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UBC Theses and Dissertations

Net carbon dioxide exchange rates in Pisum sativum L as influenced by phosphorus and nitrogen nutrition Roelants van Baronaigien, Hendrik Willem Marius 1965

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NET CARBON DIOXIDE EXCHANGE RATES IN PISUM SATIVUM L AS INFLUENCED BY PHOSPHORUS AND NITROGEN NUTRITION tor HENDRIK WTT.T.EM MARIUS ROELANTS VAN BARONAIGIEN B.S.A., University of British Columbia, 1958 A THESIS SUBMITTED IN PARTIAL FUIiFILUJENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE in the Division of Plant Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1 ? 6 U I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y * I f u r t h e r a g r e e t h a t p e r -m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t , c o p y i n g o r p u b l i -c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n s (H. W. M. R. v a n B a r o n a i g i e n ) D e p a r t m e n t o f H o r t i c u l t . u r p The U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r 8, C a n a d a D a t e -23 O c t o b e r 1964- — ABSTRACT Tiro varieties of Pi sum sativum, L, Jade and Olympic, were grown for a period of four weeks from seeding, under constant light intensity and diurnally fluctuating temperatures, while being subjected to varying-artif i c i a l l y induced-degrees of nutrient stress* Net carbon dioxide exchange rates were measured in order to determine the influence of excess or deficient nitrogen and phosphorus concentrations within the nutrient medium. Carbon dioxide assimilation rates, under various degrees of nutrient stress were highly variable, both within the same variety and between varieties tested* Microchemical determinations of chlorophylls a and b, and of inorganic phosphate, nitrate and nitrite were carried out in order to determine the relationship of some physiological aspects of mineral deficiency to carbon dioxide assimilation* Investigations as to changes in stomatal index (I) as a result of nutrient stress also showed a large amount of variation* Results obtained indicated that variety Jade was very sensitive to nitrogen deficiency within the rooting medium, as evidenced by a Large increase in stomatal index values with nitrogen deficiency* Olympic showed a greater sensitivity to phosphorus deficiency than did Jade* Neither total chlorophyll content, nor chlorophyll a/b ratio appeared to exert any deciding influence upon net carbon d ioxide assimilation* A relationship appeared to exist between CO2 assimilation and quantities of nitrate and/or nitrite present within the photosynthetic tissues of plants tested* This experiment was unable to fully explain the reason(s) for the great degree of variability of net C02 exchange rates extant even between plant specimens growing in the same nutrient medium* Same technical improvements applicable to the present experiments as v e i l as some alternative experimental procedures are diseussed* TABLE OF CONTENTS Page Introduction 1 Review of Literature 2 Materials and Methods 12 A* Determination of Net CO2 Exchange Rates 12 B* Micro-chemical Methods of Analysis 18 Results and Discussion 21 General Discussion 36 Bibliography 38 Appendix LIST OF FIGURES Figure 1: Carbon dioxide assimilation measurement apparatus Ik Figure 2« Inter relationship between nitrogen species and phosphorus i n the plants and net rates of carbon dioxide exchanged - Jade 28 Figure 3: Analysis of nitrite and nitrate concentration Influence upon net carbon dioxide exchange rates - Jade 2° Figure U : Comparison of change in chlorophyll a and b content in relation to net carbon dioxide exchange rates - Jade 31 Figure £j Comparison of the effect of nitrate and nitrite upon carbon dioxide exchange rates and chlorophyll a/b ratio of variety jade 32 Table Is LIST OF TABLES Maximum and minimum net COg exchange rates Jade and Olympic Page 22 Table l i t Average stomatal index (I) of v a r i e t i e s Jade 23 and Olympic as modified by mineral composition of nutrient solution Table I H t Summary of physically and chemically determined 25 measurements of analysis - Jade Table IV: Summary of physic a l l y and chemically determined 26 measurements of analysis - Olympic Appendix Table 1: Individual net carbon dioxide exchange rates - Jade v Table 2: Individual net carbon dioxide exchange rates - Olympic Table 3: Analysis of Stomatal Index (I - S x 100) E + S 1. IMTRODOCTION An extensive survey of pertinent literature revealed that, apart from 7/illstatter and Stoil's work(20) with carbon dioxide exchange rate differences between variegated and normal green varieties of the same species of higher plants, l i t t l e i f any investigation has been conducted to determine apparent differences in carbon dioxide assimilation between normal, ncffwvariegated varieties within any one species of higher plants* Although extensive reports exist concerning the accumulation of inorganic nitrogenous compounds in the case of phosphorus deficient plants, just as carbohydrate quantities are abnormally elevated in the case of plants growing in a nitrogen deficient medium, no evidence was discovered concerning a possible explanation of such accumulation. The possibility of the existence of both intra- and intervarietal differences in carbon dioxide absorption with regard to degrees of sensitivity to nitrogen and/or phosphorus deficiency was explored* This thesis presents results obtained during the investigation of the above-mentioned aspects of carbon dioxide assimilation by two pea varieties, Jade and Olympic, as affected by varying phosphorus-nitrogen nutrient levels. 2. REVTEff OF LITERATURE Literature cited includes various physiological and biochemical aspects of both primary and secondary reactions of the photosynthetic process* Many studies have been undertaken to establish differences in carbon dioxide assimilation by plants as measured by quantum efficiency (QE), that i s the number of quanta of the appropriate wavelengths, needed to reduce one molecule of carbon dioxide by various bacteria, algae and flower-ing plants but no conclusive evidence o£ a basic difference between the organismal quantum efficiencies has so far been found(17)• Investigations relating to differences i n assimilation number (A.N.), a quantity which i s equivalent to the amount of carbon dioxide, measured in milligram per hour per one milligram of tissue chlorophyll of normal green and (green/white) variegated forms of the same species have shown that for the latter, the assimilation number appears to be signifi-cantly higher in value than that for the former(20). when the influence of age upon the value of the assimilation number is studied, the results show that whereas the chlorophyll content of the aging leaf, continues to increase, the rate of photosynthesis also continues to increase, but to a lesser extent(21)• Studies conducted by Smillie(l4l) to determine the influence of aging upon photosynthesis and respiration showed that age had a detrimental effect upon the photophos-phorylating capacity of isolated chloroplasts of pea leaves (lib)* Smillie(li3) further demonstrated that the photosynthetic rate per unit of fresh weight at f i r s t rapidly increased, reaching a maximum intensity i n nine-day old leaves, after which i t declined slowly* On the other hand, i f the respiration rate of pea leaves was expressed per unit of fresh weight, then the rate of respiration dropped continuously during the entire aging process* Under the growth conditions employed, leaves were not fully expanded until they were twelve days old* The- greatest rates of photosynthesis and respiration were observed well before leaf expansion ceased* This observation held true for both processes* By the time the leaves were fully expanded (normally at twelve days after germination) both respiration and photo-synthesis progressively declined, even i f the chlorophyll content of the maturing leaf continued to increase for a certain length of time (h3)» In a continuation of his studies Smillie(lil) examined the respiratory and photo-synthetic enzymes and found that the dissimilar patterns for photosynthesis and respiration indicated that the cellular levels of the enzymes involved in either one of the processes under scrutiny were somewhat divergent from those obtained previously, as follows* Whereas the enzyme involved in the photo-reduction of the triphosphopyridine nucleotide (TFN+-) and ribulose - 1, $ -diphosphate carboxylase closely paralleled the results obtained for photo-synthesis; the respiratory enzymes, enolase, 6—phosphogluconic dehydrogenase and aconitase showed an i n i t i a l high value, at the leaf>s very early stage of development, but dropped soon after at a very rapid rate* In the case of the enzyme, transketolase, a catalyst, hot only involved in respiration, but also in the reformation of the carbon dioxide acceptor, Smillia(UL) found that the activity changes shown by this enzyme, up to the ninth day of development could be interpreted as being the net result of an increased action of the enzyme in regard to i t s association with photosynthesis rather than in i t s role as a respiratory enzyme* The most pronounced deviation from the photo-synthetic activity of this enzyme was found in the youngest leaves where the transketolase i s apparently mainly active as a respiratory enzyme and that consequently this enzyme in this instance parallels the degree of activity of the other respiratory enzymes, previously mentioned* k. Smillie and Krotkov(43) working with the pea variety Laxton's Progress, determined both biochemical and physiological changes occurring in developing pea leaves. They found that the moisture content of young developing leaves increased from 82.8 percent to 87.1 percent of the total weight. The number of cells per unit area decreased more rapidly than the dry weight, thus suggesting that ce l l expansion involves not only synthesis of new cellular materials but increased uptake of water as well. The fresh weight increase of the leaves continued until they had reached the age of twelve days, by which time a l l had fully expanded. At sixteen days after germination these workers noted that leaf fresh weight had diminished slightly. Determination of protein content during the leaves* period of development revealed the existence of a general tendency to decrease with age. This phenomenon is