UBC Theses and Dissertations

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UBC Theses and Dissertations

Nitrogen and conifer studies Mellor, Gary Edward 1972

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NITROGEN AND CONIFER STUDIES by GARY EDWARD MELLOR B.Sc,  U n i v e r s i t y o f Guelph, 1967  M.Sc., U n i v e r s i t y o f Guelph, 1969  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany  We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 19 72  In p r e s e n t i n g  this thesis  in p a r t i a l  f u l f i l m e n t o f the r e q u i r e m e n t s  an advanced degree at 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 , I agree the L i b r a r y I further  s h a l l make i t f r e e l y  agree t h a t p e r m i s s i o n f o r e x t e n s i v e  for scholarly by h i s of  available for  r e f e r e n c e and copying of t h i s  It  i s understood  that c o p y i n g o r  thesis  permission.  Department The U n i v e r s i t y o f B r i t i s h Col Vancouver 8, Canada  or  publication  t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my  written  that  study.  purposes may be g r a n t e d by the Head o f my Department  representatives.  for  ABSTRACT  Part  1 The  vascular  bundle  photosynthesis  is  chlorophyllous  c e l l  an  inner  bundle  usually  Gomphrena  and  sheath  d i f f e r e n t i a l to  occur  content  of  the  nitrate  content  bution  of  presence tion was  into  of  organic i n  in  with  compounds the  was  of  to  n i t r i t e  mays, studied  to  by  n i t r i t e The  higher  than  be  and the  c e l l s .  The  Ammonia  nitrate the  d i s t r i -  related  glutamate  sheath  Zea  c e l l s .  grana.  by  bundle  much  sheath  seemed  layer  reductase, i n  of  concentric  l o c a l i z a t i o n  reduction  was  C^-pathway  mesophyll  sudanense  cells  reductase  two  mesophyll  bundle  chloroplasts  localized  Part  the  by  nitrate  Nitrate  mesophyll  n i t r i t e  The  the  dehydrogenase  primarily  of  outer  Sorghum  grinding.  appears  an  enzymes,  and  with  surrounded  layer.  glutamate  globosa,  leaves  layers:  n i t r a t e - a s s i m i l a t i n g reductase,  of  to  the  incorpora-  dehydrogenase  c e l l s .  2 Four  menziesji  conifer var.  (Thuja  plicata)  (Raf.)  Sarg,  contorta)  species,  menziesii) Donn,  and  Dougl.,  (Mirb.)  Western  Lodgepole were  Douglas-fir  grown  Franco,  hemlock  pine on  (Pseudotsuga  (Pinus three  Western  (Tsuga  redcedar,  heterophylla)  contorta different  var. sources  of n i t r o g e n  ( n i t r a t e , ammonia, and  and  7:1).  ammonia  A l i n e a r r e l a t i o n s h i p was  found between l e a f area  l e a f dry weight f o r three s p e c i e s pine and Western hemlock). had  a combination of n i t r a t e  and  ( D o u g l a s - f i r , Lodgepole  Different nitrogen  treatments  no e f f e c t on t h i s r e l a t i o n s h i p .  P a r t 2-A  In t h i s p a r t the t r e e s e e d l i n g s were grown f o r  18 weeks. For D o u g l a s - f i r , Western redcedar and Western hemlock, s u r v i v a l and  growth on the n i t r a t e s o l u t i o n was  s u r v i v a l and  growth on the combination s o l u t i o n .  was  an unfavorable  s i m i l a r to Ammonia  source of n i t r o g e n f o r s u r v i v a l and  growth of D o u g l a s - f i r and Western redcedar. hemlock, ammonia was  only d e t r i m e n t a l  For Western  to s u r v i v a l .  Hemlock  s e e d l i n g s which s u r v i v e d ammonia treatment grew as w e l l as t r e e s growing on the other two  sources of n i t r o g e n .  pole pine s u r v i v e d e q u a l l y w e l l under a l l treatments was  the only s p e c i e s t h a t grew b e s t on  P a r t 2-B  At 18 weeks the s e e d l i n g s were t r a n s f e r r e d from  changed every two  At the end  The s o l u t i o n  days f o r a p e r i o d of s i x days.  pH of the s o l u t i o n s was changed.  and  ammonia.  sand c u l t u r e to l i q u i d n u t r i e n t s o l u t i o n . was  Lodge-  The  measured when the s o l u t i o n s were  of the s i x days, the s t a r c h content  of  iv the p l a n t s was measured.  The r e s u l t s i n d i c a t e t h a t these  f o r e s t s p e c i e s d i f f e r i n t h e i r t o l e r a n c e t o ammonia. Western hemlock and Lodgepole pine seem t o be able t o t o l e r a t e higher  l e v e l s o f ammonia than D o u g l a s - f i r and  Western redcedar. P a r t 2-C  In t h i s p a r t the s e e d l i n g s were grown f o r a  p e r i o d o f approximately one year. The  trends  shown i n the 18 week experiment were con-  firmed i n the longer experiment and some a d d i t i o n a l t r e a t ment e f f e c t s appeared. P a r t 2-D  In t h i s p a r t the s e e d l i n g s were grown f o r a p e r i o d  of approximately one year. Within  each s p e c i e s , an attempt was made t o c o r r e l a t e  d i f f e r e n c e s i n growth among n i t r o g e n treatments w i t h ences i n gas exchange. stomatal  resistance  I t was found from c a l c u l a t i o n s of  ( r ) t h a t the e n t r y of CO^ was not g  l i m i t i n g dry matter p r o d u c t i o n . mesophyll r e s i s t a n c e  differ-  I t i s postulated  that  (r ) may be a f a c t o r i n v o l v e d i n  c o n t r o l l i n g growth i n these t r e e s . Part 3 Two-year o l d (2-0) D o u g l a s - f i r  (Pseudotsuga  menziesii)  s e e d l i n g s were l i f t e d i n the s p r i n g and mud-packed. s e e d l i n g s were t e s t e d f o r the e f f e c t s o f v a r i o u s  These  storage  V  conditions. Mud-packed s e e d l i n g s , s t o r e d i n t h e and  subsequently  growth than  those having  other storage ature of  than  p l a n t e d , had  on  i n the  any  higher survival  other storage  field  and  light  fertilizer  of the parameters  versus and  f o r 19 and  lower  dark.  The temper-  Treatment  v e r m i c u l i t e had  measured.  days  root  conditions.  c o n d i t i o n s i n c l u d e d h i g h e r and  t h e mud-packs w i t h  effect  field  no  vi TABLE OF CONTENTS Page PART 1  The l o c a l i z a t i o n o f n i t r a t e - a s s i m i l a t i n g enzymes i n l e a v e s o f p l a n t s w i t h t h e C^-pathway o f p h o t o s y n t h e s i s .  INTRODUCTION  1  MATERIALS  3  METHODS  3  RESULTS  AND DISCUSSION  LITERATURE CITED PART 2-A  5 16  A c o m p a r i s o n o f t h e growth and s u r v i v a l of four c o n i f e r species s u p p l i e d with d i f f e r e n t forms o f n i t r o g e n .  INTRODUCTION  21  MATERIALS  22  AND METHODS  RESULTS  25  DISCUSSION  32  LITERATURE CITED  35  PART 2-B  N i t r o g e n u p t a k e and s t a r c h c o n t e n t o f c o n i f e r s s u p p l i e d with d i f f e r e n t sources of n i t r o g e n .  INTRODUCTION  36  METHODS AND MATERIALS  38  RESULTS  39  DISCUSSION  41  LITERATURE  CITED  45  vii TABLE OF CONTENTS,  cont'd. Page  PART 2-C  A comparison o f t h e growth o f f o u r coni f e r species supplied with d i f f e r e n t forms o f n i t r o g e n f o r 10 t o 12 months.  INTRODUCTION  47  METHODS AND MATERIALS  48  RESULTS  50  DISCUSSION  57  LITERATURE PART 2-D  CITED  61  Gas e x c h a n g e o f c o n i f e r s e e d l i n g s s u p p l i e d w i t h d i f f e r e n t forms o f nitrogen.  INTRODUCTION  62  SYMBOLS AND UNITS  65  THEORY  66  METHODS AND MATERIALS  67  RESULTS  68  AND DISCUSSION  LITERATURE CITED APPENDIX I  75  The r e l a t i o n s h i p b e t w e e n l e a f a r e a and l e a f d r y w e i g h t o f t h r e e c o n i f e r s p e c i e s grown on t h r e e s o u r c e s o f nitrogen.  EPILOGUE PART 3  86 A study o f storage c o n d i t i o n s l i f t e d mud-packed D o u g l a s - f i r  INTRODUCTION  for springseedlings. 87  viii  TABLE OF CONTENTS, cont'd. Page  MATERIALS AND METHODS  i  89  RESULTS AND DISCUSSION  91  LITERATURE CITED  96  ix LIST OF TABLES Page Part 1 Table  Table  1.  2.  A c t i v i t i e s of enzymes, i n m i l l i u n i t s per m i l l i g r a m of p r o t e i n , i n e x t r a c t s of mesophyll and bundle sheath t i s s u e of Zea mays, Gomphrena globosa and Sorghum sudanense  7  NO^ -N content as n moles NO^ -N per m i l l i g r a m of p r o t e i n , i n e x t r a c t s of mesophyll and bundle sheath t i s s u e of Zea mays and Gomphrena globosa  8  Part Table Table  Table  Table  Table  Table  1. 2.  3.  4;  5.  6.  2-A  Composition and c o n c e n t r a t i o n (ppm) full-strength nutrient solution...  of 23  S u r v i v a l (%) of D o u g l a s - f i r , Lodgepole p i n e , Western redcedar, and Western hemlock with d i f f e r e n t sources of nitrogen  26  T o t a l dry weight, r o o t dry weight, /R and l e a f area of D o u g l a s - f i r s u p p l i e d w i t h d i f f e r e n t sources of n i t r o g e n  27  T o t a l dry weight, r o o t dry weight, /R and l e a f dry weight of Western redcedar s u p p l i e d w i t h d i f f e r e n t sources of nitrogen  28  T o t a l dry weight, r o o t dry weight, S/R and l e a f dry weight of Western hemlock s u p p l i e d w i t h d i f f e r e n t sources of nitrogen  29  T o t a l dry weight, r o o t dry weight, /R and l e a f area of Lodgepole pine s u p p l i e d w i t h d i f f e r e n t sources of n i t r o g e n  30  S  S  S  X  LIST OF TABLES, cont'd. Page Part Table Table  1. 2.  The mean pH of the n u t r i e n t a f t e r treatment f o r 2 days  Table  Table  Table  1.  2.  3.  4.  Table  1.  2.  40  42  2-C  T o t a l , r o o t , and shoot dry weight, l e a f area and h e i g h t of D o u g l a s - f i r s u p p l i e d w i t h d i f f e r e n t sources of n i t r o g e n . . . . . . . .  52  T o t a l , r o o t , and shoot dry weight, l e a f area and h e i g h t of Western redcedar s u p p l i e d w i t h d i f f e r e n t sources of nitrogen  53  T o t a l , r o o t , and shoot dry weight, l e a f area and h e i g h t of Western hemlock s u p p l i e d w i t h d i f f e r e n t sources of nitrogen  55  T o t a l , r o o t , and shoot dry weight, l e a f area and h e i g h t of Lodgepole pine s u p p l i e d w i t h d i f f e r e n t sources of n i t r o g e n  56  Part Table  solutions  S t a r c h content of c o n i f e r s e e d l i n g s s u p p l i e d w i t h d i f f e r e n t sources of n i t r o g e n f o r 4 months (j*g/ gm dry weight) Part  Table  2-B  2-D  T o t a l dry weight of c o n i f e r s e e d l i n g s s u p p l i e d with d i f f e r e n t sources of n i t r o g e n (gm . pot )  63  Leaf area of c o n i f e r s e e d l i n g s s u p p l i e d w i t h d i f f e r e n t sources of n i t r o g e n (cm2 . p o t l )  64  -  xi LIST OF TABLES,  cont'd. Page  Table  Table  Table  3.  4.  5.  Leaf Y , Ys and Vp c o n i f e r s e e d l i n g s s u p p l i e d with d i f f e r e n t sources of n i t r o g e n (bars)  69  Water l o s s of c o n i f e r s e e d l i n g s s u p p l i e d w i t h d i f f e r e n t sources of n i t r o g e n (ml H20/24 h r . pot)  71  T r a n s p i r a t i o n r e s i s t a n c e (r + r ^)of c o n i f e r s e e d l i n g s s u p p l i e d with d i f f e r e n t sources of n i t r o g e n (sec. cm~l)  73  Part 3 Table  1.  Storage treatments and c o n d i t i o n s  90  Table  2.  S u r v i v a l and r o o t growth of mud-packed Douglas-fir seedlings stored i n various locations  93  xii LIST OF FIGURES Page Part 1 Figure  Figure  1.  2.  Schematic d i v i s i o n o f r e a c t i o n s between bundle sheath and mesophyll i n Zea and Sorghum  12  Schematic d i v i s i o n of r e a c t i o n s between bundle sheath and mesophyll i n Gomphrena  13  Appendix I Figure  1.  The r e l a t i o n s h i p between l e a f area and l e a f dry weight f o r D o u g l a s - f i r s e e d l i n g s s u p p l i e d w i t h d i f f e r e n t sources o f nitrogen 80  Figure  2.  The r e l a t i o n s h i p between l e a f area and l e a f dry weight f o r Lodgepole pine seedlings supplied with d i f f e r e n t sources o f n i t r o g e n  Figure  3.  82  The r e l a t i o n s h i p between l e a f area and l e a f d r y weight f o r Western hemlock s e e d l i n g s s u p p l i e d with d i f f e r e n t sources of n i t r o g e n 84  ACKNOWLEDGEMENTS I wish t o thank E.B. Tregunna f o r h i s help d u r i n g my stay a t UBC.  I wish t o g i v e him s p e c i a l thanks f o r t r e a t i n g  me completely  as an equal a t a l l times.  s p e c i a l thanks t o Andy Black,  I wish t o give  not only f o r s e r v i n g on my  Committee, but a l s o f o r g i v i n g me complete access  to h i s  l a b o r a t o r y and equipment and f o r the many d i s c u s s i o n s we had  concerning  P a r t 2-D i n t h i s t h e s i s .  I would a l s o  like  to thank Dr. I a i n T a y l o r , Dr. Roy T a y l o r and Dr. G.H.N. Towers f o r s e r v i n g on my Committee. Hart,  John Downton and Joe Berry  I am g r a t e f u l t o A l a n  f o r many hours o f d i s c u s s i o n  which were i n v a l u a b l e t o my p r o j e c t s .  I am a l s o  indebted  to a l l o t h e r persons with whom I came i n c o n t a c t a t UBC; they a l l c o n t r i b u t e d i n making my stay a t UBC something I w i l l never f o r g e t .  xiv  PREFACE This thesis d i f f e r s  from most theses i n t h a t i t does  not d e a l e x c l u s i v e l y with one  topic.  T h i s l a c k of  e x c l u s i v i t y i s p a r t l y a r e s u l t of p e r s o n a l p r e f e r e n c e and p a r t l y a r e s u l t of circumstance.  However a l l s t u d i e s  r e p o r t e d i n t h i s t h e s i s with the e x c e p t i o n of P a r t I are concerned w i t h the p h y s i o l o g y of whole p l a n t s . I t was  my  i n t e n t i o n on a r r i v i n g at UBC  t o get as  much r e s e a r c h experience as p o s s i b l e . The  study with C^-plants allowed me  to work on a  p r o j e c t t h a t r e q u i r e d r a p i d p u b l i c a t i o n to ensure the o r i g i n a l i t y o f d i s c o v e r i e s and a l s o allowed me more experience w i t h enzymatic  techniques.  the l o c a t i o n of n i t r i t e reductase  My d i s c o v e r y t h a t  (a NADPH2 r e q u i r i n g  enzyme) seems to be c o r r e l a t e d with the presence g r a n a l c h l o r o p l a s t s agrees with evidence  of  t h a t only g r a n a l  c h l o r o p l a s t s are capable of NADPH p r o d u c t i o n . The  study with mud-packed s e e d l i n g s allowed me  to  work on a p r o j e c t which had the p o s s i b i l i t y of immediate applicability.  My  d i s c o v e r y t h a t s p r i n g - l i f t e d mud-packs  should be s t o r e d o u t s i d e may  prove t o be of s i g n i f i c a n c e  from both a c o n s e r v a t i o n a l and a f i n a n c i a l p o i n t of view.  XV  The p r o j e c t w i t h f o r e s t s p e c i e s and n i t r o g e n sources p e r m i t t e d me t o g a i n some experience w i t h sand c u l t u r e t e c h n i q u e s , some experience  i n growing f o r e s t s p e c i e s and  some experience with psychrometric  techniques.  Evidence  which I c o l l e c t e d seems t o i n d i c a t e t h a t f o r e s t s p e c i e s vary i n t h e i r t o l e r a n c e to NH^  +  to p r e f e r e n t i a l l y metabolize NO^  r a t h e r than t h e i r o r NH^ . +  ability  This i s a  s i g n i f i c a n t f i n d i n g i n t h a t i t agrees with a new s c h o o l of  thought  a r i s i n g i n the f i e l d  I t was my i n i t i a l  of a g r i c u l t u r e .  i n t e n t i o n t o make the s e c t i o n on  n i t r o g e n n u t r i t i o n and f o r e s t s p e c i e s more p h y s i o l o g i c a l and more cohesive than i t turned out.  This p r o j e c t was  s t a r t e d as a d u a l p r o j e c t w i t h Sara Madoc-Jones.  