conclusion is based mainly on electron microscopic observations of the vesicle attachment to the plasmalemma. In the other three species studied, no clear evidence could be found to identify the timing of the process of sapwood/heartwood transformation. Some heartwood substances originated from the l i p i d 2 5 0 d r o p l e t s or lumps which appeared f r e q u e n t l y i n dying ray parenchyma c e l l s . The f i n d i n g s and r e s u l t s obtained i n t h i s study suggest some i n t e r e s t i n g i m p l i c a t i o n s with regard to heartwood fo r m a t i o n . S e v e r a l hypotheses concerning heartwood formation have been p o s t u l a t e d i n the p a s t . These hypotheses have h i g h l i g h t e d water d e f i c i e n c y , t o x i c accumulations, formation of heartwood inducing substances, and aging p r o c e s s e s . The water d e f i c i e n c y mechanism i s based on the hypothesis t h a t a low moisture content i n heartwood, combined with a l a r g e q u a n t i t y of a i r , can induce the death of ray parenchyma c e l l s . However, a hig h moisture content i n heartwood of Populus tremuloides was measured. S i m i l a r h i g h moisture contents i n heartwood were a l s o reported i n many other hardwoods (Yazawa 1960, Yazawa et a_l. 1965, Dobie and Wright 1978) and even i n softwoods (Nobuchi et a l . 1985). T h e r e f o r e , water d e f i c i e n c y alone does not support the theory of sapwood/heartwood t r a n s f o r m a t i o n , based on t h i s present study and those of o t h e r s . Stewart (1966) and Carrodus (1971) b e l i e v e d that t o x i c m a t e r i a l s are accumulated i n the inner sapwood 251 as waste p r o d u c t s of the metabol ism of sapwood ray-parenchyma c e l l s . These ray parenchyma c e l l s i n the i n n e r sapwood are presumed to d i e when the c o n c e n t r a -t i o n of t o x i c substance reaches a l e t h a l l e v e l . Bamber (1976) proposed tha t the sapwood/heartwood t r a n s f o r m -a t i o n i s i n i t i a t e d i n the sapwood ray parenchyma c e l l s by heartwood i n d u c i n g substances w i t h h o r m o n e - l i k e f u n c t i o n which move c e n t r i p e t a l l y a long the ray from the cambium. However, t h e i r p r e s e n c e / e x i s t e n c e has not been demonstrated . N o n e t h e l e s s , a l l hypotheses agree t h a t heartwood i s the r e s u l t of the death of sapwood ray parenchyma c e l l s , but they d i s a g r e e over the cause of the death of these c e l l s . In the present s t u d y , the v i t a l i t y and s u r v i v a l r a t e of sapwood ray parenchyma c e l l s d e c l i n e d from the o u t e r sapwood towards the i n n e r sapwood. The s u r v i v a l r a t e curve showed a t y p i c a l ag ing phenomenon. The changes of the m o r p h o l o g i c a l s t r u c t u r e of c e l l o r g a n e l l e s i n the i n n e r sapwood c o i n c i d e d w i t h the measurements c o l l e c t e d through l i g h t m i c r o s c o p y . For example, the lower v i t a l i t y of i n n e r sapwood ray 252 parenchyma c e l l s i s a s s o c i a t e d w i t h a s m a l l n u c l e a r e l o n g a t i o