also known to occur in tobacco(52) and sugar cane(32) as determined by measurement of total nitrogen content. Differences in protein content during the maturation process in leaves has been interpreted by Smillie and Krotkov(h3) as being indicative of a net change in the level of enzyme systems involved in a number of different metabolic pathways. In support of this hypothesis they cite the discovery of Smillie and Fuller(U2) that concentration levels of a certain group of glycolytic enzymes as a function of leaf age, were considerably different from a number of enzymes participating almost solely in the process of photosynthesis. Changes in chlorophyll content, expressed on a leaf fresh weight basis were noted; this pigment increased rapidly from an i n i t i a l low to a peak at the age of thirteen to fifteen days, after which the total chlorophyll content of the leaves slowly decreased. Nucleic acid determination of leaves during the various stages of development, revealed a 5 . continuous decline as the leaves approached f u l l maturity; the ribonucleic acid declined at a faster rate than the desaxyribonucleic acid* Study of changes in ribonucleic acid content of individual leaves, resulted in the discovery that this compound increased up to three days after germination, after which period i t declined quite rapidly* Curtis and Clark(10) state that because nitrogen i s a normal and essential constituent of chlorophyll, a low nitrogen concentration should be conducive to a low chlorophyll content in the plant. The same authors continue with the observation that even with no actual reduction in the total quantity of nitrogen within the leaf, an abundance of carbohydrates present within the leaf, may actually decrease the amount of nitrogen available for chlorophyll synthesis, due to the fact that high carbohydrate levels favour amino-acid synthesis - other than glycine - the precursor of the porphyrin ring of chlorophyll* Meyer and Anderson (30) declare that any environmental factor - such as high-light intensity, low temperature or low nitrogen favouring an accumu-lation of carbohydrates in a given plant tissue, often causes anthocyanin formation in that tissue, provided, of course, that the necessary genes for the formation of such pigments are part of the tested individual's inheritance. Eckerson(13) noted that in the absence of phosphorus the protoplasm of young cells became less refractive and that the chloroplasts disintegrated f i r s t i n the stem, and lower leaves and finally i n the upper ones* She also demonstrated that synthesis of proteins did not occur at the usual rate in phosphorus deficient plants* Experiments conducted by Aronoff et al ( U ) revealed that the amino-acids alanine and aspartic acid, as well as malic acid, were slowly labeled in the dark when algae were exposed to labeled carbon dioxide* However, the aforementioned compounds were labeled much more rapidly i f the algae were actively photosynthesising just prior to the moment of the addition of the labeled carbon dioxide* Nichiporovich(33) has presented evidence that synthesis of proteins in the chloroplasts of higher plants was greatly accelerated during photo-synthesis* The accelerated protein formation appears to take place directly from the intermediates of photosynthetic carbon dioxide reduction since the proteins were labeled when labeled carbon dioxide (C^U) was used, but NOT when labeled carbohydrates (C1^) were administered* Brachet et al(£), endeavouring to determine the effect of photo-phosphorylation upon protein synthesis found that labeled (Clii) glycine was incorporated into the micro-somal fraction of Acetobularia proteins, at rates which were independent of the influence of light* However, i f labeled carbon dioxide was fed to Acetobularia, the labeled carbon was soon incorporated into the chloro-plasts' proteins* Meyer and Anderson(31), discussing the relative quantities of pigments present in chloroplasts of higher plants, state that the ratio of chlorophyll a to chlorophyll b i s approximately three* They also declare that the proportion of chlorophyll a to that of b is higher in "shade leaves 0 of many plants, than that of "sun leaves" of the same species* This state-ment i s substantiated by Fruton and Simmonds (lf>) * Schertz(UO) showed that the factors influencing the quantity of chloroplast pigments are rainfall, soil moisture, nutrient levels in the culture medium, light intensity, temperature and humidity* Sprague and Shive(U5) working with corn, concluded that the chlorophyll content of a plant i s more closely correlated with leaf area than with dry weight of leaves* Reed's(38) work revealed that where phosphorus i s withheld from plants, the formation of phosphatides i s severely inhibited while the synthesis of triglycerides reaches abnormal proportions* Kamen(23) cites that chemical analysis of chloroplasts shows them to contain as much as forty percent of total dry weight in phospholipids* In this respect they resemble the mitochondria(27)* In Scarlet Runner bean chloroplasts six phosphatides were found, five of which have been identified as lecithin, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl inositol and phosphatidic acid* Kamen(23) also reported that the enzyme lecithinase, the agent responsible for the degradation of lecithin, is concentrated in the chloro-plastic fraction of the leaf* Garolus(d) working with cowpeas and tomatoes, grown under greenhouse conditions, showed that absence of nitrogen increases the inorganic phosphate content of c e l l sap from forty-four to one thousand forty parts per million* Nightingale(3U) discovered that in general the absorption of inorganic nitrogen was depressed when phosphorus was present i n adequate or high concentrations in the nutrient medium* Walter (1*6) immersed plants of the water weed Anacharis canadensis Rich et Mich* in various concentrations of sucrose solutions and studied the effect of this treatment upon the rate of photosynthesis. He found that the greater the concentration of the sucrose solution the less the tuxgiidity of the cells, the less the hydration of protoplasm, and the lower the rate of photosynthesis* In one experiment the rate of photosynthesis was appreciably retarded by immersion of the plants in a 0*3 molal solution and almost entirely stopped in a 0*7 molal solution of sucrose with an osmotic pressure of approximately eighteen atmospheres* However, the rate of respiration was practically unaffected by sucrose solution at any concentration up to and including a 1*0 molal solution* Pleasants(36) noted that the stomata of plants with abundant nitrogen present, opened more widely in the case of adequate levels of moisture, while 8. they closed more promptly and completely when the water supply to the plant became limited* Ziegenspeck(U8) found that in a given leaf the stomata do not appear to be formed a l l at once; on the contrary such formation occurs during a. con-siderable period of time* He was able to demonstrate the fact that two principal developmental patterns are to be distinguished: (a) i n leaves with parallel veins where the stomata are arranged in longitudinal rows, the different stages of the stomata! maturation process are observable in successively more highly differentiated parts of the leaf; the distribution of these stages following a baslpetal sequence, and (b) in leaves of netted venation* In this case, stomata of varying degrees of maturity are distributed in a much more random fashion, mature stomata being found side by side with those s t i l l i n their i n i t i a l stages of development* Salisbury(39) while conducting an extensive investigation into the stomatal frequency of the woodland flora in England, discovered the existence of a high positive correlation between the number of stomata and the number of epidermal cells per unit of leaf area* Differences in stomatal frequency found, are due to the spacing of the stomata, and not to the differences in the number of stomata formed per number of epidermal cells per unit area* Salisbury(39) then proposed a quantity which he named the stomatal index and which serves as an expression of the percentage proportion of the ultimate divisions of the leaf dermatogen that has been converted into stomata* Therefore a species under a given set of conditions wil l try to adjust to these conditions by forming a definite proportion of stomatal initials* Salisbury(39) concludes with the statement that variations i n the value of stomatal indices obtained, may be due entirely to internal changes within the plant, even i f these changes are caused by external factors of which nutritional variability i s possibly the most important one* Experiments by Burstrom(67) on the reduction of nitrate in the leaves of young wheat plants indicate that the process i s closely linked with the simultaneous reduction of carbon dioxide in the photosynthetic process, and that light i s the source of energy. No reduction of nitrate was found to take place in wheat leaves i n the dark, even when conditions appeared favourable for the process. Respiratory energy was not utilised in this mechanism* Eckerson(ll) followed some of the steps in the reduction of nitrate in the tomato plant* Young plants were transplanted to quartz sand and watered with nutrient solution lacking nitrogen compounds until such time that micro-chemical tests were negative for nitrate, nitrite, ammonia and free amino acids, while carbohydrates were s t i l l present in great abundance* Calcium nitrate was then added to the sand* The nitrate ions were rapidly absorbed and could be detected in a l l parts of the plant within twenty-four hours after the first administration* In thirty-six hours nitrites were present in considerable amounts at the tips of the stems and i n other tissues* Traces of ammonia could also be detected* By the end of forty-eight hours the quantity of nitrite had decreased, the amount of ammonium ion had increased and a small amount of asparagine was also present* Subsequently amino acid synthesis proceeded normally* Quantitative measurements estab-lished that during amino acid synthesis the carbohydrate content of the cell decreased significantly* Hageman and Flesher(l8) demonstrated that nitrate ion concentration levels of *015> M* or more inhibit the process of photo-reduction of t r i -phosphopyridine nucleotide (TPN-f-)* Their findings