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Nitrogen and conifer studies Mellor, Gary Edward 1972

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NITROGEN AND CONIFER STUDIES by GARY EDWARD MELLOR B.Sc, University of Guelph, 1967 M.Sc., University of Guelph, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 19 72 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and s tudy . I f u r t h e r agree t h a t permiss ion f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h Col Vancouver 8, Canada Department A B S T R A C T P a r t 1 T h e v a s c u l a r b u n d l e o f l e a v e s w i t h t h e C ^ - p a t h w a y o f p h o t o s y n t h e s i s i s u s u a l l y s u r r o u n d e d b y t w o c o n c e n t r i c c h l o r o p h y l l o u s c e l l l a y e r s : a n o u t e r m e s o p h y l l l a y e r a n d a n i n n e r b u n d l e s h e a t h l a y e r . T h e l o c a l i z a t i o n o f t h e n i t r a t e - a s s i m i l a t i n g e n z y m e s , n i t r a t e r e d u c t a s e , n i t r i t e r e d u c t a s e , a n d g l u t a m a t e d e h y d r o g e n a s e i n Z e a m a y s , G o m p h r e n a g l o b o s a , a n d S o r g h u m s u d a n e n s e w a s s t u d i e d b y d i f f e r e n t i a l g r i n d i n g . N i t r a t e r e d u c t i o n t o n i t r i t e a p p e a r s t o o c c u r p r i m a r i l y i n m e s o p h y l l c e l l s . T h e n i t r a t e c o n t e n t o f t h e m e s o p h y l l c e l l s w a s m u c h h i g h e r t h a n t h e n i t r a t e c o n t e n t o f t h e b u n d l e s h e a t h c e l l s . T h e d i s t r i -b u t i o n o f n i t r i t e r e d u c t a s e s e e m e d t o b e r e l a t e d t o t h e p r e s e n c e o f c h l o r o p l a s t s w i t h g r a n a . A m m o n i a i n c o r p o r a -t i o n i n t o o r g a n i c c o m p o u n d s b y g l u t a m a t e d e h y d r o g e n a s e w a s l o c a l i z e d i n t h e b u n d l e s h e a t h c e l l s . P a r t 2 F o u r c o n i f e r s p e c i e s , D o u g l a s - f i r ( P s e u d o t s u g a  m e n z i e s j i v a r . m e n z i e s i i ) ( M i r b . ) F r a n c o , W e s t e r n r e d c e d a r , ( T h u j a p l i c a t a ) D o n n , W e s t e r n h e m l o c k ( T s u g a h e t e r o p h y l l a ) ( R a f . ) S a r g , a n d L o d g e p o l e p i n e ( P i n u s c o n t o r t a v a r . c o n t o r t a ) D o u g l . , w e r e g r o w n o n t h r e e d i f f e r e n t s o u r c e s of nitrogen ( n i t r a t e , ammonia, and a combination of n i t r a t e and ammonia 7:1). A l i n e a r r e l a t i o n s h i p was found between leaf area and leaf dry weight for three species (Douglas-fir, Lodgepole pine and Western hemlock). Different nitrogen treatments had no e f f e c t on this r elationship. Part 2-A In t h i s part the tree seedlings were grown for 18 weeks. For Douglas-fir, Western redcedar and Western hemlock, su r v i v a l and growth on the n i t r a t e solution was s i m i l a r to s u r v i v a l and growth on the combination solution. Ammonia was an unfavorable source of nitrogen for s u r v i v a l and growth of Douglas-fir and Western redcedar. For Western hemlock, ammonia was only detrimental to s u r v i v a l . Hemlock seedlings which survived ammonia treatment grew as well as trees growing on the other two sources of nitrogen. Lodge-pole pine survived equally well under a l l treatments and was the only species that grew best on ammonia. Part 2-B At 18 weeks the seedlings were transferred from sand culture to l i q u i d nutrient solution. The solution was changed every two days for a period of s i x days. The pH of the solutions was measured when the solutions were changed. At the end of the s i x days, the starch content of i v the plants was measured. The results indicate that these forest species d i f f e r i n t h e i r tolerance to ammonia. Western hemlock and Lodgepole pine seem to be able to tolerate higher levels of ammonia than Douglas-fir and Western redcedar. Part 2-C In th i s part the seedlings were grown for a period of approximately one year. The trends shown i n the 18 week experiment were con-firmed i n the longer experiment and some additional treat-ment effects appeared. Part 2-D In t h i s part the seedlings were grown for a period of approximately one year. Within each species, an attempt was made to correlate differences i n growth among nitrogen treatments with d i f f e r -ences i n gas exchange. I t was found from calculations of stomatal resistance ( r g ) that the entry of CO^ was not l i m i t i n g dry matter production. I t i s postulated that mesophyll resistance (r ) may be a factor involved i n c o n t r o l l i n g growth i n these trees. Part 3 Two-year old (2-0) Douglas-fir (Pseudotsuga menziesii) seedlings were l i f t e d i n the spring and mud-packed. These seedlings were tested for the effects of various storage V c o n d i t i o n s . Mud-packed s e e d l i n g s , s t o r e d i n the f i e l d f o r 19 days and subsequently p l a n t e d , had h i g h e r s u r v i v a l and r o o t growth than those having other storage c o n d i t i o n s . The other storage 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 lower temper-ature than i n the f i e l d and l i g h t versus dark. Treatment of the mud-packs wit h f e r t i l i z e r and v e r m i c u l i t e had no e f f e c t on any of the parameters measured. v i TABLE OF CONTENTS Page PART 1 The l o c a l i z a t i o n of n i t r a t e - a s s i m i l a t i n g enzymes i n leaves of p l a n t s w i t h the C^-pathway of p h o t o s y n t h e s i s . INTRODUCTION 1 MATERIALS 3 METHODS 3 RESULTS AND DISCUSSION 5 LITERATURE CITED 16 PART 2-A A comparison of the growth and s u r v i v a l of f o u r c o n i f e r s p e c i e 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 n i t r o g e n . INTRODUCTION 21 MATERIALS AND METHODS 22 RESULTS 25 DISCUSSION 32 LITERATURE CITED 35 PART 2-B N i t r o g e n uptake and s t a r c h content of c o n i f e r 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 . INTRODUCTION 36 METHODS AND MATERIALS 3 8 RESULTS 39 DISCUSSION 41 LITERATURE CITED 45 v i i TABLE OF CONTENTS, cont'd. Page PART 2-C A comparison of the growth of fou r con-i f e r s p e c i e s s u p p l i e d w i t h d i f f e r e n t forms of n i t r o g e n f o r 10 to 12 months. INTRODUCTION 47 METHODS AND MATERIALS 48 RESULTS 50 DISCUSSION 57 LITERATURE CITED 61 PART 2-D Gas exchange 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 forms of n i t r o g e n . INTRODUCTION 62 SYMBOLS AND UNITS 65 THEORY 66 METHODS AND MATERIALS 67 RESULTS AND DISCUSSION 6 8 LITERATURE CITED 75 APPENDIX I 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 of three c o n i f e r s p e c i e s grown on thr e e sources of n i t r o g e n . EPILOGUE 86 PART 3 A study of storage c o n d i t i o n s f o r 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 . INTRODUCTION 87 v i i i 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 1. 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 milligram of protein, i n extracts of mesophyll and bundle sheath tissue of Zea mays, Gomphrena globosa and Sorghum sudanense 7 Table 2. NO^ -N content as n moles NO^ -N per milligram of protein, i n extracts of mesophyll and bundle sheath tissue of Zea mays and Gomphrena globosa 8 Part 2-A Table 1. Composition and concentration (ppm) of f u l l - s t r e n g t h nutrient solution... 23 Table 2. Survival (%) of Douglas-fir, Lodgepole pine, Western redcedar, and Western hemlock with d i f f e r e n t sources of nitrogen 26 Table 3. Total dry weight, root dry weight, S/R and leaf area of Douglas-fir supplied with d i f f e r e n t sources of nitrogen 27 Table 4; Total dry weight, root dry weight, S/R and leaf dry weight of Western redcedar supplied with d i f f e r e n t sources of nitrogen 2 8 Table 5. Total dry weight, root dry weight, S/R and leaf dry weight of Western hemlock supplied with d i f f e r e n t sources of nitrogen 29 Table 6. Total dry weight, root dry weight, S/R and leaf area of Lodgepole pine supplied with d i f f e r e n t sources of nitrogen 30 X LIST OF TABLES, cont'd. Page Part 2-B Table 1. The mean pH of the nutrient solutions after treatment for 2 days 40 Table 2. Starch content of conifer seedlings supplied with d i f f e r e n t sources of nitrogen for 4 months (j*g/ gm dry weight) 42 Part 2-C Table 1. T o t a l , root, and shoot dry weight, leaf area and height of Douglas-fir supplied with d i f f e r e n t sources of nitrogen........ 52 Table 2. Total, root, and shoot dry weight, leaf area and height of Western redcedar supplied with d i f f e r e n t sources of nitrogen 5 3 Table 3. T o t a l , root, and shoot dry weight, leaf area and height of Western hemlock supplied with d i f f e r e n t sources of nitrogen 55 Table 4. T o t a l , root, and shoot dry weight, leaf area and height of Lodgepole pine supplied with d i f f e r e n t sources of nitrogen 56 Part 2-D Table 1. Total dry weight of conifer seedlings supplied with d i f f e r e n t sources of nitrogen (gm . pot ) 6 3 Table 2. Leaf area of conifer seedlings supplied with d i f f e r e n t sources of nitrogen (cm2 . p o t - l ) 6 4 x i LIST OF TABLES, cont'd. Page Table 3. Leaf Y , Ys and Vp conifer seedlings supplied with d i f f e r e n t sources of nitrogen (bars) 69 Table 4. Water loss of conifer seedlings supplied with d i f f e r e n t sources of nitrogen (ml H20/24 hr. pot) 71 Table 5. Transpiration resistance (r + r ^ )of conifer seedlings supplied with d i f f e r e n t sources of nitrogen (sec. cm~l) 73 Part 3 Table 1. Storage treatments and conditions 90 Table 2. Survival and root growth of mud-packed Douglas-fir seedlings stored i n various locations 9 3 x i i LIST OF FIGURES Page Part 1 Figure 1. Schematic d i v i s i o n of reactions between bundle sheath and mesophyll i n Zea and Sorghum 12 Figure 2. Schematic d i v i s i o n of reactions between bundle sheath and mesophyll i n Gomphrena 13 Appendix I Figure 1. The relat i o n s h i p between leaf area and leaf dry weight for Douglas-fir seedlings supplied with d i f f e r e n t sources of nitrogen 80 Figure 2. The relat i o n s h i p between leaf area and leaf dry weight for Lodgepole pine seedlings supplied with d i f f e r e n t sources of nitrogen 82 Figure 3. The re l a t i o n s h i p between leaf area and leaf dry weight for Western hemlock seedlings supplied with d i f f e r e n t sources of nitrogen 84 ACKNOWLEDGEMENTS I wish to thank E.B. Tregunna for his help during my stay at UBC. I wish to give him spe c i a l thanks for t r e a t i n g me completely as an equal at a l l times. I wish to give sp e c i a l thanks to Andy Black, not only for serving on my Committee, but also for giving me complete access to his laboratory and equipment and for the many discussions we had concerning Part 2-D i n thi s thesis. I would also l i k e to thank Dr. Iain Taylor, Dr. Roy Taylor and Dr. G.H.N. Towers for serving on my Committee. I am gratef u l to Alan Hart, John Downton and Joe Berry for many hours of discussion which were invaluable to my projects. I am also indebted to a l l other persons with whom I came i n contact at UBC; they a l l contributed i n making my stay at UBC something I w i l l never forget. xiv PREFACE This thesis d i f f e r s from most theses i n that i t does not deal exclusively with one topic. This lack of e x c l u s i v i t y i s partly a r e s u l t of personal preference and p a r t l y a r e s u l t of circumstance. However a l l studies reported i n this thesis with the exception of Part I are concerned with the physiology of whole plants. It was my intention on a r r i v i n g at UBC to get as much research experience as possible. The study with C^-plants allowed me to work on a project that required rapid publication to ensure the o r i g i n a l i t y of discoveries and also allowed me more experience with enzymatic techniques. My discovery that the location of n i t r i t e reductase (a NADPH2 requiring enzyme) seems to be correlated with the presence of granal chloroplasts agrees with evidence that only granal chloroplasts are capable of NADPH production. The study with mud-packed seedlings allowed me to work on a project which had the p o s s i b i l i t y of immediate a p p l i c a b i l i t y . My discovery that s p r i n g - l i f t e d mud-packs should be stored outside may prove to be of si g n i f i c a n c e from both a conservational and a f i n a n c i a l point of view. X V The project with forest species and nitrogen sources permitted me to gain some experience with sand culture techniques, some experience i n growing forest species and some experience with psychrometric techniques. Evidence which I co l l e c t e d seems to indicate that forest species vary i n t h e i r tolerance to NH^+ rather than t h e i r a b i l i t y to p r e f e r e n t i a l l y metabolize NO^ or NH^ + . This i s a s i g n i f i c a n t finding i n that i t agrees with a new school of thought a r i s i n g i n the f i e l d of agriculture. It was my i n i t i a l intention to make the section on nitrogen n u t r i t i o n and forest species more physiological and more cohesive than i t turned out. This project was started as a dual project with Sara Madoc-Jones. I t was our intention that she would look at the project from an ecological point of view and that I would look at the project from a physiological point of view. However due to the death of Sara, certain parts of the project had to be abandoned. I t r i e d to salvage as much as I could. I attempted to study the c r i t e r i a which I f e l t would give me the greatest i n s i g h t into the growth responses we were observing. 