IV1£3I_J RETURNING MATERIALS: P1ace in book drop to “mamas remove this checkout from " your record. FINES win be charged if book is returned after the date stamped be1ow. GAS EXCHANGE CHARACTERISTICS OF BLACK LOCUST (ROBINIA PSEUDOACACIA L.) IN RELATION TO GROWTH, DEVELOPMENT, AND ENVIRONMENTAL FACTORS BY Tesfai Mebrahtu A DISSERTATION submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Forestry 1989 ABSTRACT GAS EXCHANGE CHARACTERISTICS OF BLACK LOCUST (ROBINIA PSEUDOACACIA L.) IN RELATION TO GROWTH, DEVELOPMENT, AND ENVIRONMENTAL FACTORS BY Tesfai Mebrahtu The responses of gas exchange, growth, and leaf traits of black locust to different external and internal plant factors were studied under controlled environment, greenhouse, and field conditions. P; of black locust was influenced by light intensity, temperature, CO2 concentration, leaf age, time of day, and moisture level, but not by nitrogen treatment. There were significant differences in P; among families in all experiments, but there were no significant correlations between Pn per unit leaf area and growth. The lack of correlation between growth and.F% per unit leaf area was largely due to variations in leaf area. Chl contents and SLW were not significantly correlated with PW.I% in black locust is limited strongly by G“I and to a lesser degree by Gs. I; reaches maximum levels early in the day. Under well watered conditions, the diurnal response of‘Pg was regulated Tesfai Mebrahtu by accumulation of carbohydrates. Under water stress conditions, the P; of black locust declines continuously due to limited water supply in the pots. Black locust maintained relatively higher'F% under water stress compared with no stress at TL exceeding 35°C and a VPD of 5.6 kPa. Pn increased with leaf age, reaching a maximum at about 16—20 days after leaf emergence, and then declined at different rates in different families. The patterns of P5 in relation to leaf age are controlled by factors affecting Gm. I% increased with increasing light intensity and did not saturate at 1900 umol.mi.sq. Pg also increased with external crx concentrations, but the increase was slower from 350 to 925 ppm. The temperature response curves of Pn were typical of C3 temperate plants. The decline in Pn at TL above optimum was attributed to decrease in internal photosynthetic processes, and not to stomatal closure. Different nitrogen treatments did not affect gas exchange, growth, or leaf traits which indicates that black locust can fix sufficient atmospheric nitrogen to support its metabolic requirements. Different water treatments did not result in differences in growth and leaf traits, but gas exchange traits were significantely different among the water treatments in greenhouse grown plants. DEDICATION This dissertation is dedicated to my friend, Kahsu Girmay and my father, Mebrahtu Negussie. ii ACKNOWLEDGMENTS I am grateful to my major adviser, Dr. James W. Hanover for giving me the opportunity, material, and encouragement when ever I needed it. He always made me feel, he has confidence in what I do. I would like to thank my guidance committee members, Dr. Thomas G. Isleib, Dr. Daniel E. Keathley, and Dr. Kurt S. Pregitzer . Thanks is also due to Dr. James A. Flore, Dr. Donald Dickmann, and Dr. Phu Nguyen for letting me use their equipments. Last but not least, I would like to thank Sue Plesko for helping me with word processing and Desmond Layne for helping do measurements. iii TABLE OF CONTENTS LIST OF TABLES ................................... LIST OF FIGURES ........ ........... LIST OF ABBREVATIONS AND SYMBOLS . INTRODUCTION ..................................... CHAPTER 1. GAS EXCHANGE RESPONSES OF BLACK LOCUST FAMILIES TO DIFFERENT LEVELS OF LIGHT, TEMPERATURE, AND C02............ Abstract ..................................... Inrroduction Materials and Methods ....... Results .... Discussion . References ................................... CHAPTER 2. EFFECT OF LEAF TEMPERATURE ON DARK RESPIRATION AND PHOTORESPIRATION OF BLACK LOCUST ........ .......................... Abstract ................................ ..... Introduction ....... ......... .... ..... ........ Materials and Methods .. ..... ...... ..... ...... Results ............ ........... . ....... ....... Discussion ............................. ...... References ............ .. ..... ......... ...... Page vi ix xii 36 37 38 39 39 46 51 CHAPTER 3: VARIATIONS AND RELATIONSHIPS IN GAS EXCHNGE, GROWTH, AND LEAF TRAITS OF HALF-SIB FAMILIES OF BLACK LOCUST. ...... Abstract .. Introduction Materials and Methods Results and Discussion ...... References iv 55 56 57 58 61 72 CHAPTER 4. DIURNAL RESPONSE OF GAS EXCHANGE OF BLACK LOCUST (ROBINIA PSEUDOACACIA L.). ...... 