confirmed the previous report by Evans and Nason ( I U ) who discovered that nitrate ion concentrations of *02 M. although optimum for nitrate reductase activity, proved sufficiently detrimental to the photo-reduction process of triphosphopyridine nucleotide 10» (TPN) to cause a seventy-five percent reduction of the photo-reductive rate in plants grown at lower nitrate ion concentration levels present in the nutrient solution* These workers also found that reduction of nitrite was inhibited "in vitro" by the presence of hydroxylamine hydrochloride (NH2OH.HCI) at 1X>~3 molar concentrations within the reaction mixture* Hageman and Flesher(l8) also discovered that the enzyme, nitrate reductase, fluctuated with both the amount of light to which the plant i s exposed, and the concentration of the nitrate ion present in the plant* The inhibitory effect of nitrate ion concentrations upon photo-reduction of triphosphopyridine nucleotide has been explained by Lundegardh(2f>), through his discovery that leaves of nitrogen deficient oat plants were much more active photosyntheticaUy than leaves of plant controls, grown on media in which adequate amounts of nitrate were present* Kraybill(2i*), studying the interaction of nitrogen and phosphorus in plants, discovered that inorganic nitrates were not reduced by tomato plants i n the absence of phosphorus, an interesting observation in the light of the findings by Evans and Nason (lit) who established that the photo-reduction of triphosphopyridine nucleotide (TPN) i s conducive to nitrate reduction in plants, but only i n the absence of gaseous oxygen; the latter observation is doubtless due to the fact that the nitrate ion can serve as a hydrogen acceptor instead of molecular oxygen* This process i s accompanied by the formation of three, high-energy adenosine triphosphate molecules per one nitrate ion reduction [N (V-plus) to N (HI plus)] for nitrite in a manner comparable to "respiratory chain* phosphorylation* Concerning the effect of molecular oxygen upon nitrate photo-reduction, McAlister and Myers (26) showed that the rate of photosynthesis in young wheat plants was about thirty to f i f t y percent higher in one half per cent oxygen than in twenty percent oxygen at high light intensity and at 11. atmospheric carbon dioxide levels of concentration* Eckerson(12.) noted that depletion of phosphates in tomato plants, caused a very rapid decrease in nitrate reducing substances in succession in leaves, stems and roots, while inorganic nitrates accumulated in the same sequence. MacGillivray(29) established that tomato plants growing in a phosphorus deficient medium, contained more total nitrogen than those grown in complete nutrient media. He also discovered that the stems of phosphate starved plants contained three times as much water soluble nitrogen and four times as much nitrate ammonia and urea as well as cf-amino acid nitrogen as the stems of the controls grown in complete nutrient media* These findings were confirmed by Hanmer(19). McCool and Weldon(28) showed conclusively that the phosphate content of plant sap i s decreased by application of sodium nitrate* Anderson and 6athurst(2) demonstrated that excess nitrate within the growth medium reduces the amount of inorganic phosphate absorbed by the plant root. Anion et al(3), Allen et al(l) and Whatley et al(ltf) discovered that chloroplasts, obtained through fractional uibra-centrifugation from cell material did not possess any aerobic oxidative metabolism in the absence of light* In other words the chloroplasts did not respire. 12 MATERIALS AND METHODS A* Determination of Net Carbon Dioxide Exchange Seeds of Pisum sativum (L.) varieties Olympic and Jade, treated with Spergon, were planted in clean, one-gallon plastic containers; twelve seeds were sown in each pot, and vermiculite (Horticultural Grade Terra-Lite, Grant Industry Ltd., Vancouver, B.C.) served as the rooting medium. After planting of the seeds each pot received sufficient distilled water (generally lf?00 ml per pot) to saturate the originally dry rooting medium* Pots thus prepared were placed in a controlled growth chamber (Percival, Boone, Iowa), at a photo period of sixteen hours per twenty-four; day temperature was maintained as close to 19»k° C as possible and night temperatures varied between lh and U>° G* Light intensity was maintained at a constant level of about 3000 foot candles, as determined with a Weston Model 756 Illumination Meter* During the f i r s t week of the plants' development in the growth chamber, the seedlings received sufficient water to maintain the saturation level of the vermiculite rooting medium* At the end of the f i r s t week, plants were thinned to seven uniformly developed plants per pot; cotyledons were removed by excision and upon com-pletion of this operation, each pot was watered with five hundred ml of the appropriate nutrient solution; whose solute composition was based upon the Hoagland nutrient solution modified by the addition of FeEDTA and varied by adding or subtracting nitrate nitrogen and/or inorganic phosphate as follows: 13. Concentration Concentration Treatment Number of Sets N/lQOO ml P/lOOO ml N +P h 1|2 ppm* 35 ppm. N - P k k2 ppm* 0 P - N h o 35 ppm. i. W* 1 21 ppm* 17.5 ppm. 2N2P 1 8ii ppm* 70 ppm* |N - P 1 21*0 ppm* 0 |P - N 1 0 17*5 ppm. 2N - P 1 81* ppm* 0 2P - N 1 0 ppm* 70 ppm* -P - N 1 0 ppm* 0 ppm. Each pot received f i v e hundred ml.of the appropriate nutrient solution twice per week u n t i l four weeks from the date of planting. I n addition to the nutrient solution treatment, plants received s u f f i c i e n t d i s t i l l e d water throughout the period t o maintain a constant water l e v e l i n the basin located beneath each pot. A f t e r the four week period of development had elapsed each individual: plant was inserted i n t o a c l e a r p l a s t i c chamber of inner dimensions 1U.6 cm by 30*2 cm by 1**2 cm ( t o t a l volume of 1852 ml) f o r a period of ten to f i f t e e n minutes, depending upon the speed with which the i n d i v i d u a l plant achieved the steady state of carbon dioxide assimilation* The a i r from the p l a s t i c carbon dioxide exchange chamber was drawn by a small e l e c t r i c pump and fed i n t o a Beckman Model ISA infra-red gas analyser, connected to an amplifier and recorder* An open system was main-tained throughout the experiment i n order to maintain as closely as possible the environmental conditions, under which these plants had been growing during t h e i r four weeks period of. development* The scheme f o r i n d i v i d u a l carbon dioxide absorption analysis i s presented i n figure 1* FANS FOR CIRCULATION OF AIR & TEMPERATURE CONTROL LIGHT SOURCE Electric Fa Chamber i - i Thermograph Amplifier Recorder F I G U R E 1. CARBON DIOXIDE ASSIMILATION MEASUREMENT APPARATUS 15. Carbon dioxide exchange as measured by absorption or evolution in the plastic exchange chamber was calculated as milligrams of carbon dioxide per l i t e r of a i r per one hour, per one square decimeter of leaf area per plant, in accordance with the following formula: 1,9769 x 7.98* x 273 x Xppm/1000 TI  Leaf Area in dm? For the purpose of this experiment the quantity ^ 2 has been discarded i n favour of the constant .9 (equivalent to £Z2) due to the fact that the 295 temperature within the plastic carbon dioxide exchange chamber was very nearly constant throughout the entire period of measurement (varying from 22*0° C to 23.5° C). The value of X in the above formula was experimental ly determined by observing the difference between the carbon dioxide content of the a i r in the exchange chamber (measured through intake A of the Figure 1) in the presence of the individual plant, tested and that of the air in the growth chamber (intake B of Figure 1). The recorder quickly indicated any change in carbon dioxide levels of the plastic chamber i n the presence of the plant. Two separate measure-ments were taken for each plant in order to ensure that any possible stimulatory effect upon respiration, due to mechanical handling (21) would be minimal* The Beckman Model l£A infra-red gas analyser was zeroed by leading pure gaseous nitrogen into the instrument (commercial grade nitrogen was used from which possible carbon dioxide had been eliminated by forcing the gas through limewater, prior to introducing i t into the instrument) just prior * The value 7.98 i s derived from 60 x 133A°°G where 133 is the flow rate per minute in milliliters; and 60 i s sixty minutes * 1 hour* The value 1*9769 stands for the weight in grams of one l i t e r carbon dioxide at 0° C and standard pressure* 16. to commencement of carbon dioxide analysis. The flow rate of air was regularly checked, by attaching intake B (of Figure 1) to a plastic hose, connected to the top of a one hundred ml pipette containing a few drops of detergent solution. Partial opening of the stop cock at the lower end of the pipette, caused a film of detergent to be formed. The time required for this film to travel the one hundred ml volume of the graduated pipette was determined with the aid of a stopwatch (1)5.06 seconds). The time value thus obtained was used to determine the flow rate per one minute time period. Prior to the commencement of each series of carbon dioxide exchange analyses, the flow rate of the air through the system was determined and, i f required, readjusted to the previously maintained value of one hundred thirty-three ml per minute. This ensured greater ease of calculations and maintained the calculated equivalent of one ppm on the recorder as a constant. After completion of each plant analysis, the a i r in the growth chamber was analyzed for carbon dioxide content via intake B, with the aid of a tridirectional stop cock, as indicated in Figure 1. Assuming the carbon dioxide content of the air in the growth chamber to be equal to 0.03 percent, i t was noted that f i f t y units of the recorder graph paper each represented an amount of six ppm of carbon dioxide. During the course of these analyses, i t was found necessary to occasionally use a different unit measure, in order to determine more accurately the sometimes minute quantities of C O 2 exchanged by the plants. After completion of each analysis of the individual specimen the total leaf area having been actively involved in the carbon dioxide exchange analysis was determined as follows. A l l leaves and bracts