1. PART 1. THE LOCALIZATION OF NITRATE-ASSIMILATING ENZYMES IN LEAVES OF PLANTS WITH THE (^-PATHWAY OF PHOTOSYNTHESIS.1 INTRODUCTION The vascular bundle of leaves with the C^-pathway of photosynthesis i s usually surrounded by two concentric c e l l layers, an inner bundle sheath layer and an outer mesophyll layer. C e l l s of the mesophyll layer contain granal chloro-plasts d i s t r i b u t e d about a large central vacuole. Surveys indicate that the bundle sheath chloroplasts of dicotyledons, c h l o r i d o i d grasses, and some panicoid grasses have w e l l -developed grana, that sugar cane and sorghum lack grana e n t i r e l y , and that corn and close r e l a t i v e s are intermedi-ate, forming only rudimentary grana (6, 14, 21, 25, 27, 32, 43). Differences i n the capacity for dye reduction (13) of agranal and granal chloroplasts of single leaves have led to the conclusion that the agranal chloroplasts are incapable of forming reductant by non-cyclic electron transport. Considerable work has been done with the enzymes involved i n the photosynthetic carbon metabolism of these C^-plants and t h e i r l o c a l i z a t i o n within the leaf (1, 5, 1 This a r t i c l e by G.E. Mellor and E.B. Tregunna appeared in Canadian Journal of Botany, Vol. 49: 137-142 (1971). E.B. Tregunna supervised the study. Subsequent developments on this topic are discussed i n addenda. 16, 17, 18, 31, 37, 39, 40, 41, 42). Although many studie have been car r i e d out on n i t r a t e reductase, n i t r i t e reductase and glutamate dehydrogenase i n corn, a C^-plant (2, 3, 4, 8, 15, 24, 35, 37, 44), there i s only one report (40) on the l o c a l i z a t i o n of such events in the leaves of C^-plants. In 1967 Ritenour et a_l. (35) studied 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 of n i t r a t e reductase, n i t r i t e reductase, and glutamate dehydrogenase i n corn and f o x t a i l leaves. They found that n i t r i t e reductase was l o c a l i z e d within the chloroplasts. This r e s u l t agreed with the findings of Ramirez et al_. (33) . Nitrate reductase was thought to be l o c a l i z e d i n the cytoplasm, although the techniques used did not eliminate the p o s s i b i l i t y that n i t r a t e reductase may be l o c a l i z e d on the external chloroplast membrane. Glutamate dehydrogenase was found i n the mitochondria. Several investigations (7, 10, 11, 34) with e t i o l a t e d seedling tissue have also indicated that glutamic acid dehydrogenase i s l o c a l i z e d i n the mitochondria. Recently Joy (23) reported that i n pea roots both soluble and part i c u l a t e (mostly mitochondria) fractions contain NADH^ and NADPH2 dependent glutamate dehydogenase. In 196 8 Leech and Kirk (28) reported that i n leaves of V i c i a  f aba, NADPEL, glutamate dehydrogenase occurred i n the chloroplast while NAD-dependent glutamate dehydrogenase 3. occurred in the mitochondria. The method of Joy (22) was used for extraction and assay of glutamate dehydrogenase i n this paper. In view of these observations I have investigated the tissue l o c a l i z a t i o n of n i t r a t e - a s s i m i l a t i n g enzymes in C^-plants with granal (Gomphrena globosa), agranal (Sorghum  sudanense), and rudimentary granal (Zea mays) bundle sheath chloroplasts. MATERIALS Zea mays, Gomphrena globosa, and Sorghum sudanense were grown on vermiculite supplemented with nutrient solution containing 100 ppm NC>3 -N as Ca (N03) 2 * 4H20. Growth conditions were 16-h day, 27/21C (day/night) temperature and 100 0 f t - c provided i n a growth chamber. Leaves of 3-week-old Zea plants, leaves of 6-week-old Sorghum, and the youngest f u l l y expanded leaves of 2-to 3-month-old Gomphrena plants were used. The age differences were necessary to obtain plants of si m i l a r s i z e . METHODS Extraction of Enzymes from Mesophyll and Bundle Sheath Tissue  Zea and Sorghum extracts, enriched i n either bundle sheath or mesophyll c e l l contents, were obtained with the use of an 'Osterizer' homogenizer. Leaves of Zea and 4 . Sorghum (2 g) were cut i n t o 1-cm p i e c e s and were ground wi t h the homogenizer i n 40 ml of the a p p r o p r i a t e b u f f e r f o r 20 sec. M i c r o s c o p i c i n s p e c t i o n r e v e a l e d t h a t , primar-i l y , mesophyll c e l l s were ruptured d u r i n g t h i s treatment w h i l e some mesophyll c e l l s and most of the bundle sheath c e l l s remained i n t a c t . T h i s e x t r a c t was t h e r e f o r e e n r i c h e d i n the contents of the mesophyll c e l l s . The remaining l e a f p i e c e s were homogenized f o r 1 min. M i c r o -s c o p i c i n s p e c t i o n of the remaining p i e c e s r e v e a l e d t h a t almost a l l of the i n t a c t c e l l s were bundle sheath c e l l s . These remaining p i e c e s were s u b j e c t e d to a very vigorous g r i n d i n g w i t h a p e s t l e i n a mortar c o n t a i n i n g g l a s s beads and g r i n d i n g media. S u f f i c i e n t g r i n d i n g p r e s s u r e was used to break the g l a s s beads, c r e a t i n g a very sharp angular g r i n d i n g m a t e r i a l . Most, but not a l l , o f the bundle sheath c e l l s were ruptured by t h i s treatment, y i e l d i n g an e x t r a c t e n r i c h e d i n the contents of bundle sheath c e l l s . The homogenizer proved u n s a t i s f a c t o r y f o r the d i f f e r e n t i a l g r i n d i n g o f Gomphrena. Gomphrena was ground as d e s c r i b e d by Berry e t aJL. (5) . The g r i n d i n g medium used f o r n i t r a t e reductase was t h a t d e s c r i b e d by Hageman and F l e s h e r (15). The g r i n d i n g medium o f Joy and Hageman (24) was used f o r n i t r i t e reductase and the g r i n d i n g medium of Joy (22) was used f o r glutamate dehydrogenase. 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 a d d i t i o n of a n o n - i o n i c detergent to a i d the breakdown of p a r t i c l e membranes. This breakdown of p a r t i c l e membranes ensured t h a t both p a r t i c u -l a t e and c y t o p l a s m i c glutamate dehydrogenase was analyzed. P l a n t m a t e r i a l was h a r v e s t e d immediately b e f o r e use a f t e r 4 h of i l l u m i n a t i o n . P r o t e i n p r e c i p i t a b l e by t r i c h l o r a c e t i c a c i d was determined by the method of Lowry (29). A s t a n -dard curve was c o n s t r u c t e d u s i n g bovine serum albumin. Enzyme assay - N i t r a t e reductase (1.6.6.1) was assayed by measuring NADI^-dependent p r o d u c t i o n of n i t r i t e (15) . N i t r i t e reductase was assayed by measuring disappearance of n i t r i t e u s i n g d i t h i o n i t e as e l e c t r o n donor, as d e s c r i b e d by Joy and Hageman (24). Glutamate dehydrogenase (1.4.1.2. and 1.4.1.4) was assayed by measuring, s p e c t r o p h o t o m e t r i -c a l l y a t 340 nm, the r a t e of o x i d a t i o n o f NAD (P) HL, depend-ent on the presence of ammonia and o<-ketoglutarate (22). Enzyme u n i t s : 1 m i l l i u n i t o f enzyme c a t a l y z e d the t r a n s f o r -mation of 1 nmole of s u b s t r a t e per minute. N i t r a t e a n a l y s i s - The method o f M i l l e r and Wideman (30) was used i n the d e t e r m i n a t i o n of n i t r a t e . RESULTS AND DISCUSSION The s p e c i f i c a c t i v i t i e s o f n i t r a t e reductase, n i t r i t e reductase, and glutamate dehydrogenase are presented i n 6 . Table 1. The t o t a l protein extracted from the leaf was about equally divided between the two extracts. 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 higher i n the mesophyll extracts of a l l three types of plants. Table 2 shows that i n both Gomphrena and Zea most of the NO^ occurs in the mesophyll extract. Thus the mesophyll extract contains high amounts of n i t r a t e reductase a c t i v i t y and high amounts of n i t r a t e . The occurrence of n i t r a t e i n the bundle sheath extract may be due to (a) improper separation of bundle sheath and mesophyll, (b) the passage of n i t r a t e from the vascular system to the mesophyll, (c) storage of some n i t r a t e i n the bundle sheath c e l l s , or (d) a combination of a l l three. In Zea and Sorghum n i t r i t e reductase a c t i v i t y i s con-fined primarily to mesophyll c e l l s (Table 1). The n i t r i t e reductase a c t i v i t y i n Gomphrena seemed to occur i n both the mesophyll and the bundle sheath c e l l s . In Zea and Sorghum the mesophyll c e l l s are capable of dye reduction and contain granal chloroplasts. The bundle sheath c e l l s of Zea and Sorghum, however, are incapable of dye reduction and contain not more than rudimentary grana (13). In Gomphrena both the mesophyll and bundle sheath c e l l s are capable of dye reduction (W. J. S. Downton, pers. TABLE 1. 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 milligram of protein, i n extracts of meso-ph y l l and bundle sheath tissue of Zea mays, Gomphrena globosa, and Sorghum sudanense. Zea mays Gomphrena globosa Sorghum sudanense Bundle Bundle Bundle Enzyme Mesophyll sheath Mesophyll sheath Mesophyll sheath Nitrate reductase 5. 50* 0. 60 6. 40 1. 53 2. 67 1. 52 3. 77* 0. 68 3. 56 0. 60 3. 84 1. 21 N i t r i t e reductase 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 58 284 71 153 64 178 52 201 57 181 61 138 NADPH 18 35 35 72 18 34 15 45 27 54 13 19 *Replicates. TABLE 2. NO- -N content, as nmoles NO- -N per milligram of protein, i n extracts of mesophyll ana bundle sheath tissue of Zea mays and Gomphrena globosa. Bundle Plant Mesophyll sheath Gomphrena globosa 5956* 170 Zea mays 715 214 *Mean of two determinations. 9. commun.)* and both contain at le a s t thylakoid overlaps (12) which are believed to be s u f f i c i e n t for non-cyclic electron flow (20). Thus the results indicate that only grana-containing chloroplasts are linked to n i t r i t e reduction. I t has been suggested that grana are necessary for photosystem II a c t i v i t y (20, Addendum 2) and that chloro-plasts d e f i c i e n t i n grana would be incapable of non-cyclic electron flow and NADP reduction. I t i s believed that n i t r i t e reductase receives electrons from ferredoxin when the l a t t e r has been reduced by ill u m i n a t i o n or by NADPH2 and a diaphorase enzyme (9, 19, 24, 33, 38). This require-ment for ferredoxin to donate electrons to n i t r i t e reductase suggests that n i t r i t e reductase should be associated with granal chloroplasts. The rudimentary grana i n bundle sheath c e l l s of Zea seem to play l i t t l e or no role i n n i t r i t e reduction. The occurrence of n i t r i t e reductase primarily i n the mesophyll of Zea agrees with the findings of Slack ejt a l . (42) . Using density f r a c t i o n a t i o n i n non-aqueous media to separate Zea mesophyll and bundle sheath chloroplasts, they found that about 80% of the n i t r i t e reductase a c t i v i t y occurred i n the mesophyll chloroplasts. In a l l three types of plants the bulk of both NADHv,and NADPILj glutamate dehydrogenase a c t i v i t y occurs i n the bundle *Research School of B i o l o g i c a l Sciences, Australian National University, Canberra, A. C. T., A u s t r a l i a . 10. sheath (Table 1). The occurrence of glutamate dehydrogenase a c t i v i t y in the bundle sheath of Gomphrena suggests that as far as glutamate dehydrogenase a c t i v i t y i s concerned, the mitochondria i n the bundle sheath are more active than the mitochondria i n the mesophyll. This r e s u l t coincides with the findings of Laetsch (25) and Downton e_t a l . (14) that i n the dicotyledons which they examined the mitochondria i n the bundle sheath are considerably larger and more developed. This d i f f e r e n t i a l development of mitochondria between the mesophyll and the bundle sheath i n t r o p i c a l grasses has never, to our knowledge, been reported. In Zea and Sorghum, however, the bulk of the glutamate dehydro-genase s t i l l occurred i n the bundle sheath. With the methods available for c e l l separation, the p o s s i b i l i t y of obtaining mesophyll extracts free of bundle sheath contamination or bundle sheath extracts free from mesophyll contamination i s high. Thus for the purpose of this discussion, i t i s assumed that the occurrence of n i -trate reductase i n the bundle sheath extracts of Zea, Sorghum, and Gomphrena, the occurrence of n i t r i t e reductase i n the bundle sheath extracts of Zea and Sorghum and the occurrence of glutamate dehydrogenase i n the mesophyll ex-tracts of Zea, Sorghum, and Gomphrena i s contamination. However, as more refined methods become available for c e l l separation, this assumption may prove incorrect and the enzyme may be found to occur i n both areas. Therefore on the basis of t h i s assumption of contamination, i t would seem that the steps i n the assimilation of n i t r a t e are separated. Thus i n Zea and Sorghum i t seems that n i t r a t e and n i t r i t e are both reduced primarily i n the mesophyll (Fig. 1) and the r e s u l t i n g ammonia i s incorporated into organic form i n the bundle sheath. Ammonia, however, i s somewhat toxic to plants because i t perhaps i n h i b i t s the production of ATP i n the mitochondria and photosynthetic electron-transport system (36). Thus i t seems probable that rapid transport of the ammonia from the mesophyll to the bundle sheath i s required. Laetsch has noted that i n sugar cane the wall separating the mesophyll c e l l s from the bundle sheath c e l l s i s trans-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 published micrographs of several other plants which have thi s type of leaf and >9-carboxylation (6, 25). Thus plas-modesmata provide a connection between the mesophyll and the bundle sheath c e l l s . Whether the transport from the mesophyll to the bundle sheath i s active or passive remains to be established. In Gomphrena, n i t r a t e i s reduced primarily i n the mesophyll (Fig. 2), while n i t r i t e can be reduced i n both NO. -> NO, CYTOPLASM NO, NH + CHLOROPLAST MESOPHYLL + NH4 + «<KETOGLUTARATE GLUTAMATE MITOCHONDRIA BUNDLE SHEATH CELL WALL Figure 1. Schematic d i v i s i o n of reactions between bundle sheath and mesophyll i n Zea and Sorghum. tsj NGy > N0 2 'CYTOPLASM CHLOROPLAST NH 4 + + <*KETOGLUTARATE MITOCHONDRIA 'MESOPHYLL BTJNDLE SHEATH CELL WALL Figure 2. Schematic d i v i s i o n of reactions between bundle sheath and mesophyll i n Gomphrena, 14. the bundle sheath and the mesophyll. Ammonia i n c o r p o r a t i o n takes p l a c e p r i m a r i l y i n the bundle sheath. Again the t r a n s p o r t of ammonia and n i t r i t e from the mesophyll to the bundle sheath c o u l d occur v i a the plasmodesmata. In c o n c l u s i o n i t should be noted t h a t when n i t r a t e -a s s i m i l a t i n g enzymes i n C ^ -plants are s t u d i e d the l o c a t i o n of the enzymes should be kept i n mind when the t i s s u e s are ground. Enzymes l o c a l i z e d i n the mesophyll should pose no problem as f a r as complete i s o l a t i o n i s concerned, s i n c e the mesophyll c e l l s are e a s i l y r u p t u r e d by normal methods of g r i n d i n g . The bundle sheath c e l l s are p a r t l y r e s i s t a n t to normal methods of g r i n d i n g . Thus f o r complete i s o l a t i o n o f enzymes l o c a l i z e d completely or p a r t l y i n the bundle sheath a more vigorous g r i n d i n g i s r e q u i r e d . ADDENDA 1. While t h i s paper was being reviewed f o r p u b l i c a t i o n , a paper appeared by J . W. M a r a n v i l l e (Influence o f n i c k e l on the d e t e c t i o n of n i t r a t e reductase a c t i v i t y i n sorghum e x t r a c t s . P l a n t P h y s i o l . 