n index and round-shaped m i t o c h o n d r i a w i t h or w i thout c r i s t a e . For a l l four t r e e s , the s i m i l a r i t y among them i n d i c a t e s the f o l l o w i n g f a c t s f o r ray parenchyma c e l l s : a common l o s s of v i t a l i t y as i n d i c a t e d by the N E I , the i n c r e a s e of m o r t a l i t y as expressed by the SR and the i n t e r n a l c e l l changes observed w i t h the TEM. T h i s suggested t h a t the death of l i v i n g sapwood ray parenchyma c e l l s i s an ag ing p r o c e s s . P a n d a l a i (1985) suggested tha t the c r a c k s a p p e a r i n g i n the s w o l l e n s t a r c h g r a n u l e s may a l l o w the e n t r y of a m y l o l y t i c enzymes. Consequent ly , such s t a r c h g r a n u l e s c o u l d be most ly c o n v e r t e d i n t o l i p i d d r o p l e t s due to enzymat ic p e n e t r a t i o n . In the p r e s e n t s t u d y , s w o l l e n and cracked s t a r c h g r a n u l e s were f r e q u e n t l y observed i n the dy ing ray parenchyma c e l l s next to the heartwood. Some heartwood s u b s t a n c e s , found w i t h e l e c t r o n m i c r o s c o p y , o r i g i n a t e d from l i p i d d r o p l e t s or l i p i d lumps. T h i s o b s e r v a t i o n and P a n a d a l a i ' s s u g g e s t i o n l eads to q u a l i f i e d support of the h y p o t h e s i s ( H i g u c h i and Fukazawa 1966, H i g u c h i 1976) t h a t 2 5 3 heartwood phenols are s y n t h e s i z e d i n s i t u i n the i n n e r sapwood ray parenchyma c e l l s from sugars and f a t s . The presen t study has demonstrated the death of many ray parenchyma c e l l s and g r a d u a l d i m i n u t i o n of s t a r c h from the middle towards the i n n e r sapwood, w e l l b e f o r e the sapwood/heartwood boundary (as i n d i c a t e d by an abrupt change i n m o i s t u r e c o n t e n t ) . I f heartwood were formed i n s i t u as a d i r e c t consequence of c e l l d e a t h , then one c o u l d expect a g r a d u a l i n c r e a s e i n heartwood e x t r a c t i v e s i n the sapwood, p r o p o r t i o n a l to the decrease i n s t a r c h and the number of e x p i r e d c e l l s . In r e t r o s p e c t , i t would have been i n s t r u c t i v e to determine the q u a l i t a t i v e and q u a n t i t a t i v e na ture of e x t r a c t i v e s a c r o s s the sapwood and i n t o the heartwood i n r e l a t i o n to the frequency of dead c e l l s . F u t u r e s t u d i e s shou ld t r a c k these e x t r a c t i v e s a c r o s s the sapwood to unders tand b e t t e r whether e x t r a c t i v e s are formed in. s i t u , or are t r a n s p o r t e d i n p r e c u r s o r form to the innermost sapwood. The v e s i c l e attachment to the plasmalemma as appeared i n P inus b a n k s i a n a s i g n i f i e d the b e g i n n i n g of c e l l w a l l f o r m a t i o n , l e a d i n g to the death of ray 254 parenchyma c e l l s . However, t h i s " i n d i c a t o r " c o u l d not be employed f o r o ther s p e c i e s due to a d i f f e r e n t s t r u c t u r e of the ray t i s s u e . 