included in the exchange chamber were detached from the stems and spread upon a pane of glass. On top of these leaves a light sensitive ozalid paper was placed and held firmly to the glass pane* The package was then exposed to a light source from a 200-watt bulb for approximately ten minutes* When the light source was removed, the ozalid paper was then subjected to the action of ammonium hydroxide vapour in a stoppered glass cylinder to allow colour development* The resulting purple leaf images represented total leaf area of the plant* The area of these images was determined with a planimeter and the total number of square inches of leaf area (planimeter units of measure) converted to sq*dm* through multiplication by the factor of conversion: 6*1J52* A l l leaves belonging to one particular set and treatment were gathered and dried at 100° C for micro-chemical analysis for inorganic phosphate, nitrate and nitrite content, as well as total chlorophyll a and b. Carbon dioxide quantities absorbed were expressed as amounts of this gas both per one gram of dry weight and per one gram of total, chloro-phyll* Values thus obtained were used to determine the possible existence of both intra- and inter-varietal differences in the varieties Jade and Olympic examined in this experiment* Stomata counts were made of both upper and lower leaf surface to determine the possible difference in numbers of stomata and/or their distribution between the two pea varieties studied* Leaf surface impressions were made by painting the upper and lower sides of each leaf with nail polish* When dry, the transparent film was removed and stomata impressions counted under X30 magnification (per predetermined area of *00035mm?)* Stomatal indices were taken of each treatment and compared for possible differences due to the influence of nutrient levels, in accordance with the procedure of Salisbury (6$)» 18. B* Micro-chemical Methods of Analysis Dried Pea Leaf Tissues* Preparation of Tissues for Analysis A l l samples were oven-dried at 100° C to constant weight. After removal from the oven each sample was weighed, then ground in an agate mortar (four inch diameter) with ca.0.1 gram calcium carbonate added(9)* After the tissue was thoroughly disintegrated, a l i t t l e 85 percent reagent grade acetone was added and grinding continued* The extract was then filtered through a Buchner funnel, fitted with low ash Watman No. 50 f i l t e r paper* The residue was washed again with solution of 85 : 15 acetone water. This procedure was continued until the tissue residue appeared colourless. The filtrates were transferred to 100 ml graduated flasks and made up to the mark with the above mentioned solvent mixture* Aliquots of fi f t y ml were pipetted into separatory funnels contain-ing f i f t y ml of petrol ether* Distilled water was carefully added until i t became apparent that a l l fat soluble pigments had entered the ether layer* Upon draining off the ether layer, the remaining H 2 O layer was saved for further analysis* The separatory funnel containing the ether solution was placed in the upper rack of support* Approximately 100 ml of water was introduced into a second separatory funnel and placed in the supporting rack below the ether containing funnels* The ether solution was allowed to run into the lower funnel* When * Analyses were carried out by Mr. W. Sargeant, of the firm of Coast ELdridge, Engineers and Chemists Ltd*, Vancouver, under the Author's supervision* a l l solution had lef t the upper separatory funnel, the latter was rinsed with a l i t t l e ether* Reversing the position of the two funnels, the funnel containing both the water and ether solutions was drained and the water layers saved* These procedures were repeated 10 times until a l l acetone was removed* The ether solutions were introduced into 100 ml volumetric flasks and made up to volume* Sixty ml reagent bottles, into which some anhydrous sodium sulphate had been introduced, were f i l l e d with ether solutions con-taining extracted chlorophyll pigments* When the solutions were optically clear, aliquots were pipetted into another dry bottle and diluted with enough dry ether to cause the value of logj, Io/l to be between 0*2 and 0*8 at wavelength 660 m/u. Aliquots were transferred to absorption cells and readings taken of Io and I at 660*0 m/u and 61*2*5 m/u, using a DK2 Beckman Spectro-photometer* Quantities of chlorophylls a and b were calculated in accordance with the following formulae: Chlorophyll a (mg/liter) a 9*93 log 1° (660) - .777 log ISL ,,x 0 d . I I (OU2.5; Chlorophyll b (mg/liter) = 17.6 log 1°. (6^2.5) " 2 , 8 1 l oS — (660) After drying, the extracted plant residues were transferred to beakers, using distilled water and the mixtures were digested at 20 degrees C for four hours with constant shaking, to extract the inorganic water soluble salts* The residue was filtered through a Buchner funnel, using a low ash f i l t e r paper, and the supernatant liquid added to the previously collected water layers, saved during the process of the chlorophyll extraction. Combined extracts were made up to constant volume and aliquots used for phosphate, nitrite and nitrate determinations* 20. Inorganic Phosphate determination(35>) The inorganic phosphate content of each sample was determined photo-coloilmetrically i n accordance with method number 2 of the Publication Number 106U of the Research Branch of the Department of Agriculture, Chemical Methods of Plant Analysis, with some modifications to conform with micro-analytical requirements* Colour developing reagents were prepared as follows: Reagent I* Ammonium molybdate - 6.6 percent solution Reagent III. Five grams of ferrous sulphate are dissolved in f i f t y ml of distilled water and after disolution was complete, one ml of 7*5 N H2S0^ i s added. Reagent ILT was freshly prepared every two hours. Readings were taken at 700 m^U, using a Number 66 Red f i l t e r . Quantities of inorganic phosphate present were expressed as ^ grams Pi per one gram dryweight of leaf tissue per average plant per set. Nitrite Nitrogen determination(37) Nitrite nitrogen was determined in accordance with method D:2£c -diazotization method - of Methods of Collection and Analysis of water samples of the Geological survey of water supply paper IkBh (I960) • Nitrate Nitrogen(37) A fresh sample of the original extracts was heated with hydrogen peroxide, to oxidize nitrite to nitrate. Nitrate nitrogen was then determined in accordance with method D25>.b.l using phenoldisulphonic acid* Differences between the former and the latter quantities represents nitrate nitrogen present in the sample. Quantities for both nitrite and nitrate nitrogen are reported in grams per one gram dry weight of leaves per plant per set. 21 RESULTS AM) DISCUSSION Carbon dioxide exchange rates, experimentally determined showed pronounced variability amongst plants subjected to the same nutrient treatment (Table 1)* The other specimens belonging to individual sets absorbed carbon dioxide in quantities somewhere in between the maximum and minimum values shown* This was found to be true for individuals belonging to both the Jade and Olympic varieties* When the average values of stomatal indices for both upper and lower leaf surface areas of the two pea varieties were determined in accordance with Salisbury's procedures (65) the following was noted* Whereas the stomatal indices (I) for the upper leaf surface areas of both varieties studied did undergo a certain change in value (Table n), those for the lower leaf areas exhibited a much more pronounced change from the pattern on the upper sur-face as follows* Although both Jade and Olympic appeared sufficiently sensitive to phosphorus deficiency in the nutrient medium to show a slight change in stomatal index values, the variety Olympic exhibited a markedly greater tolerance to nitrogen deficiency than Jade* The latter appeared highly intolerant to a lack of this mineral as was indicated by an increase of the original stomatal index value at 2ii.O to 31*. 9 an equivalent of f i f t y -eight percent* Olympic was only slightly more sensitive to phosphorus deficiency than was Jade* These results were borne out by the finding that Olympic nearly always showed symptoms of phosphorus deficiency (such as stunting, accompanied by great reduction i n intemodal distances, brittleness of leaves) more frequently and much earlier during its develop-ment than Jade when grown under identical conditions. When amounts of net carbon dioxide exchanged by specimens of either variety were expressed as milligrams of CO2 per gram of dry weight of leaf T A BLE I: MAXIMUM AND MINIMUM NET CARBONDIOXIDE EXCHANGE RATES FOR VARIETIES 22 J A D E O L Y M P I C Plants MR COo Plant! Mg C 0 2 Variety Treatmt. Set per set Max. M i n . Variety rreatmt. Set per set Max. Min. JADE N + P 1 7 1.0 .45 OLYMPIC N+P 1 5 .60 .28 2. 6 .71 .35 2 6 .94 .40 3 6 .80 .52 3 6 .63 .36 4 7 .71 .34 4 5 1.06 .39 N - P 1 7 .31 .09 N - P 1 6 .26 .11 2 6 .42 .24 2 6 .66 .19 3 5 .45 .13 3 6 1.05 .23 4 6 .71 .24 4 6 .05 .27 P - N 1 6 1.71 .63 P - N 1 6 .94 .43 2 4 .80 - . 18 2 4 .31 -.96 3 4 .71 .15 3 4 .78 .59 4 6 .84 .29 4 3 .36 -.07 4a 3 Compensat ion Point -(N+P) 1 6: .46 .15 1/2(N+P) 1 6 .55 .36 -(N+P) 1 5 .72 . 