I t was  our i n t e n t i o n t h a t she would look at the p r o j e c t from an e c o l o g i c a l p o i n t o f view and t h a t I would look at the p r o j e c t from a p h y s i o l o g i c a l p o i n t o f view. to  However due  the death o f Sara, c e r t a i n p a r t s o f the p r o j e c t had t o  be abandoned.  I tried  t o salvage as much as I c o u l d .  I  attempted t o study the c r i t e r i a which I f e l t would give me the g r e a t e s t i n s i g h t i n t o the growth responses observing.  we were  1. PART 1.  THE  LOCALIZATION OF NITRATE-ASSIMILATING ENZYMES  IN LEAVES OF PLANTS WITH THE  (^-PATHWAY OF PHOTOSYNTHESIS.  1  INTRODUCTION The  v a s c u l a r bundle of leaves with  photosynthesis  the C^-pathway of  i s u s u a l l y surrounded by two  l a y e r s , an i n n e r bundle sheath l a y e r and layer.  concentric  an outer mesophyll  C e l l s of the mesophyll l a y e r c o n t a i n g r a n a l  p l a s t s d i s t r i b u t e d about a l a r g e c e n t r a l vacuole. i n d i c a t e t h a t the bundle sheath c h l o r o p l a s t s o f c h l o r i d o i d grasses,  and  some p a n i c o i d grasses  developed grana, t h a t sugar cane and e n t i r e l y , and  t h a t corn and  32,  43).  and  Surveys  dicotyledons,  sorghum l a c k grana  (6, 14,  21,  D i f f e r e n c e s i n the c a p a c i t y f o r dye  (13) of agranal  chloro-  have w e l l -  c l o s e r e l a t i v e s are  a t e , forming only rudimentary grana  cell  intermedi25,  27,  reduction  g r a n a l c h l o r o p l a s t s of s i n g l e leaves  have l e d to the c o n c l u s i o n t h a t the agranal c h l o r o p l a s t s are i n c a p a b l e of forming reductant  by n o n - c y c l i c e l e c t r o n  transport. Considerable  work has  been done with  i n v o l v e d i n the p h o t o s y n t h e t i c C^-plants  and  the enzymes  carbon metabolism of these  t h e i r l o c a l i z a t i o n w i t h i n the l e a f  (1,  5,  1 This a r t i c l e by G.E. M e l l o r and E.B. Tregunna appeared i n Canadian J o u r n a l of Botany, V o l . 49: 137-142 (1971). E.B. Tregunna s u p e r v i s e d the study. Subsequent developments on t h i s t o p i c are d i s c u s s e d i n addenda.  16, 17, 18, 31, 37, 39, 40, 41, 42). Although many s t u d i e have been c a r r i e d out on n i t r a t e reductase, reductase and glutamate  nitrite  dehydrogenase i n corn, a C ^ - p l a n t  (2, 3, 4, 8, 15, 24, 35, 37, 44), there i s only one r e p o r t (40) on the l o c a l i z a t i o n o f such events  i n the leaves o f  C^-plants. In 1967 Ritenour e t a_l. (35) s t u d i e d the i n t r a c e l l u l a l o c a l i z a t i o n o f n i t r a t e reductase, n i t r i t e r e d u c t a s e , and glutamate  dehydrogenase i n corn and f o x t a i l l e a v e s .  They  found t h a t n i t r i t e reductase was l o c a l i z e d w i t h i n the chloroplasts.  T h i s r e s u l t agreed w i t h the f i n d i n g s o f  Ramirez e t al_.  (33) .  N i t r a t e reductase was thought  l o c a l i z e d i n the cytoplasm,  although the techniques  d i d not e l i m i n a t e the p o s s i b i l i t y t h a t n i t r a t e  t o be used  reductase  may be l o c a l i z e d on the e x t e r n a l c h l o r o p l a s t membrane. Glutamate dehydrogenase was found i n the m i t o c h o n d r i a . Several investigations  (7, 10, 11, 34) with  etiolated  s e e d l i n g t i s s u e have a l s o i n d i c a t e d t h a t glutamic dehydrogenase i s l o c a l i z e d i n the m i t o c h o n d r i a . Joy  acid Recently  (23) r e p o r t e d t h a t i n pea r o o t s both s o l u b l e and  particulate and NADPH Leech  2  (mostly mitochondria) dependent glutamate  f r a c t i o n s c o n t a i n NADH^  dehydogenase.  In 196 8  and K i r k (28) r e p o r t e d t h a t i n leaves o f V i c i a  f aba, NADPEL, glutamate  dehydrogenase o c c u r r e d i n the  c h l o r o p l a s t w h i l e NAD-dependent glutamate  dehydrogenase  3. o c c u r r e d i n the mitochondria.  The method o f Joy (22) was  used f o r e x t r a c t i o n and assay o f glutamate dehydrogenase i n t h i s paper. In view o f these o b s e r v a t i o n s  I have i n v e s t i g a t e d the  t i s s u e l o c a l i z a t i o n o f n i t r a t e - a s s i m i l a t i n g enzymes i n C^p l a n t s with g r a n a l  (Gomphrena g l o b o s a ) , a g r a n a l  sudanense), and rudimentary  granal  (Sorghum  (Zea mays) bundle  sheath c h l o r o p l a s t s .  MATERIALS Zea mays, Gomphrena globosa,  and Sorghum sudanense  were grown on v e r m i c u l i t e supplemented with n u t r i e n t s o l u t i o n c o n t a i n i n g 100 ppm NC> -N as Ca (N0 ) * 4H 0. 3  Growth c o n d i t i o n s were 16-h day,  3  27/21C  2  2  (day/night)  temperature and 100 0 f t - c p r o v i d e d i n a growth chamber. Leaves o f 3-week-old Zea p l a n t s , leaves o f 6-week-old Sorghum, and the youngest f u l l y  expanded leaves o f 2-  to 3-month-old Gomphrena p l a n t s were used. d i f f e r e n c e s were necessary  The age  to obtain plants of s i m i l a r  size.  METHODS E x t r a c t i o n o f Enzymes from Mesophyll Tissue  and Bundle Sheath  Zea and Sorghum e x t r a c t s , e n r i c h e d i n e i t h e r bundle sheath o r mesophyll c e l l contents, were obtained with the use o f an ' O s t e r i z e r ' homogenizer.  Leaves o f Zea and  4. Sorghum with for  (2 g) were c u t i n t o  1-cm  p i e c e s and were  t h e h o m o g e n i z e r i n 40 ml o f t h e a p p r o p r i a t e b u f f e r 20 s e c .  ily,  Microscopic inspection revealed that,  mesophyll  cells  were r u p t u r e d d u r i n g t h i s  while  some m e s o p h y l l  cells  remained i n t a c t .  enriched remaining  cells  o f the mesophyll  almost  a l l of the i n t a c t  These r e m a i n i n g  to  a pestle  grinding material.  an  cells  were b u n d l e  Sufficient  a very  were r u p t u r e d by t h i s  treatment,  g r i n d i n g o f Gomphrena.  Gomphrena was  The that  for  g r i n d i n g medium  o f J o y and Hageman  reductase  used  used  angular  bundle  sheath  f o r the  cells.  differential  g r o u n d as d e s c r i b e d  for nitrate  (24) was  and t h e g r i n d i n g medium  glutamate  vigorous  (5) .  d e s c r i b e d by Hageman and F l e s h e r  medium  cells.  yielding  o f bundle  unsatisfactory  B e r r y e t aJL.  sharp  Most, b u t n o t a l l , o f t h e  homogenizer proved  by  sheath  g r i n d i n g p r e s s u r e was  e x t r a c t enriched i n the contents  The  Micro-  i n a m o r t a r c o n t a i n i n g g l a s s beads  break the g l a s s beads, c r e a t i n g  cells  The  pieces revealed that  p i e c e s were s u b j e c t e d t o a v e r y  g r i n d i n g media.  sheath  cells.  p i e c e s were h o m o g e n i z e d f o r 1 min.  i n s p e c t i o n of the remaining  grinding with  sheath  T h i s e x t r a c t was t h e r e f o r e  i n the contents leaf  primar-  treatment  and most o f t h e b u n d l e  scopic  and  ground  dehydrogenase.  reductase  (15).  used  was  The g r i n d i n g  for nitrite  o f J o y (22) was  used  The e x t r a c t i o n method f o r  5 . glutamate  dehydrogenase i n c l u d e d the  detergent  t o a i d t h e breakdown o f p a r t i c l e  breakdown o f p a r t i c l e late  and  membranes e n s u r e d  c y t o p l a s m i c glutamate  P l a n t m a t e r i a l was  harvested  4 h of i l l u m i n a t i o n . a c i d was dard  determined  c u r v e was  of a non-ionic  membranes.  that both  d e h y d r o g e n a s e was  immediately  by  t h e method o f Lowry  - Nitrate  reductase  analyzed.  r e d u c t a s e was  nitrite  using dithionite  Joy  and  Hageman  and  1.4.1.4) was  cally  a t 340  e n t on  (24).  (29).  serum  A  (1.6.6.1) was  (30) was  as e l e c t r o n  analysis  reductase,  and  spectrophotometri-  o<-ketoglutarate  - The  activities  glutamate  (22).  transfor-  minute.  method o f M i l l e r  i n the d e t e r m i n a t i o n of  specific  (1.4.1.2.  o f enzyme c a t a l y z e d t h e  RESULTS AND The  of  d o n o r , as d e s c r i b e d by  by m e a s u r i n g ,  o f ammonia and  1 milliunit  used  (15) .  measuring disappearance  m a t i o n o f 1 nmole o f s u b s t r a t e p e r Nitrate  assayed  r a t e o f o x i d a t i o n o f NAD (P) HL, d e p e n d -  the  the presence  by  stan-  albumin.  Glutamate dehydrogenase  assayed  nm,  Enzyme u n i t s :  assayed  after  trichloracetic  m e a s u r i n g NADI^-dependent p r o d u c t i o n o f n i t r i t e  Nitrite  This  particu-  b e f o r e use  P r o t e i n p r e c i p i t a b l e by  constructed using bovine  Enzyme a s s a y by  addition  and  Wideman  nitrate.  DISCUSSION of nitrate  reductase,  nitrite  dehydrogenase are p r e s e n t e d  in  6.  Table 1.  The t o t a l p r o t e i n e x t r a c t e d from the l e a f was  about e q u a l l y d i v i d e d between the two e x t r a c t s . As i s seen i n Table 1 n i t r a t e reductase a c t i v i t y  was  much h i g h e r i n the mesophyll e x t r a c t s o f a l l t h r e e types of p l a n t s .  Table 2 shows t h a t i n both Gomphrena and Zea  most o f the NO^  occurs i n the mesophyll e x t r a c t .  Thus  the mesophyll e x t r a c t c o n t a i n s h i g h amounts o f n i t r a t e reductase a c t i v i t y  and h i g h amounts o f n i t r a t e .  The  occurrence o f n i t r a t e i n the bundle sheath e x t r a c t may be due to (a) improper mesophyll,  s e p a r a t i o n o f bundle sheath and  (b) the passage o f n i t r a t e from the v a s c u l a r  system t o the mesophyll,  (c) storage of some n i t r a t e i n  the bundle sheath c e l l s , o r (d) a combination o f a l l three. In  Zea and Sorghum n i t r i t e reductase a c t i v i t y  f i n e d p r i m a r i l y t o mesophyll c e l l s reductase a c t i v i t y  (Table 1 ) .  The n i t r i t e  i n Gomphrena seemed to occur i n both  the mesophyll and the bundle sheath c e l l s . Sorghum the mesophyll c e l l s  In Zea and  are capable o f dye r e d u c t i o n  and c o n t a i n g r a n a l c h l o r o p l a s t s . of  i s con-  The bundle sheath  cells  Zea and Sorghum, however, are i n c a p a b l e o f dye r e d u c t i o n  and c o n t a i n not more than rudimentary grana  (13). In  Gomphrena both the mesophyll and bundle sheath c e l l s are capable o f dye r e d u c t i o n (W. J . S. Downton, p e r s .  TABLE 1.  A c t i v i t i e s o f enzymes, i n m i l l i u n i t s per m i l l i g r a m o f p r o t e i n , i n e x t r a c t s o f mesop h y l l and bundle sheath t i s s u e of Zea mays, Gomphrena globosa, and Sorghum sudanense. Zea mays  Enzyme  Nitrate  Nitrite  reductase  reductase  Mesophyll  Bundle sheath  Gomphrena globosa  Mesophyll  Bundle sheath  Sorghum sudanense  Mesophyll  Bundle sheath  5. 50*  0. 60  6.40  1. 53  2. 67  1. 52  3. 77*  0. 68  3. 56  0. 60  3. 84  1. 21  7. 97  1. 68  2. 54  5. 06  3. 47  1. 10  6.03  1. 87  3. 83  3. 24  2. 55  0. 53  Glutamate dehydrogenase NADH  NADPH  *Replicates.  58  284  71  153  64  178  52  201  57  181  61  138  18  35  35  72  18  34  15  45  27  54  13  19  TABLE 2.  NO- -N content, as nmoles NO- -N per m i l l i g r a m o f p r o t e i n , i n e x t r a c t s o f mesophyll ana bundle sheath t i s s u e of Zea mays and Gomphrena globosa.  Plant  Gomphrena globosa Zea mays  *Mean o f two determinations.  Mesophyll  Bundle sheath  5956*  170  715  214  9. commun.)* and both c o n t a i n  at l e a s t thylakoid overlaps  (12)  which are b e l i e v e d to be s u f f i c i e n t f o r n o n - c y c l i c e l e c t r o n flow  (20). Thus the r e s u l t s i n d i c a t e t h a t only  c o n t a i n i n g c h l o r o p l a s t s are l i n k e d t o n i t r i t e  grana-  reduction.  I t has been suggested t h a t grana are necessary f o r photosystem II a c t i v i t y  (20, Addendum 2) and t h a t  p l a s t s d e f i c i e n t i n grana would be incapable e l e c t r o n flow and NADP r e d u c t i o n . nitrite  of non-cyclic  I t i s believed  reductase r e c e i v e s e l e c t r o n s  chloro-  that  from f e r r e d o x i n when  the l a t t e r has been reduced by i l l u m i n a t i o n o r by NADPH2 and  a diaphorase enzyme (9, 19, 24, 33, 38).  This  require-  ment f o r f e r r e d o x i n to donate e l e c t r o n s to n i t r i t e suggests t h a t n i t r i t e  reductase  reductase should be a s s o c i a t e d with  granal c h l o r o p l a s t s . The  rudimentary grana i n bundle sheath c e l l s  seem to p l a y l i t t l e o r no r o l e i n n i t r i t e occurrence o f n i t r i t e  o f Zea  reduction.  The  reductase p r i m a r i l y i n the mesophyll  of Zea agrees w i t h the f i n d i n g s o f Slack ejt a l . (42) . Using d e n s i t y  f r a c t i o n a t i o n i n non-aqueous media t o  separate Zea mesophyll and bundle sheath c h l o r o p l a s t s , they found t h a t about 80% of the n i t r i t e  reductase  activity  In a l l three types o f p l a n t s the bulk o f both  NADHv,and  occurred  i n the mesophyll c h l o r o p l a s t s .  NADPILj glutamate dehydrogenase a c t i v i t y occurs i n the bundle *Research School o f B i o l o g i c a l Sciences, A u s t r a l i a n U n i v e r s i t y , Canberra, A. C. T., A u s t r a l i a .  National  10. sheath  (Table 1).  activity  The occurrence of glutamate  dehydrogenase  i n the bundle sheath of Gomphrena suggests t h a t as  f a r as glutamate  dehydrogenase a c t i v i t y  i s concerned,  the  m i t o c h o n d r i a i n the bundle sheath are more a c t i v e than the m i t o c h o n d r i a i n the mesophyll. the f i n d i n g s o f L a e t s c h  This r e s u l t coincides with  (25) and Downton e_t a l . (14)  that  i n the d i c o t y l e d o n s which they examined the m i t o c h o n d r i a i n the bundle sheath are c o n s i d e r a b l y l a r g e r and more developed.  T h i s d i f f e r e n t i a l development of m i t o c h o n d r i a  between the mesophyll  and the bundle sheath i n t r o p i c a l  grasses has never, to our knowledge, been r e p o r t e d . Zea and Sorghum, however, the bulk of the glutamate genase s t i l l o c c u r r e d i n the bundle  In dehydro-  sheath.  With the methods a v a i l a b l e f o r c e l l s e p a r a t i o n , the p o s s i b i l i t y of o b t a i n i n g mesophyll  e x t r a c t s f r e e of bundle  sheath contamination or bundle sheath e x t r a c t s f r e e mesophyll  contamination i s h i g h .  from  Thus f o r the purpose  of  t h i s d i s c u s s i o n , i t i s assumed t h a t the occurrence of n i t r a t e reductase i n the bundle sheath e x t r a c t s of Zea, Sorghum, and Gomphrena, the occurrence of n i t r i t e  reductase  i n the bundle sheath e x t r a c t s o f Zea and Sorghum and occurrence of glutamate  the  dehydrogenase i n the mesophyll  ex-  t r a c t s of Zea, Sorghum, and Gomphrena i s contamination. However, as more r e f i n e d methods become a v a i l a b l e f o r c e l l s e p a r a t i o n , t h i s assumption  may  prove i n c o r r e c t and  the  enzyme may  be found t o occur i n both a r e a s .  T h e r e f o r e on  the b a s i s of t h i s assumption of contamination, i t would seem t h a t the steps i n the a s s i m i l a t i o n o f n i t r a t e are separated. Thus i n Zea and Sorghum i t seems t h a t n i t r a t e  and  n i t r i t e are both reduced p r i m a r i l y i n the mesophyll ( F i g . 1) and the r e s u l t i n g ammonia i s i n c o r p o r a t e d i n t o o r g a n i c form i n the bundle sheath. t o x i c to p l a n t s because of ATP  Ammonia, however, i s somewhat  i t perhaps  i n h i b i t s the p r o d u c t i o n  i n the m i t o c h o n d r i a and p h o t o s y n t h e t i c e l e c t r o n -  t r a n s p o r t system  (36).  