45: 591-593. 1970.) M a r a n v i l l e ' s paper i n d i c a t e s t h a t n i c k e l i n c r e a s e s the n i t r a t e reductase a c t i v i t y i n sorghum e x t r a c t s . N i c k e l was not used i n the sorghum e x t r a c t s o f t h i s paper. The a d d i t i o n o f n i c k e l to sorghum e x t r a c t s may have i n c r e a s e d the n i t r a t e reductase a c t i v i t y r e p o r t e d i n Table 1. 2. Sane et a_l. (P.V. Sane, D.J. Goodchild and R.B. Park. Biochim. Biophys. Acta 216: 162. 1970.) and Arntzen et a l . (C.J. Arntzen, R.A. D i l l e y and J. Neumann. Biochim. Biophys Acta 245:409-424. 1971.) have shown from chloroplast fragmentation studies that granal lamellae have both Photosystem I and II a c t i v i t i e s and that stromal lamellae have only Photosystem I a c t i v i t y i . e . only granal lamellae seem capable of reducing NADP. 16. LITERATURE CITED Andrews, T.J.. and M.D. Hatch. 1969. P r o p e r t i e s and mechanism of a c t i o n of pyruvate phosphate d i k i n a s e from l e a v e s . Biochem. J . 114: 117-125. Beevers, L., D. F l e s h e r , and R.H. Hageman. 1964. S t u d i e s on the 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 reductase 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 to s u l f h y d r y l l e v e l . Biochim. Biophys. A c t a , 89: 453-464. Beevers, L., D.M. Peterson, J.C. Shannon, and R.H. Hageman. 1963. Comparative e f f e c t s o f 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d on n i t r a t e metabolism i n corn and cucumber. P l a n t P h y s i o l . 38: 675-679. Beevers, L., L.E. Schrader, D. F l e s h e r , and R.H. Hageman. 1965. The r o l e o f l i g h t and n i t r a t e i n the i n d u c t i o n o f n i t r a t e reductase i n r a d i s h cotyledons and maize s e e d l i n g s . P l a n t P h y s i o l . 40: 691-698. Berry, J.A., W.J.S. Downton, and E.B. Tregunna. 19 70. The p h o t o s y n t h e t i c carbon metabolisn of Zea mays and Gomphrena globosa: the l o c a t i o n of the CC^ f i x a t i o n and the c a r b o x y l t r a n s f e r r e a c t i o n s . Can. J . Bot. 48: 777-786. B i s a l p u t r a , T., W.J.S. Downton, and E.B. Tregunna. 1969. The d i s t r i b u t i o n and u l t r a s t r u c t u r e i n leaves d i f f e r -i n g i n p h o t o s y n t h e t i c carbon metabolism. I. Wheat, Sorghum, and A r i s t i d a (Gramineae). Can. J . 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The p a r t i c i p a t i o n of phosphoenol pyruvate synthetase i n photosynthetic CO„ f i x a t i o n of t r o p i c a l grasses. Arch. Biochem. Biophys. 101: 224-225. 17. Hatch, M.D., and CR. Slack. 1969. Studies on the mechanism of ac t i v a t i o n and i n a c t i v a t i o n of pyruvate phosphate dikinase. Biochem. J. 112: 549-558. 18. Hatch, M.D., and CR. Slack. 1969. NADP-specific malate dehydrogenase and glycerate kinase i n leaves and evidence for t h e i r l ocation i n chloroplasts. Biochem. Biophys. Res. Commun. 34: 589-593. 19. Hewitt, E.J., D.P. Hucklesby, and G.F. Betts. 1968. N i t r i t e and hydroxylamine i n inorganic nitrogen metabolism with reference p r i n c i p a l l y to higher plants. In. Recent aspects of nitrogen metabolism. Edited by E.J. Hewitt and C.V. Cutting. Academic Press, New York, London, pp. 47-81. 18. 20. Honman, P.H., and G.H. Schmid. 1967. Photosynthetic reactions of chloroplasts with unusual structures. Plant Physiol. 42: 1619-1632. 21. Johnson, S i s t e r M. 1964. An electron microscope study of the photosynthetic apparatus i n plants with special reference to the Gramineae. Ph.D. Thesis, University of Texas. 22. Joy, K.W. 1969. Nitrogen metabolism of Lemna minor. II. Enzymes of n i t r a t e assimilation and some aspects of th e i r regulation. Plant Physiol. 44: 849-853. 23. Joy, K.W. 1969. Glutamate dehydrogenase i n higher plants. XI International Botanical Congress, Seattle, Washington. 24. Joy, K.W., and R.H. Hageman. 1966. The p u r i f i c a t i o n and properties of n i t r i t e reductase from higher plants and i t s dependence on ferredoxin. Biochem. J. 100: 263-273. 25. Laetsch, W.M. 1968. Chloroplast s p e c i a l i z a t i o n i n dicots possessing the C.-dicarboxylic acid pathway of photosynthetic CO„ f i x a t i o n . Amer. J. Bot. 55: 875-883. 26. Laetsch, W.M., and I. Price. 1969. Development of the dimorphic chloroplasts of cane sugar. Amer. J. Bot. 56: 77-87. 27. Laetsch, W.M., D.A. S t e t l e r , and A.J. V l i t o s . 1966. The ultrastructure of cane sugar chloroplasts. Z. Pflan-zenphysiol. 54: 472-474. 28. Leech, M.R., and P.R. Kirk. 1968. An NADP-dependent«<-glutamate dehydrogenase from chloroplasts of V i c i a  faba L. Biochem. Biophys. Res. Commun. 32: 685-690. 29. Lowry, O.H., N.J. Rosenbrough, A.L. Farr, and R.J. Randall. 19 51. Protein measurement with the f o l i n phenol reagent. J. B i o l . Chem. 193: 265-275. 30. Muller, R., and 0. Wideman. 1955. Die Bestimmung des Nitrat-Ions i n Wasser. Vom Wasser, 22:227. 31. Osmond, C.B. 1967. ^ -carboxlation during photosynthesis in A t r i p l e x . Biochim. Biophys. Acta, 141: 197-199. 19. Osmond, C.B., J.H. Troughton, and D.J. Goodchild. 1969. Physiological, biochemical and s t r u c t u r a l studies of photosynthesis and photorespiration i n two species of A t r i p l e x . Z. Pflanzenphysiol. 61: 218-237. Ramirez, J.M., F.F. Del Campo, A. Paneque, and M. Losada. 1966. Ferredoxin-nitrite reductase from spinach. Biochim. Biophys. Acta, 118: 58-71. Rautanen, N., and J.M. Tager. 1955. Ann. Acad. S c i . Fenn. Ser. A II Chem. 60: 241. Cited by B.D. Sanwal and M. Lata. 1964. Enzymes of amino acid metabolism. L-glutamic acid dehydrogenase of higher plants. Modern methods of plant analysis. Vol. VII. Springer Verlag, B e r l i n , pp. 291-292. Ritenour, G.L., K.W. Joy, J. Bunning, and R.H. Hageman. 1967. I n t r a c e l l u l a r l o c a l i z a t i o n of n i t r a t e reduc-tase, n i t r i t e reductase and glutamic acid dehydro-genase i n green leaf tissue. Plant Physiol. 42: 233-237. Salisbury, F.B., and C. Ross. 1969. Metabolism and functions of nitrogen and sulphur. Iri Plant physiology. Wadsworth Publishing Co., Belmont, C a l i f , pp. 330-348. Schrader, L.E., and R.H. Hageman. 1967. Regulation of n i t r a t e reductase a c t i v i t y i n corn seedlings by endogenous metabolites. Plant Physiol. 42: 1750-1756. Shin, M., and Y. Oda. 1966. Photosynthetic n i t r i t e reductase from spinach. Plant C e l l Physiol. 17: 643-650. Slack, CR. 1968. The photoactivation of a phospho-pyruvate synthase i n leaves of Amaranthus palmeri. Biochem. Biophys. Res. Commun. 30: 4 83-4 88. Slack, CR. 1969. Lo c a l i z a t i o n of certain photosyn-t h e t i c enzymes i n mesophyll and parenchyma sheath chloroplasts of maize and Amaranthus palmeri. Phytochemistry, 8: 1387-1391. 20. 41. Slack, C.R., and M.D. Hatch. 1967. Comparative studies on the a c t i v i t y of carboxylases and other enzymes i n re l a t i o n to the new pathway of photosynthetic carbon dioxide f i x a t i o n i n t r o p i c a l grasses. Biochem. J. 103: 660-665. 42. Slack, C.R., M.D. Hatch, and D.J. Goodchild. 1969. Di s t r i b u t i o n of enzymes i n mesophyll and parenchyma-sheath chloroplasts of maize leaves i n r e l a t i o n to the C.-dicarboxylic acid pathway of photosynthesis. Biochem. J. 114: 489-498. 43. Tregunna, E.B., B.N. Smith, J.A. Berry, and W.J.S. Downton. 1970. Some methods for studying the photosynthetic taxonomy of the angiosperms. Can. J. Bot. 48: 1209-1214. 44. Tweedy, J.A., and S.K. Ries. 1967. Effects of simazine on n i t r a t e reductase a c t i v i t y i n corn. Plant Physiol. 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 studies have been carried out to compare n i t r a t e and ammonia as sources of nitrogen for conifers. The evidence co l l e c t e d to date i s inconclusive as to which i s the most favorable form of nitrogen for conifers. This section re-ports on the growth and su r v i v a l of Douglas-fir (Pseudotsuga  menziesii var. menziesii), Lodgepole pine (Pinus contorta var. contorta), Western hemlock (Tsuga heterophylla), and Western redcedar (Thuja placata) grown for 18 weeks on n i t r a t e alone, ammonia alone or a combination of n i t r a t e and ammonia (7:1). Krajina (4) has carr i e d out some studies of nitrogen n u t r i t i o n with Douglas-fir, Western redcedar and Western hemlock. Swan (7) has carried out s i m i l a r studies with Western hemlock. In both of these works the concentration of nitrogen i n each of the treatments was d i f f e r e n t . This use of d i f f e r e n t nitrogen concentrations has led to d i f f i -culty i n in t e r p r e t i n g which was the more favorable form of nitrogen. Krajina concluded that n i t r a t e was the more favorable form of nitrogen for Douglas-fir and Western redcedar, while ammonia was the more favorable source of n i t r o g e n f o r Western hemlock. Swan concluded t h a t a combination o f n i t r a t e and ammonia (4:1) was more f a v o r a b l e to growth o f Western hemlock than n i t r a t e alone o r ammonia alone. Van Den D r i e s s c h e (8) found t h a t f o r D o u g l a s - f i r , s e e d l i n g dry weight was g r e a t e s t when ammonia and n i t r a t e were p r o v i d e d i n equal amounts. Ammonia alone r e s u l t e d i n g r e a t e r growth than n i t r a t e alone. For Western hemlock, there was no d i f f e r e n c e i n s e e d l i n g dry weight between treatments u s i n g n i t r a t e alone o r ammonia alone or a combination o f n i t r a t e and ammonia. To our knowledge, no work has been done to determine the most f a v o r a b l e source o f n i t r o g e n f o r Lodgepole p i n e . MATERIALS AND METHODS Seeds o f D o u g l a s - f i r , Lodgepole p i n e , Western hemlock and Western redcedar were o b t a i n e d from the U n i v e r s i t y o f B r i t i s h Columbia Research F o r e s t ( e l e v a t i o n o f 1000 f t ) . S t r a t i f i e d seeds were grown i n pots o f sand (20-30 mesh sand purchased from the Ottawa S i l i c a Co., Ottawa, I l l i n o i s ) The sand was moistened w i t h d i s t i l l e d water u n t i l one week a f t e r g e r mination, at which time, 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 14 ppm of N (Table 1) was a p p l i e d . Three d i f f e r e n t sources o f i n o r g a n i c n i t r o g e n were used: ammonia o n l y , n i t r a t e o n l y and a combination o f n i t r a t e and ammonia (7:1) ( h e r e a f t e r r e f e r r e d to as NH.+-N, NO ~-N and (NO ~+ NH. +)-N TABLE 1. Composition and concentration (ppm) of f u l l - s t r e n g t h nutrient solution. Element Solution N03 + NH 4 + (7:1) N03 NH 4 + N 12.2 + 1.8 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 S 15 .0 15 .0 15 .0 Cu 12.6 -4 x 10 12.6 x 10~4 12.6 -4 x 10 Zn 31.2 -4 x 10 31.2 x 10~4 31.2 x I O - 4 Mo 31.2 -4 x 10 31. 2 x 10~4 31.2 x 10~ 4 Fe 12.6 -2 x 10 12.6 x 10" 2 12. 6 x 10" 2 Mn 31.2 x 10" 3 31. 2 x I O - 3 31.2 x 10~ 3 B 31.2 x 10~ 3 31.2 x I O - 3 31.2 x 10~ 3 pH = 5.5 - 0.1 CJ r e s p e c t i v e l y ) . The composition of these n u t r i e n t s o l u t i o n s was s i m i l a r to those s o l u t i o n s used by K r a j i n a (4). However, the l e v e l of a l l n u t r i e n t s except c h l o r i d e was the same i n a l l t h r e e s o l u t i o n s . A t the end of seven weeks, 50 l i v e s e e d l i n g s were chosen a t random and t r a n s p l a n t e d i n t o p l a s t i c b u l l e t s (9) c o n t a i n i n g sand. Each n i t r o g e n treatment f o r each s p e c i e s c o n s i s t e d o f 50 b u l l e t s . The 50 b u l l e t s were h e l d i n a p l a s t i c stand. Thus there were 12 stands of t r e e s (4 s p e c i e s x 3 tre a t m e n t s ) . The s e e d l i n g s were i r r i g a t e d with f r e s h n u t r i e n t t hree times per week and d i s t i l l e d water once a week. The s e e d l i n g s were grown i n a growth room wi t h 90% r e l a t i v e humidity i n the day and 65% r e l a t i v e humidity a t n i g h t . F l u o r e s c e n t and incandescent lamps p r o v i d e d 20,000 l u x over a photoperiod of 16 hr (6 am to 10 pm). 