76 Abstract .. ....... ........ ..... ... ............ 77 Introduction ..... ............... ............. 78 Materials and Methods ........ ............ .... 79 Results . ....... ............. ........... ...... 81 Discussion . ............. . .............. ...... 99 References ............ ........ ............. .. 108 CHAPTER 5. EFFECT OF LEAF AGE ON GAS EXCHANGE OF BLACK LOCUST. ................................ 111 Abstract .. ..... ... ..... .......... ........ .... 112 Introduction ..... ......... ...... .......... ... 113 Materials ans Methods ....... ... .......... .... 113 Results ... ..... . ........... .................. 115 Discussion .............................. ..... 118 References ................................... 121 CHAPTER 6. PHOTOSYNTHESIS AND GROWTH OF BLACK LOCUST FAMILIES UNDER WATER STRESS CONDITIONS. 123 Abstract ............................ ..... .... 124 Introduction ........ . .......... . ........... .. 125 Materials and Methods ..... ................... 126 Results .. ...... ..... ........ ................. 129 Discussion ...... .............. ...... ......... 135 References .......... ................... ...... 143 CHAPTER 7. EFFECTS OF NITROGEN LEVEL ON GAS EXCHANGE, GROWTH, AND LEAF TRAITS OF BLACK LOCUST. O.......OOOOOOOOOOOOOOO000...... 146 Abstract ............. ........ ....... ..... .... 147 Introduction ................................. 148 Materials and Methods ........................ 148 Results ...................................... 150 Discussion . ................. ... .......... .... 157 References ................................... 163 CONCLUSIONS AND RECOMMENDATIONS ..... ...... ..... .... 166 LIST OF TABLES Chapter 2 1. Correlation coefficient values among Pr, true Pn, Gs, Gm, and T at 1.8 02. ...... Chapter 3 1. Mean values for gas exchange traits on three measurement days. ..................... 2. Correlations among the gas exchange traits. 3. Correlation coefficients among growth and leaf traits Pn' oooooooooooooo o ........ 0.000.... 4. Mean values for growth and leaf traits at week 15. ......... ................. ....... Chapter 6 1. Mean Pn and GS of black locust half-sib families grown under different water treatments in a greenhouse. .............................. 2. Mean Pu and GS of black locust half-sib families grown under different water treatments inthe field. O......OOOOOOOIOOOO0.0.0.0000... 3. Mean values for growth traits of black locust half-sib grown under different water treatments inagreenhouse. 00......OOOOOOOOOOIOOOO0...... 4. Mean Values for growth traits of black locust half-sib families grown under different water treatments in a greenhouse.The values are averages of 12 seedlings. ................... vi Page 45 64 65 66 67 131 131 132 132 Mean values for growth traits of black locust half-sib families grown under differnt water treatments in the field. .................... Mean values for growth traits of black locust half-sib families grown under different water treatments in the field. The values are averages of 12 seedlings. ................... Mean values for leaf traits of black locust half-sib families grown under different water treatments in a greenhouse. The values are averages of 12 seedlings. ....... ............ Chapter 7 1. Mean Pu and GS of black locust half-sib families grown under three nitrogen treatments in a greenhouse. ........ ......................... Mean PD and Gs of black locust half-sib families grown under three nitrogen treatments in a greenhouse. The values are averages of 15 seedlings. ......... ......................... Mean Pn and GS of black locust half-sib families grown under three nitrogen treatments in the field. The values are averages of 15 seedlings. Mean values for growth traits of black locust half-sib families grown under three nitrogen treatments in a greenhouse. ................. Mean values for growth traits of black locust half-sib families grown under three nitrogen treatments in a greenhouse. The values are averages of 15 seedlings. ......... .......... Mean values for growth traits of black locust half-sib families grown under three notrogen treatments in the field. .................... Mean values for growth traits of black locust half-sib families grown under three nitrogen treatments in the field. The values are averages of 15 seedlings. ................... Mean values for leaf traits of black locust half-sib families grown under three nitrogen treatments in a greenhouse. ................. vii 133 133 134 151 151 152 152 153 153 154 154 Mean values for leaf traits of black locust half-sib families grown under three nitrogen treatments in a greenhouse. The values are averages of 15 seedlings. .................. 