Thus i t seems p r o b a b l e t h a t  rapid  t r a n s p o r t o f the ammonia from the mesophyll to the bundle sheath i s r e q u i r e d . Laetsch has noted t h a t i n sugar cane the w a l l the mesophyll c e l l s  separating  from the bundle sheath c e l l s i s t r a n s -  versed by many plasmodesmata (26).  Plasmodesmata l i n k i n g  the mesophyll c e l l s to the bundle sheath c e l l s can be seen i n p u b l i s h e d micrographs o f s e v e r a l o t h e r p l a n t s which have t h i s type o f l e a f and >9-carboxylation (6, 25).  Thus p l a s -  modesmata p r o v i d e a c o n n e c t i o n between the mesophyll the bundle sheath c e l l s .  and  Whether the t r a n s p o r t from the  mesophyll to the bundle sheath i s a c t i v e o r p a s s i v e remains to be  established. In Gomphrena, n i t r a t e i s reduced p r i m a r i l y i n the  mesophyll  ( F i g . 2), w h i l e n i t r i t e can be reduced i n both  NO.  -> NO,  NO,  NH +  NH + + «<KETOGLUTARATE 4  GLUTAMATE  CYTOPLASM  CHLOROPLAST  MITOCHONDRIA  MESOPHYLL  BUNDLE SHEATH  CELL WALL F i g u r e 1.  Schematic d i v i s i o n o f r e a c t i o n s between bundle sheath and mesophyll i n Zea and Sorghum. tsj  NGy  >  N0  2  NH  'CYTOPLASM  CHLOROPLAST  'MESOPHYLL  + 4  + <*KETOGLUTARATE  MITOCHONDRIA  BTJNDLE SHEATH CELL WALL  F i g u r e 2.  Schematic d i v i s i o n o f r e a c t i o n s between bundle sheath and mesophyll i n Gomphrena,  14. the  b u n d l e s h e a t h and  takes place transport  o f ammonia and  In c o n c l u s i o n assimilating the  of  f a r as  mesophyll grinding.  from the  be  be  The  are  Again  n o t e d t h a t when are  i n the  studied  mesophyll  isolation  easily  the  cells  by  are  completely  or p a r t l y i n the is  pose  no  since  partly resistant  of  grinding  are  n o r m a l methods  Thus f o r c o m p l e t e  a more v i g o r o u s  location  should  grinding.  sheath  the  tissues  t o n o r m a l methods o f enzymes l o c a l i z e d  to  nitrate-  i s concerned,  ruptured  bundle sheath  the  mesophyll  k e p t i n mind when t h e  complete  cells  incorporation  plasmodesmata.  enzymes i n C ^ - p l a n t s  Enzymes l o c a l i z e d  p r o b l e m as the  nitrite  i t should  Ammonia  bundle sheath.  o c c u r v i a the  enzymes s h o u l d  ground.  mesophyll.  p r i m a r i l y i n the  bundle sheath could  of  the  isolation bundle  required.  ADDENDA 1.  While t h i s  p a p e r was  a p a p e r a p p e a r e d by on  the  detection  extracts.  Plant  Maranville  P h y s i o l . 45:  of  sorghum e x t r a c t s  may  reported  (Influence  n i c k e l increases  this  paper.  1.  activity  the  N i c k e l was The  have i n c r e a s e d  i n Table  for publication,  591-593. 1970.)  i n sorghum e x t r a c t s .  sorghum e x t r a c t s  activity  J . W.  reviewed  of n i t r a t e reductase  paper i n d i c a t e s that activity  being  of  nickel  i n sorghum Maranville's  n i t r a t e reductase not  used  in  the  a d d i t i o n of n i c k e l to the  n i t r a t e reductase  2.  Sane e t a_l. (P.V. Sane, D.J. Goodchild  Biochim. (C.J.  Biophys.  Arntzen,  and R.B. Park.  A c t a 216: 162. 1970.) and Arntzen  R.A. D i l l e y  and J . Neumann.  Biochim.  et a l . Biophys  A c t a 245:409-424. 1971.) have shown from c h l o r o p l a s t fragmentation  s t u d i e s t h a t g r a n a l l a m e l l a e have both  Photosystem I and II a c t i v i t i e s have only Photosystem I a c t i v i t y seem capable  o f reducing NADP.  and t h a t stromal  lamellae  i . e . only g r a n a l l a m e l l a e  16. LITERATURE  CITED  Andrews, T . J . . and M.D. H a t c h . 1969. P r o p e r t i e s and mechanism o f a c t i o n o f p y r u v a t e p h o s p h a t e d i k i n a s e f r o m l e a v e s . B i o c h e m . J . 114: 117-125. B e e v e r s , L., D. F l e s h e r , and R.H. Hageman. 1964. S t u d i e s on t h e p y r i d i n e n u c l e o t i d e s p e c i f i c i t y o f n i t r a t e r e d u c t a s e i n h i g h e r p l a n t s and i t s r e l a t i o n s h i p t o s u l f h y d r y l l e v e l . B i o c h i m . B i o p h y s . A c t a , 89: 453464. B e e v e r s , L., D.M. P e t e r s o n , J . C . Shannon, and R.H. Hageman. 1963. 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Bot. 48: 1209-1214.  44.  Tweedy, J.A., and S.K. R i e s . 1967. E f f e c t s o f simazine on n i t r a t e reductase a c t i v i t y i n corn. P l a n t P h y s i o l . 42: 280-282.  21.  PART 2-A.  A COMPARISON OF THE GROWTH AND SURVIVAL OF FOUR CONIFER SPECIES SUPPLIED WITH DIFFERENT FORMS OF NITROGEN.  INTRODUCTION Few s t u d i e s have been c a r r i e d out t o compare n i t r a t e and  ammonia as sources o f n i t r o g e n  for conifers.  The evidence  c o l l e c t e d to date i s i n c o n c l u s i v e as to which i s the most f a v o r a b l e form o f n i t r o g e n f o r c o n i f e r s .  This s e c t i o n r e -  p o r t s on the growth and s u r v i v a l o f D o u g l a s - f i r m e n z i e s i i v a r . m e n z i e s i i ) , Lodgepole pine v a r . c o n t o r t a ) , Western hemlock Western redcedar n i t r a t e alone, and  ammonia  (Pinus  (Pseudotsuga contorta  (Tsuga h e t e r o p h y l l a ) , and  (Thuja p l a c a t a ) grown f o r 18 weeks on  ammonia alone o r a combination o f n i t r a t e  (7:1).  Krajina  (4) has c a r r i e d out some s t u d i e s o f n i t r o g e n  n u t r i t i o n with D o u g l a s - f i r , Western redcedar and Western hemlock.  Swan  (7) has c a r r i e d out s i m i l a r s t u d i e s  Western hemlock.  with  In both o f these works the c o n c e n t r a t i o n  o f n i t r o g e n i n each o f the treatments was d i f f e r e n t . use o f d i f f e r e n t n i t r o g e n c o n c e n t r a t i o n s  This  has l e d t o d i f f i -  c u l t y i n i n t e r p r e t i n g which was the more f a v o r a b l e form o f nitrogen.  K r a j i n a concluded t h a t n i t r a t e was the more  f a v o r a b l e form o f n i t r o g e n f o r D o u g l a s - f i r and Western redcedar, while  ammonia was the more f a v o r a b l e source o f  nitrogen  f o r Western hemlock.  combination to  of nitrate  Swan c o n c l u d e d  and ammonia  (4:1)  was more f a v o r a b l e  growth o f Western hemlock than n i t r a t e  alone.  V a n Den D r i e s s c h e  s e e d l i n g d r y weight  (8) f o u n d  g r e a t e r growth than n i t r a t e t h e r e was no d i f f e r e n c e  combination To  that  a l o n e o r ammonia for Douglas-fir,  was g r e a t e s t when ammonia a n d n i t r a t e  were p r o v i d e d i n e q u a l amounts.  treatments  that a  alone.  i n seedling  using nitrate of nitrate  Ammonia a l o n e r e s u l t e d i n F o r Western d r y weight  hemlock, between  a l o n e o r ammonia a l o n e o r a  and ammonia.  o u r k n o w l e d g e , no work h a s been done t o d e t e r m i n e  t h e most f a v o r a b l e s o u r c e o f n i t r o g e n f o r L o d g e p o l e  pine.  MATERIALS AND METHODS Seeds o f D o u g l a s - f i r ,  Lodgepole  p i n e , Western hemlock  and W e s t e r n r e d c e d a r were o b t a i n e d f r o m British  Columbia  Stratified  seeds  sand purchased The  Forest  with d i s t i l l e d water u n t i l time, n u t r i e n t  ( T a b l e 1) was a p p l i e d .  o n l y and a c o m b i n a t i o n  (hereafter referred  of nitrate  Illinois) one week  solution contain-  Three  s o u r c e s o f i n o r g a n i c n i t r o g e n were u s e d : nitrate  (20-30 mesh  f r o m t h e O t t a w a S i l i c a Co., O t t a w a ,  g e r m i n a t i o n , a t which  14 ppm o f N  ( e l e v a t i o n o f 1000 f t ) .  were grown i n p o t s o f s a n d  s a n d was m o i s t e n e d  after ing  Research  the University o f  different  ammonia o n l y , and ammonia (7:1)  t o as NH. -N, NO ~-N and (NO ~+ N H . ) - N +  +  TABLE 1.  Composition and c o n c e n t r a t i o n  (ppm) o f f u l l - s t r e n g t h n u t r i e n t  Element  Solution N0  N  solution.  3  + NH  12.2  (7:1)  + 4  + 1.8  N0  NH  3  + 4  14 .0  14 .0  P  11 .8  11 .8  11 .8  K  22 .0  22 .0  22 .0  Ca  20 .0  20 .0  20 .0  Mg  9.0  9.0  9 .0  15 .0  15 .0  15 .0  S  -4  x 10~  4  31.2  x 10~  4  31.2  x IO  31. 2 x 1 0 ~  4  31.2  x 10~  4  12.6  2  12. 6 x 1 0 "  2  - 3  31.2  x 10~  3  - 3  31.2  x 10~  3  12.6  Zn  31.2  Mo  31.2  Fe  12.6  x 10  Mn  31.2  x 10"  3  31. 2 x I O  B  31.2 x 1 0 ~  3  31.2  x 10 x 10 x 10  -4 -4 -2  -4  12.6  Cu  x 10"  x IO  12.6  x 10 - 4  pH = 5.5 - 0.1 CJ  respectively).  The c o m p o s i t i o n  was  similar  the  l e v e l of a l l n u t r i e n t s except  all  three At  to those  solutions  of these used  nutrient  by K r a j i n a ( 4 ) .  c h l o r i d e was  weeks,  50 l i v e  containing  sand.  Each n i t r o g e n treatment  c o n s i s t e d o f 50 b u l l e t s . plastic  stand.  species  x 3 treatments).  The 50 b u l l e t s  p e r week and d i s t i l l e d  and i n c a n d e s c e n t  a photoperiod  twilight  o f 16 h r  of trees  fresh  i n which the l i g h t s p e r a t u r e c y c l e was  humidity  The  relative at night.  20,000 l u x  (6 am t o 10 pm).  Dawn and  by s t a g g e r i n g t h e o r d e r  came on and went o f f . used  (4  nutrient  90%  lamps p r o v i d e d  e f f e c t s were p r o d u c e d  species  w a t e r o n c e a week.  i n t h e day and 65% r e l a t i v e  (9)  were h e l d i n a  s e e d l i n g s were grown i n a g r o w t h room w i t h  Fluorescent  bullets  f o r each  Thus t h e r e were 12 s t a n d s  s e e d l i n g s were i r r i g a t e d w i t h  three times  over  t h e same i n  s e e d l i n g s were  c h o s e n a t random a n d t r a n s p l a n t e d i n t o p l a s t i c  humidity  However,  solutions.  t h e end o f s e v e n  The  solutions  A 12 h r tem-  (8 am t o 8 pm a t 24C; 8 pm t o  8 am a t 2 0 C ) . E i g h t e e n weeks a f t e r was  determined,  and  total  ratio  the s u r v i v i n g  dry weight,  seedling survival  s e e d l i n g s were  harvested  r o o t d r y w e i g h t and s h o o t / r o o t  (S/R) were r e c o r d e d .  oven d r i e d  germination,  P l a n t m a t e r i a l was  a t 80C o r l y o p h i l i z e d ;  either  t h e methods gave  equal  results. species  Leaf area except  determine results  (Appendix  I) was d e t e r m i n e d  f o r Western redcedar;  i t was i m p o s s i b l e t o  a c c u r a t e l y the area o f the cedar  except  for a l l  s u r v i v a l were a n a l y z e d w i t h  scales. A l l one way a n a l y s e s  o f v a r i a n c e f o l l o w e d by d e t e r m i n a t i o n o f t h e l e a s t ficant  difference  (LSD).  signi-  S u r v i v a l d a t a were a n a l y z e d by  means o f t h e C h i - s q u a r e - 2 x n  contingency  table.  RESULTS The  data on p l a n t s u r v i v a l  dry weights, in  Tables  leaf  are given i n Table  2.  The  a r e a a n d S/R f o r t h e s u r v i v o r s a r e g i v e n  3, 4, 5, and 6 f o r D o u g l a s - f i r , W e s t e r n  W e s t e r n h e m l o c k and L o d g e p o l e  pine  redcedar,  respectively.  Douglas-fir The  s e e d l i n g s i n t h e NC> ~-N a n d ( N 0 ~ + NH^ ) -N t r e a t +  3  3  ments h a d s i m i l a r p e r c e n t  survival  had  percent  a significantly  Seedlings total  +  4  survival  r e c e i v i n g NH -N produced +  4  (Table 2 ) .  significantly  lower  d r y w e i g h t and r o o t d r y w e i g h t and a s i g n i f i c a n t l y  h i g h e r S/R t h a n was  lower  and t h e t r e e s i n NH -N  t h e o t h e r two t r e a t m e n t s  no s i g n i f i c a n t  difference  (Table 3).  i n l e a f a r e a among t h e t h r e e  treatments. Western The  There  Redcedar s e e d l i n g s i n t h e (NO ~ + NH. )-N +  a n d NO ~-N  TABLE 2.  S u r v i v a l (%) o f D o u g l a s - f i r , Lodgepole pine, Western redcedar and Western hemlock s u p p l i e d with d i f f e r e n t sources o f n i t r o g e n . Source of  Species  Nitrogen  N0 -  NH  98  90  64*  Western redcedar  100  100  20*  Western hemlock  100  92  64*  92  86  88  N0 ~ + N H 3  Douglas-fir  Lodgepole  pine  * D i f f e r e n c e among means w i t h i n a species  + 4  (7:1)  3  s i g n i f i c a n t a t the 5% l e v e l .  + 4  TABLE 3.  T o t a l dry weight, r o o t dry weight, S/R and l e a f area o f D o u g l a s - f i r s u p p l i e d with d i f f e r e n t sources of nitrogen.1 Source o f N i t r o g e n N0 ~ + N H 3  T o t a l dry weight Root dry weight  (gm/plant) (gm/plant)  S/R T o t a l l e a f s u r f a c e area  Values  (cm / p l a n t )  + 4  (7:1)  NC>~ 3  NH  + 4  1019  1192  0787  0498  0554  0271  1.12  1.32  2.13  12.86  13.19  10.74  connected by the same l i n e do not d i f f e r a t the 5% l e v e l by LSD,  TABLE 4.  T o t a l dry weight, r o o t dry weight, S/R and l e a f dry weight o f Western redcedar s u p p l i e d with d i f f e r e n t sources o f n i t r o g e n . 1 Source o f N i t r o g e n N0 ~ + N H 3  T o t a l dry weight Root dry weight  (gm/plant) (gm/plant)  S/R Leaf dry weight  + 4  (7:1)  3  NH  + 4  .1015  .1440  .0265  .0358  .0429  .0121  2.19 (gm/plant)  N0 -  .0792  Values connected by the same l i n e do not d i f f e r a t the 5% l e v e l  2.48 .0939  by LSD.  2.06 .0142  TABLE 5.  T o t a l dry weight, r o o t dry weight, S/R and l e a f area o f Western hemlock s u p p l i e d with d i f f e r e n t sources o f nitrogen.1 Source N0 ~ 3  T o t a l dry weight Root dry weight  (gm/plant) (gm/plant)  S/R 2 (cm / p l a n t )  + 4  (7:1)  NC> ~ 3  NH  + 4  .0672  .0523  . 0518  .0190  .0150  .0142  2. 58  T o t a l l e a f s u r f a c e area  Values  + NH  of Nitrogen  15.28  3.00  11. 39  connected by the same l i n e do not d i f f e r a t the 5% l e v e l by LSD.  3.10  10 . 64  TABLE 6.  T o t a l dry weight, r o o t dry weight, S/R and l e a f area o f Lodgepole s u p p l i e d with d i f f e r e n t sources o f n i t r o g e n . 1  pine  Source o f N i t r o g e n N0 ~ + N H 3  T o t a l dry weight Root dry weight  (gm/plant) (gm/plant)  S/R  T o t a l l e a f s u r f a c e area  2 (cm / p l a n t )  + 4  (7:1)  N0 ~ 3  NH  + 4  . 1214  . 1119  .1677  .0 580  .0526  .0653  1.14  1. 34  12.10  10.97  Values connected by the same l i n e do not d i f f e r a t the 5% l e v e l by LSD.  1.94 17.52  31. treatments  h a d 100% s u r v i v a l ,  while those  t r e a t m e n t h a d o n l y 20% s u r v i v a l  (Table far  f o l l o w e d by t h e (NO^  4).  Those on t h e N H - N  significantly  Western  +  Trees  l o w e r amounts o f l e a f  i n NH -N +  4  produced  treatments.  Hemlock receiving  a n d 92% s u r v i v a l  ( N 0 ~ + N H ) - N a n d NC> ~-N h a d +  3  4  respectively  3  (Table 2).  +  4  dry weight  (NC>  3  (Table 5 ) .  + N H ) - N produced  difference  The s e e d l i n g s grown  a significantly  +  4  Seedlings  T o t a l d r y weight,  a n d S/R showed no s i g n i f i c a n t  among t h e t h r e e t r e a t m e n t s with  treatment  and r o o t d r y w e i g h t  r e c e i v i n g N H - N h a d o n l y 64% s u r v i v a l . root  total  T h e r e was no s i g n i f i c a n t  i n S/R among t h e t h r e e  Seedlings 100%  the largest  + NH^ )-N  dry weight.  t h a n t h e o t h e r two t r e a t m e n t s . difference  The s e e d l i n g s  t r e a t m e n t p r o d u c e d by  +  4  the lowest t o t a l  +  (Table 2 ) .  r e c e i v i n g NO^ -N t r e a t m e n t p r o d u c e d dry weight  i n t h e NH^ -N  larger  leaf  area than  s e e d l i n g s grown o n t h e o t h e r two t r e a t m e n t s .  Lodgepole  Pine  T h e r e was l i t t l e three treatments sulted  difference  (Table 2).  i n significantly  i n survival  Treatments  larger  total  among t h e  with NH -N r e +  4  d r y weight,  leaf  a r e a a n d S/R t h a n t h e o t h e r two t r e a t m e n t s  (Table 6 ) .  