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 hr tem-p e r a t u r e c y c l e was used (8 am to 8 pm a t 24C; 8 pm to 8 am a t 20C). Eighte e n weeks a f t e r germination, s e e d l i n g s u r v i v a l was determined, the s u r v i v i n g s e e d l i n g s were h a r v e s t e d and t o t a l dry weight, r o o t dry weight and s h o o t / r o o t r a t i o (S/R) were recorded. P l a n t m a t e r i a l was e i t h e r oven d r i e d a t 80C or l y o p h i l i z e d ; the methods gave equal r e s u l t s . Leaf area (Appendix I) was determined f o r a l l s p e c i e s except f o r Western redcedar; i t was im p o s s i b l e t o determine a c c u r a t e l y the area of the cedar s c a l e s . A l l r e s u l t s except s u r v i v a l were analyzed w i t h one way analyses of v a r i a n c e f o l l o w e d by de t e r m i n a t i o n of the l e a s t s i g n i -f i c a n t d i f f e r e n c e (LSD). S u r v i v a l data were analyzed by means of the Chi-square-2xn contingency t a b l e . RESULTS The data on p l a n t s u r v i v a l are giv e n i n Table 2. The dry weights, l e a f area and S/R f o r the s u r v i v o r s are giv e n i n Tables 3, 4, 5, and 6 f o r D o u g l a s - f i r , Western redcedar, Western hemlock and Lodgepole p i n e r e s p e c t i v e l y . D o u g l a s - f i r The s e e d l i n g s i n the NC>3~-N and (N0 3~ + NH^ + ) -N t r e a t -ments had s i m i l a r p e r c e n t s u r v i v a l and the t r e e s i n NH 4 +-N had a s i g n i f i c a n t l y lower percent s u r v i v a l (Table 2). Seedlings r e c e i v i n g NH 4 +-N produced s i g n i f i c a n t l y lower t o t a l dry weight and r o o t dry weight and a s i g n i f i c a n t l y h i g h e r S/R than the oth e r two treatments (Table 3). There was no s i g n i f i c a n t d i f f e r e n c e i n l e a f area among the three treatments. Western Redcedar The s e e d l i n g s i n the (NO ~ + NH. + )-N and NO ~-N TABLE 2. Survival (%) of Douglas-fir, Lodgepole pine, Western redcedar and Western hemlock supplied with d i f f e r e n t sources of nitrogen. Species N03~ + NH 4 + Source (7:1) of Nitrogen N0 3- NH 4 + Douglas-fir 98 90 64* Western redcedar 100 100 20* Western hemlock 100 92 64* Lodgepole pine 92 86 88 *Difference among means within a species s i g n i f i c a n t at the 5% l e v e l . TABLE 3. Total dry weight, root dry weight, S/R and leaf area of Douglas-fir supplied with d i f f e r e n t sources of nitrogen.1 Source of Nitrogen N03~ + NH 4 + (7:1) NC>3~ NH 4 + Total dry weight (gm/plant) 1019 1192 0787 Root dry weight (gm/plant) 0498 0554 0271 S/R 1.12 1.32 2.13 Total leaf surface area (cm /plant) 12.86 13.19 10.74 Values connected by the same li n e do not d i f f e r at the 5% l e v e l by LSD, TABLE 4. Total dry weight, root dry weight, S/R and le a f dry weight of Western redcedar supplied with d i f f e r e n t sources of nitrogen.1 Source of Nitrogen N03~ + NH 4 + (7:1) N0 3- NH4 + Total dry weight (gm/plant) .1015 .1440 .0265 Root dry weight (gm/plant) .0358 .0429 .0121 S/R 2.19 2.48 2.06 Leaf dry weight (gm/plant) .0792 .0939 .0142 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. TABLE 5. Total dry weight, root dry weight, S/R and leaf area of Western hemlock supplied with d i f f e r e n t sources of nitrogen.1 Source of Nitrogen N03~ + NH 4 + (7:1) NC>3~ NH4 + Total dry weight (gm/plant) .0672 .0523 . 0518 Root dry weight (gm/plant) .0190 .0150 .0142 S/R 2. 58 3.00 3.10 2 Total leaf surface area (cm /plant) 15.28 11. 39 10 . 64 Values connected by the same li n e do not d i f f e r at the 5% l e v e l by LSD. TABLE 6. Total dry weight, root dry weight, S/R and leaf area of Lodgepole pine supplied with d i f f e r e n t sources of nitrogen.1 Total dry weight (gm/plant) Root dry weight (gm/plant) S/R 2 Total leaf surface area (cm /plant) Source of Nitrogen N03~ + NH 4 + (7:1) N0 3~ NH 4 + . 1214 . 1119 .1677 .0 580 .0526 .0653 1.14 1. 34 1.94 12.10 10.97 17.52 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. 31. treatments had 100% s u r v i v a l , w h i l e those i n the NH^+-N treatment had o n l y 20% s u r v i v a l (Table 2). The s e e d l i n g s r e c e i v i n g NO^ -N treatment produced the l a r g e s t t o t a l dry weight f o l l o w e d by the (NO^ + NH^ +)-N treatment (Table 4). Those on the NH 4 +-N treatment produced by f a r the lowest t o t a l dry weight. Trees i n NH 4 +-N produced s i g n i f i c a n t l y lower amounts of l e a f and r o o t dry weight than the o t h e r two treatments. There was no s i g n i f i c a n t d i f f e r e n c e i n S/R among the three treatments. Western Hemlock See d l i n g s r e c e i v i n g (N0 3~ + NH 4 +)-N and NC>3~-N had 100% and 92% s u r v i v a l r e s p e c t i v e l y (Table 2). Se e d l i n g s r e c e i v i n g NH 4 +-N had only 64% s u r v i v a l . T o t a l dry weight, r o o t dry weight and S/R showed no s i g n i f i c a n t d i f f e r e n c e among the three treatments (Table 5). The s e e d l i n g s grown with (NC>3 + NH 4 +)-N produced a s i g n i f i c a n t l y l a r g e r l e a f area than s e e d l i n g s grown on the other two treatments. Lodgepole Pine There was l i t t l e d i f f e r e n c e i n s u r v i v a l among the thre e treatments (Table 2). Treatments with NH 4 +-N r e -s u l t e d i n s i g n i f i c a n t l y l a r g e r t o t a l dry weight, l e a f area and S/R than the oth e r two treatments (Table 6). There was no d i f f e r e n c e i n r o o t dry weight among the three treatments. DISCUSSION On the basis of growth and su r v i v a l data i t would seem that Douglas-fir, Western redcedar and Western hemlock sur-vived and grew well with either NO^  -N or (NO^ + NH 4 +)-N. The NH4+-N treatment was detrimental to both the growth and sur v i v a l of Douglas-fir and Western redcedar, but only to the s u r v i v a l of Western hemlock. Western hemlock seedlings which survived on the NH4+-N treatment grew as well as trees growing on the other two sources of nitrogen. Lodge-pole pine survived equally well with a l l nitrogen treatments and was the only species that grew best on NH4+-N. The lower su r v i v a l of Western hemlock seedlings i n the NH4+-N treatment may not be very s i g n i f i c a n t in the natural f i e l d environment i f excessive seedling production occurs. With Douglas-fir and Western redcedar the small size of seedlings grown on NH4+-N may lead to increased mortality in the f i e l d due to competition. Many workers have proposed that a low S/R i s preferable for s u r v i v a l of conifers (1, 3, 5, 6). However, Hermann (2) has shown that s u r v i v a l for Douglas-fir seedlings with a small root system was s i g n i f i c a n t l y lower than for seedlings with a large root system, regardless of the size of the shoot. A consideration of the data reported here for e f f e c t of nitrogen treatment on su r v i v a l , root weight and S/R, would indicate that: 1) For Douglas-fir, a low S/R and a l a r g e r o o t system are both i n d i c a t i v e o f hig h s u r v i v a l (Tables 2 and 3). 2) For Western redcedar, development o f a l a r g e r o o t system by the s u r v i v o r s i s more i n d i c a t i v e o f h i g h s u r v i v a l than i s a low S/R (Tables 2 and 4). 3) For Western hemlock, n e i t h e r r o o t development nor a low S/R of the s u r v i v o r s i s i n d i c a t i v e o f s u r v i v a l (Tables 2 and 5). 4) For Lodgepole pine, development o f a l a r g e r o o t system i s more i n d i c a t i v e o f high s u r v i v a l than development o f a low S/R. With Lodgepole p i n e , the high S/R of the NH 4 +-N treatment (Table 6) was not d e t r i m e n t a l to s u r v i v a l (Table 2) , p o s s i b l y because t h i s treatment produced s e e d l i n g s with as l a r g e a r o o t system as t h a t produced by s e e d l i n g s grown on the othe r treatments. D o u g l a s - f i r s e e d l i n g s grown on NH 4 +-N a l s o had a high S/R (Table 3), but had low s u r v i v a l (Table 2). In t h i s case the i n c r e a s e d S/R was a r e s u l t o f decreased r o o t development. The s i m i l a r l e a f area produced by D o u g l a s - f i r (Table 3) on the three treatments i n d i c a t e s t h a t p h o t o s y n t h e t i c area was not the l i m i t i n g f a c t o r i n the low p r o d u c t i o n o f t o t a l dry weight on the NH 4 +-N treatment. Western hemlock s e e d l i n g s produced only 1/3 to 1/2 the dry matter produced by D o u g l a s - f i r or Lodgepole p i n e i n s p i t e o f the f a c t t h a t these three s p e c i e s had s i m i l a r l e a f areas (Tables 3, 5, and 6). 3 4 . There seems to be a relationship between where the trees occur i n the f i e l d and the a b i l i t y of the trees to metabolize ammonia. Young Douglas-fir and Western redcedar, which occur on s o i l s where n i t r a t e i s expected to be the predominant form of nitrogen ( 4 ), grew and survived poorly on NH4+-N as compared to growth and su r v i v a l on the other nitrogen treatments. Western hemlock and Lodgepole pine, which occur on s o i l s where ammonia i s expected to be the predominant form of nitrogen (4) grew as well or better on NH4+-N than on the other two sources of nitrogen. Van Den Driessche recently reported (8) that Douglas-fir grew better on NH4+-N alone than on NO^ -N alone. This obser-vation i s i n contrast to the data reported here and the results of Krajina (4) and indicates that the relationship between where the trees occur i n the f i e l d and the a b i l i t y of the trees to metabolize NH^+-N or NO^ -N i s not as cle a r -cut as we have stated. LITERATURE CITED George, E.G. 1939. Tree planting on the d r i e r sections of the northern great p l a i n s . J. Forestry 37: 695-698. Hermann, R.K. 19 64. Importance of the top-root ratios for s u r v i v a l of Douglas-fir seedlings. Tree Planters Notes 64: 7-11. U.S. Forest Service, U.S. Dept. of Agriculture, Washington, D.C. Karstian, C F . 1925. Forest planting i n the i n t e r -mountain region. B u l l e t i n No. 1264. U.S. Forest Service, U.S. Department of Agriculture, Washington, D.C. Krajina, V.J. 1969. Ecology of forest trees i n B r i t i s h Columbia. Ecology of Western North America 2: 1-146. Botany Department, University of B r i t i s h Columbia. Pearson, G.A. 1950. Management of Ponderosa pine i n the southwest. Agriculture Monograph No. 6. U.S. Forest Service, U.S. Department of Agriculture, Washington, D.C . Stoeckeler, J.H. 1950. Can nurserymen produce white pine seedling stock comparable to transplants. Technical note 339: Lake States Forestry Experimental Station, U.S. Forest Service, St. Paul 1, Minn. Swan, H.S.D. 1960. The mineral n u t r i t i o n of Canadian pulpwood species. Pulp and Paper Research Institute of Canada. Woodlands Research Index No. 116. Montreal, Canada. Van Den Driessche, R. 1971. Response of conifer seedlings to n i t r a t e and ammonium sources of nitrogen. Plant and S o i l 34: 421-439. Walters, J. 1968. Planting gun and b u l l e t . J. American Society of 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 plants appear to u t i l i z e ammonia in preference to n i t r a t e and vice versa. Potatoes, r i c e , buckwheat (7), some conifer species (17) and low bushberry (16), are examples of plants that seem to u t i l i z e ammonia i n preference to n i t r a t e , while beets, wheat (7), tomatoes, mustard, rye, oats (10) and apple (6) trees are examples of plants that seem to u t i l i z e n i t r a t e i n preference to ammonia. In previous work (Part 2-A) i t was concluded that Western hemlock and Lodgepole pine survived and grew better when supplied with ammonia rather than n i t r a t e . The opposite results were found for Douglas-fir and Western redcedar. However, two pieces of information prevented me from conclud-ing that Western redcedar and Douglas-fir 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 that Western hemlock and Lodgepole pine u t i l i z e ammonia p r e f e r e n t i a l l y : a) Van Den Driessche (17) concluded that Douglas-fir seedlings grow better when supplied with ammonia and b) the results of a 1 year study (Part 2-C) indicate that growth of Lodgepole pine and Western hemlock was less on n i t r a t e than on either ammonia or a combination of n i t r a t e and ammonia (7:1) where n i t r a t e was the major source of nitrogen. 37. Recent work with o t h e r p l a n t s suggested a s o l u t i o n to t h i s apparent c o n t r a d i c t i o n of r e s u l t s . Harada e t a l . (8) and B l a i r e_t al_. (2) have shown t h a t with some p l a n t s , t h e r e i s no d i f f e r e n c e between growth on n i t r a t e o r ammonia, when low c o n c e n t r a t i o n s o f n i t r o g e n are s u p p l i e d . T h i s suggests t h a t p l a n t s may d i f f e r i n t h e i r a b i l i t y to t o l e r a t e v a r i o u s l e v e l s o f ammonia r a t h e r than t h e i r a b i l i t y to u t i l i z e e i t h e r ammonia or n i t r a t e p r e f e r e n t i a l l y . T h i s t o l e r a n c e may be expressed i n s e v e r a l ways a) perhaps by r e g u l a t i n g the amount o f ammonia taken up and/or b) by a m e t a b o l i c r e g u l a t i o n once the ammonia i s taken up. I t has been shown t h a t wheat s e e d l i n g s (15, 18), rye grass (13) and some c o n i f e r s (17) take up more ammonia than n i t r a t e when the two forms are p r e s e n t together. In c o n i f e r s (17) ammonia was taken up e x c l u s i v e l y when the two sources were presen t together. Ammonia i n t o x i c l e v e l s i s thought to i n h i b i t photo-p h o s p h o r y l a t i o n (12) and subsequently to i n h i b i t carbon d i o x i d e f i x a t i o n (5). Therefore i f ammonia i s presen t at an i n h i b i t o r y c o n c e n t r a t i o n , the amount of s t a r c h would be expected to be low. Two r e p o r t s (4, 9) have i n d i c a t e d t h a t s t a r c h content o f ammonia-treated D o u g l a s - f i r i s lower than i n n i t r a t e - t r e a t e d D o u g l a s - f i r . In a d d i t i o n Bassham (1) has shown t h a t ammonia has 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. influenced metabolism from sugar synthesis to protein synthesis. An experiment was undertaken with Douglas-fir, West-ern redcedar, Lodgepole pine and Western hemlock to 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 starch content of the seedlings was related to the source of available nitrogen. METHODS AND MATERIALS Details of growing conditions, culture solutions, c u l -ture technique, dry weight and su r v i v a l have been described previously (Part 2-A). The seedlings were grown i n b u l l e t s and were supplied with 1 of 3 sources of nitrogen: n i t r a t e only (NO^ -N), ammonia only (NH^+-N) or a combination of n i t r a t e and ammonia (7:1)((N0 3 + NH 4 +)-N). At 4 months