155 viii LIST OF FIGURES Chapter 1 1. Effect of PAR on Pn,<%, and Galof greenhouse grown black locust half-sib families measured at 25°Cfmdand 350 ppm C02.The values are averages of four seedlings. .................. Effect of TL on Pn, Gm, and T of greenhouse grown black locyst half-sib families measured at 1400 umol.m' .s'iDAR and 350 ppm C02. The values are averages of four seedlings. ....... Effect of C02 on P“, Gs, and Gm of greenhouse grown hflf-sib families measured at 1000 umol.m' .s'iDAR and 30°C TL. The values are averages of four seedlings. ................. Effect of PAR on Pn,(%, and Gnlof field grown black locust half-sib families measured at 25°C inland 350 ppm C02.The values are averages of four seedlings. ..... ........................ Effect of TL on Pn, G5, and Gm of field grown black cu t half-sib families measured at 1400 umol.m' .5“ AR and 350 ppm C02. The values are averages of four seedlings. ................. Chapter 2 1. Effect of TL on Rd, % reduction of Pn by Rd, and Gsin the dark of field grown black locust half-sib families measured at 350 ppm C02.The values are averages of four seedlings. ...... ix Page 13 15 17 19 21 4O Effect of TL on Pr, true P", and Pa of field grown black logyst half-sib families measured at 1900 umol.nf .s'PAR. The values are averages of four plants. .................... Effect of TL on % reduction of Pn by P, and photosynthesis of field grown black locust half—sib families. The photosynthesis values were calculated after removing the effects of Rd and Pr. The values are averages of four seedlings. .... .......... .................... Chapter 4 1. Diurnal patterns of P“, Gm ,and Gs of greenhouse grown black locust half-sib families measured inside a greenhouse. The values are averages of two plants. .......... Diurnal patterns of T, PAR, VPD, and TL of greenhouse grown black locust seedlings measured inside a greenhouse. The values are averages of two plants. ......... ...... ...... Diurnal patterns of Pn,(%, and (Gad of greenhouse grown black locust seedlings measured outdoors. The values are averages of two plants. .............................. Diurnal patterns of T, PAR, VPD, and TL of greenhouse grown black locust seedlings measured outdoors. The values are averages of two plants. .............................. Diurnal Patterns of Pn,(%, and Gn,of greenhouse grown black locust seedlings measured outdoors? T e plants were kept under 700 umol.nf .s’ AR overnight. The values are averages of two plants. .......... Diurnal patterns of T, PAR, VPD, and TL of greenhouse grown black locust seedlings measured oufdo rs. The plants were kept under 700 umol.nf .s' AR overnight. The values are averages of two plants. ...................... Diurnal patterns of P“, Gs, and Gm of greenhouse grown black locust seedlings measured outdoors. The plants were not watered for two days before measurement. The values are averages of two plants. ...................... 43 44 84 85 86 87 88 89 9O 10. 11. Diurnal patterns of T, PAR, VPD, and TL of greenhouse grown black locust seedlings measured outdoors. The plants were not watered for two days before measurement. The values are averages of two plants. ...................... Diurnal patterns of Pa, Gs , PAR, and TL of field grown black locust seedlings measured on September 24, 1988. The values are averages of three plants. ............................... Diurnal patterns of P“, Gs, PAR, and TL of field grown black locust seedlings measured on October 14, 1988. The values are averages of three plants. ...... . ....... ................. Diurnal patterns of P“, Gs, PAR, and TL of field grown black locust seedlings measured on October 15, 1988. The values are averages of three plants. .................... ........ Chapter 5 1. Effect of leaf age on Pn,(%, and (Gun, of black locust seedlings measured on J ne 11 1988 at a TL of 29°C and 1700 umol.m' .s'PAR. The values are averages of two plants. ...... xi 91 95 96 97 117 ATP Chl RuBiSCO SLW T TL Tr VPD LIST OF ABBREVATIONS AND SYMBOLS adenosine triphosphate chlorophyll intercellular Cchoncentration mesophyll conductance stomatal conductance photosynthetically active radiation net photosynthetic rate photorespiration dark respiration ribulose bisphosphate carboxylase/oxygenase specific leaf weight transpiration rate leaf temperature transpiration ratio vapor pressure difference xii INTRODUCTION Black locust (Robinia pseudoacacia L.) is a tree species native to the Appalachian Mountains and the Ozark Plateau of the USA. Now it is naturalized in almost all states in the USA