T h e r e was no d i f f e r e n c e  among t h e  three  treatments.  i n r o o t d r y weight  DISCUSSION On  the b a s i s o f growth and s u r v i v a l data i t would seem  t h a t D o u g l a s - f i r , Western redcedar and Western hemlock  sur-  v i v e d and grew w e l l with e i t h e r NO^ -N o r (NO^ + NH )-N. +  4  The  NH -N treatment was d e t r i m e n t a l +  4  t o both the growth and  s u r v i v a l o f D o u g l a s - f i r and Western redcedar, but only t o the s u r v i v a l o f Western hemlock.  Western hemlock  seedlings  which s u r v i v e d on the NH -N treatment grew as w e l l as +  4  t r e e s growing on the other  two sources o f n i t r o g e n .  pole pine s u r v i v e d e q u a l l y w e l l with a l l n i t r o g e n and was the only s p e c i e s The  treatments  t h a t grew b e s t on NH -N. +  4  lower s u r v i v a l o f Western hemlock s e e d l i n g s  NH -N treatment may not be very +  4  field  Lodge-  environment i f e x c e s s i v e  i n the  s i g n i f i c a n t i n the n a t u r a l  seedling production  occurs.  With D o u g l a s - f i r and Western redcedar the s m a l l s i z e o f seedlings  grown on NH -N may l e a d t o i n c r e a s e d +  4  mortality  i n the f i e l d due t o competition. Many workers have proposed t h a t a low S/R i s p r e f e r a b l e for  survival of conifers  (1, 3, 5, 6).  However, Hermann (2)  has  shown t h a t s u r v i v a l f o r D o u g l a s - f i r s e e d l i n g s with a  s m a l l r o o t system was s i g n i f i c a n t l y lower than f o r  seedlings  w i t h a l a r g e r o o t system, r e g a r d l e s s o f the s i z e o f the shoot.  A c o n s i d e r a t i o n o f the data r e p o r t e d here f o r e f f e c t  of n i t r o g e n  treatment on s u r v i v a l , r o o t weight and S/R,  would i n d i c a t e t h a t :  1) F o r D o u g l a s - f i r , a low S/R and a  large  r o o t system a r e both  (Tables  2 and 3 ) .  i n d i c a t i v e o f high  2) F o r W e s t e r n r e d c e d a r ,  development o f  a l a r g e r o o t s y s t e m by t h e s u r v i v o r s i s more of high  survival  than  i s a low S/R  survival  (Tables  indicative  2 and 4 ) .  3) F o r W e s t e r n h e m l o c k , n e i t h e r r o o t d e v e l o p m e n t n o r a low  S/R o f t h e s u r v i v o r s i s i n d i c a t i v e  2 and 5 ) . root  4) F o r L o d g e p o l e p i n e ,  s y s t e m i s more i n d i c a t i v e  of survival  (Tables  development o f a l a r g e  o f high  survival  than  d e v e l o p m e n t o f a low S/R. With Lodgepole p i n e , treatment  (Table  the high  +  4  6) was n o t d e t r i m e n t a l  2) , p o s s i b l y b e c a u s e t h i s with  S/R o f t h e N H - N  treatment  to survival  produced  grown o n N H - N +  4  treatments.  survival  (Table  was  a result  o f decreased  The  similar  3) o n t h e t h r e e  2).  leaf  S/R ( T a b l e  In this root  case  3 ) , b u t had  t h e i n c r e a s e d S/R  development.  a r e a p r o d u c e d by D o u g l a s - f i r  treatments  a r e a was n o t t h e l i m i t i n g total  Douglas-fir seedlings  a l s o had a high  low  indicates that factor  d r y w e i g h t on t h e NH -N +  4  i n t h e low p r o d u c t i o n o f treatment. 1/3 t o 1/2  d r y m a t t e r p r o d u c e d by D o u g l a s - f i r o r L o d g e p o l e s p i t e o f the fact  leaf  areas  (Tables  that these  (Table  photosynthetic  Western hemlock s e e d l i n g s p r o d u c e d o n l y  in  seedlings  as l a r g e a r o o t s y s t e m as t h a t p r o d u c e d by s e e d l i n g s  grown o n t h e o t h e r  the  (Table  three  3, 5, a n d 6 ) .  pine  s p e c i e s had s i m i l a r  34.  There seems t o be a r e l a t i o n s h i p between where the t r e e s occur i n the f i e l d metabolize ammonia.  and the a b i l i t y o f the t r e e s t o  Young D o u g l a s - f i r and Western redcedar,  which occur on s o i l s where n i t r a t e i s expected predominant form o f n i t r o g e n  t o be the  ( 4 ) , grew and s u r v i v e d  poorly  on NH -N as compared t o growth and s u r v i v a l on the o t h e r +  4  nitrogen  treatments.  Western hemlock and Lodgepole  pine,  which occur on s o i l s where ammonia i s expected t o be the predominant form o f n i t r o g e n  ( 4 ) grew as w e l l o r b e t t e r on  NH -N than on the o t h e r two sources o f n i t r o g e n . +  4  Driessche recently reported  Van Den  ( 8 ) t h a t D o u g l a s - f i r grew  b e t t e r on NH -N alone than on NO^ -N alone. +  4  This  obser-  v a t i o n i s i n c o n t r a s t t o the data r e p o r t e d here and the results of Krajina  ( 4 ) and i n d i c a t e s t h a t the r e l a t i o n s h i p  between where the t r e e s occur i n the f i e l d and the a b i l i t y o f the t r e e s t o metabolize NH^ -N o r NO^ -N i s not as c l e a r +  cut as we have s t a t e d .  LITERATURE CITED George, E.G. 1939. Tree p l a n t i n g on the d r i e r s e c t i o n s of the n o r t h e r n great p l a i n s . J . F o r e s t r y 37: 695698. Hermann, R.K. 19 64. Importance o f the t o p - r o o t r a t i o s f o r s u r v i v a l o f D o u g l a s - f i r s e e d l i n g s . Tree P l a n t e r s Notes 64: 7-11. U.S. F o r e s t S e r v i c e , U.S. Dept. o f A g r i c u l t u r e , Washington, D.C. K a r s t i a n , C F . 1925. F o r e s t p l a n t i n g i n the i n t e r mountain r e g i o n . B u l l e t i n No. 1264. U.S. F o r e s t S e r v i c e , U.S. Department o f A g r i c u l t u r e , Washington, D.C. K r a j i n a , V . J . 1969. Ecology o f f o r e s t t r e e s i n B r i t i s h Columbia. Ecology o f Western North America 2: 1-146. Botany Department, U n i v e r s i t y o f B r i t i s h Columbia. Pearson, G.A. 1950. Management o f Ponderosa pine i n the southwest. A g r i c u l t u r e Monograph No. 6. U.S. F o r e s t S e r v i c e , U.S. Department o f A g r i c u l t u r e , Washington, D.C . S t o e c k e l e r , J.H. 1950. Can nurserymen produce white pine s e e d l i n g stock comparable t o t r a n s p l a n t s . T e c h n i c a l note 339: Lake States F o r e s t r y Experimental S t a t i o n , U.S. F o r e s t S e r v i c e , S t . Paul 1, Minn. Swan, H.S.D. 1960. The m i n e r a l n u t r i t i o n o f Canadian pulpwood s p e c i e s . Pulp and Paper Research I n s t i t u t e o f Canada. Woodlands Research Index No. 116. Montreal, Canada. Van  Den D r i e s s c h e , R. 1971. Response o f c o n i f e r s e e d l i n g s to n i t r a t e and ammonium sources o f n i t r o g e n . P l a n t and S o i l 34: 421-439.  Walters, J . 1968. P l a n t i n g gun and b u l l e t . J . American S o c i e t y o f A g r i c u l t u r a l Engineers 49: 336-339.  36. PART 2-B.  NITROGEN UPTAKE AND STARCH CONTENT OF CONIFERS SUPPLIED WITH DIFFERENT SOURCES OF NITROGEN.  INTRODUCTION Some p l a n t s appear to u t i l i z e n i t r a t e and v i c e v e r s a . c o n i f e r species  ammonia i n p r e f e r e n c e to  Potatoes, r i c e , buckwheat  (17) and low bushberry  of p l a n t s t h a t seem t o u t i l i z e  (7), some  (16), are examples  ammonia i n p r e f e r e n c e t o  n i t r a t e , w h i l e beets, wheat (7), tomatoes, mustard, r y e , oats  (10) and apple  (6) t r e e s are examples o f p l a n t s  seem to u t i l i z e n i t r a t e i n p r e f e r e n c e In previous  that  to ammonia.  work (Part 2-A) i t was concluded  Western hemlock and Lodgepole pine s u r v i v e d  that  and grew b e t t e r  when s u p p l i e d with ammonia r a t h e r than n i t r a t e .  The opposite  r e s u l t s were found f o r D o u g l a s - f i r and Western redcedar. However, two p i e c e s o f i n f o r m a t i o n ing  prevented me from  conclud-  t h a t Western redcedar and D o u g l a s - f i r u t i l i z e n i t r a t e  p r e f e r e n t i a l l y and t h a t Western hemlock and Lodgepole utilize  ammonia p r e f e r e n t i a l l y : a) Van Den D r i e s s c h e  concluded t h a t D o u g l a s - f i r s e e d l i n g s  pine (17)  grow b e t t e r when  s u p p l i e d w i t h ammonia and b) the r e s u l t s o f a 1 year study (Part 2-C) i n d i c a t e t h a t growth o f Lodgepole pine and Western hemlock was l e s s on n i t r a t e than on e i t h e r ammonia or a combination o f n i t r a t e and ammonia (7:1) where n i t r a t e was the major source o f n i t r o g e n .  37. R e c e n t work w i t h o t h e r this and is low  apparent Blair  plants  suggested a s o l u t i o n to  contradiction of results.  H a r a d a e t a l . (8)  e_t al_. (2) have shown t h a t w i t h  there  no d i f f e r e n c e b e t w e e n g r o w t h on n i t r a t e o r ammonia, when concentrations  that plants  of nitrogen  may d i f f e r  are supplied.  i n their  ability  o f ammonia r a t h e r  either  ammonia o r n i t r a t e p r e f e r e n t i a l l y .  may be e x p r e s s e d  than t h e i r  This  suggests  to tolerate  levels  the  some p l a n t s ,  ability  various  to u t i l i z e This  tolerance  i n s e v e r a l ways a) p e r h a p s by r e g u l a t i n g  amount o f ammonia t a k e n up a n d / o r b) by a m e t a b o l i c  r e g u l a t i o n o n c e t h e ammonia i s t a k e n up. I t h a s b e e n shown t h a t wheat s e e d l i n g s grass  (13) a n d some c o n i f e r s  (17) t a k e up more ammonia  n i t r a t e when t h e two forms a r e p r e s e n t conifers two  Ammonia  has  (5). Therefore  i n h i b i t o r y concentration,  starch in  together.  (12) and s u b s e q u e n t l y  fixation  expected  In  i n t o x i c l e v e l s i s thought t o i n h i b i t  phosphorylation  an  together.  than  (17) ammonia was t a k e n up e x c l u s i v e l y when t h e  s o u r c e s were p r e s e n t  dioxide  (15, 1 8 ) , r y e  t o be low.  to i n h i b i t  carbon  i f ammonia i s p r e s e n t  at  t h e amount o f s t a r c h w o u l d be  Two r e p o r t s  (4, 9) have i n d i c a t e d  content o f ammonia-treated D o u g l a s - f i r  nitrate-treated Douglas-fir. shown t h a t  photo-  i s lower  that than  I n a d d i t i o n Bassham (1)  ammonia h a s a r e g u l a t o r  e f f e c t on C h l o r e l l a ,  38. i n f l u e n c e d metabolism from sugar s y n t h e s i s to p r o t e i n synthesis. An experiment was undertaken with D o u g l a s - f i r , Western redcedar, Lodgepole pine and Western hemlock t o determine a) i f ammonia was taken up p r e f e r e n t i a l l y over n i t r a t e and b) i f the s t a r c h content  o f the s e e d l i n g s was r e l a t e d to the  source o f a v a i l a b l e n i t r o g e n .  METHODS AND  MATERIALS  D e t a i l s o f growing c o n d i t i o n s , c u l t u r e s o l u t i o n s , c u l t u r e technique, previously  dry weight and s u r v i v a l have been d e s c r i b e d  (Part 2-A).  and were s u p p l i e d with only  The s e e d l i n g s were grown i n b u l l e t s 1 o f 3 sources o f n i t r o g e n :  (NO^ -N), ammonia only  n i t r a t e and ammonia a f t e r germination, b u l l e t s , adhering placed i n jars  nitrate  (NH^ -N) o r a combination of +  (7:1)((N0  + NH )-N). +  3  4  A t 4 months  the s e e d l i n g s were removed from the sand was washed o f f and the r o o t s were  (2 s e e d l i n g s per j a r ) o f n u t r i e n t s o l u t i o n  c o n t a i n i n g the a p p r o p r i a t e  n i t r o g e n treatment  (eg. seed-  l i n g s s u p p l i e d with NO^ -N i n the b u l l e t s were p l a c e d i n NO^ -N n u t r i e n t s o l u t i o n ) .  The j a r s were aerated  constantly  and  the n u t r i e n t s o l u t i o n was changed every second day.  The  pH o f the n u t r i e n t s o l u t i o n s was measured f o r s i x  consecutive  days.  A f t e r s i x days on the n u t r i e n t s o l u t i o n the s e e d l i n g s  39 .  were l y o p h i l i z e d . sonication  of  Starch  the  and  extracted  was  .1 gm  lyophilized  solid  from  perchloric  Soluble carbohydrate  the  acid  was  e x t r a c t e d by  m a t e r i a l i n 20  r e s i d u e were r e t a i n e d .  r e s i d u e and  hydrolyzed  u s i n g hydrogen peroxide  and  ethanol.  Starch  was  to glucose  a c c o r d i n g t o McCready et_ al_.  determined  ml  with  (14) .  Glucose  sulfuric  acid  a c c o r d i n g t o D u b o i s e_t a l . (3) . All by  d a t a was  a n a l y z e d by  d e t e r m i n a t i o n o f t h e LSD  analyses  a t 5%  of variance followed  level.  RESULTS D e t a i l e d d e s c r i p t i o n s of dry weight, r o o t d r y w e i g h t were r e p o r t e d i n P a r t pH  area  and  2-A.  Data The  pH  initial  after  pH  o f the n u t r i e n t  2 days exposure t o the  With Western redcedar +  NH  leaf  4  —  -N  and  pH  than  pole pine was  + NH^  3  from  t h e NO^  significantly  treatment  The  roots i s given i n Table  W e s t e r n h e m l o c k , t h e pHs  ) -N  treatment  each o t h e r  -N  t h e pH  s o l u t i o n b u t was  5.5.  of  1.  the  +  (NC>  tinguishable  and  s o l u t i o n was  treatment.  of the  (NO^  h i g h e r than still  solution.  and  s o l u t i o n s were  have a s i g n i f i c a n t l y  W i t h D o u g l a s - f i r and + NH )-N +  4  t h e pH  much l o w e r  indis-  treatment  of the NH -N  than  +  4  t h e pH  lower  Lodge-  solution treatment  o f t h e NO^  -N  TABLE 1.  The mean pH of the n u t r i e n t  Species  s o l u t i o n s a f t e r treatment f o r 2 days.  1,2  Source o f N i t r o g e n NH -N +  4  (N0 ~ 3  + NH )-N +  4  N0 " -N 3  Douglas-fir  4.20  4.69  6. 17  Western redcedar  4.15  4.27  6. 10  Western hemlock  4.22  4. 39  5. 79  Lodgepole  3.85  4.87  6. 02  pine  "'"Initial pH o f n u t r i e n t s o l u t i o n s 5.5. 2 Values  connected  by the same l i n e do not d i f f e r at the 5% l e v e l by LSD.  41. Starch  Content  D o u g l a s - f i r and Western redcedar t r e a t e d with NH^ -N +  had the lowest s t a r c h content compared t o the o t h e r n i t r o g e n treatments  (Table 2 ) . The (NC> ~ + NH )-N treatment  pro-  +  3  4  duced l e v e l s o f s t a r c h equal t o t h a t o f the n i t r a t e ment.  With Lodgepole  treat-  pine and Western hemlock, there was  no d i f f e r e n c e i n s t a r c h p r o d u c t i o n among the three t r e a t ments .  DISCUSSION When NO^ -N i s taken up, the pH o f a n u t r i e n t  solution  r i s e s , probably as a r e s u l t o f the e x p u l s i o n o f OH HC0 ~ 3  (2,10).  and/or  When NH -N i s taken up the pH o f the +  4  n u t r i e n t s o l u t i o n decreases, probably as a r e s u l t o f the expulsion of H  (2,10).  +  The d i f f e r e n c e i n pH between the NH -N treatment and +  4  the pine  (N0  + N H ) - N treatment o f D o u g l a s - f i r and Lodgepole +  3  4  (Table 1) may be due to  from the (N0  1) l e s s uptake o f NH -N +  4  + NH )-N treatment with concomitant +  3  expulsion of H  4  +  and/or  2) simultaneous  lower  uptake o f s m a l l  amounts o f N0 ~-N w i t h NH -N from the (N0 ~ + N H ) - N +  3  and thus r e l e a s e o f both H i t can be concluded  +  4  3  and OH .  +  4  For a l l four species,  t h a t NH -N was the predominant source +  4  o f n i t r o g e n when s u p p l i e d as (N0  + NH )-N. +  3  4  These r e s u l t s  agree with Van Den D r i e s s c h e ' s work with c o n i f e r s  ( 2 ) . The  TABLE 2.  Starch content o f c o n i f e r s e e d l i n g s s u p p l i e d w i t h d i f f e r e n t f o r 4 months( g/gm dry w e i g h t ) . !  Species  Source of  sources o f n i t r o g e n  Nitrogen + NH )-N  N0  118.1  107.3  106.7  143. 7  131.0  Western hemlock  173.3  179.7  181.0  Lodgepole pine  117.0  114.4  103. 4  NH -N +  4  Douglas-fir Western  redcedar  Values connected  52.1  (N0  +  3  4  by the same l i n e do not d i f f e r a t the 5% l e v e l by LSD.  3  -N  NH -N  content  +  4  12.25  o f t h e ( N 0 ~ + N H ) - N was 1.75 ppm N w h i l e +  3  ppm o f NC> -N was p r e s e n t . 