35 2(N+P) 1 5 .60 .40 1/2(N+P) 1 5 .59 .36 2(N+P) 1 4 .63 .49 1/2P-N 1 6 .67 .36 2P-N 1 5 .45 .11 1/2 P-N 1 4 .78 .38 2 P-N 1 4 .45 .21 1/2N-P 1 6 ,78 .52 2N-P 1 4 .64 .58 1/2N-P 1 5 .78 .33 2N-P 1 4 .62 .33 23 T A B L E II: AVERAGE STOMATAL INDEX OF VARIETIES JADE AND OLYMPIC AS MODIFIED BY MINERAL COMPOSITION OF NUTRIENT SOLUTION Variety Treatmt. AVERAGE I Variety Treatmt. AVERAGE I Dorsal Ventral Dorsal Ventral JADE N + P 21.7 24.0 OLYMPIC N + P 17.1 23.5 N - P 22.7 28.0 N - P 20.6 29.1 P - N 25.4 34.9 P - N 22.1 26.6 a* tissue and then compared (Tables i n and IV), the resultant values revealed that Jade variety, while much more vigourous in physical appearance, was apparently less able to absorb carbon dioxide under adverse nutritional conditions than Olympic, An exception was the latter 1s behaviour in relation to carbon dioxide exchanged, when grown in nitrogen deficient nutrient media containing s tandard quantities of phosphorus, A further observation was that from a total of sixteen experi-mental sets of Jade, four showed a pronounced deficiency of nitrate N (V plus) within their tissues (Tables III and IV), % e n these sets were compared with respect to net carbon dioxide uptake, i t became apparent that in the absence of phosphorus within the nutrient medium the quantities of carbon dioxide assimilated by plants grown in complete Hoagland solution < (i.e. Jade Table III - sets 2 and 3 for (N + P) - and set 2 of N - P) treatments)* were slightly higher for the latter treatment. In the case of Jade plants (Table i n ) grown in twice the normal amount of phosphorus, but lacking an EXTERNAL source of nitrate i t was interesting to note that the nitrite N (III plus) was found to be present in plants, in sufficient quantities to be determined micro-chemical ly and in amounts equal to one half the quantity found in plants grown i n nutrient media, deficient in nitrate (N (V plus) but containing standard concentra-tions of phosphorus. This observation did not hold true for the Olympic variety (Table IV) for which i t was found that the nitrate content of those plants (grown in (P - N) media) which had reached the Compensation Point, was approximately equal to twice the amount of nitrate in those plants, whose net carbon dioxide exchange rates had reached values lying above the compensation point. T A B L E IH':. SUMMARY OF PHYSICAL AND CHEMICALLY DETERMINED MEASUREMENTS OF ANALYSIS J A D E Treat-Average M g C 0 2 D M 2 Leaf Total. D. Wt. Average Dry Wt. Plant No. of CO2X10-4 Mg per IMG D. W. M g C02 Average M g P i Average Mg N°+ /Plant Average Mg N3+ /Plant Mg Chlorophyll /Plant/GR Drv Weight Ratio A Mg Pi +N3+ Mg P« +N5+ Mg Pi +N3+ +N5+ GR C O 2 Plant Total Chlor-ophyll " Chlorophyll SET ment Mg Plants IG D. W. /Plant A B B Plant Plant Plant GR Chi Plant DW D. W.-2 N+P . 5019 5526 921 6 5.4 .54 .23 0 .97 .405 14 3.7 1.20 - - .97 .545 .55 3 . 7115 8549 1221 7 5,8 .58 .64 0 .54 .421 12 3.5 1.18 - 1. 31 .541 .54 4 . 5502 2673 534 5 10.3 1.03 .95 .92 0 .600 17 3.5 - 1.87 - • .71 . 770 . 7 7 2 N«P . 3228 3013 502 6 6.4 .64 .18 0 l»-4 .436 135 3.2 1.58 - - .50 .571 .57 3 . 2401 1270 254 5 9.4 .94 .30 2.0 0 .576 178 3.2 - 2. 3 - .35 .754 .75 4 .4563 2110 351 6 12.9 1.29 .26 .81 . 0 .507 145 3.9 - 1.09 - .64 . 652 .65 2 P-N . .2068 959 239 4 8.6 .86 .50 .12 . 3 .305 177 1.7 .80 .62 .92 .42 .482 .48 3 .4073 1826 456 4 8.9 .80 1.10 .57 1.7 .497 172 2.9 2. 80 1. 67 3, 37 .56 .669 .67 4 .5007 1829 304 6 16.4 1.64 .50 1.4 0 .293 095 3.0 li-90 1.29 . 388 .39 1 1/2P^ .5317 2126 354 6 15.0 1.50 .25 .75 0 .218 070 3.1 - 1.00 - 1. 84 .288 .29 1 2P-N .2407 2715 543 5 4.4 .44 .40 0 .8 .234 , 208 1.1 1.20 - - .54 .442 .44 1 1/2N-F .6319 3268 544 6 11.6 1.16 .15 1.0 0 .348 141 2.4 1.15 - 1.29 .489 .49 1 2N-P . 6044 2379 594 4 10.1 1.01 0 1.6 0 . 925 192 4.9 - 1.6-P .54 1.117 1.12 1 - -.N-P .3637 4390 731 6 4.9 .49 .36 .35 .23 .386 105 3.6 .59 .94 .74 .491 .49 1 1/2N 1/2P .4639 4319 719 6 6.4 .64 .15 .15 .81 360 151 2.3 .96 .30 1.11 .90 .511 .51 1 2N2P . 4992 4410 882 5 5.6 .56 .26 .28 .46 .740 154 4.9 .72 .54 1.00 .56 .894 .89 T A B L E I V : SUMMARY OF PHYSICALLY AND CHEMICALLY DETERMINED MEASUREMENTS OF ANALYSIS Treat-Average Ma C02 D M * Leaf Total D. Wt. Average Dry wt. No. of CO 2 xl0-4 Mg per Mg C 0 2 IG D. W. Average M g Pi Average Mg N 3 * Average Mg N~+ MgChlorophyll /Plant/GR D. W. Ratio MgJ>i + N J + Mg. Pi M s Pi +N3+ + N 5 + GR C 0 2 Plant Total Chlor-ophyll Plant DW Chlorophyll SET ment Mg Plant Plants IMg D, W, /plant /Plant /Plant a b a/b Plant Plant Plant GR Chl. D . W. 2 N+P .5932 3935 655 6 9.0 .90 .50 .61 « 0 .372 .101 3.5 1.11 - -• 1.25 .473 .47 3 .4681 3761 626 6 7.4 .74 .76 .95 0 .555 .160 3,4 1.71 - - ..64 .715 .72 4 .6595 1540 308 5 21,4 2.14 1.10 1. 38 0 .620 .178 3.4 2.48 - - .82 .798 .80 2 N-P .3804 2535 422 6 6.0 .90 .60 .86 0 .460 .115 4.0 1.46 - - .93 «5*75 .58 3 .5059 2430 405 6 12.4 1.24 .15 .86 0 .460 .125 3.6 1. 01 - - ,86 .585 * 59 4 .4839 2212 368 6 13.1 1. 31 .16 1.11 0 .540 .140 3.8 1.27 - - .71 .680 .68 2 P-N -.3671 1220 305 4 -12.0 -1.20 1.17 2. 35 0 .265 .097 2.5 3.52 - - - .362 .36 3 .6755 995 248 4 27.2 2.72 .60 2. 62 0 .912 .287 3.1 3.22 - - .56 1.199 1. 20 4 .3428 1260 420 3 8.2 .82 .88 2.73 0 .560 .200 2.8 3.61 - - .45 .760 .76 1 1/2P-N .5642 2189 547 4 10.3 1.03 .82 1, 37 0 .412 .120 3.4 2.19 - - 1.05 .532 .53 1 1/2N-P . 5407 1684 336 5 16.0 1. 60 .40 1.46 0 .608 .205 3.0 1,86 - - . .66 .813 .81 1 2P-N .3614 1873 468 4 7.7 .77 1.12 1.52 0 .487 .130 3.7 2. 64 - - .58 .617 .62 1 2N-P .4377 1938 484 4 9.1 .91 .43 1.45 0 .947 .237 3.9 1.88 - - .36 1.184 1. 20 1 -N-P .5139 1995 399 5 12.8 1.28 .26 1.18 0 .600 .152 3.9 1.44 - - .68 .752 .75 1 1/2N 1/2P ,4782 2457 491 5 9.7 .97 .30 .99 0 .488 .136 3.4 1.29 - .76 .624 .62 1 2N2P .5765 2111 527 4 10.9 1.09 1.10 1.35 0 .830 .212 3.9 2.45 - - .55 1.042 1. 04 4 P-N 0 725 241 3 i 0 1.06 5.10 0 .520 .196 2.6 6.16 - - 0 .716 .72 * NOTE: „2 U Gram pex Gram Dry Weight Extract ro ON 27 Whenever the NET rate of carbon dioxide exchanged became high in relation to other sets of the same nutrient treatment (Table III)» i t was very nearly always accompanied by a high concentration of the nitrate nitrogen species, while the nitrite nitrogen was either completely absent or else present in very small quantities* The fact that in the case of Jade, some sets did not contain either nitrate N (V plus) or nitrite N (III plus) in DETECTABLE amounts AT THE SAME TIME, furnished the opportunity to compare the function of either nitrogen species with respect to carbon dioxide assimilation* This was approached from tac aspects - one being the function of either nitrate or nitrite in regard to its role in carbon dioxide assimilation IN CON-JUNCTION with inorganic phosphorus present, the other involving separate analyses of the role of nitrate (or nitrite) ions and inorganic phosphorus. The resultant graphs showed the following interesting relationships. For Jade ( Figure 2) increased concentrations of nitrite and phosphorus with-in the plant resulted in an apparent increase in the amount of carbon dioxide absorbed* Higher concentrations of nitrate and phosphorus reduced the net rate of carbon dioxide exchange* The presence of BOTH nitrate and nitrite as well as phosphorus showed some stimulatory action upon carbon dioxide assimilation at increased levels within the plant* Using the second method of approach (Figure 3) when the phosphorus and nitrogen species were separated, i t was found that increased amounts of phosphorus tended to reduce the amount of carbon dioxide assimilated in the presence of nitrate ions within the plant* In the case of nitrite being exclusively present within the plant, increased concentrations of phosphorus resulted in an apparent increase in net carbon dioxide exchange rates* •a 53 T3 o u bo nl s > F I G U R E 2. I N T E R R E L A T I O N S H I P B E T W E E N N I T R O G E N S P E C I E S A N D P H O S P H O R U S IN T H E P L A N T S A N D N E T R A T E S OF C A R B O N D I O X I D E E N C H A N G E D - J A D E . 2X l-O-1.1-l.l, 'i l-X l.o -'I -.(, -•1 -LEGEND -x>— • t>«** c o 2 Assimilation r NITRITE present only. — — » B U ? v COo Assimilation " H NITRATE present only. — -•pM**Kr* C O s Assimilation NITRATE and NITRITE both present. - >< •— . Visually fitted — Lines. »•• ft-P I * I.O 1.1* '•1* i.0 m e P N 3+ and/or N 5 + —I ,3'37 per GRAM of Leaf Dry Weight ro CO LEGEND •a M T3 E & o u 60 s <u 60 n! a > < F I G U R E 3. A N A L Y S I S OF N I T R I T E A N D N I T R ' A T E C O N C E N T R A T I O N I N F L U E N C E U P O N N E T C A R B O N D I O X I D E E X C H A N G E R A T E S - J A D E — A — a — X -C02 Assimilation NITRITE present only. Phosphorus associated With NITRITE NITRATE present only. Phosphorus associated with NITRATE Visually fitted lines of © OP —x-D-Of I— >X<S -So ~\— •7* —r~ t.o — i — —r— J .6 — I ug N ug N ugP 3+ 5+ per gram of leaf dry weight ro ?-30 ""hen the data for Olympic (Table IV) were examined, a difference concerning the results of the chemical analysis became apparent* At NO TIME was any nitrite nitrogen found to be present in sufficient quantities to be measured* In addition i t wi l l b e noted that at NO TIME had nitrate nitrogen disappeared* Consequently i t was not possible to follow the same analytical procedures as had been applied to the Jade variety, due to the fact that a l l samples tested contained only nitrate ions, with phosphorus* Comparison of the chlorophyll a/b ratio (Table III) showed that deficiency of nitrogen i n plantsgenerally tended to reduce the value of this expression* It was noted that chlorophyll b content increased in those plants grown in nitrogen deficient media containing twice the standard amounts of phosphorus, while chlorophyll a was reduced to the extent as to be nearly equal to that of chlorophyll b* It was also observed from these measurements that the influence of a lack of nitrate in the nutrient medium on chlorophyll a concentrations was much more severe than that on chlor-ophyll b levels* The latter may have received a stimulus from the presence of excess amounts of phosphorus provided that the nutrient solution was deficient i n nitrogen* This observation was exemplified by the two extremes where for the (2F - N) nutrient treatment, the amount of chlorophyll b reached i t s highest value while chlorophyll a was at its next to lowest concentration* In the case of the (2N - F) treatment, the chlorophyll a concen-tration reached itsmaximum value - a large increase over that found for the previously mentioned ( 2P - N) treatment* Chlorophyll b changed but l i t t l e i n concentration in plantsgrown i n either (2N - P) or (2P - N) nutrient media. Analysis of chlorophyll a and b concentration fluctuations in Olympic (Table IV) as a consequence of nutrient treatments did not appear z.o H SI F I G U R E 4. C O M P A R I S O N OF C H A N G E IN C H L O R O P H Y L L a A N D b C O N T E N T IN R E L A T I O N T O N E T C A R B O N D I O X I D E E X C H A N G E R A T E S - J A D E . LEGEND . o(ixPtY*0 Chlorophyll a «(t«? 11 **) Chlorophyll I b T 3 E y 6 0 > 1.8-f.y -(.1 -•I J \ OIF-") •IN-P) I 4?