and Canada, and extensive black locust plantations are found in South Korea, China, Hungary, and other countries in eastern and western Europe (Keresteszi, 1988). Black locust is a drought tolerant leguminous species that fixes atmospheric nitrogen and a species with a potential to be used for multiple purposes including lumber, poles, bee forage, wood fiber, chemicals, and animal feed. Due to these and other desirable charactristics, black locust is the second most widely planted species in the world. It is easily propagated by tissue culture (Han and Keathley, 1988), root cuttings (Prentice, 1987), stem cuttings (Puri e; a;., 1988), root sprouts, coppicing and from seed (Keresteszi, 1988). Selection and breeding through progeny testing have been the basis for the improvement of forest trees. Half-sib progenies of over 400 trees of black locust collected from a wide range were tested for several traits including height growth at 2 sites in southern Michigan (Mebrahtu and 2 Hanover, in press). As in any other tree species not subjected to selective breeding, the genetic variations for all traits were extensive. This was the result of data collected after one growing season. Even though preliminary investigation of data from the second growing season showed similar results to the data from the first growing season, the extent of the variation after 5 or more growing seasons awaits evaluation. Because of the long generation time of trees, selection for growth characteristics of mature trees have been usually made on juvenile trees. This approach has not always been successful so far (Zobel and Talbert, 1984), probably due to low correlation for growth traits at juvenile and mature stages. One alternative method for early evaluation that has been studied for the last 20 years or more is the use of photosynthesis as a screening method to detect genotypes with maximal growth. The dry weight production of a plant is a function of its Pn during light conditions and Rd during dark conditions. Therefore, it seems logical to use photosynthesis as a selection criterion in breeding programs. However, the relationships between.F; and growth have been variable in those species studied so far: positive, negative, or no relationships were reported. The conclusions from these studies were reached by doing photosynthetic measurements one or more times at given environmental conditions and doing correlations with growth. The problem with this approach is that growth is the product 3 of various processes occurring over months or years, while the photosynthetic measurements reflect the potential of a plant at a given time. Photosynthesis is a complex process affected by internal and external factors such as light, temperature, moisture, nutrition, genetics, and age (Salisbury and Ross, 1985). Due to changes in the external and internal factors affecting photosynthesis during a day, season, and developmental age, the photosynthetic rates of plants change diurnally, seasonally, and with plant age. That is why single measurements of photosynthesis at a given set of conditions usually do not reflect the growth of individual plants. This study was necessary for at least two reasons: first, there is no literature on the photosynthetic properties of black locust. The information on the photosynthetic capacity of a species, and major factors that affect it,is vital for the culture and management of a species. Second, the old question of the relationship between photosynthesis and growth had to be tested for black locust since different species may have different life strategies. Even if there is no apparent relationship between photosynthesis and growth, it is important to understand the causes of the lack of that relationship. The main objective of the study was to determine the factors that limit.F% under field growing conditions in Michigan, and how that information can be used to improve the growth 4 of black locust. The study attempted to answer the following questions: a) Is the fast growth of black locust in the field due to higher PR? b) How do external and internal plant factors affect Pn in black locust? c) What is the diurnal pattern of P3 in black locust? d) What limits P; of black locust? e) Is there any possibility of using gas exchange as selection criteria or in any other way to facilitate black locust breeding programs? The entire study was done on half-sib families evaluated for growth performance earlier (Mebrahtu and Hanover, in press). The decision to study half-sib families was not to estimate genetic parameters such as heritability or genetic gains. Large numbers of families and individual trees are