3  When N H - N  i s present  +  4  hibits  carbon  tion  (1).  were s u p p l i e d w i t h (Table 2).  NH -N +  4  taken  (1.75  of N0  -N.  pine  3  +  4  production t o p r o t e i n produc-  mechanisms w o u l d  +  i n this  treatment  +  3  produced  high  ppm N) o f N H - N  3  -N o n l y .  levels  These r e s u l t s would i n d i c a t e  +  than  pro-  (14 ppm N) and low  and h i g h  +  4  4  amounts o f  and W e s t e r n h e m l o c k s t a r c h  that  (14 ppm N) Lodgepole  t o l e r a n c e t o ex-  D o u g l a s - f i r and W e s t e r n  However D o u g l a s - f i r and W e s t e r n r e d c e d a r a r e o f low amounts o f N H - N . +  4  dry matter p r o d u c t i o n  +  4  explained.  In a d d i t i o n , the high  ( P a r t 2-A) o f L o d g e p o l e p i n e and  W e s t e r n h e m l o c k on e i t h e r N H - N easily  (N0 +NH )-N,  form o f n i t r o g e n  t o amounts p r o d u c e d w i t h N 0  4  tolerant  redcedar  4  s u p p l i e s o f NH -N  redcedar.  with  14 ppm N H - N , s t a r c h p r o d u c t i o n was  and W e s t e r n h e m l o c k have a h i g h e r  ternal  interfere  When D o u g l a s - f i r and W e s t e r n  d u c t i o n was t h e same on b o t h (1.75  +  4  s e e d l i n g s were s u p p l i e d w i t h  With Lodgepole pine  levels  NH -N i n -  (5) and N H - N h a s a l s o  ppm N) was t h e p r e d o m i n a n t  Trees  equal  levels,  On t h e o t h e r h a n d , when D o u g l a s - f i r and  redcedar  up.  starch  sugar  Both o f these  starch production.  Western  i n toxic  dioxide fixation  b e e n shown t o s w i t c h  low  4  In fact,  o r (N0  + NH )-N +  3  4  i s now  the seedlings supplied with  (NC>  +'NH  3  The may  4  )-N were u t i l i z i n g  tolerance  NH^ -N.  o f D o u g l a s - f i r t o low l e v e l s  e x p l a i n why V a n Den D r i e s s c h e  The may  the  variation  occur  i n tolerance  explained  i n nature.  2-A) o c c u r  predominant  selection NH -N +  4  and  NH -N  i n terms o f s e l e c t i o n  by where t h e  D o u g l a s - f i r and W e s t e r n a t high  levels  levels  considered  necessary.  redcedar,  o f NH -N +  4  3  form o f n i t r o g e n tolerance  (11).  In these  or protection  has n o t been n e c e s s a r y .  o f NH -N occur +  4  on s o i l s  t o be t h e predominant  these species  t o ammonia  o n s o i l s where NC> -N i s t h o u g h t t o be  f o r ammonia  toxicity  of the seedlings  selection  species,  against  Lodgepole  W e s t e r n h e m l o c k w h i c h grow and s u r v i v e w e l l  at high  rather  +  4  3  w h i c h grow and s u r v i v e p o o r l y (Part  that  NC> ~-N.  be e a s i l y  trees  +  4  (17) c o n c l u d e d  D o u g l a s - f i r grew b e t t e r when s u p p l i e d w i t h than with  o f NH -N  ( P a r t 2-A)  where N H - N i s +  4  form o f n i t r o g e n  f o r ammonia  pine  tolerance  (11). In  has been  45. LITERATURE CITED 1.  Bassham, J . 1971. The c o n t r o l o f p h o t o s y n t h e t i c metabolism. Science 172: 526-534.  carbon  2.  B l a i r , G., M.H. M i l l e r and W.A. M i t c h e l l . 1969. N i t r a t e and ammonium as sources o f n i t r o g e n f o r corn and t h e i r i n f l u e n c e on the uptake o f i o n s . Agronomy J . 62: 530-532.  3.  Dubois, M., K.A. G i l l e s , J.K. Hamilton, P.A. Rebers and Fred Smith. 19 56. C o l o r i m e t r i c method f o r determination o f sugars and r e l a t e d substances. A n a l y t i c a l Chemistry 28: 350-356.  4.  E b e l l , L.F. and E.E. McMullan. 19 70. Nitrogenous substances a s s o c i a t e d with d i f f e r e n t i a l cone responses o f D o u g l a s - f i r to ammonium and n i t r a t e f e r t i l i z a t i o n , Can. J . Bot. 48: 2169-2177.  5.  Gaffron, F.C.  6.  Gransmanis, V.O. and D.J.D. N i c h o l a s . 1966. Uptake o f n i t r a t e by Jonathan apple t r e e s . P l a n t and S o i l 25: 461-462.  7.  Goring, C.A.I. 1956. The n i t r o g e n n u t r i t i o n o f p l a n t s . Down to E a r t h . Spring, 7-9.  8.  Harada, T., H. Takaki and Y. Yamada. 1968. E f f e c t o f n i t r o g e n source on the chemical components i n young p l a n t s . S o i l S c i . and PI. Nut. 14: 47-55.  9.  Hart, A.L. and G.E. M e l l o r . 1971. Leaf s t r u c t u r e o f D o u g l a s - f i r s u p p l i e d w i t h d i f f e r e n t forms o f n i t r o g e n . Proceedings o f the Canadian S o c i e t y o f P l a n t P h y s i o l o g i s t s p. 35. Toronto.  H. 1960. Energy storage. In P l a n t P h y s i o l o g y . Steward (ed.). Academic Press, New York.  10.  K i r b y , E.A. 1968. Ion uptake and i o n i c balance i n p l a n t s i n r e l a t i o n t o the form o f n i t r o g e n . In. E c o l o g i c a l aspects o f the mineral n u t r i t i o n o f p l a n t s . I.H. Rorison (ed.). B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s , Oxford and Edinburgh.  11.  K r a j i n a , V.J. 1969. Ecology o f f o r e s t t r e e s i n B r i t i s h Columbia. Ecology o f Western North America 2: 1-146. Botany Department, U n i v e r s i t y o f B r i t i s h Columbia.  46. 12.  Krogmann, D.W. , A.T. Jagendorf and M. Avron. 1959. Uncouplers o f spinach c h l o r o p l a s t photophosphory l a t i o n . 34: 272-  13.  Lycklama, J.C. 19 63. Th.e a b s o r p t i o n o f ammonium and n i t r a t e by p e r e n n i a l r y e grass. A c t a Bot. N e e r l . 12: 316-323.  14.  McCready, R.M., J . Guggolz, V. S i l i v i e r a and H. Owens. 19 50. Determination o f s t a r c h and amylose i n v e g e t a b l e s . A n a l . Chem. 22: 1156-1158.  15.  M i n o t t i , P.L., D.C. W i l l i a m s and W.A. Jackson. 1969. N i t r a t e uptake by wheat as i n f l u e n c e d by ammonium and o t h e r c a t i o n s . Crop Science 9: 9-14.  16.  Townsend, L.R. 1970. E f f e c t o f form o f n i t r o g e n and pH on n i t r a t e reductase a c t i v i t y i n lowbush B l u e b e r r y leaves and r o o t s . Can. J . P l a n t S c i . 50: 603-605.  17.  Van Den D r i e s s c h e , R. 1971. Response o f c o n i f e r seedl i n g s t o n i t r a t e and ammonium sources o f n i t r o g e n . P l a n t and S o i l 34: 421-439.  18.  Weissman, G.S. 1951. Nitrogen metabolism o f wheat s e e d l i n g s as i n f l u e n c e d by the ammonium:nitrate r a t i o and the hydrogen-ion c o n c e n t r a t i o n . Amer. J . Bot. 38: 162-174.  PART 2-C.  A COMPARISON OF  THE  GROWTH OF FOUR CONIFER  SPECIES SUPPLIED WITH DIFFERENT FORMS OF NITROGEN FOR  10 TO  12 MONTHS.  1  INTRODUCTION Previously and  (Part 2-A)  i t was  shown t h a t the s u r v i v a l  growth of c o n i f e r s e e d l i n g s were a f f e c t e d by the  of n i t r o g e n .  source  For D o u g l a s - f i r and Western hemlock, s u r v i v a l  and  growth on a n i t r a t e s o l u t i o n were s i m i l a r to s u r v i v a l  and  growth on a combination of n i t r a t e and  Ammonia was and  ammonia  an unfavorable source of n i t r o g e n  for survival  growth of D o u g l a s - f i r and Western redcedar.  hemlock, ammonia treatment was Hemlock t r e e s which s u r v i v e d as t r e e s growing on the other  (7:1).  only d e t r i m e n t a l  For Western to s u r v i v a l .  ammonia treatment grew as w e l l two  sources of  nitrogen.  Lodgepole pine s u r v i v e d e q u a l l y w e l l under a l l treatments and was  the only s p e c i e s  Swan (2) r e p o r t e d p e r i o d , Jack pine  that grew b e s t on  ammonia.  t h a t over a four month growing  (Pinus banksiana) and White spruce  (Picea  glauca) grew b e t t e r when s u p p l i e d w i t h ammonia r a t h e r than  1 T h i s s e c t i o n deals w i t h work i n i t i a t e d by S. MadocJones but continued by the author a f t e r the untimely death of S. Madoc-Jones. A l s o the data i n t h i s s e c t i o n represents p a r t of a paper presented at the XII P a c i f i c Science Congress, Canberra, 19 71. Ammonia and N i t r a t e i n the Nitrogen Economy of Some C o n i f e r s Growing i n D o u g l a s - f i r Communities of the P a c i f i c North-West America by V.J. K r a j i n a , S. Madoc-Jones and G.E. M e l l o r .  48. nitrate. for  Durzan and Steward  another  (1) continued Swan's  12 months and found t h a t n i t r a t e now gave the  b e s t r e s u l t s f o r White p i n e , while Jack pine s t i l l when s u p p l i e d w i t h ammonia. in  experiment  grew b e s t  The o b j e c t of the work presented  t h i s s e c t i o n was to determine  the e f f e c t of s u p p l y i n g four  c o n i f e r s p e c i e s w i t h d i f f e r e n t sources o f n i t r o g e n f o r a p e r i o d o f 10 t o 12 months.  METHODS AND  MATERIALS  Seed source, growing c o n d i t i o n s and n u t r i e n t were d e s c r i b e d p r e v i o u s l y (Part 2-A).  Seeds o f D o u g l a s - f i r ,  Western redcedar, Western'hemlock and Lodgepole sown  solution  pine were  (38 per 1 g a l l o n crock) i n sand and the crocks were  irrigated initially water.  seven times a day w i t h d e m i n e r a l i z e d  I r r i g a t i o n with n u t r i e n t s o l u t i o n s was begun one  week a f t e r germination.  Each crock was i r r i g a t e d automa-  t i c a l l y and r e c e i v e d 250 ml o f n u t r i e n t f o u r times a day. For the f i r s t seven weeks a f t e r germination the concentrat i o n of the n u t r i e n t s o l u t i o n was t h a t used i n P a r t 2-A (i.e.  14 ppm N).  A t seven weeks a f t e r germination the  c o n c e n t r a t i o n o f the n u t r i e n t s o l u t i o n was doubled N) and remained so u n t i l h a r v e s t . treatments were used as i n P a r t 3-B (NO_  + NH. )-N). +  (28 ppm  The same n i t r o g e n (NH -N, NO^ -N and +  4  The crocks were p l a c e d i n the growth  room i n a r a n d o m i z e d c o m p l e t e b l o c k d e s i g n F i v e weeks to  after  f i v e per crock.  germination  (3 b l o c k s ) .  t h e p l a n t s were  T h i r t e e n weeks a f t e r  thinned  germination  D o u g l a s - f i r and L o d g e p o l e  p i n e were t h i n n e d t o two p l a n t s  per crock.  after  redcedar per  F i f t e e n weeks  germination  a n d W e s t e r n h e m l o c k were t h i n n e d t o two p l a n t s  crock.  The two l a r g e s t D o u g l a s - f i r a n d  p i n e s e e d l i n g s were c h o s e n t o r e m a i n . justified  vival  Lodgepole  This choice  on t h e g r o u n d s t h a t s i n c e b o t h  shade i n t o l e r a n t largest  Western  was  species are  i n t h e a r e a o f t h e i r p r o v e n a n c e t h e two  s e e d l i n g s w o u l d have t h e g r e a t e s t c h a n c e o f s u r -  i n nature.  The W e s t e r n r e d c e d a r  and t h e W e s t e r n  h e m l o c k s e e d l i n g s c h o s e n t o r e m a i n were t h e l a r g e s t the s m a l l e s t s e e d l i n g s because both  and  s p e c i e s a r e shade  tolerant. Western redcedar months a f t e r pine,  germination  then  Leaf  Shoot h e i g h t  t h e t r e e s were h a r v e s t e d  l e a v e s and b r a n c h e s .  80C t o c o n s t a n t  Lodgepole was  and d i v i d e d  into  The t r e e s were o v e n d r i e d a t  area o f D o u g l a s - f i r , Western redcedar,  random f r o m  10  weight.  W e s t e r n h e m l o c k was at  and W e s t e r n h e m l o c k and  12 months a f t e r g e r m i n a t i o n .  determined, roots,  and D o u g l a s - f i r were h a r v e s t e d  determined  each t r e e .  10 g r o u p s o f 10 n e e d l e s  by c h o o s i n g  These needles  each.  and  100 l i v e  needles  were d i v i d e d  The a r e a o f t h e s e  into  needles  50.  was  determined  times  by m u l t i p l y i n g  a correction  described  factor  i n Appendix  tions.  After  of needles, After was  determined  leaf  a r e a had  was  average  tree  then  and  constant weight. determined total  All  The  Thus t h e  and leaf  leaf  determina-  10  groups). the  leaves  from the  total  dried  each  area to  dry weight of  a knowledge o f t o t a l  a r e a c o u l d be  leaves  area of Western  to determine  a r e a o f 1 gm  group  weight.  dry weight,  l e a v e s were t h e n  again with  needles  f o r each  o f the  Leaf  width  as  dry weight o f  photocopying  d a t a were a n a l y z e d w i t h  f o l l o w e d by  chlorotic  to constant  determined. by  age  i n these  (average  using a planimeter  the Xerox c o p i e s .  weight,  used  a r e a o f 1 gm  f o r each t r e e  determined  of this  been d e t e r m i n e d  a knowledge o f t o t a l  redcedar  was  were n o t  a r e a o f a t r e e was  l e n g t h times  N e c r o t i c and  t h e g r o u p s were d r i e d  d r y i n g the  Thus w i t h  of  leaf  f o r needles  I.  or p o r t i o n s of needles  together  leaves  leaf  dry  determined. analyses  d e t e r m i n a t i o n o f t h e LSD  of  a t the  variance  5%  level.  RESULTS Douglas-fir The seedlings  (N0 ~ 3  + NH )-N treatment +  4  f o l l o w e d by  t h e NO^  -N  produced  treatment  the  and  tallest  the  NH -N +  4  51. treatment duced  (Table 1 ) .  S e e d l i n g s s u p p l i e d with NH -N pro+  4  the s h o r t e s t p l a n t s , and the lowest shoot d r y weight,  t o t a l dry weight and l e a f area.  The NO^ -N s e e d l i n g s had a  l a r g e r t o t a l dry weight than the (NC>  3  + NH^ )-N  T h i s d i f f e r e n c e i n t o t a l dry weight was mostly to d i f f e r e n c e s i n r o o t dry weight.  seedlings.  +  attributable  In s p i t e o f a d i f f e r e n c e  i n t o t a l dry weight 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 i n l e a f area between the N0 ~-N and the (N0 ~ + NH^ )-N  seed-  +  3  lings.  3  The d i f f e r e n c e i n d r y matter p r o d u c t i o n between the  NH -N and (NC> +  4  3  + NH )-N  treatments was a r e s u l t o f  +  4  d i f f e r e n c e i n shoot dry weight.  The t i p s o f the o l d e r  needles were f r e q u e n t l y c h l o r o t i c and n e c r o t i c .  Dr. V . J .  K r a j i n a i d e n t i f i e d these c h a r a c t e r i s t i c s as symptoms o f magnesium and c a l c i u m d e f i c i e n c y .  S e v e r a l NH -N s e e d l i n g s +  4  l o s t many l e a v e s b e f o r e h a r v e s t . Western Redcedar S e e d l i n g s on the N0  3  -N treatment had a l a r g e r dry  matter p r o d u c t i o n than those on the (N0  + NH )-N +  3  4  treat-  ment and t h i s d i f f e r e n c e was due t o a d i f f e r e n c e i n shoot dry weight  (Table 2 ) .  The NH -N treatment produced the +  4  s h o r t e s t s e e d l i n g s , the s m a l l e s t l e a f area and the s m a l l e s t total,  r o o t and shoot dry weight.  There was a d i r e c t  r e l a t i o n s h i p between l e a f area and dry matter p r o d u c t i o n ( i . e . the l a r g e r the l e a f area, the l a r g e r the dry matter production).  The o l d e r l e a v e s o f the NH. -N t r e a t e d +  TABLE 1.  T o t a l , r o o t , and shoot dry weight, l e a f area and h e i g h t o f D o u g l a s - f i r s u p p l i e d with d i f f e r e n t sources o f nitrogen.1 Source o f N i t r o g e n N0 ~ 3  T o t a l d r y weight (gm/crock) Root dry weight Shoot dry weight  (gm/crock) (gm/crock)  T o t a l s u r f a c e area o f leaves Shoot h e i g h t  (cm)  2  3  + 4  (7:1)  NH  + 4  167.4  133.4  68.8  64.0  42.0  27. 3  103 . 4 (cm /plant)  NC>~ + N H  91.5  41.5  5855.1  4280.9  1621.8  74.0  90.2  43.2  Values connected by the same l i n e do not d i f f e r a t the 5% l e v e l by LSD.  TABLE 2.  