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The f i n e s t r u c t u r e of v e s i c l e s a s s o c i a t e d w i t h the c e l l s u r f a c e i n H e l i a n t h u s shoot t i s s u e . C a n . J . B o t . 45 :2103-2108. Wardrop, A . B . and J . Cronshaw. 1962. Format ion of p h e n o l i c substances i n the ray parenchyma of ang iosperms . Nature 193:90-92. 280 W i l c o x , H . 1962 Cambial growth c h a r a c t e r i s t i c s . In "Tree Growth" e d . by T . T . K o z l o w s k i , 57-88. Ronald Press C o . , New Y o r k , NY. W i s n i e w s k i , M. and E . N . Ashworth . 1986. A comparison of s e a s o n a l u l t r a s t r u c t u r a l changes i n stem t i s s u e s of peach (Prunus p e r s i c a ) t h a t e x h i b i t c o n t r a s t i n g mechanisms of c o l d h a r d i n e s s . B o t . Gaz . 147:407-417. Yamamoto, K. 1982. Y e a r l y and s easona l proces s of m a t u r a t i o n of ray parenchyma c e l l s i n P inus s p e c i e s . Res . B u l l . C o l l . Exp . F o r . , Hokkaido U n i v . 39(2) :245-296. Yamamoto, K . , K. Fukazawa and S. I s h i d a . 1978. Study on the c e l l w a l l development of ray parenchyma i n genus P i n u s . 2. Seasonal changes of c e l l w a l l t h i c k e n i n g and l i g n i f i c a t i o n i n P inus s t r o b u s . Res . B u l l . C o l l . Exp . F o r . , Hokkaido U n i v . 35(1) : 99-108. Yamamoto, K . , K.Fukazawa and S. I s h i d a . 1979a. Study on the c e l l w a l l development of ray parenchyma i n genus Pinus u s i n g u l t r a v i o l e t m i c r o s c o p y . Res . B u l l . C o l l . E x p e r . F o r e s t s , Hokkaido U n i v . 36(1) : 79-96. 281 Yamamoto, K . , K.Fukazawa and S. I s h i d a . 1979b. Study on the c e l l w a l l development of ray parenchyma i n genus P i n u s . Seasonal changes of c e l l w a l l t h i c k e n i n g and l i g n i f i c a t i o n i n P inus b a n k s i a n a . Res . B u l l . C o l l . Exp . F o r . , Hokkaido U n i v . 36(4) :451-458. Yang , K . C . , G. Hazenberg , G . E . B r a d f i e l d and J . R . Maze. 1985. V e r t i c a l v a r i a t i o n o f sapwood t h i c k n e s s i n P inus banks iana Lamb, and L a r i x l a r i c i n a (Du Roi ) K. K o c h . Can . J . F o r . Res . 15:822-828. Yang , K . C . 1986. A p r o p o s a l f o r a new i n d i c a t o r f o r e x p r e s s i n g the m e t a b o l i c a c t i v i t y of l i v i n g sapwood ray parenchyma c e l l s . IAWA B u l l . n . s . 7 ( 1 ) : 1 7 - 2 0 . Yang , K . C . 1987. M e t a b o l i c a c t i v i t y of l i v i n g sapwood r a y c e l l s i n P i c e a mariana expressed by a new i n d i c a t o r . Can. J . B o t . 65:1775-1778. Yazawa, K. 1960. The s e a s o n a l water content of sapwood and heartwood of b r o a d - l e a v e d t r e e s , e s p e c i a l l y of beech (Fagus c r e n a t a B . ) . J . J a p . Wood Res . Soc . 6 (4 ) :170-175 . 282 Yazawa, K . , S. I s h i d a and H . M i y a j i m a . 1965. 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K e l l i s o n . 1968. M o i s t u r e c o n t e n t of southern p i n e t r e e s . Techn . Rept . No. 37. Sch . F o r . R e s . , NC S t a t e U n i v . , R a l e i g h , NC. 44 pp. Z y c h a , H . 