required to do any genetic analysis. Half-sib families of known growth potential were used for the following reasons: (1) since the growth perfrmance of these families has already been tested, it is possible to study if the the variation in growth in the field is due to variations in P"; (2) since the families are genetically different, it is possible to study if there are inherent differences in gas exchange associated with their genetic variation; and (3) since the families are collected from a broader range, it is possible to study variation in.Pg and related gas exchange traits associated with adaptation to different environments. The P" of plants grown in controlled environments is different from field grown plants. Therefore, some of the experiments were done in both field and greenhouse grown plants. The First chapter is on the effects of PAR, TR, and C02cn1£%. Light and temperature are two of the major environmental factors mediating life processes in plants, and the response of black locust to these factors had to be tested to determine the PAR and TL under which the other studies should be done. The second chapter deals with Rdanxi I’ the processes by which plants lose energy. The ,: relationship betweeniawand.growth of black locust is discussed in chapter 3. Variations in diurnal response and leaf age are some of the sources for the lack of relationship between growth and.Fg. The effect of these factors is discussed in chapters 4 & 5. The last two chapters deal with the effect of watering and nitrogen fertilization on gas exchange, growth, and leaf traits of black locust. CHAPTER 1 GAS EXCHANGE RESPONSES OF BLACK LOCUST FAMILIES TO DIFFERENT LEVELS OF LIGHT, TEMPERATURE, AND C02 ABSTRACT The response of Enand related gas exchange traits to temperature, light, and Cszere studied using greenhouse and field grown black locust seedlings. The saturation light intensity forin1was higher than for most woody plants. The response to temperature ofimqof greenhouse grown plants was similar to other temperate woody plants with the optimum temperature between 200 and 25°C. The optimum temperature for Pn shifted to lower TL in field grown plants. Pn of black locust increased rapidly with increased C02 up to 350 ppm and gradually increased to the highest concentration (925ppm). The increase injmqwith increased light and C02 levels was due to increased ATP production and carboxylation, respectively. The response of PntxJ temperature was not regulated by diffussion processes. Both Gs and Gm were not highly correlated with Pa in greenhouse grown plants. The response oflmqto temperature could have been the result of direct temperature effects on electron transport, carboxylation, and mitochondrial respiration. Pn was significantly different among the half-sib families in the PAR and TL experiments, while there were no significant family x PAR, family X C02, and family X TL interactions. INTRODUCTION The temperature, light, and COzresponse curves for Pn have been studied for some tree species (Ledig and Clark, 1977; Lawrence and Oechel, 1983; Jurik gt g;., 1984). Such studies provide preliminary information on the ecological requirements of a species and the productivity potential of that species at different site conditions. Most important of all, the characterization of the gas exchange responses of a species to different levels of light, temperature, and 002 must be done before starting studies on treatment effects on gas exchange (eg. genetic variability, diurnal response). Measurements of gas exchange characteristics have not been done for black locust. Temperature affects the three partial processes of photosynthesis: (1) electron transport and photophosphorylation (Stidham gt gt., 1982); (2) diffusion of COzfrom outside the plant to the site of carboxylation (Moon gt g;., 1987); and (3) COzfixation by the photosynthetic carbon reductive cycle (Monson gt gl., 1982). Higher temperatures were implicated in midday depression of photosynthetic rate (Schulze gt gt., 1974) and freezing temperatures decrease photosynthetic rates (DeLucia and 9 Smith, 1987). The amount of light absorbed by the leaves drives the electron transport system while the short wave length blue light is believed to be involved in stomatal opening (Salisbury and Ross, 1985). Of course, COzis the rate limiting element in the photosynthetic carbon reduction cycle, and much interest in the effect of C02 on Pn has resulted from the predicted rise in atmospheric C02 content in the future (Jurik gt. gt., 1984). This phenomenon is especially important in forestry where the rotation age of many tree species exceeds 50 years. The main goals