T o t a l , r o o t , and shoot dry weight, l e a f area and h e i g h t o f Western s u p p l i e d with d i f f e r e n t sources of nitrogen.1 Source N0 ~ 3  Total  dry weight  Root dry weight  (gm/crock) (gm/crock)  Shoot dry weight Total  (gm/crock)  2 s u r f a c e area of leaves (cm / p l a n t )  Shoot h e i g h t  (cm)  N0 ~ 3  redcedar  of N i t r o g e n  + NH  + 4  (7:1)  NH^  201. 5  160. 7  37. 6  47. 1  40. 7  8. 2  154. 4  120. 0  29. 6  6410. 0  5060. 0  1410. 0  76. 7  77. 0  42. 6  Values connected by the same l i n e do not d i f f e r at the 5% l e v e l by  LSD.  5.4. s e e d l i n g s were n e c r o t i c and c h l o r o t i c and  magnesium d e f i c i e n c y a c c o r d i n g  Western  o f the treatments  resulted and  t o Dr. K r a j i n a ) .  Hemlock  T h e r e were no s i g n i f i c a n t any  (symptoms o f c a l c i u m  (Table  differences i n height  3).  The NO^ -N  i n the lowest  leaf  area  dry weight.  With  the exception  shoot  among  treatment  and s m a l l e s t t o t a l , of leaf  root  area,  t h e r e was no d i f f e r e n c e i n any o f t h e p a r a m e t e r s m e a s u r e d between t h e N H - N  and ( N 0 ~ + N H ) - N  +  3  might have expected production  treatments.  +  4  4  a difference i n total  between t h e N H - N  and ( N 0  +  4  dry matter  + NH )-N 4  ments c o n s i d e r i n g t h e l a r g e d i f f e r e n c e i n l e a f l e a v e s o f s e e d l i n g s on t h e N H - N a n d ( N 0 +  4  dark green, areas. being  area.  The  + NH )-N +  3  4  were  however a few showed some n e c r o t i c a n d c h l o r o t i c  These areas indicative  supplied with  Lodgepole  treat-  +  3  One  were i d e n t i f i e d  by D r . V . J . K r a j i n a as  o f magnesium d e f i c i e n c y .  N0  3  The s e e d l i n g s  -N p r o d u c e d y e l l o w i s h g r e e n  leaves.  Pine  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 i n h e i g h t between the  three  N0  -N t r e a t m e n t  3  total +  (Table 4).  produced  d r y w e i g h t and l e a f  NH )-N 4  treatments  s e e d l i n g s produced  w e i g h t and t h u s  The s e e d l i n g s o n t h e  the lowest area.  shoot  The N 0  similar  3  dry weight,  -N a n d ( N 0  3  +  amounts o f r o o t d r y  differences i n total  d r y w e i g h t between  TABLE 3.  T o t a l , r o o t , and shoot dry weight l e a f area and h e i g h t o f Western s u p p l i e d with d i f f e r e n t sources o f n i t r o g e n .  hemlock  Source o f N i t r o g e n NO.  Total  dry weight  Root dry weight  (gm/crock) (gm/crock)  Shoot dry weight  (gm/crock) 2  Total  s u r f a c e area o f leaves  Shoot h e i g h t  (cm / p l a n t )  N0 ~ + N H 3  + 4  (7:1)  NH,  107.6  235.4  212.2  26.3  51.4  49.6  81.3  184.0  162.5  9078.2  22496.5  12341.9  49.6  47.6  44.3  (cm)  Values connected by the same l i n e do not d i f f e r a t the 5% l e v e l by LSD.  TABLE 4.  T o t a l , r o o t , and shoot dry weight, l e a f area and h e i g h t o f Lodgepole s u p p l i e d with d i f f e r e n t sources o f n i t r o g e n . 1 Source N0 3  T o t a l d r y weight Root dry weight  (gm/crock) (gm/crock)  Shoot dry weight Total  (gm/crock)  s u r f a c e area o f leaves  Shoot h e i g h t (cm)  2 (cm / p l a n t )  of Nitrogen  N0 ~ + N H 3  pine  + 4  (7:1)  NH  + 4  113. 2  210.0  229.7  48.6  67. 6  82.2  65.6  142.4  147. 5  2251.7  6460.8  6789.8  36.7  29.0  33.7  Values connected by the same l i n e do not d i f f e r a t the 5% l e v e l by LSD.  57.  these two treatments were due t o d i f f e r e n c e s i n shoot dry weight.  There was no d i f f e r e n c e i n any o f the parameters  measured between the NH^ -N and (NO^ +  The  leaves o f s e e d l i n g s  + N H ) - N treatments. +  4  t r e a t e d with NH -N and (NO^ + +  4  NH^ )-N were dark green while many leaves o f the n i t r a t e +  treatment were y e l l o w i s h green over t h e i r e n t i r e l e n g t h .  DISCUSSION The  experiment d e s c r i b e d  i n t h i s p a r t extends the  growth p e r i o d from the experiment d e s c r i b e d  i n P a r t 2-A.  There were, however, c e r t a i n d i f f e r e n c e s i n c u l t u r e techniques  between the two experiments: r e c e p t a c l e s f o r  growing, c o n c e n t r a t i o n o f n i t r o g e n , and the frequency o f nitrogen application. Both D o u g l a s - f i r and Western redcedar s e e d l i n g s on the NH -N treatment grew l e a s t over both the 4 month +  4  growing p e r i o d and over the 10 month growing p e r i o d . A t 4 months the N0 ~-N and (N0 ~ + N H ) - N Douglas+  3  fir  3  4  s e e d l i n g s were i n d i s t i n g u i s h a b l e from each o t h e r , b u t  a f t e r 10 months c e r t a i n d i f f e r e n c e s became n o t i c e a b l e . The  N0  higher due  3  -N treatment produced t a l l e r s e e d l i n g s and a t o t a l d r y weight.  This d i f f e r e n c e i n dry weight was  t o d i f f e r e n c e s i n root d r y weight. A t both 4 and 10 months, the trends  among n i t r o g e n  treatments with Western redcedar were e x a c t l y the same with the e x c e p t i o n  t h a t a d i f f e r e n c e i n l e a f area was  58.  d e t e c t a b l e b e t w e e n t h e N0 ~-N a n d t h e ( N 0 ~ + N H ) - N +  3  treatments periods,  3  a t t h e e n d o f 10 months.  the N0  3  4  Over both  -N t r e a t m e n t p r o d u c e d  treatment  the largest dry  weight. At  4 months, t h e o n l y e f f e c t  f o r W e s t e r n h e m l o c k was on l e a f t h e e n d o f t h e 12 month the N0 and  leaf  area.  —  t h e NH^ -N a n d ( N 0  3  by f a r t h e l o w e s t d r y m a t t e r  + NH^ )-N t r e a t m e n t s were except  treatment produced  +  4  s e e d l i n g s on  +  f r o m one a n o t h e r  NH )-N  At  O v e r b o t h t r e a t m e n t p e r i o d s , s e e d l i n g s on  +  able  a r e a and s u r v i v a l .  treatment period,  -N t r e a t m e n t p r o d u c e d  3  o f source o f n i t r o g e n  for leaf  area.  indistinguish-  The ( N 0  3  seedlings with the largest  + leaf  area. At  t h e e n d o f t h e 4 month t r e a t m e n t p e r i o d ,  N H - N was +  4  t h e most f a v o r a b l e s o u r c e o f n i t r o g e n f o r L o d g e p o l e Seedlings  from  t h e N0 ~-N a n d t h e ( N 0 ~ + N H ) - N +  3  were i n d i s t i n g u i s h a b l e  3  f r o m one a n o t h e r .  12 month t r e a t m e n t p e r i o d ,  the N0  3  +  4  guishable In  from  + NH )-N +  3  4  treatment  A t t h e end o f t h e  -N t r e a t m e n t  t h e l o w e s t d r y w e i g h t and s m a l l e s t l e a f the NH -N and (N0  4  pine.  area.  produced S e e d l i n g s on  t r e a t m e n t s were now  indistin-  one a n o t h e r .  c o n c l u s i o n t h e t r e n d s shown i n t h e 4 month  ments were c o n f i r m e d  i n the longer experiment  additional  treatment  effects  separating  the treatments with  experi-  and some  appeared, p a r t i c u l a r l y i n (N0  + NH )-N +  3  4  from  treat-  merits i n which only one form o f n i t r o g e n was present. A t the c o n c e n t r a t i o n s  o f n i t r o g e n used  (14 o r 28 ppm),  NO^ -N i s the most f a v o r a b l e form o f n i t r o g e n f o r Douglasfir  and Western redcedar  w h i l e NH. -N i s the most f a v o r a b l e 4 +  form o f n i t r o g e n f o r Lodgepole pine and Western hemlock. In P a r t 2-B, i t was shown t h a t NH -N was the predomi+  4  nant form o f n i t r o g e n taken up from the treatment.  (NO^  + NH^"") -N 1  A t t h i s low c o n c e n t r a t i o n o f NH -N +  4  D o u g l a s - f i r and Western redcedar  (1.75 ppm),  s u r v i v e d and grew b e t t e r  than when s u p p l i e d with a high r a t e o f NH -N +  4  At the end o f 10 months, Western redcedar  (14.0 ppm).  i n the lower  c o n c e n t r a t i o n o f NH -N a l s o gave b e t t e r growth than i n +  4  the higher c o n c e n t r a t i o n o f NH -N.  However a t both 4 and  +  4  10 months, the low NH -N treatment produced l e s s dry +  4  weight than the NO^ -N treatment.  The q u a n t i t y o f NH -N +  4  in  the low NH -N treatment may s t i l l  to  o b t a i n the optimum growth r a t e o f the s e e d l i n g s .  +  4  have been too  high  A t 4 months D o u g l a s - f i r s e e d l i n g s on the low NH -N and NO^ -N treatments were i n d i s t i n g u i s h a b l e from +  4  each other; however a t 10 months the NO^ -N treatment produced a g r e a t e r amount o f d r y matter than the low NH -N treatment. +  4  A t the end o f the 12 month p e r i o d the  q u a n t i t y o f NH -N i n the low NH -N treatment may s t i l l +  4  +  4  have been too high t o o b t a i n the optimum growth r a t e o f the s e e d l i n g s .  60.  With Lodgepole pine and Western hemlock at the end of 4 months, the NO^  -N treatment with one e x c e p t i o n produced  amounts o f dry matter s i m i l a r o t h e r two treatments.  However, a t the end of 12 months,  the NC> -N treatment produced 3  o t h e r two treatments.  to those produced on the  l e s s dry matter than the  The s e e d l i n g s may  to metabolize enough NO^  have been able  -N t o supply t h e i r needs when  they were s m a l l , but as the s e e d l i n g s i n c r e a s e d i n s i z e , they might not have been a b l e to a s s i m i l a t e or c o n v e r t enough NO.,  -N to meet t h e i r needs.  61. LITERATURE CITED 1.  Durzan, D.J. and F.C. Steward. 1967. The n i t r o g e n metabolism of P i c e a glauca and Pinus banksiana as i n f l u e n c e d by m i n e r a l n u t r i t i o n . Can. J . Bot. 45: 695-710.  2.  Swan, H.S.D. 1960. The m i n e r a l n u t r i t i o n o f Canadian pulpwood s p e c i e s . Pulp and Paper Research I n s t i t u t e o f Canada. Woodlands Research Index No. 116, Montreal, Canada.  PART 2-D.  GAS EXCHANGE OF CONIFER SEEDLINGS SUPPLIED WITH DIFFERENT FORMS OF NITROGEN.  INTRODUCTION In P a r t 2-C i t was shown t h a t D o u g l a s - f i r s e e d l i n g s t r e a t e d w i t h NH -N produced the lowest dry weight and l e a f +  4  area  (Tables 1 and 2 ) .  between the N 0  3  There was no d i f f e r e n c e i n l e a f  -N and (NO^  + NH^"*) -N s e e d l i n g s  area  although  1  the NO^ -N s e e d l i n g s produced a h i g h e r dry weight. With Western redcedar,  there was a d i r e c t  between t o t a l dry weight and l e a f area  relationship  ( i . e . the l a r g e r the  l e a f area, the l a r g e r the dry matter p r o d u c t i o n ) .  The  NO^ -N t r e a t e d s e e d l i n g s produced the l a r g e s t l e a f and the l a r g e s t amount o f dry matter, (N0 ~ 3  + N H ) - N treatment +  4  area  f o l l o w e d by the  and then the NH -N +  4  treatment.  NH -N and (N0 ~ + N H ) - N t r e a t e d Western hemlock +  +  4  3  4  s e e d l i n g s produced s i m i l a r amounts o f dry matter,  although  there was a l a r g e d i f f e r e n c e i n l e a f area.  3  The N0 -N  t r e a t e d s e e d l i n g s produced the l e a s t amount o f dry matter and l e a f  area.  Lodgepole pine s e e d l i n g s s u p p l i e d with N0 the lowest dry weight and l e a f area.  -N produced  3  There was no d i f f e r -  ence i n dry weight and l e a f area between the NH -N and +  4  (NO ~ + N H „ ) - N +  treatments.  TABLE 1.  T o t a l dry weight (gm.pot~l).1  of c o n i f e r s e e d l i n g s s u p p l i e d w i t h d i f f e r e n t sources o f n i t r o g e n  Species  Nitrogen NO3-  Treatment  N 0 ~ + NH 3  + 4  (7:1)  NH  + 4  Douglas-fir  167.4  133. 4  68.8  Western redcedar  201.5  160. 7  37.6  Lodgepole  113.2  210.0  229.7  107.6  235. 4  212.2  pine  Western hemlock  Values connected by the same l i n e do not d i f f e r a t the 5% l e v e l by LSD.  TABLE 2.  Leaf area of c o n i f e r s e e d l i n g s s u p p l i e d with d i f f e r e n t (cm2.pot~l).1  Species  sources o f n i t r o g e n  N i t r o g e n Treatment N0 3  N0 ~ + N H 3  + 4  (7:1)  NH  + 4  Douglas-fir  11,710.2  8,561.8  3,243.6  Western redcedar  12,820.0  10,120.0  2 ,820.0  4,503.4  12,921.6  13,579.6  18,156.4  44 ,993.0  24,683.8  Lodgepole pine Western hemlock  Values connected by the same l i n e do not d i f f e r a t the 5% l e v e l by LSD.  The  l a c k o f c o r r e l a t i o n between l e a f area and  dry weight w i t h D o u g l a s - f i r t h a t the of the  nitrogen  leaves.  and  Western hemlock suggested  treatment was  The  plant  a f f e c t i n g the  gas  exchange  r e s u l t s presented i n t h i s paper suggest  trends i n stomatal r e s i s t a n c e water r e l a t i o n s and  (r ) which would a f f e c t both s exchange.  gas  SYMBOLS AND (C)„) z a  External  ^ 2^c  Chloroplast  C 0  Co_  UNITS  concentration  2.  Transpiration  (gm  _3  (gm  cm  sec  )  C0  RH r a  R e l a t i v e humidity Boundary l a y e r d i f f u s i o n r e s i s t a n c e  r  m  Mesophyll d i f f u s i o n r e s i s t a n c e  r  g  Stomatal d i f f u s i o n r e s i s t a n c e  (H„0)  flux  (gm  cm  )  -1  P  2  )  -1  cm  -2  cm  _3 •  CO2 c o n c e n t r a t i o n -2  T  (gm  min  ) (sec cm  (sec cm (sec  cm  Water vapor c o n c e n t r a t i o n  of a i r (gm  (H 0)  Water vapor c o n c e n t r a t i o n (gm cm"3)  of stomatal  V  T o t a l water p o t e n t i a l  ¥  Gravitational potential  g H* m  Matric  2, 2  a  potential  Pressure p o t e n t i a l ¥  s  Solute p o t e n t i a l  (bar)  (bar) (bar) (bar)  (bar)  "*")  cm  -3  )  cavity  "*")  THEORY I n t e r n a l water s t r e s s may energy s t a t u s  of the  ( *¥ ) ( 1 ) .  systems, H* i s g i v e n by  The  s  T  i n terms of  contained water, u s u a l l y  t o t a l water p o t e n t i a l  T  be d e s c r i b e d  the  p  matric p o t e n t i a l  (Y  expressed  In both p l a n t  and  as  soil  equation  m  1  the  T  g  ) and  gravitational potential  (  m are u s u a l l y  g  n e g l e c t e d f o r p l a n t systems and  be w r i t t e n  +  c  ¥  fa  Transpiration _  T  (ID  (H 0)  s  2  9  s  -  (H_0) 2  + r  (H 0)  -  o  ,  a  .  (III)  a (H_0)  i s calculated  ments of r e l a t i v e humidity, assuming 100% stomates.  L i k e w i s e the  flow of CX^  RH  from measureinside  i s c o n t r o l l e d by  '"" S  usua  (CO  )  -  2  a  (CO  ) £_£ r + r + r s a m  ^-'-y assumed to be  f o r P can be  calculated  ( I V )  zero.  The  from T,  resistances  taking  i n t o account  the d i f f e r e n t d i f f u s i o n c o e f f i c i e n t s of water vapor C0  the  relationship P =  + r  may  i s g i v e n by  For p l a n t s  r  (I)  p  r  ^^2^c  equation  as  V = T  similar  )  in air.  and  METHODS AND MATERIALS Four c o n i f e r s p e c i e s , D o u g l a s - f i r , Western hemlock, Lodgepole pine and Western redcedar sand. The  were grown i n pots o f  A f t e r t h i n n i n g , each p o t contained  2 seedlings.  s e e d l i n g s were s u p p l i e d with complete n u t r i e n t s o l u -  t i o n i n which only the source  o f n i t r o g e n was v a r i e d .  Nitrogen was s u p p l i e d as ammonia o n l y , n i t r a t e only o r as a combination o f n i t r a t e and ammonia (7:1). these and  (Hereafter  treatments w i l l be r e f e r r e d t o as NH -N, NO^ -N +  4  (N0  + NH )-N). +  3  4  The n i t r o g e n content  s o l u t i o n s was 14 ppm N f o r the f i r s t thereafter.  (Part 2-C).  s e e d l i n g s were grown i n a growth room with 90%  RH i n the day and 65% a t n i g h t . descent  7 weeks and 2 8 ppm  Exact d e t a i l s o f the c u l t u r e technique are  d e s c r i b e d elsewhere The  o f the  lamps p r o v i d e d  16 h r (6 am t o 10 pm).  F l u o r e s c e n t and i n c a n -  20,000 lux over a photoperiod o f Dawn and t w i l i g h t e f f e c t s were  produced by s t a g g e r i n g the order i n which the l i g h t s came on and went o f f .  A 12 h r temperature c y c l e was used  (8 am t o 8 pm a t 2 4C;  8 pm t o 8 am a t 20C).  