1948. The f o r m a t i o n of heartwood and a l l i e d p r o c e s s e s i n Faqus s y l v a t i c a . F o r s t w i s s . 67 (2 ) :80 -109. APPENDIX 1 Scientific name of Species 283 Abies balsamea ( L . ) M i l l . A b i e s s a c h a l i n e n s i s F r . Schm. A c a c i a n i l o t i c a D e b i l e Acer p l a t a n o i d e s L . Acer rubrum L . A e s c u l u s t u r b i n a t a Blume Alnus q l u t i n o s a G a e r t n . Anqophora c o s t a t a Domin. A n t i a r i s A f r i c a n a E n g l . B e i l s c h m i e d i a tawa (A. Cunn. ) Benth . et Hook. f . ex K i r k B e t u l a pendula Roth B r i d e l i a r e t u s a Spreng. Carya l a c i n i o s a (Michx. f . ) L o u d . Cephalo taxus h a r r i n q t o n i a K. Koch C e r c i d i p h y l l u m japonicum S i e b . e t Z u c c . Chamaecyparis obtusa E n d l . C o n v o l u l u e arevens L . C r y p t o m e r i a j a p o n i c a D. Don E u c a l y p t u s e l a l o p h o r a L . E u c a l y p t u s l ' a t i f o l i a F . E u c a l y p t u s t e r e t i c o r n i s Smith Fagus q r a n d i f o l i a E h r h . Fagus c r e n a t a Blume F r a x i n u s e x c e l s i o r L . J u g l a n s n i g r a L . J u n i p e r u s r i q i d a S i e b . et Z u c c . L a q e r s t r o e m i a i n d i c a L . L a r i x l e p t o l e p i s G o r d . L i r i o d e n d r o n t u l i p i f e r a L . M a g n o l i a q r a n d i f l o r a L . Mansonia a l t i s s i m a A . Chev. 284 M e l i a azedarach L . Quercus q l a u c a M. and T . Quercus robur L . P i c e a a b i e s K a r s t . P i c e a q l e h r i i M. e t H . P i c e a mariana ( M i l l . ) B . S . P . P inus b a n k s i a n a Lamb. P inus d e n s i f l o r a S i e b . e t Z u c c . P inus p e n t a p h y l l a C a r r . P inus ponderosa D o u g l . ex Laws P inus r a d i a t a D. Don P inus r e s i n o s a A i t . P inus s t r o b u s L . P inus s y l v e s t r i s L . Podocarpus m a c r o p h y l l a D. Don v a r . maki ( S i e b . et Zucc) E n d l . Podocarpus n a q i Z o l l . et M o r i t z . Populus euramer ica (Dode) G u i n i e r CV. Robusta Populus t r e m u l o i d e s M i c h x . Prunus avium L . Prunus s e r o t i n a E h r h . Pseudotsuqa j a p o n i c a B e i s s n . P t e r o c a r y a r h o i f o l i a S i e b . et Z u c c . R o b i n i a pseudoacac ia L . S a l i x f r a q i l i s L . S c i a d o p i t y s v e r t i c i l l a t a S i e b . et Z u c c . Sequoia semperv irens (D. Don) E n d l . T a i w a n i a c r y p t o m e r i o i d e s Hayata Tamarix a p h y l l a ( L . ) K a r s t . Taxus c u s p i d a t a S i e b . e t Z u c c . Tec tona g r a n d i s L . f . Thuja o r i e n t a l i s L . Thuj a p l i c a t a Donn ex D. Don T h u j o p s i s d o l a b r a t a S i e b . e t Z u c c . T i l i a micruel iana Maxim. Tsuqa s i e b o l d i i C a r r . Zea mays L . 286 APPENDIX 2 An example of the procedure used f o r data d e r i v a t i o n . S p e c i e s : P inus b a n k s i a n a . Data c o l l e c t e d (the number of growth r i n g s i n sapwood): May(24) , June ( 2 4 ) , J u l y (24) , August (23 ) , October (24) , December (25) . Procedure 1. The l a r g e s t growth r i n g number i n the sapwood i s 25. 