of this study were: (1) to characterize the response ofimqof black locust to a wide range of light, temperature, and COzlevels; (2) to compare the response patterns of different families with contrasting growth habits; (3) to evaluate the relationships among the different gas exchange traits; and (4) to compare the response curves of greenhouse and field grown plants. MARTERIALS AND METHODS Plant material A. Greenhouse Seedlings from 9 half-sib families selected for contrasting growth performance were grown from seed in 7.4 litre pots in a greenhouse. The potting mix was peat moss, perlite, and vermiculite (3:1:1). The seedlings were watered daily and fertilized with Peters slow release fertilizer. The greenhouse was maintained at 27°C and 17°C day and night 10 temperatures,respectively. The plants were grown for 9 weeks under a 20 hr photoperiod before the first measurement. B. Field Seed from nine half-sib families with different growth performances in a progeny test (Mebrahtu and Hanover, in press) were sown in small pots on June 23, 1988 in a greenhouse. The seeds germinated in 5 to 7 days and were transplanted to 7.4 litre pots with a potting mix of peat moss, vermiculite, and perlite (3:1:1). The seedlings were allowed to grow in the greenhouse for about 2 weeks. After 2 weeks, the seedlings were transferred outdoors to grow under natural conditions until the measurement time. Both macro- and micronutrients were added at the time the plants were being moved outside. The July and August growing conditions were very harsh with temperatures from 3d°to 35°C. Plants had to be watered more than once a day. Temperatures suddenly dropped in September and night temperatures as low as 4°C were observed. Gas exchange Measurements of COzand water vapor exchange were performed using an open gas exchange system described by Sams and Flore (1982). Four plants from each family were used for each experiment (light, temperature, and C02) . Each morning before measurement, the 9 week old seedlings were watered and transported from the greenhouse in a van to the 11 laboratory where the gas exchange system is housed. Since I was able to do measurements on 8 plants in one day, two families were measured in a single day. Two plants each from two families were measured in the morning hours and the remaining two plants from each family were measured in the afternoon. This was necessary because of possible diurnal variations, even though the measurements were done under controlled environment conditions. Leaflets ranging in number from 7 to 13 from fully matured attached leaves were enclosed in the leaf chamber during measurement. Following each measurement, leaflets were excised and leaf areas were determined using a portable leaf area meter (LI-3000, LiCor Inc.). Light response was determined at a TL of 25°C and ambient C02 concentration of 350 ppm. The air temperature of the chamber was 2§%2with 50% relative humidity. The light intensities and their sequences in the gas exchange measurements were: 1900, 1400, 1000, 550, 400, 150, and 0 2.5'1The different light intensities except the dark umol.m' (0) were obtained by placing a combination of screens and plastics over the leaf chambers. The temperature response measurements were done at PAR of 1400 umol.m'2.s'5nd ambient C02 concentration of 350 ppm. The lowest TL was 10°C because of condensation problems in the gas lines. TL up to 40°C were achieved by varying the chamber temperature. ngwere increased progressively from the lowest temperature. No attempt was made to hold VPD 12 constant. The C02 response was determined at a TL of 30°C and 1000 umol.nf2.s'PAR. This PAR is not the saturating light intensity for black locust, but it was the maximum obtainable under the experimental condition. For C02 concentrations below 350 ppm, the C02 in the air flow system was scrubbed and a known amount added from a tank. To obtain the C02 concentrations above 350 ppm, known amounts of C02 were added from a tank to the ambient laboratory air (350 PPm)- Pn,(%,- Mm 408 'l (mmol.m'2.s'1) 20 ’ -— 1m 01 o l l l l L l -200 O 200 400 600 800 1000 1200 C02 CONC. (PPM) Figure 3. Effect of C02 on P", Gs, and CuI of greenhguse grown half-sib families measured at 1000 umol.m° .s PAR and 30°C TL. The values are averages of four seedlings. 