aft e r germination, the seedlings were removed from the b u l l e t s , adhering sand was washed o f f and the roots were placed i n jars (2 seedlings per jar) of nutrient solution containing the appropriate nitrogen treatment (eg. seed-lings supplied with NO^ -N i n the b u l l e t s were placed i n NO^ -N nutrient solution). The jars were aerated constantly and the nutrient solution was changed every second day. The pH of the nutrient solutions was measured for six consecutive days. After s i x days on the nutrient solution the seedlings 39 . were l y o p h i l i z e d . S o l u b l e carbohydrate was e x t r a c t e d by s o n i c a t i o n o f .1 gm l y o p h i l i z e d m a t e r i a l i n 20 ml e t h a n o l . S t a r c h and the s o l i d r e s i d u e were r e t a i n e d . S t a r c h was e x t r a c t e d from the r e s i d u e and h y d r o l y z e d to glucose with p e r c h l o r i c a c i d a c c o r d i n g to McCready et_ al_. (14) . Glucose was determined u s i n g hydrogen peroxide and s u l f u r i c a c i d a c c o r d i n g to Dubois e_t a l . (3) . A l l data was analyzed by analyses 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 the LSD a t 5% l e v e l . RESULTS D e t a i l e d d e s c r i p t i o n s of dry weight, l e a f area and r o o t dry weight were r e p o r t e d i n P a r t 2-A. pH Data The i n i t i a l pH o f the n u t r i e n t s o l u t i o n was 5.5. The pH a f t e r 2 days exposure to the r o o t s i s given i n Table 1. With Western redcedar and Western hemlock, the pHs o f the + — + NH 4 -N and (NC>3 + NH^ ) -N treatment s o l u t i o n s were i n d i s -t i n g u i s h a b l e from each other and have a s i g n i f i c a n t l y lower pH than the NO^ -N treatment. With D o u g l a s - f i r and Lodge-po l e pine the pH of the (NO^ + NH 4 +)-N treatment s o l u t i o n was s i g n i f i c a n t l y h i g h e r than the pH of the NH 4 +-N treatment s o l u t i o n but was s t i l l much lower than the pH of the NO^ -N treatment s o l u t i o n . TABLE 1. The mean pH of the nutrient solutions af t e r treatment for 2 days. 1,2 Species Source of Nitrogen NH4+-N (N03~ + NH 4 +)-N N0 3" -N 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 pine 3.85 4.87 6. 02 "'"Initial pH of nutrient solutions 5.5. 2 Values connected by the same line do not d i f f e r at the 5% l e v e l by LSD. 41. Starch Content Douglas-fir and Western redcedar treated with NH^+-N had the lowest starch content compared to the other nitrogen treatments (Table 2). The (NC>3~ + NH4 + )-N treatment pro-duced leve l s of starch equal to that of the n i t r a t e t r e a t -ment. With Lodgepole pine and Western hemlock, there was no difference i n starch production among the three t r e a t -ments . DISCUSSION When NO^ -N i s taken up, the pH of a nutrient solution r i s e s , probably as a r e s u l t of the expulsion of OH and/or HC03~ (2,10). When NH4+-N i s taken up the pH of the nutrient solution decreases, probably as a r e s u l t of the expulsion of H + (2,10). The difference i n pH between the NH4+-N treatment and the (N03 + NH 4 +)-N treatment of Douglas-fir and Lodgepole pine (Table 1) may be due to 1) less uptake of NH4+-N from the (N03 + NH 4 +)-N treatment with concomitant lower expulsion of H + and/or 2) simultaneous uptake of small amounts of N03~-N with NH4+-N from the (N03~ + NH 4 +)-N and thus release of both H + and OH . For a l l four species, i t can be concluded that NH4+-N was the predominant source of nitrogen when supplied as (N03 + NH 4 +)-N. These results agree with Van Den Driessche's work with conifers (2). The TABLE 2. Starch content of conifer seedlings supplied with d i f f e r e n t sources of nitrogen for 4 months( g/gm dry weight).! Species Source of Nitrogen NH4+-N (N03 + NH 4 +)-N N0 3 -N Douglas-fir 52.1 118.1 107.3 Western redcedar 106.7 143. 7 131.0 Western hemlock 173.3 179.7 181.0 Lodgepole pine 117.0 114.4 103. 4 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. NH 4 +-N content o f the (N0 3~ + NH 4 +)-N was 1.75 ppm N w h i l e 12.25 ppm of NC>3 -N was present. When NH 4 +-N i s presen t i n t o x i c l e v e l s , NH 4 +-N i n -h i b i t s carbon d i o x i d e f i x a t i o n (5) and NH 4 +-N has a l s o been shown to switch sugar p r o d u c t i o n t o p r o t e i n produc-t i o n (1). Both of these mechanisms would i n t e r f e r e w i t h s t a r c h p r o d u c t i o n . When D o u g l a s - f i r and Western redcedar were s u p p l i e d w i t h 14 ppm NH 4 +-N, s t a r c h p r o d u c t i o n was low (Table 2). On the other hand, when D o u g l a s - f i r and Western redcedar s e e d l i n g s were s u p p l i e d w i t h (N0 3 +NH 4 +)-N, NH 4 +-N (1.75 ppm N) was the predominant form of n i t r o g e n taken up. Trees i n t h i s treatment produced amounts of s t a r c h equal to amounts produced w i t h N0 3 -N onl y . With Lodgepole pine and Western hemlock s t a r c h pro-d u c t i o n was the same on both h i g h (14 ppm N) and low l e v e l s (1.75 ppm N) of NH 4 +-N and high l e v e l s (14 ppm N) of N0 3 -N. These r e s u l t s would i n d i c a t e t h a t Lodgepole pine and Western hemlock have a h i g h e r t o l e r a n c e to ex-t e r n a l s u p p l i e s o f NH 4 +-N than D o u g l a s - f i r and Western redcedar. However D o u g l a s - f i r and Western redcedar are t o l e r a n t of low amounts of NH 4 +-N. In a d d i t i o n , the high dry matter p r o d u c t i o n (Part 2-A) of Lodgepole pine and Western hemlock on e i t h e r NH 4 +-N or (N0 3 + NH 4 +)-N i s now e a s i l y e x p l a i n e d . In f a c t , the s e e d l i n g s s u p p l i e d w i t h (NC>3 +'NH4 )-N were u t i l i z i n g NH^ -N. The t o l e r a n c e of D o u g l a s - f i r t o low l e v e l s o f NH 4 +-N may e x p l a i n why Van Den Dr i e s s c h e (17) concluded t h a t 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 with NH 4 +-N r a t h e r than with NC>3~-N. The v a r i a t i o n i n t o l e r a n c e o f the s e e d l i n g s to ammonia may be e a s i l y e x p l a i n e d i n terms of s e l e c t i o n by where the t r e e s occur i n nature. D o u g l a s - f i r and Western redcedar, which grow and s u r v i v e p o o r l y at high l e v e l s o f NH 4 +-N (Part 2-A) occur on s o i l s where NC>3 -N i s thought t o be the predominant form o f n i t r o g e n (11). In these s p e c i e s , s e l e c t i o n f o r ammonia t o l e r a n c e or p r o t e c t i o n a g a i n s t NH 4 +-N t o x i c i t y has not been necessary. Lodgepole pine and Western hemlock which grow and s u r v i v e w e l l (Part 2-A) at h i g h l e v e l s of NH 4 +-N occur on s o i l s where NH 4 +-N i s consi d e r e d to be the predominant form o f n i t r o g e n (11). In these s p e c i e s s e l e c t i o n f o r ammonia t o l e r a n c e has been necessary. 45. LITERATURE CITED 1. Bassham, J. 1971. The control of photosynthetic carbon metabolism. Science 172: 526-534. 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. Nitrate and ammonium as sources of nitrogen for corn and th e i r influence on the uptake of ions. 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. Colorimetric method for determination of sugars and related 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 sub-stances associated with d i f f e r e n t i a l cone responses of Douglas-fir 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, H. 1960. Energy storage. In Plant Physiology. F.C. Steward (ed.). Academic Press, New York. 6. Gransmanis, V.O. and D.J.D. Nicholas. 1966. Uptake of n i t r a t e by Jonathan apple trees. Plant and S o i l 25: 461-462. 7. Goring, C.A.I. 1956. The nitrogen n u t r i t i o n of plants. Down to Earth. Spring, 7-9. 8. Harada, T., H. Takaki and Y. Yamada. 1968. E f f e c t of nitrogen source on the chemical components in young plants. S o i l S c i . and PI. Nut. 14: 47-55. 9. Hart, A.L. and G.E. Mellor. 1971. Leaf structure of Douglas-fir supplied with d i f f e r e n t forms of nitrogen. Proceedings of the Canadian Society of Plant Physiologists p. 35. Toronto. 10. Kirby, E.A. 1968. Ion uptake and io n i c balance i n plants i n r e l a t i o n to the form of nitrogen. In. Ecological aspects of the mineral n u t r i t i o n of plants. I.H. Rorison (ed.). Blackwell S c i e n t i f i c Publications, Oxford and Edinburgh. 11. Krajina, V.J. 1969. Ecology of forest trees i n B r i t i s h Columbia. Ecology of Western North America 2: 1-146. Botany Department, University of B r i t i s h Columbia. 46. 12. Krogmann, D.W. , A.T. Jagendorf and M. Avron. 1959. Uncouplers of spinach chloroplast photophosphor-y l a t i o n . 34: 272-13. Lycklama, J.C. 19 63. Th.e absorption of ammonium and ni t r a t e by perennial rye grass. Acta Bot. Neerl. 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 of starch and amylose i n vegetables. Anal. Chem. 22: 1156-1158. 15. Minotti, P.L., D.C. Williams and W.A. Jackson. 1969. Nitrate uptake by wheat as influenced by ammonium and other cations. Crop Science 9: 9-14. 16. Townsend, L.R. 1970. E f f e c t of form of nitrogen and pH on n i t r a t e reductase a c t i v i t y i n lowbush Blueberry leaves and roots. Can. J. Plant S c i . 50: 603-605. 17. Van Den Driessche, R. 1971. Response of conifer seed-lings to n i t r a t e and ammonium sources of nitrogen. Plant and S o i l 34: 421-439. 18. Weissman, G.S. 1951. Nitrogen metabolism of wheat seedlings as influenced by the ammonium:nitrate r a t i o and the hydrogen-ion concentration. 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 (Part 2-A) i t was shown that the s u r v i v a l and growth of conifer seedlings were affected by the source of nitrogen. For Douglas-fir and Western hemlock, s u r v i v a l and growth on a n i t r a t e solution 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 (7:1). Ammonia was an unfavorable source of nitrogen for s u r v i v a l and growth of Douglas-fir and Western redcedar. For Western hemlock, ammonia treatment was only detrimental to s u r v i v a l . Hemlock trees which survived ammonia treatment grew as well as trees growing on the other two sources of nitrogen. Lodgepole pine survived equally well under a l l treatments and was the only species that grew best on ammonia. Swan (2) reported that over a four month growing period, Jack pine (Pinus banksiana) and White spruce (Picea glauca) grew better when supplied with ammonia rather than 1 This section deals with work i n i t i a t e d by S. Madoc-Jones but continued by the author after the untimely death of S. Madoc-Jones. Also the data i n t h i s section represents part of a paper presented at the XII P a c i f i c Science Congress, Canberra, 19 71. Ammonia and Nitrate i n the Nitrogen Economy of Some Conifers Growing i n Douglas-fir Communities of the P a c i f i c North-West America by V.J. Krajina, S. Madoc-Jones and G.E. Mellor. 48. n i t r a t e . Durzan and Steward (1) continued Swan's experiment for another 12 months and found that n i t r a t e now gave the best results for White pine, while Jack pine s t i l l grew best when supplied with ammonia. The object of the work presented in this section was to determine the e f f e c t of supplying four conifer species with d i f f e r e n t sources of nitrogen for a period of 10 to 12 months. METHODS AND MATERIALS Seed source, growing conditions and nutrient solution were described previously (Part 2-A). Seeds of Douglas-fir, Western redcedar, Western'hemlock and Lodgepole pine were sown (38 per 1 gallon crock) i n sand and the crocks were i r r i g a t e d i n i t i a l l y seven times a day with demineralized water. I r r i g a t i o n with nutrient solutions was begun one week afte r germination. Each crock was i r r i g a t e d automa-t i c a l l y and received 250 ml of nutrient four times a day. For the f i r s t seven weeks af t e r germination the concentra-tion of the nutrient solution was that used i n Part 2-A (i. e . 14 ppm N). At seven weeks after germination the concentration of the nutrient solution was doubled (28 ppm N) and remained so u n t i l harvest. The same nitrogen treatments were used as i n Part 3-B (NH4+-N, NO^ -N and (NO_ + NH. + )-N). The crocks were placed i n the growth room i n a randomized complete b l o c k design (3 b l o c k s ) . F i v e weeks a f t e r germination the p l a n t s were th i n n e d to f i v e per crock. T h i r t e e n weeks a f t e r germination D o u g l a s - f i r and Lodgepole pine were th i n n e d to two p l a n t s per crock. F i f t e e n weeks a f t e r germination Western redcedar and Western hemlock were thinned to two p l a n t s per crock. The two l a r g e s t D o u g l a s - f i r and Lodgepole pine s e e d l i n g s were chosen to remain. T h i s choice was j u s t i f i e d on the grounds t h a t s i n c e both s p e c i e s are shade i n t o l e r a n t i n the area of t h e i r provenance the two l a r g e s t s e e d l i n g s would have the g r e a t e s t chance of s u r -v i v a l i n nature. The Western redcedar and the Western hemlock s e e d l i n g s chosen to remain were the l a r g e s t and the s m a l l e s t s e e d l i n g s because both s p e c i e s are shade t o l e r a n t . Western redcedar and D o u g l a s - f i r were harvested 10 months a f t e r germination and Western hemlock and Lodgepole p i n e , 12 months a f t e r g ermination. Shoot h e i g h t was determined, then the t r e e s were ha r v e s t e d and d i v i d e d i n t o r o o t s , leaves and branches. The t r e e s were oven d r i e d a t 80C to con s t a n t weight. Leaf area of D o u g l a s - f i r , Western redcedar, and Western hemlock was determined by choosing 100 l i v e needles at random from each t r e e . These needles were d i v i d e d i n t o 10 groups of 10 needles each. The area of these needles 50. was determined by m u l t i p l y i n g t o g e t h e r 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 f o r needles o f t h i s age as d e s c r i b e d i n Appendix I. N e c r o t i c and c h l o r o t i c needles or p o r t i o n s of needles were not used i n these determina-t i o n s . A f t e r l e a f area had been determined f o r each group of needles, the groups were d r i e d to c o n s t a n t weight. A f t e r d r y i n g the average area of 1 gm dry weight o f leaves was determined f o r each t r e e (average o f the 10 groups). Thus wi t h a knowledge of t o t a l l e a f dry weight, the t o t a l l e a f area o f a t r e e was determined. Leaf area of Western redcedar was determined by photocopying leaves from each t r e e and then u s i n g a p l a n i m e t e r to determine the area of the Xerox c o p i e s . The leaves were then d r i e d to constant weight. Thus the area of 1 gm dry weight of leaves was determined and again with a knowledge of t o t a l l e a f dry weight, t o t a l l e a f area c o u l d be determined. A l l data were analyzed with analyses of 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 of the LSD a t the 5% l e v e l . RESULTS D o u g l a s - f i r The (N0 3~ + NH 4 +)-N treatment produced the t a l l e s t s e e d l i n g s f o l l o w e d by the NO^ -N treatment and the NH 4 +-N 51. treatment (Table 1). Seedlings supplied with NH4+-N pro-duced the shortest plants, and the lowest shoot dry weight, t o t a l dry weight and leaf area. The NO^ -N seedlings had a larger t o t a l dry weight than the (NC>3 + NH^+)-N seedlings. This difference i n t o t a l dry weight was mostly attributable to differences i n root dry weight. In spite of a difference in t o t a l dry weight there was no s i g n i f i c a n t difference i n leaf area between the N03~-N and the (N0 3~ + NH^+)-N seed-l i n g s . The difference i n dry matter production between the NH4+-N and (NC>3 + NH 4 +)-N treatments was a r e s u l t of difference i n shoot dry weight. The tips of the older needles were frequently c h l o r o t i c and necrotic. Dr. V.J. Krajina 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 of magnesium and calcium deficiency. Several NH4+-N seedlings l o s t many leaves before harvest. Western Redcedar Seedlings on the N0 3 -N treatment had a larger dry matter production than those on the (N03 + NH4 + )-N t r e a t -ment and thi s difference was due to a difference i n shoot dry weight (Table 2). The NH4+-N treatment produced the shortest seedlings, the smallest leaf area and the smallest t o t a l , root and shoot dry weight. There was a d i r e c t relationship between leaf area and dry matter production (i . e . the larger the leaf area, the larger the dry matter production). The older leaves of the NH.+-N treated TABLE 1. Total, root, and shoot dry weight, leaf area and height of Douglas-fir supplied with d i f f e r e n t sources of nitrogen.1 Source of Nitrogen N03~ NC>3~ + NH 4 + (7:1) NH 4 + Total dry weight (gm/crock) 167.4 133.4 68.8 Root dry weight (gm/crock) 64.0 42.0 27. 3 Shoot dry weight (gm/crock) 103 . 4 91.5 41.5 Total surface area of leaves (cm 2/plant) 5855.1 4280.9 1621.8 Shoot height (cm) 74.0 90.2 43.2 Values connected by the same li n e do not d i f f e r at the 5% l e v e l by LSD. TABLE 2. Total, root, and shoot dry weight, leaf area and height of Western redcedar supplied with d i f f e r e n t sources of nitrogen.1 Source of Nitrogen N0 3~ N0 3~ + NH 4 + (7:1) NH^ Total dry weight (gm/crock) 201. 5 160. 7 37. 6 Root dry weight (gm/crock) 47. 1 40. 7 8. 2 Shoot dry weight (gm/crock) 154. 4 120. 0 29. 6 Total surface area of leaves 2 (cm /plant) 6410. 0 5060. 0 1410. 0 Shoot height (cm) 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 (symptoms o f c a l c i u m and magnesium d e f i c i e n c y a c c o r d i n g t o Dr. K r a j i n a ) . Western Hemlock There were no s i g n i f i c a n t d i f f e r e n c e s i n h e i g h t among any o f the treatments (Table 3). The NO^ -N treatment r e s u l t e d i n the lowest l e a f area and s m a l l e s t t o t a l , r o o t and shoot dry weight. With the ex c e p t i o n o f l e a f area, there was no d i f f e r e n c e i n any of the parameters measured between the NH 4 +-N and (N0 3~ + NH 4 +)-N treatments. One might have expected a d i f f e r e n c e i n t o t a l dry matter p r o d u c t i o n between the NH 4 +-N and (N0 3 + NH 4 +)-N t r e a t -ments c o n s i d e r i n g the l a r g e d i f f e r e n c e i n l e a f area. The leaves o f s e e d l i n g s on the NH 4 +-N and (N0 3 + NH 4 +)-N were dark green, however a few showed some n e c r o t i c and c h l o r o t i c areas. These areas were i d e n t i f i e d by Dr. V.J. K r a j i n a as being i n d i c a t i v e o f magnesium d e f i c i e n c y . The s e e d l i n g s s u p p l i e d w i t h N0 3 -N produced y e l l o w i s h green l e a v e s . Lodgepole Pine There was no s i g n i f i c a n t d i f f e r e n c e i n h e i g h t between the three treatments (Table 4). The s e e d l i n g s on the N0 3 -N treatment produced the lowest shoot dry weight, t o t a l dry weight and l e a f area. The N0 3 -N and (N0 3 + NH 4 +)-N s e e d l i n g s produced s i m i l a r amounts of r o o t dry weight and thus d i f f e r e n c e s i n t o t a l dry weight between TABLE 3. Total, root, and shoot dry weight leaf area and height of Western hemlock supplied with d i f f e r e n t sources of nitrogen. NO. Source of Nitrogen N0 3~ + NH 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 Total dry weight (gm/crock) Root dry weight (gm/crock) Shoot dry weight (gm/crock) 2 Total surface area of leaves (cm /plant) Shoot height (cm) 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. TABLE 4. Total, root, and shoot dry weight, leaf area and height of Lodgepole pine supplied with d i f f e r e n t sources of nitrogen.1 N0 3-Source of Nitrogen N0 3~ + NH 4 + (7:1) NH4 + Total dry weight (gm/crock) 113. 2 210.0 229.7 Root dry weight (gm/crock) 48.6 67. 6 82.2 Shoot dry weight (gm/crock) 65.6 142.4 147. 5 Total surface area of leaves 2 (cm /plant) 2251.7 6460.8 6789.8 Shoot height (cm) 36.7 29.0 33.7 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 7 . these two treatments were due to differences i n shoot dry weight. There was no difference i n any of the parameters measured between the NH^+-N and (NO^ + NH 4 +)-N treatments. The leaves of seedlings treated with NH4+-N and (NO^ + NH^+)-N were dark green while many leaves of the n i t r a t e treatment were yellowish green over t h e i r entire length. DISCUSSION The experiment described i n this part extends the growth period from the experiment described i n Part 2-A. There were, however, certain differences i n culture techniques between the two experiments: receptacles for growing, concentration of nitrogen, and the frequency of nitrogen application. Both Douglas-fir and Western redcedar seedlings on the NH4+-N treatment grew least over both the 4 month growing period and over the 10 month growing period. At 4 months the N03~-N and (N0 3~ + NH 4 +)-N Douglas-f i r seedlings were indistinguishable from each other, but after 10 months certain differences became noticeable. The N0 3 -N treatment produced t a l l e r seedlings and a higher t o t a l dry weight. This difference i n dry weight was due to differences i n root dry weight. At both 4 and 10 months, the trends among nitrogen treatments with Western redcedar were exactly the same with the exception that a difference i n leaf area was 58. d e t e c t a b l e between the N0 3~-N and the (N0 3~ + NH 4 +)-N treatments a t the end of 10 months. Over both treatment p e r i o d s , the N0 3 -N treatment produced the l a r g e s t dry weight. A t 4 months, the only e f f e c t of source o f n i t r o g e n f o r Western hemlock was on l e a f area and s u r v i v a l . A t the end of the 12 month treatment p e r i o d , s e e d l i n g s on the N0 3 -N treatment produced by f a r the lowest dry matter and l e a f area. Over both treatment p e r i o d s , s e e d l i n g s on + — + the NH^ -N and (N0 3 + NH^ )-N treatments were i n d i s t i n g u i s h -a b l e from one another except f o r l e a f area. The (N0 3 + NH 4 +)-N treatment produced s e e d l i n g s w i t h the l a r g e s t l e a f area. A t the end of the 4 month treatment p e r i o d , NH 4 +-N was the most f a v o r a b l e source of n i t r o g e n f o r Lodgepole p i n e . Seedlings from the N0 3~-N and the (N0 3~ + NH 4 +)-N treatment were i n d i s t i n g u i s h a b l e from one another. A t the end of the 12 month treatment p e r i o d , the N0 3 -N treatment produced the lowest dry weight and s m a l l e s t l e a f a r e a . S e e d l i n g s on the NH 4 +-N and (N0 3 + NH 4 +)-N treatments were now i n d i s t i n -g u i s h a b l e from one another. In c o n c l u s i o n the trends shown i n the 4 month e x p e r i -ments were confirmed i n the longer experiment and some a d d i t i o n a l treatment e f f e c t s appeared, p a r t i c u l a r l y i n s e p a r a t i n g the treatments with (N0 3 + NH 4 +)-N from t r e a t -merits i n which only one form of nitrogen was present. At the concentrations of nitrogen used (14 or 28 ppm), NO^ -N i s the most favorable form of nitrogen for Douglas-f i r and Western redcedar while NH.+-N i s the most favorable 4 form of nitrogen for Lodgepole pine and Western hemlock. In Part 2-B, i t was shown that NH4+-N was the predomi-nant form of nitrogen taken up from the (NO^ + NH^ "1") -N treatment. At this low concentration of NH4+-N (1.75 ppm), Douglas-fir and Western redcedar survived and grew better than when supplied with a high rate of NH4+-N (14.0 ppm). At the end of 10 months, Western redcedar i n the lower concentration of NH4+-N also gave better growth than i n the higher concentration of NH4+-N. However at both 4 and 10 months, the low NH4+-N treatment produced less dry weight than the NO^ -N treatment. The quantity of NH4+-N in the low NH4+-N treatment may s t i l l have been too high to obtain the optimum growth rate of the seedlings. At 4 months Douglas-fir seedlings on the low NH4+-N and NO^ -N treatments were indistinguishable from each other; however at 10 months the NO^ -N treatment produced a greater amount of dry matter than the low NH4+-N treatment. At the end of the 12 month period the quantity of NH4+-N i n the low NH4+-N treatment may s t i l l have been too high to obtain the optimum growth rate of the seedlings. 60. With Lodgepole pine and Western hemlock at the end of 4 months, the NO^ -N treatment with one exception produced amounts of dry matter si m i l a r to those produced on the other two treatments. However, at the end of 12 months, the NC>3 -N treatment produced less dry matter than the other two treatments. The seedlings may have been able to metabolize enough NO^ -N to supply t h e i r needs when they were small, but as the seedlings increased i n siz e , they might not have been able to assimilate or convert enough NO., -N to meet t h e i r needs. 61. LITERATURE CITED 1. Durzan, D.J. and F.C. Steward. 1967. The nitrogen metabolism of Picea glauca and Pinus banksiana as influenced by mineral n u t r i t i o n . Can. J. Bot. 45: 695-710. 2. Swan, H.S.D. 1960. The mineral n u t r i t i o n of Canadian pulpwood species. Pulp and Paper Research Insti t u t e of Canada. Woodlands Research Index No. 116, Montreal, Canada. PART 2-D. GAS EXCHANGE OF CONIFER SEEDLINGS SUPPLIED WITH DIFFERENT FORMS OF NITROGEN. INTRODUCTION In Part 2-C i t was shown that Douglas-fir seedlings treated with NH4+-N produced the lowest dry weight and leaf area (Tables 1 and 2). There was no difference i n leaf area between the N0 3 -N and (NO^ + NH^ "1*) -N seedlings although the NO^ -N seedlings produced a higher dry weight. With Western redcedar, there was a di r e c t relationship between t o t a l dry weight and leaf area ( i . e . the larger the leaf area, the larger the dry matter production). The NO^ -N treated seedlings produced the largest leaf area and the largest amount of dry matter, followed by the (N03~ + NH 4 +)-N treatment and then the NH4+-N treatment. NH4+-N and (N03~ + NH 4 +)-N treated Western hemlock seedlings produced s i m i l a r amounts of dry matter, although there was a large difference i n leaf area. The N0 3 -N treated seedlings produced the least amount of dry matter and leaf area. Lodgepole pine seedlings supplied with N0 3 -N produced the lowest dry weight and lea f area. There was no d i f f e r -ence i n dry weight and leaf area between the NH4+-N and (NO ~ + NH„ +)-N treatments. TABLE 1. Total dry weight of conifer seedlings supplied with d i f f e r e n t sources of nitrogen (gm.pot~l).1 Species NO3-Nitrogen Treatment N 0 3 ~ + NH 4 + (7:1) NH 4 + Douglas-fir 167.4 133. 4 68.8 Western redcedar 201.5 160. 7 37.6 Lodgepole pine 113.2 210.0 229.7 Western hemlock 107.6 235. 