D o u g l a s - f i r and Western redcedar harvested  10 months a f t e r germination  s e e d l i n g s were while  pine and Western hemlock were harvested germination.  Lodgepole  12 months a f t e r  68.  Leaf M* and  H*  were measured w i t h a three wire  thermocouple psychrometer ( 2 ) . d e s c r i b e d by E h l i g (II).  (3).  The  was  techniques used  determined  Leaf samples were taken at 9 am  p o i n t o f the t r e e s . T r a n s p i r a t i o n was  Each t r e e was  determined  are  from e q u a t i o n  from the growing  sampled 3 times.  by c o v e r i n g the pot w i t h a  sheet of P a r a f i l m and measuring the l o s s of water over a 24 h r p e r i o d .  A f t e r d e t e r m i n a t i o n of t r a n s p i r a t i o n ,  the t r e e s were h a r v e s t e d and oven d r i e d at  RESULTS AND Leaf  T,  V , s  V  80C.  DISCUSSION  p  S e n s i t i v i t y of the thermocouples ranged .29 /tv per bar.  from  .23 to  For r o u t i n e determinations w i t h the  psychrometer the standard e r r o r of o b s e r v a t i o n of s t a n dard KCl s o l u t i o n s was There was  about - .5 bar.  no d i f f e r e n c e i n l e a f  V,  y  and  H*  s among the d i f f e r e n t n i t r o g e n treatments. value of l e a f  V  y  and s  species  (Table 3 ) .  Y  was  p  Therefore  determined  one  f o r each  p  The mean values of l e a f V f o r Douglas-  f i r , Western redcedar, Lodgepole  pine and Western hemlock  were -21.2, -11.8, -10.6  and -18.8  bars w i t h  e r r o r s of .8, .5, .3 and  .7 b a r s .  The  standard  standard e r r o r of  Leaf V , (bars).  TABLE 3.  *V  s  and  of c o n i f e r  Species  seedlings supplied with  d i f f e r e n t sources of n i t r o g e n  *s  Douglas-fir  -21.2  -34.0  +12. 8  Western redcedar  -11. 8  -21. 4  +9.7  Lodgepole  -10.6  -24.5  +13.9  -18. 8  -29.4  +10.6  pine  Western hemlock Nutrient NH  Solution -.77  + 4  N 0 ~ + NH 3  NO3-  + 4  -.65 -.43  VO  70.  l e a f *¥$ f o r D o u g l a s - f i r , W e s t e r n r e d c e d a r , and  W e s t e r n h e m l o c k were 1.1, 3 c o n t a i n s *f f o r t h e  Table The  of V  values  reported »j>  reported i n Table  -16  conifers  .4  t o -31 (8) .  and  .6  3 are s i m i l a r (4, 5,  pine  respectively.  respective nutrient  f o r o t h e r woody s p e c i e s  of from  green  .6,  Lodgepole  6).  solutions. to  values  Values  of  b a r s have b e e n r e p o r t e d f o r e v e r o f *f  Values  r e p o r t e d here  fall  close  s to this has  range.  been The  within  similar  Table  leaf  4* among t h e n i t r o g e n  species indicates  t h a t t h e r e was  between the n i t r o g e n  no  amounts o f w a t e r l o s t  sampling  difference  in  f o r the  leaf  from  potential  the pots  at the  time  of  s t u d i e s are given i n  4.  treatment  lost  treatment  and  3  treatments  treatments.  W i t h D o u g l a s - f i r and W e s t e r n r e d c e d a r ,  (N0 ~  for trees  Uptake The  leaf  v a l u e o f Hf  o u r k n o w l e d g e no  reported.  water d e f i c i t Water  To  1/3 1/2  + NH )-N +  4  Lodgepole approximately treatments.  t o 1/4 t o 1/3  treatment pine  1/2  the the  the  +  4  amount o f w a t e r o f t h e NO^ amount o f w a t e r o f  the  (Table 4).  s e e d l i n g s s u p p l i e d w i t h NO^  the  NH -N  -N  amount o f w a t e r o f t h e o t h e r  lost two  -N  TABLE 4.  Water l o s s o f c o n i f e r s e e d l i n g s s u p p l i e d w i t h d i f f e r e n t sources of n i t r o g e n (ml H 0/24 hr.pot) . 2  Species  Nitrogen Treatment NO3-  N 0 ~ + NH 3  + 4  (7:1)  NH . 4  +  Douglas-fir  683.7  403.2  180. 3  Western  627.2  554.2  203.2  Lodgepole pine  222.2  486.3  395.1  Western hemlock  482.4  553.6  324.7  redcedar  Western hemlock s e e d l i n g s t r e a t e d w i t h l o s t the most water f o l l o w e d by the NO^  -N  (NC>  + NH )-N +  3  4  and NH -N t r e a t +  4  ments . Since t r a n s p i r a t i o n i s considered  an unavoidable  evil  (7) which o f t e n r e s u l t s i n water d e f i c i t s and because was  no d i f f e r e n c e i n water d e f i c i t  n i t r o g e n treatments,  we  there  (Table 3) among the  suggest t h a t d i f f e r e n c e s i n growth  among n i t r o g e n treatments w i t h i n s p e c i e s cannot be a t t r i b u t e d d i r e c t l y t o d i f f e r e n c e s i n water r e l a t i o n s . l e a f *? may  have been maintained  by c o n t r o l l i n g T v i a r . g  As p r e v i o u s l y s t a t e d stomatal CO  i s determined from T.  2  p l a n t s may  have had  However s i m i l a r  r e s i s t a n c e t o flow  of  Thus the water r e l a t i o n s of  an i n d i r e c t e f f e c t on y i e l d i . e . by  l i m i t i n g the r a t e of e n t r y of C0 .  Thus r  culated according  In t h i s experiment  o  r  a  i s considered  to equation  III.  of r  + r S  o  c  i s considered  t o be s m a l l .  a.  because  and  Thus c a l c u l a t i o n a l l variability  + r cl  + r  shows t h a t f o r Western redcedar, p i n e , there was  cal-  s i s attributable to r . A s a s (Table 5) with dry weight (Table 1)  .  w i t h i n s p e c i e s between r S  was  a i r c i r c u l a t i o n i n the  gives an approximation of r  comparison of r  + r  to be constant w i t h i n s p e c i e s and  of the s m a l l s i z e of leaves and growth room, r  these  D o u g l a s - f i r and  l i t t l e difference in r  Lodgepole  + r among n i t r o g e n s a treatments but a l a r g e d i f f e r e n c e i n y i e l d . With Western  TABLE 5.  T r a n s p i r a t i o n r e s i s t a n c e (r + r ) of c o n i f e r s e e d l i n g s d i f f e r e n t sources of n i t r o g e n ( s e c e r n ! ) .  supplied with  -  Species  Nitrogen  Treatment  N0 ~  N0 ~ + N H  Douglas-fir  3.3  4.0  3.3  Western redcedar  3.4  3.9  3.2  Lodgepole pine  3.9  5.0  6.5  Western hemlock  7.2  15.5  14.4  3  3  + 4  (7:1)  NH  + 4  hemlock, t h e r e was  a d i r e c t r e l a t i o n s h i p between r + r ^ s a and y i e l d i . e . the h i g h e r the v a l u e of r + r , the S  h i g h e r the y i e l d .  to support c a u s a l i t y . case i s r  s  + r  a  cl  An i n v e r s e r e l a t i o n s h i p would be needed These r e s u l t s i n d i c a t e that i n no  r e s t r i c t i n g dry matter p r o d u c t i o n .  These  r e s u l t s suggest t h a t the e f f e c t of n i t r o g e n treatment  on  r ^ had an e f f e c t on dry matter p r o d u c t i o n .  I t has been  r e p o r t e d f o r Pinus h a l p e n s i s t h a t l i g h t and  temperature  a f f e c t e d CO,, uptake and Kozlowski  through  S i m i l a r l y , Wuenscher  (10) found t h a t f o r three woody s p e c i e s at  3000 f t - c the d i f f e r e n c e s i n r differences i n r resistance.  (9) .  m  were more important than  i n determining t o t a l C 0  9  transfer  LITERATURE CITED B a r r s , H.D. 1968. Determination of water d e f i c i t s i n p l a n t t i s s u e , pp. 235-368. I_n T.T. Kozlowski (ed.) Water d e f i c i t and p l a n t growth, V o l . I. Academic P r e s s , New York. Chow, T.L. and J . de V r i e s . 1972. Dynamic measurement of s o i l and l e a f water p o t e n t i a l w i t h a double loop p e l t i e r type thermocouple psychrometer. Western S o c i e t y S o i l Science meeting a t Oregon June 12-15. E h l i g , G.F. 1961. Measurement of the energy s t a t u s of water i n p l a n t s w i t h a thermocouple psychrometer. P l a n t P h y s i o l . 37: 288-290. Kaufmann, M.R. 196 8. E v a l u a t i o n of the pressure chamber technique f o r e s t i m a t i n g p l a n t water p o t e n t i a l o f f o r e s t t r e e s p e c i e s . F o r e s t S c i . 14: 369-374. Kaufmann, M.R. 1968. Water r e l a t i o n s of pine s e e d l i n g s i n r e l a t i o n t o r o o t and shoot growth. P l a n t P h y s i o l . 43: 281-288. Klepper, B. 196 8. D i u r n a l p a t t e r n s o f water p o t e n t i a l i n woody p l a n t s . P l a n t P h y s i o l . 43: 1931-1934. Kramer, P. 1969. T r a n s p i r a t i o n pp. 296-346. In P l a n t and s o i l water r e l a t i o n s h i p s : a modern s y n t h e s i s . McGraw-Hill Book Company. P i s e k , A. 1956. Der wasserhaushalt der mesa und Hygrophyten. Enclop. P l a n t P h y s i o l . 3: 825-853. Whiteman, P.C. and D. K o l l e r . 1965. Environmental cont r o l of p h o t o s y n t h e s i s and t r a n s p i r a t i o n i n Pinus h a l e p e n s i s . I s r a e l J . Bot. 13: 166-176. Wuenscher, J.E. and T.T. Kozlowski. 19 . Carbon d i o x i d e t r a n s f e r r e s i s t a n c e as a f a c t o r i n shade t o l e r a n c e of t r e e s e e d l i n g s . Can. J . Bot. 48: 453-456.  76. APPENDIX I The r e l a t i o n s h i p between l e a f a r e a a n d l e a f d r y w e i g h t o f t h r e e c o n i f e r s p e c i e s grown on t h r e e s o u r c e s o f n i t r o g e n . 1  Determination  of leaf  area  for p h y s i o l o g i c a l experiments. conifer small this  l e a f area  size  trees,  and  a r e a v a i l a b l e (1, 2, 4, 5, 6, 7 ) , b u t t h e  (4).  area  dry weight  of fascicles.  i s required  the other  tionship  Once t h i s  t o determine  hand, B r i x  d i d not occur  area  varying  effect  of different  between l e a f  surface  of fascicle  surface  for Douglas-fir seedlings light  In our study,  surface  area  area  rela-  between  the leaf  surface  the r e l a -  and l e a f d r y w e i g h t o f  An a d d i t i o n a l s t u d y  sources  area.  intensities  o f e a c h s e e d l i n g was m e a s u r e d t o d e t e r m i n e  18-week-old s e e d l i n g s .  relation-  (3) h a s shown t h a t a l i n e a r  temperatures.  t i o n s h i p between l e a f  in  dry weight  initial  fascicle  with  C a b l e (4)  the determination  ages 65 a n d 100 d a y s grown a t v a r y i n g and  area  F o r mature Ponderosa p i n e ,  a l i n e a r r e l a t i o n s h i p between f a s c i c l e  surface  make  a l a r g e number o f  method i s t o c o r r e l a t e l e a f  s h i p has been e s t a b l i s h e d o n l y  On  shape o f t h e l e a v e s  F o r experiments using  the simplest  l e a f d r y weight found  S e v e r a l methods o f e s t i m a t i n g  and t h r e e - d i m e n s i o n a l  difficult.  i s a f u n d a m e n t a l measurement  of nitrogen  was made o n t h e  on t h e r e l a t i o n s h i p  and l e a f d r y w e i g h t o f e n t i r e  1 T h i s a r t i c l e by G.E. M e l l o r Can. J . F o r . Res. i n P r e s s .  and E.B. T r e g u n n a w i l l  appear  seedlings. Three c o n i f e r s p e c i e s , D o u g l a s - f i r  (Pseudotsuga  m e n z i e s i i v a r . m e n z i e s i i ) , Lodgepole pine v a r . contorta)  and Western hemlock  (Pinus  contorta  (Tsuga h e t e r o p h y l l a )  were grown f o r 18 weeks i n sand i n a growth chamber under conditions described previously  (8).  The  s e e d l i n g s were  given n u t r i e n t s o l u t i o n i n which only the source of n i t r o g e n and the c o n c e n t r a t i o n o f c h l o r i d e were v a r i e d . d i f f e r e n t sources  of n i t r o g e n  ( n i t r a t e o n l y , ammonia o n l y ,  and a combination of n i t r a t e and ammonia A l l treatments contained germination,  14 ppm  N.  (7:1) were used.  A t 18 weeks a f t e r  f i v e s e e d l i n g s of each s p e c i e s from each  n i t r o g e n source were s e l e c t e d a t random and were removed.  The  o f the leaves was  the  leaves  l e n g t h and width of the leaves were  measured by means o f a micrometer.  The  t o t a l surface  area  determined by m u l t i p l y i n g l e n g t h times  width times a c o r r e c t i o n f a c t o r . accounts f o r l o n g i t u d i n a l and The  Three  The  correction factor  c r o s s e c t i o n a l curvature.  c o r r e c t i o n f a c t o r s f o r D o u g l a s - f i r and Western hemlock  were taken from Barker Lodgepole pine was  (2) and  the c o r r e c t i o n f a c t o r f o r  taken from Smith  of each s e e d l i n g was  (9).  Leaf dry weight  determined by d r y i n g the leaves i n  an oven at 80 C to constant weight. between l e a f dry weight and  The r e l a t i o n s h i p  l e a f s u r f a c e area was  mined by c a l c u l a t i n g the c o r r e l a t i o n c o e f f i c i e n t  deter(r).  The  78.  l e a f s u r f a c e area t o l e a f d r y weight r e l a t i o n s h i p among n i t r o g e n treatments  w i t h i n s p e c i e s was analyzed by t e s t i n g  f o r homogeneity o f r v a l u e s . With a l l three c o n i f e r s p e c i e s , no d i f f e r e n c e was found between r values determined  f o r the three n i t r o g e n  sources.  Thus a pooled r value was determined  species.  The pooled r values were: Lodgepole pine  Western hemlock  f o r each  (.967) and D o u g l a s - f i r (.927).  (.949),  These  values are s i g n i f i c a n t a t the 5% l e v e l and i n d i c a t e a l i n e a r r e l a t i o n s h i p between l e a f d r y weight and l e a f of e n t i r e s e e d l i n g s .  area  A f t e r the l i n e a r r e l a t i o n s h i p was  e s t a b l i s h e d a r e g r e s s i o n c o e f f i c i e n t was determined and the r e g r e s s i o n l i n e drawn u s i n g the formula y = bx ( F i g s . 1,  2, 3) .  This l i n e a r r e l a t i o n s h i p between t o t a l l e a f area and l e a f dry weight p r o v i d e s an easy  and q u i c k method f o r  d e t e r m i n a t i o n o f l e a f area, once the i n i t i a l is  found.  relationship  A l i n e a r r e l a t i o n s h i p indicates that i n experi-  ments comparing p h o t o s y n t h e t i c o r t r a n s p i r a t i o n r a t e s o f s e e d l i n g s grown on d i f f e r e n t sources o f n i t r o g e n , the d i f f e r e n c e s i n r e s u l t s between n i t r o g e n treatments  will  be o f the same magnitude whether u n i t s o f l e a f dry weight or l e a f area a r e used.  Figure  1.  The  r e l a t i o n s h i p between l e a f  l e a f dry weight supplied with nitrogen.  area  for Douglas-fir  different  sources  and  seedlings of  LEAF  SURFACE  AREA  (cm ) 2  Figure  2.  The leaf  relationship  between l e a f  area  dry weight  f o r Lodgepole  pine  seedlings sources of  supplied with nitrogen.  different  and  Figure  3.  The r e l a t i o n s h i p  between  leaf  area  l e a f d r y w e i g h t f o r Western hemlock seedlings sources  supplied with  of nitrogen.  different  and  LEAF  DRY  WEIGHT  85.  LITERATURE CITED 1.  Baker, F.S. 194 8. A s h o r t method o f determining l e a f area and volume growth i n pine t r e e s . H i l g a r d i a 18: No. 8.  2.  Barker H. 196 8. Methods o f measuring l e a f s u r f a c e area of some c o n i f e r s . Canadian F o r e s t r y Branch Department P u b l i c a t i o n No. 1212 6 p.  3.  B r i x , H. 1967. An a n a l y s i s o f dry matter p r o d u c t i o n o f D o u g l a s - f i r s e e d l i n g s i n r e l a t i o n t o temperature and l i g h t i n t e n s i t y . Can. J . Bot. 45: 2063-2072.  4.  Cable, D.R. 1958. E s t i m a t i n g s u r f a c e area o f Ponderosa pine f o l i a g e i n c e n t r a l A r i z o n a . F o r . S c i . 4: 45-49.  5.  Kozlowski, T.T., and F. Schumacher. 1943. E s t i m a t i o n o f stomated f o l i a r s u r f a c e o f p i n e . P l a n t P h y s i o l . 18: 122-127.  6.  Kramer, P.J. 19 37. An improved p h o t o - e l e c t r i c apparatus f o r measuring l e a f area. Amer. J . Bot. 24: 375-376.  7.  Madgwick, H.A.I. 1964. E s t i m a t i o n o f s u r f a c e area o f pine needles with s p e c i a l r e f e r e n c e to Pinus r e s i n o s a . J . F o r e s t 62: 636.  8.  Madoc-Jones, S. 1970. The n i t r o g e n n u t r i t i o n o f some c o n i f e r s o f B r i t i s h Columbia, pp. 16-23 In Progress Report: N a t i o n a l Research C o u n c i l Grant No. A-92. V . J . K r a j i n a (ed). Botany Department, U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, B.C., Canada.  