-p) , I N - P ) J (N*p) • ( P - h > ' ( P-N/ I « 0 N )(M-P) O(H«-P) •(r*-p) (lp-n)»lO(2p-n) O ( N - P ) O ( N t P ) 0(Mt?) O(itUP) i.O Average mg chlorophyll a, b per gram of leaf dry weight C O M P A R I S O N OF T H E E F F E C T OF N I T R A T E A N D N I T R I T E U P O N C A R B O N D I O X I D E E X C H A N G E R A T E S A N D C H L O R O P H Y L L a / b R A T I O - J A D E . LEGEND ( x) Chlorophyll a/b ratio C0 2 assimilation in presence of NITRATE only. C0 2 assimilation in presence of NITRITE only. {3.0 (3.0> P . H (3.2) N-P (Id) lit* 4N-P C i t ) H - P 1.1 ^ l . f c a . i — 7 A v e r a g e jig NOo o r NO^ per gram ofileaf dry weight N-P (vs) ZP-rt H.0 M-P C v i ro 33 to show the same behaviour for this quality as that of Jade* When nutrient treatment (P - N) (set 2) chlorophyll content was compared to that of (N - P) (set 2) l i t t l e variation in chlorophyll b content was noted; chlorophyll a concentrations showed a considerable decrease in these sets* It was observed that, contrary to the fluctuations in chlorophyll a/b ratios obtained for Jade when grown under various degrees of nutrient stress, those of the Olympic variety remain nearly the same, even under severe nutrient stress, as was evidenced by the nutrient deficiency signs* Further exploration* with regard to variations in both chlorophyll;, a and b, in relation to net carbon dioxide exchange rates by plants of both varieties (Figures h and 5) when grown under various nutrient stress con-ditions, revealed that concentrations of chlorophyll a varied to a much greater extent (which was generally in accordance with nitrogen concentrations of the nutrient medium) than chlorophyll b, under identical conditions of nutrient stress* An example w i l l illustrate this observation* Comparison of chlorophyll b concentrations of variety Olympic (Table IV) sets two of nutrient treatments (N - p) and (2N - P) showed an 2 increase in the latter treatment of only *032 mg pa r one gram of leaf tissue dry weight, while chlorophyll a content increases from .6 mg for the former set of treatment, to *9U7 mg for the latter series; a difference of nearly •3 mg* Analysis of Jade (Table III) for chlorophyll a and b content for the same nutrient treatments, showed chlorophyll a content to increase by nearly .6 mg, whereas chlorophyll b increased only by *0J> mg per one gram of leaf tissue dry weight* The relationship of chlorophyll a content with carbon dioxide absorption by plants of the Jade variety when subjected to nutrient stress were subsequently examined* From the resultant graph (Figure h) i t became 3li apparent that the relationship of chlorophyll a content to the carbon dioxide exchange rate could not be determined with any degree of precision WITHOUT due consideration of the presence of either nitrate or nitrite within the plant's photosynthetic tissues* This conclusion was borne out by the observation that a nutrient solution, containing one half the standard amount of phosphorus, while being mono-deficient i n nitrate, was MORE beneficial with respect to CO2 assimilation in the presence of nitrate than (2P - N) nutrient grown plants, whose tissues contained only nitrite ions* ?his finding was especially interesting when i t was noted that very l i t t l e difference existed between the chlorophyll a contents of plants grown in (|p - N) and (2P - N) nutrient media* When the chlorophyll a/b ratios for set 3 and k of complete (N + P) treatment for variety Jade (Table HI) were compared, i t was found that these ratios were identical* However, the net carbon dioxide exchange rates of these two sets were NOT the same* Further perusal revealed that the net carbon dioxide exchange rate i n the exclusive INTERNAL presence of nitrate ions was very nearly double the amount of C O 2 absorbed when only nitrite was present within the plant (Figure f>). when the table containing a summary of experimental data (Table 17) for Olympic was examined i t was found that some of the "erratic 1 1 sets of the Hoagland (P - N) nutrient treatment contained comparatively high quantities of either phosphorus and/or nitrate nitrogen* This was especially noticeable for that group of plants whose net rate of carbon dioxide exchanged was found to be equal to the Compensation Point where nearly five mg of nitrate nitrogen was found to be present within the photosynthetic tissues - a fact which implies that nitrate ions in this case do not appear to exert an enhancing effect upon net carbon 35 dioxide exchange rates* This i s contrary to that found for plants belonging to the Jade variety, for which the exclusive internal presence of nitrate was found to promote carbon dioxide assimilation* 36 GENERAL DISCUSSION Results obtained during the course of this investigation tended to be inconclusive with respect to establishing the reason for the existence of either intra-, or intervarietal differences of specimens belonging to the same species of Pi sum sativumX* Apart from the fact that the variability Ctf results might possibly be due to errors in technique, such as mechanical stimulation of respiration as the result of handling, some of the results obtained in measuring net carbon dioxide exchange rates of individual plants belonging to the pea variety Jade, showed the possible existence of some relationship with the internal presence of nitrite and/or nitrate within the photosynthetic tissues of the individual specimens. In addition, i t was found that l i t t l e i f any relationship existed between chlorophyll a, or alternatively the chlorophyll a/b ratio and the net ralie of carbon dioxide exchanged. Of the physical characteristics, the stomatal index of ventral surfaces of leaves tended to vary more extensively as the result of nutrient stress than the dorsal sides of the same leaves. Another possibility for the variability in carbon dioxide exchanged by individual plants, not con-sidered for the purposes of this experiment, was the existence of endogenous rhythmic variations with respect to carbon dioxide absorption. The facfc that some plants grown in nitrate deficient media, sometimes contained appreciable amounts of either nitrate or nitrite appears to indicate that the growth medium (Horticultural Grade Vermiculite) was somewhat less inert tHnamwas supposed. An alternative to the above would be that some nitrate, i n i t i a l l y present within the cotyledons^ of the pea plants had found its way into the plants, prior to excision. 37 The latter difficulty may be overcome by micro-chemical analysis for nitrate content of the intact seeds prior to germination, followed by analysis of nitrate of the excised colyledons. The resultant difference may then be used as a correctinn factor to be incorporated in the final nitrate analysis of the photosynthetic tissues at the conclusion of the experiment. Finally two alternatives to this experiment are suggested as follows: A greater number of plants belonging to the same species should be tested with respect to variations in net carbon dioxide exchanve rates as the result of nutrient stress, and each analysis to be followed immediately by establishing the value of the stomatal index of both ventral and dorsal side of the leaves of each plant. Such procedure would offer a possibility of establishing a positive correlation between the net rate of carbon dioxide exchanged, and its stomatal index, thus establishing at least partly, the reason for variations in CO2 assimilation. In addition, each plant should be analysed for both nitrate and/or nitrite content of its photosynthetic tissues. Such procedure would possibly result in proving a positive correlation between the nitrate/nitrite content of leaves and net carbon dioxide exchange rates by each plant. A second method involving variability of carbon dioxide exchange rates, as caused by nutrient stress, would be to analyse a smaller number of plants of different varieties belonging to the same species for net rate of carbon dioxide exchanged, but on an hourly basis for a continuous period in order to establish i f endogenous rhythmic variations, when extant, are possibly subject to change as a result of nutrient stress. Although a continuous analysis of nitrate.nitrite would be highly desirable, each time a CO2 analysis is completed, technical difficulties presently extant would 38 prevent such ananalysis to be carried out, without detaching the leaves from the plant under the above mentioned conditions of this experiment. BIBLIOGRAPHY 1. Allen, M.B.; Arnon, D.I.; Capindale, J.B."; Whatley, F.R.and Durham, L.J. 1955 - Photosynthesis inisolated chloroplasts III, Evidence for complete photosynthesis, J. Amer. Chem. Sec. J_7_ : klk9 2. Anderson, F.G. and Bathurst, A.C. 1938 - Stikstof en fosfor in lemoene, Boerderey, S.A. 13_ : 3^ 9 3. Arnon, D.I.; Whatley, F.R. and Allen, M.B. 193k - Photosynthesis in isolated chloroplasts II, Photophosphorylation -the conversion of light into phosphate bond energy, J. Amer. Chem. Soc. J_6 : 6324 k, Aronoff, S,; Benson, A.A.; Hassid. W.Z. and Calvin, M. 19k7 - Distribution of carbon (ik) in photosynthesizing barley seedlings, Science 105 : 66k 5. Brachet, J.; Chautrerme, H. and Vanderhaeghe, F. 1955 - Recherches sur les interactions biochimiques entre le noyau et le cytoplasme chez les organismes unicellulaires II, Acetobularia mediterranea, Biochem et Biophys. Acta 18 : 'jkk 6. Burstrom, H. 19^ 3 - Photosynthesis and assimilation of nitrate by wheat leaves, Ann. Agr. Coll. Sweden 11 : 1 7. Ibidem 19^ 5 - The nitrate nutrition of plants, Ann. Agr. Coll. Sweden 1^ : 1 8. Carolus, R.L. 1935 - Experiences with rapid chemical tests for determination of nutrient deficiencies in vegetable crops, Proc. Am. Soc. Hort. Sci. 3J. : 579 9. Canada Department of Agriculture 1962 - Chemical Methods of Plant Analysis, Canada Dept. of Agriculture Research Branch Publication No. 106h, Phosphorus - Method 2. 10. Curtis, 0.; and Clark, D.G. 1950 - An introduction to Plant Physiology, McGraw-Hill Co., New York, p. 60 11. Eckerson, S. 192^  - Protein synthesis in plants I, nitrate reduction, Bot. Gaz. 21 : 377 12. Ibidem I929 - Influence of phosphorus deficiency on the metabolism of tomato, Abstr. Am. J. Botany 16 : 852 13. Ibidem 1931 - Influence of phosphorus deficiency on the metabolism of tomato plants (Lycopersicon esculentum), Contr. Boyce-Thompson Inst. _1 : 197 lk, Evans, H.J. and Nason, A. 1953 - Pyridineunucleotide - nitrate reductase from extracts of higher plants, Plant Physiol. 28 : 233 15. Fruton, J.S. and Simmonds, S. 1959 - General Biochemistry, second Ed. second print, J. Wiley & Sons Inc., New York, p. 182 16. Ibidem - p. 871 17. Giese, A.C. 1960 - Cell Physiology, W.B. Saunders Co., Philadelphia, Chapter 19, App. 1, p. 356 18. Hageman, R.H. and Flasher, P. 1960 - Nitrate reductase activity in corn seedlings as affected by light and nitrate content of nutrient media, Plant Physiol. 