2. One datum was c r e a t e d and added f o r the months of May, June , J u l y and O c t o b e r . 3. Two data were c r e a t e d and added f o r the month of A u g u s t . 4. The new data were c r e a t e d from the average of the o b s e r v a t i o n s of the two adjacent growth r i n g s where the death of sapwood ray parenchyma c e l l s began. 5. The m a r g i n a l ray parenchyma c e l l s began to d i e at the seventh growth r i n g i n P inus b a n k s i a n a . 2 8 7 The data f o r the e i g h t h growth r i n g was d e r i v e d , from the average of the NEI i n the seventh growth r i n g and the cont iguous r i n g immediate ly i n s i d e i t . In Augus t , two new r i n g s were needed to t o t a l the number 25. The f i r s t datum was c r e a t e d as s t a t e d i n Step 6. The second datum was d e r i v e d from the average of the NEI i n the o r i g i n a l e i g h t h and o r i g i n a l n i n t h growth r i n g s . The f i r s t d e r i v e d growth r i n g serves as the e i g h t h growth r i n g . The o r i g i n a l e i g h t h growth r i n g serves as the n i n t h growth r i n g . The second d e r i v e d r i n g s erves as the t e n t h growth r i n g . The o r i g i n a l n i n t h growth r i n g serves as the e l e v e n t h growth r i n g and so on . The o r i g i n a l se t and a d j u s t e d se t of growth r i n g s i n August i s shown as below. The () i n d i c a t e s the d e r i v e d r i n g s . Original Set Adjusted Set 2 8 9 APPENDIX 3 Summary of u l t r a s t r u c t u r a l changes of f i v e c e l l o r g a n e l l e s i n l i v i n g sapwood ray parenchyma c e l l s due to age and seasona l v a r i a t i o n i n P inus b a n k s i a n a , P i c e a m a r i a n a , Ab ie s balsamea and Populus t r e m u l o i d e s . 290 ABBREVIATIONS A - absent D - d i s p e r s e d i s t r i b u t i o n F - few o b s e r v a t i o n s H - h i g h d e n s i t y L - low d e n s i t y La - l a r g e s i z e Lm - lump M - medium d e n s i t y Mi - medium s i z e N - numerous d i s t r i b u t i o n s P - patchy d i s t r i b u t i o n R - rough s u r f a c e Rd - round shape Ro - r o d - l i k e shape S - smooth s u r f a c e Sa - s a c c u l a r c r i s t a Sm - s m a l l s i z e TI - l o n g t u b u l a r c r i s t a Ts - s h o r t t u b u l a r c r i s t a VL - v e r y l a r g e s i z e VN - v e r y numerous Pinus banksiana Cell Sampling Month Organelle Variable Location May June July Aug. Oct. Dec. Chromatin Outer-sap P/D P/D D D D D Distribution Mid-sap NA NA D D D NA Inner-sap D P P P P P Nuclear Outer-sap S/R R S S S/R S z Envelope Mid-sap NA NA S R S/R NA Surface Inner-sap S R R S/R S/R S 6. 13 Size of Outer-sap Sm A Sm/Mi A Sm/Mi A i l Starch Mid-sap A La Mi/L Mi Mi/La A Granule Inner-sap VL Mi/L La A Mi/La A Outer-sap Mi Sm/L Sm Sm/Mi Sm/Mi Sm/Lm Size Mid-sap A Sm/M Sm Sm Sm/Mi Sm/Mi Inner-sap Sm/L Sm/La Sm/Mi/Lm Sm/Lm Sm/Lm Sm/Lm Outer-sap M M M H M M Density Mid-sap NA H L/H L/H L/H L / H Inner-sap M/H H L/H H L/H L / H Outer-sap VN N VN F/N VN F Distribution Mid-sap F F N VN N F •S Inner-sap F F F F F F c Outer-sap Rd/Ro Rd Ro Rd/Ro Rd Rd p Shape Mid-sap Rd Rd Rd/Ro Rd/Ro Rd/Ro Rd Inner-sap Rd Rd Rd Rd Rd Rd