9.  Smith, J.H.G. 1970. Weight, s i z e , and p e r s i s t a n c e o f needles o f D o u g l a s - f i r , Western hemlock, Western redcedar and o t h e r B r i t i s h Columbia c o n i f e r s . Mimeographed r e p o r t , F o r e s t r y Department, U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, B.C., Canada.  86.  EPILOGUE In P a r t 2, I s t u d i e d the e f f e c t s o f NH -N and N0 ~-N +  4  on four s p e c i e s o f c o n i f e r s .  I have concluded  s t u d i e s t h a t Western hemlock and Lodgepole  3  from  these  pine are more  t o l e r a n t o f NH -N than D o u g l a s - f i r and Western redcedar. +  4  The exact l e v e l o f NH -N t h a t these s p e c i e s w i l l  tolerate  +  4  should be determined.  T h i s experiment  should be c a r r i e d  out w i t h a w e l l - s t i r r e d l i q u i d c u l t u r e i n which t h e l e v e l of n i t r o g e n i s monitored  daily.  In t h i s manner the l e v e l  of n i t r o g e n i n c o n t a c t w i t h the r o o t s u r f a c e would n o t vary t o any great  degree.  A study o f the n i t r a t e - r e d u c i n g enzymes i n these s p e c i e s should a l s o be 'carried out.  forest  Such a study would  i n d i c a t e the a b i l i t y o f these s p e c i e s t o metabolize NO^ -N. A b r i e f attempt was made t o e x t r a c t n i t r a t e However i t was n o t p o s s i b l e t o e x t r a c t p r o t e i n  reductase.  precipitable  by t r i c h l o r a c e t i c a c i d from D o u g l a s - f i r , l e t alone reductase.  nitrate  One ml o f a D o u g l a s - f i r e x t r a c t was added t o a  very a c t i v e n i t r a t e reductase p r e p a r a t i o n from c o r n .  The  n i t r a t e reductase from c o r n was completely i n h i b i t e d by a d d i t i o n o f the D o u g l a s - f i r e x t r a c t . In a d d i t i o n , a study o f the amino a c i d p o o l s i n these s p e c i e s might g i v e f u r t h e r i n s i g h t i n t o the a b i l i t y o f these s p e c i e s t o t o l e r a t e v a r i o u s l e v e l s o f ammonia.  87.  PART 3.  A STUDY OF STORAGE CONDITIONS FOR MUD-PACKED DOUGLAS-FIR SEEDLINGS.  SPRING-LIFTED 1  INTRODUCTION In B r i t i s h Columbia, r e f o r e s t a t i o n by p l a n t i n g makes use  of 1-0  and  2-0  seedlings.  The  s e e d l i n g s are grown i n  n u r s e r i e s , l i f t e d between November and A p r i l , i n the dark and  then p l a n t e d i n the  stored at  2C  field.  A c u r r e n t method of p l a n t i n g employs mud-packed seedlings.  Mud-packing i s done by surrounding  the  root  system of the s e e d l i n g s with a mixture o f peat moss, c l a y and water formed i n t o a c y l i n d e r .  The mud  i s then d r i e d  i n a stream of warm a i r u n t i l i t i s s e m i - r i g i d .  In  c o n d i t i o n the r o o t system i s thought to be p r o t e c t e d d e s s i c a t i o n and p h y s i c a l damage.  The  by punching a h o l e i n the ground with  s e e d l i n g s are  the mud-capsule i s p l a c e d i n the  l e a v i n g the stem and ground.  from planted  a d i b b l e or s t e e l  to the same depth as the l e n g t h of the mud-capsule. i s withdrawn and  this  rod  The  rod  hole,  the needles of the s e e d l i n g above the  S o i l i s tramped down t i g h t l y a g a i n s t the mud-pack.  A c r i t i c a l p e r i o d i n the establishment the time between l i f t i n g and  of s e e d l i n g s i s  subsequent r e g e n e r a t i o n  of a  1 T h i s s e c t i o n which i s p a r t of an a r t i c l e by G.E. M e l l o r , R.A. K e l l e r , and E.B. Tregunna w i l l appear i n Canadian J o u r n a l of F o r e s t Research. E.B. Tregunna s u p e r v i s e d t h i s study.  88.  new  r o o t system.  Water s t r e s s due  p l a n t d e s s i c a t i o n may of s e e d l i n g s  occur  for planting.  to r o o t , shoot, or whole  i n the p r e p a r a t i o n Water s t r e s s may  and  also  storage occur  a f t e r p l a n t i n g when, because of r o o t i n j u r y or d i s r u p t i o n of the s o i l - p l a n t - a i r continuum, t r a n s p i r a t i o n exceeds water uptake  (1).  In t h i s study, the e f f e c t of v a r i o u s on s u r v i v a l and  root regeneration  storage  of s p r i n g - l i f t e d  packed D o u g l a s - f i r s e e d l i n g s i s examined. shown t h a t D o u g l a s - f i r s e e d l i n g s l i f t e d are damaged by the f i e l d  f o u r weeks of storage  lings.  after April  at 2C.  He  years,  1st  reduced by  seedlings  A p r i l 1st have been used f o r mud-packing.  has  found t h a t  the s u r v i v a l of unstored  During the l a s t two  mud-  Winjum (4)  s u r v i v a l of s t o r e d s e e d l i n g s was  to 90% when compared with  conditions  30  seed-  lifted  after  In many i n s t a n c e s  these mud-packed s e e d l i n g s have been p l a c e d i n storage a p e r i o d of two study was  to three weeks at 2C.  The  o b j e c t of  for  this  to determine the best method of s t o r i n g s p r i n g -  l i f t e d mud-packed D o u g l a s - f i r s e e d l i n g s .  As  mentioned  p r e v i o u s l y , a c r i t i c a l p e r i o d i n the establishment  of seed-  l i n g s i s the time between l i f t i n g and r e g e n e r a t i o n  of a  new  r o o t system.  Thus v a r i o u s storage  c o n d i t i o n s were  chosen to determine i f r o o t r e g e n e r a t i o n  c o u l d be enhanced.  89. MATERIALS AND METHODS During the week o f A p r i l 19th, 1970, 2-0  Douglas-fir  t r e e s grown a t Green Timbers F o r e s t Nursery from seed l o t (92G14/B2/1020/201) were l i f t e d and mud-packed a t P e l t o n R e f o r e s t a t i o n Company.  Immediately a f t e r mud-packing, 220  s e e d l i n g s were d i v i d e d i n t o 11 storage r e p l i c a t i o n s x 10 t r e e s )  (Table 1 ) .  t r e e s i n t h e i r r e s p e c t i v e storage  treatments (2  Before p l a c i n g the  treatments, the mud-  packed p o r t i o n o f the s e e d l i n g was soaked f o r 10 min i n water o r 1/2-strength Hoagland's s o l u t i o n .  After  the mud-packs were d r i e d t o t h e i r o r i g i n a l condition.  The s e e d l i n g s  The s e e d l i n g s  were p l a c e d  s t o r e d i n the l i g h t  i n order  (25C and f i e l d )  to keep the t r e e s i n an u p r i g h t  The p o r t i o n o f each s e e d l i n g above the mud-pack  was completely exposed.  F o r the v e r m i c u l i t e  wet v e r m i c u l i t e was added t o the bags. i n the f i e l d were p l a c e d  treatments,  The t r e e s  stored  i n a s h e l t e r which had only the  top covered with p o l y e t h y l e n e s l i g h t l y opaque and p r o v i d e d and  Reforestation  i n p l a s t i c bags which were then t r a n s f e r r e d t o  a r i g i d container position.  semi-rigid  s t o r e d i n the dark were p l a c e d  i n p l a s t i c bags, as i s done by the P e l t o n Company.  soaking,  plastic.  The p l a s t i c was  s h e l t e r from both d i r e c t sun  rain. The  mud-packs kept i n cartons  t h e i r moisture d u r i n g the storage mud-packs l o s t moisture.  i n the dark r e t a i n e d p e r i o d w h i l e the exposed  The exposed mud-packs a t 25C had  TABLE 1.  Storage treatments and c o n d i t i o n s .  2C Room (dark)  IOC Room (dark)  Field '  - fertilizer  - fertilizer  - vermiculite - f e r t i l i z e r  - vermiculite - f e r t i l i z e r  + light  + fertilizer  + fertilizer  + vermiculite - f e r t i l i z e r  + vermiculite - f e r t i l i z e r  + light  + vermiculite + f e r t i l i z e r  + vermuculite + f e r t i l i z e r  + light  - vermiculite - f e r t i l i z e r  - light  a  25C Room  b  c  Field  r e f e r s to the roof o f the Bio-Sciences B u i l d i n g , U n i v e r s i t y o f B r i t i s h  Field  conditions  Ave.  during  Maximum d a i l y Range  25C Room c o n d i t i o n s  Columbia.  storage  Temperature 12.6C Ave. Minimum d a i l y Range  9.5C-18.0C during  storage  1000 f t - c a n d l e s , 16 hour day  Ave. RH 65%  Temperature 5.6C  2.5C-9.0C  Ave. RH 7 5%  91.  to be moistened d a i l y and the mud-packs i n the f i e l d had to be moistened d a i l y or every other day depending on the weather c o n d i t i o n s .  The mud-packs were moistened w i t h  a f i n e spray o f water.  Holes f o r drainage were c u t i n the  bottom o f the bags.  In s p i t e of the f a c t that as l i t t l e  water as p o s s i b l e was used f o r m o i s t e n i n g , water drainage from the bags was poor.  This was p a r t i c u l a r l y true of  the v e r m i c u l i t e treatments. During storage  (8th, 11th, 14th and 18th days) r o o t  growth and s u r v i v a l were measured.  A s e e d l i n g was con-  s i d e r e d t o be dead when i t s needles were brown and dropping and the stem was d r y , b r i t t l e Root growth was evaluated protruding  and e a s i l y broken.  by measuring the amount o f r o o t  from the mud-pack.  A f t e r 19 days s t o r a g e ,  the s e e d l i n g s were p l a n t e d i n  the UBC B o t a n i c a l Garden a t 10 i n c h i n t e r v a l s f t apart.  Each row c o n s i s t e d o f one r e p l i c a t e .  to minimize the e f f e c t o f moisture s t r e s s , moistened p e r i o d i c a l l y out.  i n rows 2 In order  the s o i l was  t o prevent the mud-packs from d r y i n g  A f t e r 44 days, the s e e d l i n g s were dug up and were  evaluated  on the b a s i s of s u r v i v a l  In e v a l u a t i n g the r e s u l t i n g  and r o o t growth.  data,  were used followed by Tukey s -latest 1  RESULTS AND  analyses  of v a r i a n c e  («=*= .05).  DISCUSSION  F e r t i l i z e r and v e r m i c u l i t e treatment of the mud-packs  had no e f f e c t on any o f the parameters measured.  Therefore  the i n d i v i d u a l treatments w i t h i n each storage l o c a t i o n were combined except i n the 2 5C treatments. ments the (+) l i g h t treatments the  (-) l i g h t  With the 25C t r e a t -  are r e p o r t e d s e p a r a t e l y from  treatments.  A f t e r 19 days i n storage there were no r o o t s v i s i b l e on the o u t s i d e o f the mud-packs from any of the storage  locations.  S u r v i v a l o f the t r e e s a t the end of the storage treatment i s shown i n Table 2. produced  Storage o f the t r e e s a t 25C  significantly  treatments.  (+) l i g h t  lower storage s u r v i v a l than the o t h e r  A l l o t h e r storage treatments were s t a t i s t i c a l l y  indistinguishable. The maximum d a i l y temperature p e r i o d ranged  ranged  from 4.0C t o 20C with  (Table 2) was computed on the b a s i s o f  the number of s e e d l i n g s p l a n t e d . f i e l d had by f a r the h i g h e s t f i e l d storage treatments  had a f i e l d  The t r e e s s t o r e d i n the survival.  A l l other  s u r v i v a l o f l e s s than 53%.  low s u r v i v a l of s p r i n g - l i f t e d s e e d l i n g s a t 2C, agrees  w i t h the f i n d i n g s of Winjum field  (4). In a d d i t i o n t o s t u d y i n g  and storage s u r v i v a l i n d i v i d u a l l y the combined  survival 2).  o f 19C.  of 9.0C.  Field survival  The  the p l a n t e d  from 13.5C t o 30.0C with an average  The minimum d a i l y temperature an average  throughout  ( t o t a l s u r v i v a l ) should a l s o be c o n s i d e r e d  The f i e l d  s t o r e d t r e e s had the h i g h e s t t o t a l  (Table  survival.  TABLE 2.  S u r v i v a l and r o o t growth o f mud-packed D o u g l a s - f i r s e e d l i n g s locations.  Storage l o c a t i o n  Field survival %  Total survival  Root growth cm  96.6 a  75.5 a  75.0 a  36. 2 a  2C room  100.0 a  50.0 b  50.0 b  22.2 b  IOC room  100.0 a  47.5 b  47.5 b  25.0 b  25C room ( + )l i g h t  63. 3 b  53.2 b  41.6 b  26. 8 b  25C room (-) l i g h t  100.0 a  10.0 c  10.0 c  0.8 c  Field  Storage s u r v i v a l %  stored i n various  Means marked by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t . i  Root growth i s expressed as cm of r o o t p r o t r u d i n g  from the mud-pack per s u r v i v i n g s e e d l i n g .  i  U)  A l l other The  treatments had a t o t a l s u r v i v a l of 55% or l e s s . f i e l d s t o r e d t r e e s had the g r e a t e s t amount o f  r o o t growth  (Table  2).  This l e n g t h o f r o o t was made up o f  numerous roots and r e p r e s e n t s  an e x t e n s i v e  o b t a i n i n g moisture and n u t r i e n t s . considerable  capacity f o r  These p l a n t s had a  area o f c o n t a c t with the s o i l beyond the  s u r f a c e o f the mud-pack.  Trees s t o r e d a t 2C, IOC, and  25C (+) l i g h t produced i n t e r m e d i a t e  amounts o f r o o t growth  w h i l e the t r e e s s t o r e d a t 25 (-) l i g h t produced the lowest amount o f r o o t growth.  There was a d i r e c t r e l a t i o n s h i p  between r o o t growth and f i e l d did  survival.  Seedlings  which  not s u r v i v e the p l a n t i n g p e r i o d produced no r o o t  growth. Stone  This observation  agrees with the f i n d i n g s o f  (2). He noted t h a t at 60 days a f t e r p l a n t i n g , bare-  r o o t D o u g l a s - f i r s e e d l i n g s which had d i e d , had produced no new r o o t s . Since  an e f f o r t was made t o reduce the e f f e c t o f  moisture s t r e s s i n t h i s study, s u r v i v a l as noted not be e x t r a p o l a t e d behaviour.  should  i n an e f f o r t t o p r e d i c t a c t u a l  On the other hand, Stone e t a l .  t h a t the r o o t r e g e n e r a t i n g  field  (3) noted  p o t e n t i a l i s a measure o f the  p h y s i o l o g i c a l c o n d i t i o n o f the s e e d l i n g and under many field  c o n d i t i o n s , one might reasonably expect i t to be  reflected i n f i e l d  survival.  In summary, i t would seem from the parameters  measured i n t h i s study, t h a t s p r i n g - l i f t e d , mud-packed D o u g l a s - f i r s e e d l i n g s should be s t o r e d i n the f i e l d . Storage results.  a t 2C, IOC and 25C (+) l i g h t produced  intermediate  Storage a t 25C (-) l i g h t was the l e a s t s a t i s -  factory .  ADDENDUM On January Can.  12th a f t e r p u b l i c a t i o n o f t h i s paper i n  J . F o r . Res., a l e t t e r was r e c e i v e d from M. Crown,  F o r e s t e r with the P a c i f i c Logging Company L i m i t e d . Crown s u p p l i e d some unpublished s u r v i v a l data  Mr.  concerning  s p r i n g - l i f t e d mud-packs which had been s t o r e d a t 2C.  In  a d d i t i o n , he added "We read w i t h i n t e r e s t your paper on the moisture s t a t u s o f mud-packs.  The r e s u l t s would  appear to g i v e the reason why o p e r a t i o n a l experience w i t h s p r i n g - l i f t e d mud-packs has g e n e r a l l y been l e s s satisfactory  than w i n t e r - l i f t e d mud-packs."  LITERATURE CITED Kozlowski, T.T. 1968. I n t r o d u c t i o n , pp. 1-22. In T.T. Kozlowski (ed.) Water d e f i c i t and p l a n t growth, V o l . I. Academic Press, New York. Stone, Edward. 19 55. Poor s u r v i v a l and the p h y s i o l o g i c a l c o n d i t i o n o f p l a n t i n g stock. F o r e s t S c i . 1: 90-94. Stone, Edward, J.L. Jenkinson, and S.L. Krugman. 1962. Root-regenerating p o t e n t i a l o f D o u g l a s - f i r s e e d l i n g s l i f t e d a t d i f f e r e n t times o f the year. F o r e s t S c i . 8 288-297. Winjum, J.K. 1963. E f f e c t s of l i f t i n g date and storage on 2-0 D o u g l a s - f i r and N o b l e - f i r . J . of F o r e s t r y 61: 648-654.  

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