3J? s 700 19. Hammer, K.C. 1936 - Effects of nitrogen supply on rates of photosynthesis and respiration in plants, Bot. Gaz. 97_ : Jkk 20. H i l l , R. and Whittingham, CP. 1956 - Methuen monographs on Biochemical subjects, J. Wiley & Sons, Inc. New York, Photosynthesis, p. 57• 21. Ibidem - p. 59 22. Holden, M. 1952 - The fractionation and enzymic breakdown of some phosphorus compounds in leaf tissues, Biochem. J. £1 : 433 23. Kamen, M.D. 1963 - Primary Processes in Photosynthesis, Acad. Press, N. York, p. 31 2k. Kraybell, H.R. 1930 - Plant metabolism studies as an aid in determining fertilizer requirements I, Ind. and Eng. Chem. 22 : 275 25. Lundegardh, H. 1932 - Die Nahrstoff Aufhahme der Pflanze, Jena. 26. McAllister, H.D. and Meyers, J. 19^ 0 - The timecourse of photosynthesis and fluorescence observed simultaneously, Smithonian Misc. Coll. 29 s 6 27. MacArthur, C.A. 1961 - Lecture Series - Phospholipids, the University of Saskatchewan. 28. McCool, M.M. and Weldon, M.D. 1930 - The effect of sodium nitrate on the composition of the expressed sap of small grains, J. Am. Soc. Agron. 22 : h3k 29. MacGillivray, X.H. 1926 m Importance of phosphorus in the production of seed and'non-seed portions of the tomato fruit, Proc. Am. Soc. Hort. Sci. 22 : 37k 30. Mayer, B.S. and Anderson, D.B. 1956 - Plant Physiology second Ed. kth print, D. Van Nostrand Co. New York, p. 60 31. Ibidem - p. 305 32. Mothes, K.; Bottger, I. and Wollgiehn, R. 1958 - Uhtersuchungen uber den Zu Sammenhang zwischen Nuklein saure und Eiweiszstoffwechsel in grunen blattern, Naturwiss, V? : 316 33. Nichiporovich, A.A. 1955 - Tracer atoms used to study the products of photosynthesis as' depending on the conditions in which the process takes place, Proc. First Geneva Conf. on Peaceful Uses of Atomic Energy. 3I+. Nightingale, G.T. 19^2 - Nitrate and carbonhydrate reserves in relation to nitrogen nutrition in Pineapple, Bot. Gaz. 103 : k09 35. I960 - Official Methods of Analysis of the Association of Official Agricultural Chemists, Ninth Ed. ACAC Washington, D.C. p. 92. Method 6.100 36. Pleasants, A.L. 1930 - The effdct of inorganic nitrate fertilizers on stomatal behaviour, Elisha Mitchell, Sci. Soc._h6> : 95 37. Rainwater, F.H. and Thatcher, L.L. i960 - Methods for collection and analysis of water samples, Geological survey of water supply paper ihjk, U.S. Government Printing Office, Washington, D.C, Methods 25b and 25c, p. 216. 38. Reed, H.S. 1907 - The value of certain nutritive elements to the plant cel l , Ann. Bot. 21 : 501 39« Salisbury, E.J. 1927 - On the causes and ecological significance of stomatal frequency with special reference to the woodland flora, Phil. Trans. Roy. Soc. B216 : 1 kO. Schertz, F.M. 1929 - The effect of potassium, nitrogen and phosphorus upon the chloroplast pigments upon the mineral contents of the leaves and upon production in crop plants, Plant Physiol. 4 : 269 kl. Smillie, R.M. 1962 - Photosynthetic and respiratory activities of Growing Pea Leaves, Plant Physiol. 31 : 7l6 k2, Smillie, R.M. and Fuller, R.C. 1960 - Photosynthetic and respiratory enzymes in leaves, Federation Proc. 19 : 328 43. Smillie, R.M. and Krotkov, G. 1961 - Changes in the dry weight, protein, nucleic acid and chlorophyll contents of growing pea leaves, Can. J . Bot. 22 : 891 kk Ibidem 1959 - Enzymic activities of sub-cellular particles from Leaves, IV, Photosynthetic phosphorylation and photosynthesis by isolated chloroplasts from pea leaves, Can. J. Bot. 3X : 1217 45. Sprague, H.B. and Shive, J.W. 1929 - A study of the relations between chloroplast pigments and dry weights of tops in Dent Corn, Plant Physiol. 4 : 165 46. Walter, H. 1929 - Plasmaquellung und assimilation, Protoplasma 6 : 113 kT, Whatley, F.R.; Allen, M.B. and Arnon, D.I. 1955 - Photosynthetic phosphorylation as an anaerobic process, Biochem. et Biophys. Acta 16 : 605 48. Ziegenspeck, H. 1944 - Vergleichende untersuchungen der Entwicklung der Spaltoffnungen von monokotyledonen und Dilkotyledonen im lichte der Polariskopia und Dichroskopie, Protoplasma 38 : 197 APPENDIX TABLE 1. INDIVIDUAL PLANTS NET CARBONDIOXIDE EXCHANGE RATES J A D E JADE COMPLETE (N+P) MODIFIED (FeiiDTA) HOAGLAND SOLUTION M B dm* Mg Soln/lOOOcc Plant ppm ppm Leaf Mg FlOWE/^^ SET REP c o 2 TEMP. C0 2 /hr Area C ° 2 1dm 2 Average D. Wt. xn xp xn xp 1 1 67 21 1.0573 .6 1.7625 42 35 133 2 29 .4577 .5 .9152 3 18 .2838 .4 .7095 4 27 22 . 4261 .8 .5326 5 29 .4577 .6 .7629 29 .4577 6 12 .1814 .3 .6046 12 .1814 7 12 22.5 .1814 .4 .4535 12 .1814 .8201 2 1 16 21.5 .2515 .4 .6312 42 35 133 15 .2367 2 6 22.0 .09467 .2 .4730 6 . 09467 3 9 .1421 .2 .7105 8 .1260 4 7 .1104 .3 .3681 8 .1260 5 12 .1814 .4 .4735 10 .1578 6 9 .1421 .4 .3552 9 .1421 .5019 5526 4.5 3 1 15 21.5 .2367 .31 .7635 42 35 133 15 .2367 2 10 .1578 .30 .5260 9 .1421 3 21 21.9 .3314 .42 .7785 18 .2838 4 17 22.0 .2683 .39 .6879 17 .2683 5 12 .1814 .28 .6478 10 .1578 6 17 .2683 .31 .8654 0 1.4 15 .2367 .7115 8549 SET REP. ppm C 0 2 TEMP, C0 2/nr, Leaf Area d m 2 Mg Average D. Wt. Soln/lOOOcc Plant ppm F l o w j / ^ xn xp xn xp - ^ M i n . 4 1 26 23.0 .4103 .77 .5302 42 35 133 26 .4103 2 8 .1260 .225 .5600 9 .1421 3 9 .1421 .20 .7105 12 .1814 4 3 .04733 .137 .3454 5 28. 3 .07888 5 9 .1421 .23 .5743 8 .1260 6 7 .1104 .19 .5810 7 5502 2673 4.6 4.7 TOTAL AVERAGE FOUR SETS . 6459 Mg C 0 9 9 DM Leaf Surface Area APPENDIX TABLE 1. JADE MODIFIED (FeEDTA) HOAGLAND SOLUTION (-P) Leaf Mg Soln/lOOOcc Plant ppm ppm M g / U Area C0 2 /hr D. Wt SET REP. C 0 2 TEMP C0 2 /hr . d m z 1 d m 2 Average xn xp xn xp 1 1 3 21.5 .04733 .5 . 0947 42 0 133 3 .04733 2 4 . 06310 .6 .1051 6 .09467 3 7 22 . .1104 .5 .2200 7 .1104 4 4 .06310 .4 .1573 5 .07888 5 3 22.5 .04733 .3 .1570 3 .04733 6 5 .07888 .3 ,2620 4 .06310 7 5 07888 .3 .2620 4 .06310 .1892 1 2 1 4 22. .06310 .21 . 3004 42 0 133 6 .09467 2 4 .06310 .17 .3710 4 .06310 3 4 .06310 .21 .3004 6 . 09467 4 5 .07888 .18 .4271 4 .06310 5 3 .04733 .16 .2961 2 .031S0 6 2 .03150 .13 .2420 .3228 1.1 2 .03150 3013 0 3 1 2 . 23. ( .03156 .14 .2254 42 0 133 2 .03156 2 1 .01578 .12 .1315 1 .01578 3 2 .03156 .069 .4573 3 .04733 4 .5 .00789 .058 .1361 .5 .00789 5 1 .01578 .063 .2504 10.2 1.5 2 .03156 . 2401 .1270 6 SET REP. ppm co 2 TEMP, Mg C 0 2 / h r . Leaf Area d m 2 Mg CO2/&L 1 d m z Average D. Wt. Soln/lOOOcc Plant ppm Flowr^——-xn xp xn xp 4 1 5 23.5 .07888 .11 .7171 42 0 133 4 .06310 2 4 .06310 .12 .5258 3 .04733 3 5 .07888 .18 .4382 6 . 09467 4 2 .03156 .13 .2427 2 .03156 5 2 .03156 .068 .4641 1 .01578 6 1 .01578 .063 .2504 1 .01578 .4563 2110 4.9 1.6 TOTAL AVERAGE FOUR SETS .3021 Mg co 2 2 DM Leaf Surface Area APPENDIX TABLE 1. JADE MODIFIED (FeEDTA) HOAGLAND SOLUTION (=N) SET REP. ppm C 0 2 TEMP Mg C 0 2 / h r Leaf Area d m 2 Mg C 0 2 / n r 1 dm 2 Plant Average Plant Total Mg D. Wt. Soln/lOOOcc xn xp PJantt ppm Flow? xp 17 18 22 18 14 12 10 12 9 9 19 21 21.5 22.0 22.5 .2683 .2838 .3472 .2838 .2209 .1894 .1578 .1894 .1421 .1421 .09467 .1260 .3 .2 .2 .2 .2 .2 .8943 1.736 1.104 .7890 .7105 .4733 35 ,8487 133 1 2 3 4 -1 -1 +2 +2 -1 -1 +5 +5 22. -.01578 -.01578 +.03156 +.03156 - . 01578 -.01578 +.07888 +.07888 .12 .095 .085 .097 -.1308 +. 3314 -.1856 +.8123 35 .2068 959 2.0 133 1* 6 8 6 6 5 4 2 1 23.5 .09467 .1260 .09467 .09467 .07888 .06310 .03156 .01578 .27 .16 .11 .20 .3505 .4041 .7171 .1578 35 .4073 1826 2.3 4.5 133 2 2 3 2 3 3 1 2 .5 .5 4 5 23.5 23.5 .03156 .03156 .04733 .03156 .04733 .04733 .01578 .03156 .00789 .00789 .06310 .07888 .064 .056 .12 .054 .08 .16 .4931 .8451 .3944 .2921 .5916 .3943 35 .5007 1829 3.2 133 * GIANT FORM TOTAL AVERAGE FOUR SETS .4916 Mg C O s / D M z Leaf Area t ppm co 2 Mg C02/hr. Leaf Area d m 2 Mg C0 2/hF, 1 d m 2 Mg D. Wt. Soln/lOOOcc Plant ppm F l o w r ^ ' ^ M i n . SET REP TEMP. Average xn xp xn xp t N„+1/2P 1 1 2 5 4 2 2 23.5 .07888 .06310 .03156 .03156 .124 .087 .6361 .3627 0 17.5 133 ! N1/2P 3 4 5 3 2 3 3 4 3 .04733 .03156 .04733 .04733 .06310 .04733 .092 .129 .093 .5144 .3669 .6744 t • 6 4 4 .06310 .06310 .10 .6310 .5318 2126 4. 5 1.5 N.+2P 1 1 2 2 23.5 .03156 .03156 .07 .4508 0 70 133 N2P 2 3 4 3 5 1 1 1 2 .04733 .07888 .01578 .01578 .01578 .03156 .16 .135 .14 .2958 .1168 .1127 5 3 2 . 04733 .03156 .164 .2276 .2407 2715 0 2.1 + 1 1/2N.-P ! 1 1 2 5 4 6 6 23.5 .07888 .06310 .09467 .09467 .1 .148 .7888 .6396 21 0 133 ' 1/2N-P 3 7 6 .1104 . 09467 .19 .5816 4 6 6 . 09467 .09467 .169 .5601 5 5 3 .07888 .09733 .15 .5258 • 6 6 . 09467 .136 .6961 .6319 3268 6. 0 0. 9 SET REP. ppm co 2 TEMP Mg C02/hr. Leaf Area dm2 Mg Average Mg D. Wt. Soln/lOOOcc Plant ppm Flowr^—• '"Min. xn xp xn xp +2N, -F 1 1 2 6 6 7 7 23.5 .09467 .09467 .1104 .1104 .158 .169 .5991 .5810 84 0 133 2N-P 3 4» 9 9 6 5 .1421 .1421 . 09467 .07888 .22 .16 .6459 .5916 .6044 2379 6.4 0 -N-P 1 1 2 5 5 2 2 23.5 .07888 .07888 .03156 .03156 .21 .21 .3756 .1502 0 0 133 -N-P 3 4 5 6 4 3 4 4 6 6 6 5 .06310 .04733 .06310 .06310 .09467 .09467 'i 09467 .07888 .135 .21 .212 .214 .4674 .3004 ' .4456 .4423 .3637 4390 2.1 2.2 +1/2N, +1/2P 1 1 2 7 8 7 7 23.5 .1104 .1260 .1104 .1104 .2 .23 .5520 .4800 21 17.5 133 1/2N 1/2P 3 4 5 6 7 7 6 5 3 4 9 9 .1104 .1104 .09467 .07888 .04733 .06310 ,1421 . 1421 .22 .20 .13 .32 .5018 .4733 .3640 .4125 .4639 4319 .9 .9 -i-2N 2P 1 1 9 9 23.5 .1421 .1421 „3 ' : .4763 84 70 133 2N 2P 2 3 4 5 8 7 10 10 12 12 9 10 .1260 .1104 .1578 .1578 .1814 .1814 .1421 .1578 .23 .26 .39 .35 .5478' .6069 .4651 .4060 .4992 4410 1.4 1.3 * Nos, 5 & 6 died before testing APPENDIX TABLE 2, INDIVIDUAL PLANTS NET CARBONDIOXIDE EXCHANGE RATES O L Y M P I C Leaf Mg Total Soln/lOOOcc Pla nt pprr ppm Area C0 2 /hr . Mg Mg Flovjj,— SET REP. co2 TEMP. C 0 2 / n r . d m 6 1 d m 2 Average D. Wt. xn xp xn xp " ^ M i n . 1 1 30 22.8 .4734 .8 . 5917 42 35 133 27 .4261 2 20 22.9 .3156 .6 .5261 20 .3156 3 15 .2367 .4 .5917 15 .2367 .4 .5917 4 16 23 .2525 .4 .6315 15 .2367 5 9 .1421 .5 .2842 9 .1421 .5250 2 1 8 22.0 .1260 .29 .4348 42 35 133 8 .1260 2 9 .1421 .15 .9472 7 .1104 3 12 .1894 .32 .5918 10 .1578 4 5 • .07888 .14 .5633 4 .04733 5 5 .07888 .159 . 4961 6 . 09467 6 10 .1578 .30 .5260 9 .1421 .5932 3935 3.7 3.0 3 1 9 22.0 .1421 .36 .3944 42 35 133 11 .1736 2 6 .09467 .15 .6311 5 .07888 3 6 . 09467 .19 .4982 5 .07888 4 -• 6 .09467 .17 .5568 " 6 .09467 5 3 .04733 .13 . 3644 4 .06310 6 6 . 09467 .26 .3641 6 .09467 .4681 3761 5.7 4.6 SET REP. ppm CO2 TEMP, Mg C02/nr. Leaf Arei dmz Mg C02/hr. 1 dm2 Average Mg D. wt. Soln/lOOOcc Plant ppm Flowp^' Mg 2n xp xn xp 4 1 3 23.5 .04738 .12 .3948 42 35 133 5 .07888 2 7 .1104 .15 .7360 7 .1104 3 6 .09467 .15 .6311 5 .07888 4 5 .07888 .074 1. 