Outer-sap Sa Sa T l T l Sa Ts Crista Mid-sap Sa Sa T l T l T l A Inner-sap Ts Ts Sa Sa Sa A Outer-sap R R R S R S/R 1 Surface Mid-sap R R R R S/R S 5 Inner-sap R R R R S/R S i Vesicle Outer-sap No No Yes No Yes No H Attachment Mid-sap Yes Yes No/Yes Yes No/Yes No Inner-sap Yes Yes Yes Yes No/Yes No Picea mariana Cell Sampling Month Organelle Variable Location May June July Auq. Oct. Dec. Chromatin Outer-sap D D D D D D Distribution Mid-sap P P P D D P Inner-sap P P P P P P CD Nuclear Outer-sap S/R S S S S/R S 25 Envelope Mid-sap S S S S S R/S Surface Inner-sap S S broken broken N/A broken 0 i9 Size of Outer-sap Sm/La Mi/La A A Sm/Mi A i i Starch Mid-sap La Mi/La A A A La Granule Inner-sap La La A A A A Outer-sap Sm/Mi Sm/Mi Sm/L Sm/L Sm/Mi Sm/M Size Mid-sap Sm/Mi Sm/Mi Mi/La Mi Mi Mi/La Inner-sap Mi/La Mi/Lm Mi/Lm La/Lm La/Lm Sm/La Outer-sap H H H H H L/H It Density- Mid-sap H H L/M L/H L/H L / H Inner-sap H L/H L/H H H L / H Outer-sap VN VN VN VN VN N Distribution Mid-sap VN N F N N N Inner-sap N N F F F F e Outer-sap Rd/Ro Rd/Ro Rd/Ro Rd/Ro Rd Rd 5 Shape Mid-sap Rd Rd/Ro Rd/Ro Rd/Ro Rd Rd Inner-sap Rd A/Rd Rd Rd Rd Rd Outer-sap Sa TI TI TI Ts Sa Cris ta Mid-sap Sa TI TI Ts Sa Sa Inner-sap Ts Sa A A A Ts Outer-sap R R S S R R i Surface Mid-sap R R S R R S i Inner-sap broken broken broken broken broken R 1 Vesicle Outer-sap No No No No No No 1 Attachment Mid-sap No No No No No/Yes No Inner-sap No No No No No No Plaatalertrra Mibocrxndria Lipid Droplets Anyio-plasts Nucleus 9 1 H -cf <J ct (D PJ cn e o ro CD ct 3 5 a c t CD I CD i-i cn H. I PJ I cn ts cn pj pj P *c 2 2 3 o o o 2 2 2 o o o % % % o o o Z 2 Z o o o 2 2 2 O O O 2 2 2 O O O 5 a ct CD 1 CD t-i cn i-i 1 cu 1 cn 'p cn I Hi PJ O CD W W \ CO W td cn cn W W W cn w \ w w O W W CD w w w H-cn ct PJ 5 & ct CD I CD i-i cn t-i I PJ I cn ti cn p *a H3 i-3 i-3 cn cn cn i-3i-3»-3 cn cn cn cn >p i-3 i-3 i-3 i-3 i-3 01 P H •3 I-3 cn H •-3 i-3 I-3 m cn cn •8 ? 2 Cb Ct 3 ^ t-t cn 1 PJ cn >p W W \ & & » O www Ch Ch & W W W a & a & & a w & W W \ PJ O i W O cn ct n 8" ct H -O 3 cn ct >< 3 5 & ct CD I ~ i-i cn 1 PJ 03 >P h3 CDH. I 0) PJ 73 W H-N CD I T*' CD t-( cn i-i 1 PJ 1 cn 73 cn 73 w 2 W w w W 2 2 2 2 s m s a a a ^ a a as jr< a a a s jr^a a a a a Q W W l"( Ct H-PJ PJ N 3 H (D c a l - O CD Hi HI ro PJ H 0 0 ro 73 ro &2 a. ct cn *p fa OJ ct I H. cn i-i 1 PJ 1 cn 73 cn PJ 73 cn 73 PJ ^ p 7 i ? . > > > w w w w w w w w w w w w w w w w w w cn p" ct R H 0 Ct H' 3 a ct W W W w w w w w w w o w w w w > I I I E6Z' Populus tremuloides Cell Sampling Month Organelle Variable Location May June July Aug. Oct. Nov. Chromatin Outer-sap D D D D D D Distribution Mid-sap D D D D D D ni Inner-sap D P D/P D/P D/P D/P Nuclear Outer-sap S S S S S S Envelope Mid-sap S/R R S S S S Surface Inner-sap S S S S S S o B Size of Outer-sap Sm Sm Sm A Sm A II Starch Mid-sap Sm Sm Sm La Sm A Granule Inner-sap Sm Sm A Sm/Mi Sm A Outer-sap Sm S/La Sm/La Sm/La Sm/La Sm/Mi Size Mid-sap Sm/Mi Sm/Lm Sm/Lm Sm/Mi Sm/La Sm/La Inner-sap Sm/La Lm Sm/VL Sm/Lm Sm/La Sm/La CU Outer-sap H H H H M H a c r Density Mid-sap H H H H H H M P Inner-sap H L/H L/H H H H Outer-sap N F F F F F Distribution Mid-sap N F N N F F Inner-sap F