4 212.2 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. TABLE 2. Leaf area of conifer seedlings supplied with d i f f e r e n t sources of nitrogen (cm2.pot~l).1 Species N0 3-Nitrogen Treatment N03~ + NH 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 Lodgepole pine 4,503.4 12,921.6 13,579.6 Western hemlock 18,156.4 44 ,993.0 24,683.8 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. The lack of c o r r e l a t i o n between leaf area and plant dry weight with Douglas-fir and Western hemlock suggested that the nitrogen treatment was a f f e c t i n g the gas exchange of the leaves. The results presented i n this paper suggest trends i n stomatal resistance (r ) which would a f f e c t both s water relations and gas exchange. SYMBOLS AND UNITS _ 3 (C)„) External Co_ concentration (gm cm ) z a 2. _3 • ^ C 0 2 ^ c Chloroplast CO2 concentration (gm cm ) - 2 - 1 T Transpiration (gm cm sec ) - 2 - 1 P C 0 2 flux (gm cm min ) RH Relative humidity r Boundary layer d i f f u s i o n resistance (sec cm "*") a r m Mesophyll d i f f u s i o n resistance (sec cm "*") r g Stomatal d i f f u s i o n resistance (sec cm - 3 (H„0) Water vapor concentration of a i r (gm cm ) 2, a (H 2 0) Water vapor concentration of stomatal cavity (gm cm"3) V Total water potential (bar) ¥ Gravitational potential (bar) g H* Matric potential (bar) m Pressure potential (bar) ¥ s Solute potential (bar) THEORY Internal water stress may be described i n terms of the energy status of the contained water, usually expressed as t o t a l water pote n t i a l ( *¥ ) (1). In both plant and s o i l systems, H* i s given by the equation T s T p 1 m T g The matric pot e n t i a l (Y ) and g r a v i t a t i o n a l potential ( ) m g are usually neglected for plant systems and equation (I) may be written as V = T c + ¥ ( I D fa p Transpiration i s given by (H 90) - (H_0) , . T _ 2 s 2 a (III) r + r s a For plants (Ho0) - (H_0) i s calculated from measure-ments of r e l a t i v e humidity, assuming 100% RH inside the stomates. Likewise the flow of CX^ i s controlled by a s i m i l a r relationship (CO ) - (CO ) P = £_£ ( I V ) r + r + r s a m ^ ^ 2 ^ c '""S u s u a^-'-y assumed to be zero. The resistances r + r for P can be calculated from T, taking into 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 and C0 2 i n a i r . METHODS AND MATERIALS Four conifer species, Douglas-fir, Western hemlock, Lodgepole pine and Western redcedar were grown i n pots of sand. After thinning, each pot contained 2 seedlings. The seedlings were supplied with complete nutrient solu-t i o n i n which only the source of nitrogen was varied. Nitrogen was supplied as ammonia only, n i t r a t e only or as a combination of n i t r a t e and ammonia (7:1). (Hereafter these treatments w i l l be referred to as NH4+-N, NO^ -N and (N03 + NH 4 +)-N). The nitrogen content of the solutions was 14 ppm N for the f i r s t 7 weeks and 2 8 ppm thereafter. Exact d e t a i l s of the culture technique are described elsewhere (Part 2-C). The seedlings were grown i n a growth room with 90% RH i n the day and 65% at night. Fluorescent and incan-descent lamps provided 20,000 lux over a photoperiod of 16 hr (6 am to 10 pm). Dawn and t w i l i g h t effects were produced by staggering the order i n which the li g h t s came on and went o f f . A 12 hr temperature cycle was used (8 am to 8 pm at 2 4C; 8 pm to 8 am at 20C). Douglas-fir and Western redcedar seedlings were harvested 10 months after germination while Lodgepole pine and Western hemlock were harvested 12 months after germination. 68. Leaf M* and H* were measured with a three wire thermocouple psychrometer (2). The techniques used are described by E h l i g (3). was determined from equation (II). Leaf samples were taken at 9 am from the growing point of the trees. Each tree was sampled 3 times. Transpiration was determined by covering the pot with a sheet of Parafilm and measuring the loss of water over a 24 hr period. After determination of t r a n s p i r a t i o n , the trees were harvested and oven dried at 80C. RESULTS AND DISCUSSION Leaf T, V s, V p S e n s i t i v i t y of the thermocouples ranged from .23 to .29 /tv per bar. For routine determinations with the psychrometer the standard error of observation of stan-dard KCl solutions was about - .5 bar. There was no difference i n leaf V , y and H* s p among the d i f f e r e n t nitrogen treatments. Therefore one value of leaf V y and Y was determined for each s p species (Table 3). The mean values of l e a f V for Douglas-f i r , Western redcedar, Lodgepole pine and Western hemlock were -21.2, -11.8, -10.6 and -18.8 bars with standard errors of .8, .5, .3 and .7 bars. The standard error of TABLE 3. Leaf V , (bars). * V s and of conifer seedlings supplied with d i f f e r e n t sources of nitrogen Species *s Douglas-fir -21.2 -34.0 +12. 8 Western redcedar -11. 8 -21. 4 +9.7 Lodgepole pine -10.6 -24.5 +13.9 Western hemlock -18. 8 -29.4 +10.6 Nutrient Solution NH4 + N 0 3 ~ + NH 4 + -.77 -.65 NO3- -.43 VO 70. l e a f *¥$ f o r D o u g l a s - f i r , Western redcedar, Lodgepole pine and Western hemlock were 1.1, .6, .4 and .6 r e s p e c t i v e l y . Table 3 c o n t a i n s *f f o r the r e s p e c t i v e n u t r i e n t s o l u t i o n s . The v a l u e s of V r e p o r t e d i n Table 3 are s i m i l a r t o values r e p o r t e d f o r other woody s p e c i e s (4, 5, 6). Values of »j> of from -16 to -31 bars have been r e p o r t e d f o r ever-green c o n i f e r s (8) . Values of *f r e p o r t e d here f a l l c l o s e s to t h i s range. To our knowledge no value of Hf f o r t r e e s has been r e p o r t e d . The s i m i l a r l e a f 4* among the n i t r o g e n treatments w i t h i n s p e c i e s i n d i c a t e s t h a t there was no d i f f e r e n c e i n water d e f i c i t between the n i t r o g e n treatments. Water Uptake The amounts of water l o s t from the pots at the time of l e a f sampling f o r the l e a f p o t e n t i a l s t u d i e s are given i n Table 4. With D o u g l a s - f i r and Western redcedar, the NH 4 +-N treatment l o s t 1/3 to 1/4 the amount of water of the NO^ -N treatment and 1/2 to 1/3 the amount of water of the (N0 3~ + NH 4 +)-N treatment (Table 4). Lodgepole pine s e e d l i n g s s u p p l i e d w i t h NO^ -N l o s t approximately 1/2 the amount o f water o f the other two treatments. TABLE 4. Water loss of conifer seedlings supplied with d i f f e r e n t sources of nitrogen (ml H20/24 hr.pot) . Species NO3-Nitrogen Treatment N 0 3 ~ + NH 4 + (7:1) NH . + 4 Douglas-fir 683.7 403.2 180. 3 Western redcedar 627.2 554.2 203.2 Lodgepole pine 222.2 486.3 395.1 Western hemlock 482.4 553.6 324.7 Western hemlock seedlings treated with (NC>3 + NH 4 +)-N l o s t the most water followed by the NO^ -N and NH4+-N tr e a t -ments . Since t r a n s p i r a t i o n i s considered an unavoidable e v i l (7) which often results i n water d e f i c i t s and because there was no difference i n water d e f i c i t (Table 3) among the nitrogen treatments, we suggest that differences i n growth among nitrogen treatments within species cannot be attributed d i r e c t l y to differences i n water r e l a t i o n s . However si m i l a r leaf *? may have been maintained by c o n t r o l l i n g T v i a r g . As previously stated stomatal resistance to flow of CO2 i s determined from T. Thus the water relations of these plants may have had 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 rate of entry of C0 o. Thus r + r was c a l -culated according to equation I I I . In t h i s experiment r i s considered to be constant within species and because a c of the small size of leaves and a i r c i r c u l a t i o n i n the growth room, r i s considered to be small. Thus c a l c u l a t i o n a. of r + r gives an approximation of r and a l l v a r i a b i l i t y S o . s within species between r + r i s attributable to r . A s a s comparison of r + r (Table 5) with dry weight (Table 1) S cl shows that for Western redcedar, Douglas-fir and Lodgepole pine, there was l i t t l e difference i n r + r among nitrogen s a treatments but a large difference i n y i e l d . With Western TABLE 5. Transpiration resistance (r + r ) of conifer seedlings supplied with d i f f e r e n t sources of nitrogen (secern -!). Species Nitrogen Treatment N03~ N03~ + NH 4 + (7:1) NH 4 + 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 hemlock, there 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 higher the value of r + r , the S cl higher the y i e l d . An inverse r e l a t i o n s h i p would be needed to support causality. These results indicate that i n no case i s r + r r e s t r i c t i n g dry matter production. These s a results suggest that the e f f e c t of nitrogen treatment on r ^ had an e f f e c t on dry matter production. I t has been reported for Pinus halpensis that l i g h t and temperature affected CO,, uptake through (9) . S i m i l a r l y , Wuenscher and Kozlowski (10) found that for three woody species at 3000 f t - c the differences i n r m were more important than differences i n r i n determining t o t a l C0 9 transfer resistance. LITERATURE CITED Barrs, H.D. 1968. Determination of water d e f i c i t s i n plant tissue, pp. 235-368. I_n T.T. Kozlowski (ed.) Water d e f i c i t and plant growth, Vol. I. Academic Press, New York. Chow, T.L. and J. de Vries. 1972. Dynamic measurement of s o i l and leaf water p o t e n t i a l with a double loop p e l t i e r type thermocouple psychrometer. Western Society S o i l Science meeting at Oregon June 12-15. E h l i g , G.F. 1961. Measurement of the energy status of water i n plants with a thermocouple psychrometer. Plant Physiol. 37: 288-290. Kaufmann, M.R. 196 8. Evaluation of the pressure chamber technique for estimating plant water pote n t i a l of forest tree species. Forest S c i . 14: 369-374. Kaufmann, M.R. 1968. Water relations of pine seedlings i n r e l a t i o n to root and shoot growth. Plant Physiol. 43: 281-288. Klepper, B. 196 8. Diurnal patterns of water pote n t i a l i n woody plants. Plant Physiol. 43: 1931-1934. Kramer, P. 1969. Transpiration pp. 296-346. In Plant and s o i l water r e l a t i o n s h i p s : a modern synthesis. McGraw-Hill Book Company. Pisek, A. 1956. Der wasserhaushalt der mesa und Hygro-phyten. Enclop. Plant Physiol. 3: 825-853. Whiteman, P.C. and D. K o l l e r . 1965. Environmental con-t r o l of photosynthesis and transpiration i n Pinus  halepensis. I s r a e l J. Bot. 13: 166-176. Wuenscher, J.E. and T.T. Kozlowski. 19 . Carbon dioxide transfer resistance as a factor i n shade tolerance of tree seedlings. 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 area and l e a f dry weight o f three c o n i f e r s p e c i e s grown on three sources o f n i t r o g e n . 1 Determination of l e a f area i s a fundamental measurement f o r p h y s i o l o g i c a l experiments. S e v e r a l methods o f e s t i m a t i n g c o n i f e r l e a f area are a v a i l a b l e (1, 2, 4, 5, 6, 7), but the s m a l l s i z e and th r e e - d i m e n s i o n a l shape of the leaves make t h i s d i f f i c u l t . F or experiments u s i n g a l a r g e number of t r e e s , the s i m p l e s t method i s to c o r r e l a t e l e a f area with l e a f dry weight (4). For mature Ponderosa p i n e , Cable (4) found 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 dry weight and s u r f a c e area o f f a s c i c l e s . Once t h i s i n i t i a l r e l a t i o n -s h i p has been e s t a b l i s h e d o n l y the de t e r m i n a t i o n o f f a s c i c l e dry weight i s r e q u i r e d to determine f a s c i c l e s u r f a c e area. On the oth e r hand, B r i x (3) has shown t h a t a l i n e a r r e l a -t i o n s h i p d i d not occur f o r D o u g l a s - f i r s e e d l i n g s between ages 65 and 100 days grown at v a r y i n g l i g h t i n t e n s i t i e s and v a r y i n g temperatures. In our study, the l e a f s u r f a c e area o f each s e e d l i n g was measured to determine the r e l a -t i o n s h i p between l e a f s u r f a c e area and l e a f dry weight of 18-week-old s e e d l i n g s . An a d d i t i o n a l study was made on the e f f e c t of d i f f e r e n t sources o f n i t r o g e n on the r e l a t i o n s h i p between l e a f s u r f a c e area and l e a f dry weight 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 and E.B. Tregunna w i l l appear i n Can. J . For. Res. i n P r e s s . seedlings. Three conifer species, Douglas-fir (Pseudotsuga  menziesii var. menziesii), Lodgepole pine (Pinus contorta var. contorta) and Western hemlock (Tsuga heterophylla) were grown for 18 weeks in sand in a growth chamber under conditions described previously (8). The seedlings were given nutrient solution i n which only the source of nitrogen and the concentration of chloride were varied. Three d i f f e r e n t sources of nitrogen (nitrate only, ammonia only, and a combination of n i t r a t e and ammonia (7:1) were used. A l l treatments contained 14 ppm N. At 18 weeks afte r germination, f i v e seedlings of each species from each nitrogen source were selected at random and the leaves were removed. The length and width of the leaves were measured by means of a micrometer. The t o t a l surface area of the leaves was determined by multiplying length times width times a correction factor. The correction factor accounts for longitudinal and crossectional curvature. The correction factors for Douglas-fir and Western hemlock were taken from Barker (2) and the correction factor for Lodgepole pine was taken from Smith (9). Leaf dry weight of each seedling was determined by drying the leaves i n an oven at 80 C to constant weight. The relationship between leaf dry weight and leaf surface area was deter-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 ( r ) . The 78. leaf surface area to leaf dry weight relationship among nitrogen treatments within species was analyzed by testing for homogeneity of r values. With a l l three conifer species, no difference was found between r values determined for the three nitrogen sources. Thus a pooled r value was determined for each species. The pooled r values were: Lodgepole pine (.949), Western hemlock (.967) and Douglas-fir (.927). These values are s i g n i f i c a n t at the 5% l e v e l and indicate a li n e a r r e l a t i o n s h i p between leaf dry weight and leaf area of entire seedlings. Afte r the l i n e a r r e l a t i o n s h i p was established a regression c o e f f i c i e n t was determined and the regression l i n e drawn using the formula y = bx (Figs. 1, 2, 3) . This l i n e a r relationship between t o t a l leaf area and leaf dry weight provides an easy and quick method for determination of leaf area, once the i n i t i a l r elationship i s found. A l i n e a r relationship indicates that in experi-ments comparing photosynthetic or transpiration rates of seedlings grown on d i f f e r e n t sources of nitrogen, the differences i n results between nitrogen treatments w i l l be of the same magnitude whether units of leaf dry weight or leaf area are used. F i g u r e 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 with d i f f e r e n t sources of n i t r o g e n . L E A F S U R F A C E A R E A ( c m 2 ) F i g u r e 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 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 . Figure 3. 