0648 5 .07888 5 2 .03156 .067 . 4710 2 .03156 .6595 1540 6. 9 5.5 TOTAL AVERAGE FOUR SETS . 5614 MG CO2 9 , r „ r 1 DM Leaf Surface Area APPENDIX TABLE 2 OLYMPIC - MODIFIED (FeEDTA) HOAGLAND SOLUTION (-P) Leaf Mg Soln /1000cc Plant ppm ppm Mg area co 2/V Mg Mg Flowr^-^" SET REP. C0 2 TEMP. C02/hr dm2 1 dm2 Average D. Wt. xn xp xn xp ^ M i n . 1 1 6 23.0 .09467 .7 .1333 42 0 133 I 6 .09467 2 6 .09467 .5 .1893 6 .09467 3 3 . 04733 .4 .1143 25 .03945 4 5 .07888 .3 .2629 3 .04733 5 3 .04733 .4 .1143 4 .06310 6 4 .06310 .5 .1262 3 .04733 .1567 2 1 5 22.0 .07888 .157 .5024 42 0 4 .06310 2 4 .06310 .194 .3258 3 .04733 3 3 .04733 .186 .2547 2 .03156 4 2 .03156 .161 .1960 2 .03156 5 3 .04733 .141 .3366 2 .03156 6 3 .04733 .071 .6674 3.6 2 .03156 .3804 2535 5.2 3 1 4 22.0 .06310 .12 .5258 42 0 4 .06310 2 5 .07888 .16 .4930 5 .07888 3 3 . 04733 .20 .2369 3 .04733 4 2 .03156 .11 .2878 4 .06310 5 4 .06310 .06 1.0516 2 .03156 6 2 .03156 .07 .4508 .5059 2430 5.2 .9 2 .03156 SET REP. ppm c o 2 TEMP. Mg C 0 2 A r . Leaf Area dmz Mg 1 dm* Average Mg D. Wt. Soln/lOOOcc Plant ppm Flowr^— ^ M i n . Mg xn xp xn xp 4 1 5 23.5 .07888 .12 . 6573 42 0 133 6 .09467 2 3 .04733 .09 .5258 3 ,04733 3 3 .04733 .11 .4302 2 .03156 4 2 23.5 .03156 .117 .2765 3 .04733 .085 .5568 3 .04733 6 2 .03156 .069 .4573 2 .03156 .4839 2212 6.7 1.0 TOTAL AVERAGE FOUR SETS . 3817 Mg C 0 2 DM2 Leaf Surface Area APPENDIX . T A B L E 2 OLYMPIC - MODIFIED (FeEDTA) HOAGLAND SULUTION (-N) Leaf Area, dm Mg COo/ Rr 1 am2 Soln/lOOOcc Plant ppm SET REP ppm c o 2 TEMP. Mg C 0 2 / h r . Average Mg D. Wt. Mg xn xp xn xp Flowr^_—-1 1 2 3 4 5 6 24 24 16 15 14 16 11 10 15 16 11 13 21 21.5 22 .3788 . 3788 .2525 .2367 .2209 .2525 .1736 .1578 .2367 .2525 .1736 .2051 .4 • 3 .3 .4 .3 .2 .9470 .8416 .7363 .4340 .7890 .8680 .7695 0 35 133 2 1 2 3 -3 ~2 +2 +1 -3 0 22.0 -.04733 -.03156 +.03156 +.01578 -.04733 0 .11 .10 .125 -.4307 +. 3784 -.3784 0 35 133 4 -6 -6 -.09467 -.09467 .098 -.9660 -.3671 1220 10.5 4.7 3 1* 2 3 7 6 4 • 3 4 5 22 .1104 .09467 .06310 .04733 .06310 .07888 .19 .09 .08 .5810 .7011 .7887 0 35 133 4 2 2 .03156 .03156 .05 . .6312 .6755 995 9.4 2.4 4 1 2 -.5 -.5 3 3 22. -.00789 -.00789 .04733 .04733 .10 .147 -.07888 .3212 0 35 133 3 3 3 .04733 .04733 .13 .3644 1260 8.2 2.6 4 0 0 0 .061 0 5 0 0 0 0 .044 0 6 0 0 0 .064 0 .3428 725 15.3 3.2 TOTAL AVERAGE FOUR SETS . 3551 Mg C0 2/DM 2 Leaf Area A P P E N D I X T A B L E 2 OLYMPIC - MODIFIED (FeEDTA) HOAGLAND SOLUTION Leaf Mg Soln/lOOOcc Plant ppm SET REP ppm C 0 2 TEMP. Me C 0 2 / n r . Area d m 2 Average Mg D. Wt. Mg xn xp xn xp F l o w r ^ ^ M i n . •N+1/2P 1 1 3 3 21.5 .04733 .04733 .088 .5378 0 17.5 133 1 •N+1/2P 2 3 5 3 4 3 22.0 .07888 .04733 .06310 .09733 .144 .08 .5477 .7887 4 6 6 .09467 . 09467 .247 .3832 .5642 2189 5.5 3.3 •N+2P 1 1 4 5 23.0 .06310 .07888 .164 .3847 •• 0 70 133 f •N+2P 2 3 2 3 5 5 .03156 .04733 .07888 .07888 .147 .172 .2147 .4586 4 3 3 .04733 .04733 .122 .3877 .3614 1873 6.1 4.5 +1/2N/-P 1 1 3 2 23.0 .04733 .03156 .10 .4733 21 0 133 1/2N-P 2 4 5 .06310 .07888 .109 .5789 3 3 4 .04733 .06310 .09 .5258 4 3 4 .04733 .06310 .06 .7888 5 3 2 .04733 .03156 .14 . 3370 .5407 1684 7.3 1.9 +2N-P 1 1 3 3 23.5 .04733 .04733 .131 .3613 84 0 133 2N-P 2 3 6 5 3 2 .09467 .07888 .04733 .03156 .22 .14 .4303 .3380 4 3 2 .04733 .03156 .076 .6227 .4377 1938 5.8 1.7 Leaf Mg Soln, /lOOOcc Plant ppm SET REP ppm co 2 TEMP Mg , C02/hr. Area dm2 CO2 / hr. 1 dm2 Average Mg D. Wt. Mg xn xp xn xp Flowr/'^ /^Min. -N-P 1 1 3 3 21.5 .04733 .04733 .07 .6761 0 0 133 -N-P 2 3 4 4 3 2 2 6 5 .06310 .04733 .03156 .03156 .09467 .07888 .16 .13 .3943 .7282 5 4 6 .06310 .09467 .15 .4206 .5139 1995 5.9 1.3 +1/2N, +1/21, 1 1 1 3 3 22.0 .04733 .04733 .13 .3641 21 17.5 133 1/2N 1/2P 2 3 4 3 3 6 6 6 6 .04733 .04733 .09467 . 09467 .09467 .09467 .08 .19 .20 .5916 .4982 .4733 5 5 5 .07888 .07888 .17 .4640 .4782 2457 4.9 1.5 +2N, +2P I 1 1 6 5 22.0 .09467 .07888 .16 .5916 84 70 133 J2N 2P 2 3 6 5 5 6 . 09467 .07888 .07888 .09467 .16 .16 .5916 .4930 4 8 7 .1260 .1104 .20 .6300 .5765 2111 5.4 4.4 APPENDIX TADLE 3. ANALYSIS OF STOMATAL INDEX (I = x 100) Variety Treat-ment Leaf Side No, Stomata S Ho.. E Epid Cells S+E I Variet) Treat-ment Leaf Side No. S (Stomata] No. -E Epid Cells S -i- E I 1 Olympic N+P Upper 2 8 10 20.0 Jade N+P Uppei 3 6 9 33. 3 2 3 8 11 27..3 1 7 8 12..5 3 3 8 11 27..3 3 7 10 30.0 4 1 7 8 12.5 1 7 8 12.5 5 4 7 11 36.4 1 6 7 14..2 6 2 8 10 20,0 2 7 9 22.2 7 1 8 9 11.1 2 8 10 20..0 8 3 8 11 27..3 3 8 11 27..3 9 3 9 12 25..0 2 7 9 22.2 10 3 6 9 33.3 2 6 8 25..0 11 2 7 9 22.2 2 5 7 28.4 12 1 7 8 12.5 2 7 9 22.2 13 2 10 12 16.-6. 1 6 7 14.-2 14 1 8 9 11.1 2 6 8 25.-0 15 1 9 10 10.0 2 7 9 22.2 16 1 9 10 10.0 1 6 7 14.2 17 1 10 11 9.1 2 7 9 22.-2 18 2 13 15 13.-2 3 7 10 30.0 19 1 9 10 10.0 4 10 14 28.4 20 1 11 12 8.33 1 6 7 14.-2 21 1 9 10 10.0 3 7 10 30.0 22 1 7 8 12.5 1 5 6 16. -6 23 1 7 8 12.5 2 7 9 22.2 24 1 6 7 14.2 2 7 9 22.-2 25 1 6 7 14.2 3 7 10 30.-0 26 2 6 8 25.0 2 6 8 25.0 27 1 6 7 14. .2 1 7 8 12.5 28 1 6 7 14.2 4 8 12 33.-3 29 1 4 5 20.0 2 8 10 20.0 30 1 5 6 16.6 1 9 10 10.0 31 1 6 7 14.2 1 7 8 12.5 OLYMPIC (AVERAGE) JADE (AVERAGE) Standard DORSAL Stomatal Index 17.1 Standard DORSAL Stomatal Index 21.7 - Variety Treat-ment Leaf Side No. S (Stomata) No. E (Epid Cells) S + E - I Variety Treat? merit Leaf Side No. S Stomata) No. E (Epid Cells) S + E I 1 Olympic P-N Uppei 1 5 6 16.6 Jade P-N - Uppe : 3 6 ' 9 33.3 2 '" 1 '• ; 5 6 16. 6 3 8 11 27.3 3 3 6 9 33.3 2 6 8 25.0 4 2 8 10 20.0 3 7 10 30.0 5 1 6 7 14.2 2 7 9 27.2 6 1 6 7 14.2 3 6 9 33.3 7 2 5 7 28.4 1 7 8 12.5 8 3 8 11 27.3 3 7 10 30.0 9 2 6 18 25.0 2 8 10 20.0 10 3 8 11 27.3 3 8 11 27.3 11 1 7 8 12.5 1 6 7 14.2 12 2 7 9 22.2 2 7 9 22.2 13 2 8 10 20.0 1 6 7 14.2 14 2 6 8 25.0 2 7 9 22.2 15 2 7 9 22.2 4 9 13 30.4 16 2 5 7 28.4 3 9 12 25.0 17 1 6 7 14.2 2 6i. 8 25.0 18 1 6 7 14.2 2 6 8 25.0 19 2 6 8 25.0 2 7 9 22.2 20 2 7 9 22.2 4 7 11 39.6 21 3 7 10 30.0 3 6 9 33. 3 22 2 6 8 25.0 1 7 8 12.5 23 2 9 11 18. 2 1 7 8 12.5 24 1 6 7 14.2 2 5 7 28.4 25 2 6 8 25.0 2 6 8 25.0 26 2 6 8 25.0 1 5 6 16..6 27 2 8 10 30.0 2 5 7 28.4 28 2 7 9 22. 2 3 9 12 25.0 29 2 6 8 25.0 3 7 10 30.0 30 2 8 10 20/0 3 6 9 33.3 OLYMPIC (AVERAGE JADE (AVERAGE) Stomatal Index (P-N) 22.1 Stomatal Index (P-N) 25.4 Variety Treat-ment Leaf side No, S (Stomata) No.. E (Epid . Cells) S + E i Variety Treat-ment Leaf Side No. S. (Stomata) No. E (Epid Cells) S + E I 1 Olympic N-P Uppei 4 17 21 19.9 Jade N-P Upper 2 8 10 20 . 2 3 12 15 20.0 2 9 11 18.2 3 3 13 16 18,7 2 10 12 16.6 4 2 13 15 13,2 2 9 11 18.2 5 . 6 16 22 27,3 1 9 10 16.0 6 5 15 20 25,0 2 7 9 22.2 7 5 13 18 27,5 1 9 10 10.0 8 4 12 16 25.0 3 6 9 33. 3 9 4 14 18 22.2 1 6 7 14.2 10 4 14 18 22, 2 4 6 10 40.0 11 4 12 16 25.0 2 7 9 22.2 12 4 14 18 22.2 1 6 7 14.2 13 3 14 17 17.4 3 8 11 27.3 14 4 14 18 22,2 3 9 12 25.0 15 3 15 18 16,5 1 8 9 11.1 16 4 13 17 23,2 3 9 12 25.0 17 2 13 15 13,2 1 8 9 11.1 18 2 13 15 13.2 2 18 loi-.; 20.0 19 5 15 20 25.0 4 8 12 33.3 20 3 12 15 20.0 3 7 10 30.0 21 4 15 19 20.8 4 7 11 36.4 22 4 16 18 11.1 2 7 9 22.2 23 2 16 18 11.1 4 7 11 36.4 24 4 15 19 20.8 2 7 9 22.2 25 2 8 10 20.0 3 7 10 30.0 26 1 5 6 16.6 2 6 8 25.0 27 2 6 8 25.0 4 9 13 30.4 28 2 7 9 22.2 1 7 8 12.5 29 2 6 8 25.0 1 6 7 14.2 30 2 9 ... 11 18.2 3 7 10 30.0 OLYMPIC (AVERAGE) JADE (AVERAGE) Stomatal Index (N-P) 20, 6 Stomatal Index (N-P) 22.7 Variety Treat-; ment Leaf Side No, S (Stomata) No. E (Epid Cells) S + E I Variety Treat-ment Leaf Side No. S (Stomata No. E (Epid Cells) S + E I 1 Olympic N+P Lowei 1 3 4 25.0 Jade N+P Lower 1 6 7 14.2 2 1 4 5 20. 0 3 5 8 37.5 3 1 4 5 20.0 3 8 11 27'i 3 4 2 5 7 28.4 1 6 7 14.2 5 1 5 6 16.6 3 7 10 30.0 6 1 6 7 14.2 2 5 7 28.4 7 1 5 6 16.6 2 6 8 25.0 ' 8 1 4 5 20,0 2 6 8 25. 0 9 2 4 6 33.3 2 5 .7 28,4 10 2 4 6 33.3 3 7 10 30.0 11 1 3 4 25.0 1 5 6 16.6 12 1 4 5 20.0 3 6 9 33. 3 13 2 4 6 33. 3 1 5 6 16.6 14 2 8 10 20'. 0 3 7 10 30.0 15 1 4 5 20.0 2 5 7 28.4 16 1 4 5 20. 0 2 6 8 25.0 17 1 4 5 20.0 1 8 9 11.1 18 2 4 6 33.3 3 8 11 27. 3 19 1 4 5 20.0 2 7 9 22. 2 20 1 4 5 20.0 3 7 10 30.0 21 1 4 5 20. 0 1 6 7 14. 2 22 2 3 5 40.0 3 6 9 33.3 23 1 3 4 25.0 2 5 7 28.4 24 1 4 5 20. 0 2 7 9 22.2 25 1 3 4 25.0 2 6 8 25.0 26 1 5 6 16.6 1 6 7 14.2 27 1 3 4 25.0 3 7 10 30.0 28 1 3 4 25.0 2 6 8 25.0 29 1 3 4 25.0 1 7 8 12.5 30 1 1 3 4 25.0 1 5 6 16.6 OLYMPIC (AVERAGE). Standard VENTRAL Stomatal Index 23.5 JADE (AVERAGE) Standard VENTRAL Stomatal Index 24.0 Variety Treat-ment" Leaf Side No. S (Stomata) No. E (Epid • Cells) S + E I Variety Treat-m ent Leaf Side No. S (Stomata) No. E (Epid Cells) S + E I 1 Olympic P-N Lower 2 5 7 28.4 Jade P-N Lower 2 6 8 25.0 2 1 5 6 16i6 2 4 6 33.3 3 3 4 7 42.6 3 6 9 33.3 4 2 3 5 40.0 3 3 6 50/0 5 2 4 6 33.3 3 5 8 37.5 6 1 5 6 16. 6 3 4 7 42.6 7 1 5 6 16.6 2 5 7 28.4 8 1 3 4 25.0 3 7 10 30.0 9 2 3 5 40.0 2 4 6 16. 6 10 1 3 4 25.0 1 2 3 33.3 11 1 5 6 16.6 1 2 3 33.3 12 3 4 7 42.6 1 3 4 25.0 13 2 5 7 28.4 2 4 6 33.3 14 1 4 5 20.0 2 2 4 50.0 15 1 4 5 20.0 3 4 7 42.6 16 1 5 6 16.6 2 4 6 33.3 17 1 4 5 20.0 2 7 9 22.2 18 2 5 7 28.4 2 6 8 25.0 19 1 5 6 16.6 4 :'.8 10 •. 40.0 20 2 3 5 40.0 3 4 7 42.6 21 1 5 6 16.6 2 4 6 33. 3 22 2 4 6 33.3 1 4 5 20.0 23 1 4 5 20.0 3 6 9 33.3 24 1 4 5 20.0 4 6 10 40.0 25 1 4 5 20.0 2 4 6 33.3 26 2 4 6 33.3 3 3 6 50.0 27 2 6 8 25.0 2 1 3 66.6 28 3 4 7 42.6 1 3 4 25.0 29 3 7 10 30.0 2 4 6 33.3 OLUMPIC (AVERAGE)) JADE (AVERAGE) Stomatal Index (P-N) 26.6 Stomatal Index (P-N) 34.9 Variety Treat ment Leaf Side No. S [Stomata) No. E (Epid Cells) S + E I Variety Treat-ment Leaf Side No. S [Stomata) No. E (Epid Cells S + E I 1 Olympic N-P Lower 1 5 6 16.6 Jade N-P Lower 3 5 8 37.5 2 3 6 9 33.3 i:.- 4 5 20.0 3 3 7 10 30.0 2 6 8 25.0 4 2 4 6 33.3 1 3 4 25.0 5 1 4 5 20.0 3 4 7 42.6 6 1 4 5 20.0 2 5 7 28.4 7 4.-. 7 i i 36.3 - 1 4 5 20.0 8 1 4 5 20.0 1 5 6 16.6 9 2 5 7 28.4 2 6 8 25.0 10 1 4 5 20.0 2 5 7 28.4 11 1 3 4 25.0 1 4 5 20.0 12 1 3 4 25.0 2 3 5 40.0 13 1 2 3 33.3 1 3 4 25.0 14 1 3 4 25.0 1 4 5 , 20.0 15 2 4 6 33.3 1 3 4 25.0' 16 1 3 4 25.0 1 2 3 33.3 17 2 4 6 33.3 2 3 5 40.0 18 3 5 8 37.5 1 4 5 20.0 19 1 3 4 25.0 2 5 7 28.4 20 1 3 4 25.0 1 3 4 25.0 21 1 4 5 20.0 1 4 5 20.0 22 1 4 5 20.0 1 3 4 25.0 23 2 4 6 33.3 2 6 8 25.0 24 3 3 6 50.0 1 4 5 20.0 25 4 4 8 50.0 2 4 6 33.3 26 1 5 6 16.6 2 4 6 33.'3 27 1 2 3 33,3 1 3 4 25.0 28 2 3 5 40.0 2 3 5 40.0 29 2 3 5 40.0 2 4 6 33.3 30 1 3 4 25.0 - -J 2 3 5 40.0 OLYMPIC (AVERAGE) JADE (AVERAGE) Stomatal Index (N-P) 29,1 Stomatal Index (N-P) 28.0 

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