18 B. FIELD GROWN PLANTS Response to light. F5 in all of the families increased with increasing PAR and did not saturate at 1900 umolmmgefeFigure 4). The slopes of the lines were steeper in some families than others at the upper PAR levels. Light compensation occurred at about 25 umol‘m'z's'PAR. Rd ranged from 5 to 14 percent of IF" were significantly different among Pn at 1900 umol'm'z's’ the families (.01 probability level), while family x PAR interaction was non-significant. (% increased with increasing PAR in all families, but the slopes were steeper in 2 families (Figure 4). The correlations between Pu and Gs were weak but significant at all PAR levels except the highest (1900) and the lowest (0). There was no significant genetic variation among the families (.05 probability level) and family x PAR interaction was non-significant. Gm followed the same pattern as Pn with increasing PAR (Figure 4) . The correlations between Pu and Gm were very high (r = .91 - .95) and significant (.01 probability level) at each PAR level except in the dark. There was significant genetic variation in G",(.01 probability level) among the families and the family x PAR interaction was non- significant. T increased with increasing PAR in all families (data not shown). Even in the dark, T was about one-third of the T 2 at 1900 umol'm' s'l.There was significant genetic variation l9 3O 7" 25 — u "-2 20 » ‘T' c e 15 » 9. '°. 10 ~ _ 0 U P. m E o -5 200 '0, 150 — o" '7'. ii 100 - 0-1 2. 5 50 "‘ o 100 —‘— FAM 208 80 - + FAM'808 TU) '* FAM 385 a -<> FAM 416 0-1 E 40 r- 20 *- 0 l l -500 o 500 1000 1500 2000 2500 PAR (umol.m'2.s'1) Figure 4. Effect of PAR on P", Gs, and Gm of field grown , black locust half-sib families measured at 25 C T and 350 ppm C02. The values are averages of four seed'lings. 20 among the families (.01 probability level) and family x PAR interaction was non-significant. The correlation between T and Pn was non-significant. The correlation between Gs and T was high and significant at PAR below 1000 umol'm'z's'l Correlation analyses were done for each PAR level. Cidecreased with increasing PAR and either remained constant or increased up to the highest PAR (data not shown). The correlation betweenifi1and Ciwas very weak, but Ciwas significantly correlated with Gs and T at all PAR levels. A significant variation was observed among the families and family x PAR interaction was non-significant. Response to temperature PM increased with increasing 11,and declined rapidly from the peak (Figure 5). The temperature optima for Pn were between 15 and 25°C.]filwas significantly different among the families (.01 probability level) and family x TL interaction was non-significant. One of the families (308) had a much higherlm1than the other families. Gs of 2 families showed a two-peaked response to TL, the first at 10°C and the second at 30°C (Figure 5) . Gs of the other 4 families increased with increased.11,and peaked at 30°C before they declined rapidly. The Gs values were very high and similar except for one family (408). The variation iJ1(%'was non-significant among the families (.05 probability level) and family x:T1,interaction was non- significant. Gs was not significantly correlated with P“ and 21 01 O b O I 0 O I M O I P" (mg C02. dm'2.hr") 8 O _ __ _ h— __ 300 PAM 208 PAM 308 ‘ PAM 385 250 — _,_ -l(- u) 20° ” -8- FAM 386 .94— 6 PAM 408 PAM 418 LEAF TEMPERATURE (DEGREE C) Figure 5. Effect of T on P, Gs, and G"I of field grown_ black locust half-sib families measuredm at 1400 umol. m .s PAR and 350 ppm C02. The values are averages of four seedlings. 22 weakly but significantly correlated with T. The maximum Gm was observed at 15°C TL except in one family which peaked at 20°C (Figure 5) . Gm declined from the peak gradually with increase TL. The variation in Gm among the families was significant (.01 probability level) and family x TL interaction was non-significant. The correlation between Pu and Gm were significant and high except at the two lowest TL (data not shown). T increased with increased TL up to 35°C in all families, but in 3 families T was higher at 10°C than at 15°C (data not shown). At 40°C TL, T was higher, lower or the same as T at 35°C TL. T was significantly different among the families (.01 probability level) and family x TL interaction was significant (.05 probability level). The correlations between Pu and T were non-significant except at the 10°C TL (data not shown). Ci decreased at 15°C TL and then increased to a peak at 25° to 30°C and then declined (data not shown). There was no significant difference among the families in Ci(.05 probability level) and family x TL interaction was significant (.01 probability level). The correlations with Pn were weak and non-significant (data not shown). DISCUSSION The Rd for black locust were similar to other tree species (Lawrence and Oechel, 1983) , but when Rd was expressed as percentage of P“, black locust had a very low 23 rate. Light saturation of’Ph_in‘most tree species occurs at PAR values between 