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 Western hemlock 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 . L E A F D R Y W E I G H T 85. LITERATURE CITED 1. Baker, F.S. 194 8. A short method of determining leaf area and volume growth i n pine trees. Hilgardia 18: No. 8. 2. Barker H. 196 8. Methods of measuring leaf surface area of some conifers. Canadian Forestry Branch Depart-ment Publication No. 1212 6 p. 3. Brix, H. 1967. An analysis of dry matter production of Douglas-fir seedlings in r e l a t i o n to 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. Estimating surface area of Ponderosa pine foliage in central Arizona. For. S c i . 4: 45-49. 5. Kozlowski, T.T., and F. Schumacher. 1943. Estimation of stomated f o l i a r surface of pine. Plant Physiol. 18: 122-127. 6. Kramer, P.J. 19 37. An improved photo-electric apparatus for measuring leaf area. Amer. J. Bot. 24: 375-376. 7. Madgwick, H.A.I. 1964. Estimation of surface area of pine needles with special reference to Pinus resinosa. J. Forest 62: 636. 8. Madoc-Jones, S. 1970. The nitrogen n u t r i t i o n of some conifers of B r i t i s h Columbia, pp. 16-23 In Progress Report: National Research Council Grant No. A-92. V.J. Krajina (ed). Botany Department, University 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 persistance of needles of Douglas-fir, Western hemlock, Western redcedar and other B r i t i s h Columbia conifers. Mimeographed report, Forestry Department, University of B r i t i s h Columbia, Vancouver 8, B.C., Canada. 86. EPILOGUE In Part 2, I studied the eff e c t s of NH4+-N and N03~-N on four species of conifers. I have concluded from these studies that Western hemlock and Lodgepole pine are more tolerant of NH4+-N than Douglas-fir and Western redcedar. The exact l e v e l of NH4+-N that these species w i l l tolerate should be determined. This experiment should be ca r r i e d out with a w e l l - s t i r r e d l i q u i d culture i n which the l e v e l of nitrogen i s monitored d a i l y . In thi s manner the l e v e l of nitrogen i n contact with the root surface would not vary to any great degree. A study of the nitrate-reducing enzymes i n these forest species should also be 'carried out. Such a study would indicate the a b i l i t y of these species to metabolize NO^ -N. A b r i e f attempt was made to extract n i t r a t e reductase. However i t was not possible to extract protein p r e c i p i t a b l e by t r i c h l o r a c e t i c acid from Douglas-fir, l e t alone n i t r a t e reductase. One ml of a Douglas-fir extract was added to a very active n i t r a t e reductase preparation from corn. The ni t r a t e reductase from corn was completely i n h i b i t e d by addition of the Douglas-fir extract. In addition, a study of the amino acid pools i n these species might give further insight into the a b i l i t y of these species to tolerate various levels of ammonia. 87. PART 3. A STUDY OF STORAGE CONDITIONS FOR SPRING-LIFTED MUD-PACKED DOUGLAS-FIR SEEDLINGS.1 INTRODUCTION In B r i t i s h Columbia, reforestation by planting makes use of 1-0 and 2-0 seedlings. The seedlings are grown in nurseries, l i f t e d between November and A p r i l , stored at 2C in the dark and then planted i n the f i e l d . A current method of planting employs mud-packed seedlings. Mud-packing i s done by surrounding the root system of the seedlings with a mixture of peat moss, clay and water formed into a cylinder. The mud i s then dried i n a stream of warm a i r u n t i l i t i s semi-rigid. In this condition the root system i s thought to be protected from dessication and physical damage. The seedlings are planted by punching a hole i n the ground with a dibble or s t e e l rod to the same depth as the length of the mud-capsule. The rod i s withdrawn and the mud-capsule i s placed i n the hole, leaving the stem and the needles of the seedling above the ground. S o i l i s tramped down t i g h t l y against the mud-pack. A c r i t i c a l period i n the establishment of seedlings i s the time between l i f t i n g and subsequent regeneration of a 1 This section which i s part of an a r t i c l e by G.E. Mellor, R.A. K e l l e r , and E.B. Tregunna w i l l appear in Canadian Journal of Forest Research. E.B. Tregunna supervised t h i s study. 88. new root system. Water stress due to root, shoot, or whole plant dessication may occur i n the preparation and storage of seedlings for planting. Water stress may also occur a f t e r planting when, because of root injury or disruption of the s o i l - p l a n t - a i r continuum, transpiration exceeds water uptake (1). In this study, the e f f e c t of various storage conditions on s u r v i v a l and root regeneration of s p r i n g - l i f t e d mud-packed Douglas-fir seedlings i s examined. Winjum (4) has shown that Douglas-fir seedlings l i f t e d a f t e r A p r i l 1st are damaged by four weeks of storage at 2C. He found that the f i e l d s u r v ival of stored seedlings was reduced by 30 to 90% when compared with the s u r v i v a l of unstored seed-l i n g s . During the l a s t two years, seedlings l i f t e d a f t e r A p r i l 1st have been used for mud-packing. In many instances these mud-packed seedlings have been placed i n storage for a period of two to three weeks at 2C. The object of t h i s study was to determine the best method of storing spring-l i f t e d mud-packed Douglas-fir seedlings. As mentioned previously, a c r i t i c a l period i n the establishment of seed-lings i s the time between l i f t i n g and regeneration of a new root system. Thus various storage conditions were chosen to determine i f root regeneration could be enhanced. 89. MATERIALS AND METHODS During the week of A p r i l 19th, 1970, 2-0 Douglas-fir trees grown at Green Timbers Forest Nursery from seed l o t (92G14/B2/1020/201) were l i f t e d and mud-packed at Pelton Reforestation Company. Immediately a f t e r mud-packing, 220 seedlings were divided into 11 storage treatments (2 re p l i c a t i o n s x 10 trees) (Table 1). Before placing the trees i n t h e i r respective storage treatments, the mud-packed portion of the seedling was soaked for 10 min i n water or 1/2-strength Hoagland's solution. A f t e r soaking, the mud-packs were dried to t h e i r o r i g i n a l semi-rigid condition. The seedlings stored i n the dark were placed in p l a s t i c bags, as i s done by the Pelton Reforestation Company. The seedlings stored i n the l i g h t (25C and f i e l d ) were placed i n p l a s t i c bags which were then transferred to a r i g i d container i n order to keep the trees in an upright p o s i t i o n . The portion of each seedling above the mud-pack was completely exposed. For the vermiculite treatments, wet vermiculite was added to the bags. The trees stored in the f i e l d were placed in a shelter which had only the top covered with polyethylene p l a s t i c . The p l a s t i c was s l i g h t l y opaque and provided shelter from both d i r e c t sun and r a i n . The mud-packs kept i n cartons i n the dark retained t h e i r moisture during the storage period while the exposed mud-packs l o s t moisture. The exposed mud-packs at 25C had TABLE 1. Storage treatments and conditions. 2C Room (dark) IOC Room (dark) F i e l d a ' b 25C Roomc - f e r t i l i z e r - f e r t i l i z e r - vermiculite - f e r t i l i z e r - vermiculite - f e r t i l i z e r + l i g h t + f e r t i l i z e r + f e r t i l i z e r + vermiculite - f e r t i l i z e r + vermiculite - f e r t i l i z e r + l i g h t + vermiculite + f e r t i l i z e r + vermuculite + f e r t i l i z e r + l i g h t - vermiculite - f e r t i l i z e r - l i g h t F i e l d refers to the roof of the Bio-Sciences F i e l d conditions during storage Ave. Maximum daily Temperature 12.6C Ave. Range 9.5C-18.0C 25C Room conditions during storage 1000 ft-candles, 16 hour day Ave. RH 65% Building, University of B r i t i s h Columbia. Minimum d a i l y Temperature 5.6C Ave. RH 7 5% Range 2.5C-9.0C 91. to be moistened da i l y and the mud-packs i n the f i e l d had to be moistened da i l y or every other day depending on the weather conditions. The mud-packs were moistened with a fine spray of water. Holes for drainage were cut i n the bottom of the bags. In spite of the fact that as l i t t l e water as possible was used for moistening, water drainage from the bags was poor. This was p a r t i c u l a r l y true of the vermiculite treatments. During storage (8th, 11th, 14th and 18th days) root growth and su r v i v a l were measured. A seedling was con-sidered to be dead when i t s needles were brown and dropping and the stem was dry, b r i t t l e and e a s i l y broken. Root growth was evaluated by measuring the amount of root protruding from the mud-pack. After 19 days storage, the seedlings were planted i n the UBC Botanical Garden at 10 inch inte r v a l s i n rows 2 f t apart. Each row consisted of one r e p l i c a t e . In order to minimize the e f f e c t of moisture stress, the s o i l was moistened p e r i o d i c a l l y to prevent the mud-packs from drying out. After 44 days, the seedlings were dug up and were evaluated on the basis of su r v i v a l and root growth. In evaluating the r e s u l t i n g data, analyses of variance were used followed by Tukey 1 s -latest («=*= .05). RESULTS AND DISCUSSION F e r t i l i z e r and vermiculite treatment of the mud-packs had no e f f e c t on any of the parameters measured. Therefore the i n d i v i d u a l treatments within each storage location were combined except i n the 2 5C treatments. With the 25C tr e a t -ments the (+) l i g h t treatments are reported separately from the (-) l i g h t treatments. After 19 days i n storage there were no roots v i s i b l e on the outside of the mud-packs from any of the storage locations. Survival of the trees at the end of the storage treatment i s shown i n Table 2. Storage of the trees at 25C (+) l i g h t produced s i g n i f i c a n t l y lower storage s u r v i v a l than the other treatments. A l l other 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 throughout the planted period ranged from 13.5C to 30.0C with an average of 19C. The minimum da i l y temperature ranged from 4.0C to 20C with an average of 9.0C. F i e l d s u r v i v a l (Table 2) was computed on the basis of the number of seedlings planted. The trees stored i n the f i e l d had by far the highest f i e l d s u r v i v a l . A l l other storage treatments had a f i e l d s u r v ival of less than 53%. The low sur v i v a l of s p r i n g - l i f t e d seedlings at 2C, agrees with the findings of Winjum (4). In addition to studying f i e l d and storage s u r v i v a l i n d i v i d u a l l y the combined sur v i v a l (total survival) should also be considered (Table 2). The f i e l d stored trees had the highest t o t a l s u r v i v a l . TABLE 2. Survival and root growth of mud-packed Douglas-fir seedlings stored i n various locations. Storage location Storage survival F i e l d s u r vival Total s u r v i v a l Root growth % % cm F i e l d 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 ( + ) li 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 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 root protruding from the mud-pack per surviving seedling. i U ) A l l other treatments had a t o t a l s u rvival of 55% or le s s . The f i e l d stored trees had the greatest amount of root growth (Table 2). This length of root was made up of numerous roots and represents an extensive capacity for obtaining moisture and nutrients. These plants had a considerable area of contact with the s o i l beyond the surface of the mud-pack. Trees stored at 2C, IOC, and 25C (+) l i g h t produced intermediate amounts of root growth while the trees stored at 25 (-) l i g h t produced the lowest amount of root growth. There was a d i r e c t r e l a t i o n s h i p between root growth and f i e l d s u r v i v a l . Seedlings which did not survive the planting period produced no root growth. This observation agrees with the findings of Stone (2). He noted that at 60 days afte r planting, bare-root Douglas-fir seedlings which had died, had produced no new roots. Since an e f f o r t was made to reduce the e f f e c t of moisture stress i n this study, s u r v i v a l as noted should not be extrapolated i n an e f f o r t to predict actual f i e l d behaviour. On the other hand, Stone e t a l . (3) noted that the root regenerating p o t e n t i a l i s a measure of the physi o l o g i c a l condition of the seedling and under many f i e l d conditions, one might reasonably expect i t to be refl e c t e d i n f i e l d s u r v i v a l . In summary, i t would seem from the parameters measured i n th i s study, that s p r i n g - l i f t e d , mud-packed Douglas-fir seedlings should be stored i n the f i e l d . Storage at 2C, IOC and 25C (+) l i g h t produced intermediate r e s u l t s . Storage at 25C (-) l i g h t was the lea s t s a t i s -factory . ADDENDUM On January 12th afte r publication of this paper i n Can. J. For. Res., a l e t t e r was received from M. Crown, Forester with the P a c i f i c Logging Company Limited. Mr. Crown supplied some unpublished s u r v i v a l data concerning s p r i n g - l i f t e d mud-packs which had been stored at 2C. In addition, he added "We read with i n t e r e s t your paper on the moisture status of mud-packs. The results would appear to give the reason why operational experience with s p r i n g - l i f t e d mud-packs has generally been less s a t i s f a c t o r y than w i n t e r - l i f t e d mud-packs." LITERATURE CITED Kozlowski, T.T. 1968. Introduction, pp. 1-22. In T.T. Kozlowski (ed.) Water d e f i c i t and plant growth, Vol. I. Academic Press, New York. Stone, Edward. 19 55. Poor s u r v i v a l and the physiological condition of planting stock. Forest 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 of Douglas-fir seedlings l i f t e d at d i f f e r e n t times of the year. Forest S c i . 8 288-297. Winjum, J.K. 1963. Effects of l i f t i n g date and storage on 2-0 Douglas-fir and Noble-fir. J. of Forestry 61: 648-654. 

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