F A A A A Outer-sap Rd Rd Rd/Ro Rd/Ro Rd Rd Shape Mid-sap Rd Rd Rd Rd Rd Rd Inner-sap Rd A/Rd A A A A Outer-sap Sa Sa T l T l Sa A/Sa Crista Mid-sap Sa Sa Ts Ts Ts A/Sa Inner-sap A/Sa A/Sa A A A A Outer-sap S S/R S/R S/R S S Surface Mid-sap S S R R S S i Inner-sap NA S/broken R R/broken broken S Vesicle Outer-sap No No No No No No 1 Attachment Mid-sap No No No No No No Inner-sap No No No No No No PUBLICATIONS Book Yang, K.C. and Y.S.H. Yang 1987. Minute Structure of Taiwanese Woods. Hua Shiang Yuan'Publishing Co., Taipei, Taiwan, R.O.C 179 pp. Papers Yang, K.C. 1987a. Wood properties, wood qualities and s i l v i c u l t u r a l treatments. Quart. J.C. For. 20(2): 7-28 Yang, K.C. 1987b. Growth ring contrast enhancement and the differentiation of sapwood,and heartwood zones. Wood and Fiber Science 19(4): 339-342 Yang, K.C. 1987c. Metabolic activity of l i v i n g sapwood ray cells in Picea mariana (Mill.) B.S.P. expessed by a new indicator. Can. J . Bot. 65: 1775-1778. Yang, K.C. 1987d. Solid wood products and new products in "Forest Crop Planning" Twenty-two Exp. Views. Canadian Pulp and Paper Association, invited paper, p. 3.1-3.2 Yang, K.C. and G. Hazenberg. 1987. Geogaphical variation of wood properties in Larix laricina juvenile wood in northern Ontario. Can. J. For. Res. 17: 648-653 Yang, K.C, C Benson and J.K. Wong. 1986 Distribution of juvenile wood in two stems of Larix l a r i c i n a (Du Roi) K. Koch. Can. J . For. Res. 16: 1041-1049 Yang, K.C. 1986. A proposal for a new indicator f o r expressing the metabolic activity of living sapwood ray parenchyma cells in softwoods and hardwoods. IAWA New Series 7(1): 17-20 Yang, K.C. 1986. The ultrastructure of pits in Paulownia  tomentosa. Wood and Fiber Science 18(1): 118-126 Yang, K.C, G. Hazenberg, G.E. Bradfield and J.R. Maze. 1985 Vertical variation of sapwood thickness in Pinus  banksiana Lamb, and Larix laricina (Du Roi) K. Koch. Can. J . For. Res. l5:'822-828. Oliveira, L., A. Burns, T. Bisalputra and K.C. Yang. 1983. The use of an ultra-low viscosity medium (VCD/HXSA) in the embedding of plant ce l l s for electron microscopy. J . of Microscopy 132(2): 105-202 Yang, K.C. 1981. TROCHODENDRON - A hardwood without vessels. Quart. J.C. For. 14(2): 11-19 Yang, K.C. 1980. Preliminary evaluation of 12 hybrid solar kilns . Solar Energy Society of Canada Inc. Conference Proceeding. Aug. 6-10, 1980. SESCI Vancouver, B.C. 14.4 p. 420-423 Yang, K.C. 1980. A solar k i l n performance at a high latitude. N-48 N. For. Prod. J . 30(3): 37-40 Yang, K.C. 1979. A better lumber drying process with a non-greenhouse solar k i l n . Solar Energy Society of Canada Inc. Conference proceeding. Vol. 2. 79-86, 8 pages Yang, K.C. 1978. The fine structure of the pit in yellow birch. International Association of Wood Anatomists 4: 71-77 Yang, K.C. 1978. Feasibility of using solar energy for lumber drying in Northwestern Ontario. Solar Energy Society of Canada Inc., Conference Proceedings, Aug. 20-24, 1978, London, Ontario, Vol. 1. 4-1-3, 18 pages Koran, Z. and K.C. Yang. 1972. Gum distribution in yellow birch. Wood Science 5(2): 95-101 

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