400 and 1100 umol.uf2.s'(Lawrence and Oechel, 1983: Nelson and Ehlers, 1984; Koike, 1987). Higher light saturation levels similar to black locust in this study were reported only for lodgepole pine (Kramer and Decker, 1944) and horticultural crops such as cassava (Pereira gt _l., 1986), peanut and sunflower (Salisbury and Ross, 1985), and field grown cotton (Patterson gt gt., 1977). Even in cotton, light saturation of PnIIf chamber lAt optimum grown plants occurred at about 750 umol.nf2.s' temperatures and ambient COzcnncentrations, light saturation of Pn in C3 plants results from substrate limitations in carboxylation. No explanation is available from past studies as to why different C3species or plants reach light saturation ofimqat different PAR. Photosynthetic rates in plants with the C4 metabolism do not saturate even at full sun light due to their C02 concentrating mechanisms. Higher chl content was observed in both cassava (Pereira gt _;., 1986) and black locust (Chapter 3) while Ledig and Clark (1977) reported the doubling of the saturating light intensity of pitch pine (Pingg rigidg Mill.) by fertilization. Obviously, work towards understanding the mechanisms that lead to different light saturation PAR is needed. Higher Pn per unit leaf area for field grown plants than growth chamber or greenhouse grown plants was observed for many plants (Patterson gt g;., 1982; Nelson and Ehlers, 24 1984; McMillen and McCleod, 1983). In all studies, the difference in Pn between the field and controlled environment grown plants were minimized or removed when Pn was calculated per unit leaf fresh weight, leaf dry weight, or mesophyll volume. The difference in aner unit leaf area was attributed to higher SLW in field plants (Nelson and Ehlers, 1984), more carboxylating enzyme in field plants (Patterson gt gt., 1977), and more mesophyll area/leaf area (Nobel gt gt., 1975). These and other characteristics which are typical of sun and shade leaves are summarized by Givnish (1988). In my work with black locust, I was not able to observe obvious differences in Pa per unit leaf area as those described above between field greenhouse grown plants which were measured under identical conditions. Actually, the Pnretes in the greenhouse grown plants were slightly higher than the field grown plants. At least two factors are responsible for the higher’szrates in the greenhouse grown plants: 1) the field plants were measured at ages 3 to 4 weeks older than the greenhouse grown plants and there could be age related differences such as those observed by Ledig and Clark (1977); and 2) the field grown plants were measured at the fourth week of September, a month in which we had more cloudy days than sunny, with night temperatures below 10°C. Yet, SLW were higher in the field grown plants. Light saturation of’Fm in chamber grown cotton plants occurred at 750 umol'm'z's'vihile the light saturation of Pa of 25 field grown plants occurred at 1500 umol'm'z's'(Patterson gt 31., 1977). This upward shift in the saturation PAR of field grown plants was also observed in poplars (Nelson and Ehlers, 1984). The acclimation of plants to growth light intensity was demonstrated by Bjorkman gt gt. (1972). They grew Atriplgx gatula in growth chambers under three light 2's'l) .The light response intensities (92, 290, and 920 umolmf curve ofifinper unit leaf area of these plants showed a difference in the light saturation PAR. Pn saturated at a higher PAR in the plants grown at 920 micromol‘m'z's'aind at 2 1 lower PAR in those grown under 92 umol'm'z's'lthe 290 umol'm' ‘5' plants being intermediate. In the higher and intermediate PAR plants, the light saturation PAR was greater than the growth PAR. Differences in the light saturation PAR of these 3 groups of plants were also observed when the data of Bjorkman _e_t g1. (1972) were reanalyzed to produce Pn per unit leaf dry weight and Pn per unit soluble protein (Givnish, 1988). What causes the shift in the light saturation PAR of P5? Although conclusions can be reached from the typical morphological and anatomical differences between field grown and growth chamber or greenhouse grown plants, it is appropriate to use studies which observed the shift in the saturation PAR by applying some treatment other than growth PAR. Ledig and Clark (1977) reported the doubling of the light saturation PAR of lodgepole by increased fertilization. A higher light saturation was also observed in shoots from fertilized stands compared to the 26 control in Douglas-fir (Brix, 1971). Therefore, the higher light saturation PAR in field grown plants may be due to increased carboxylation which results from higher RuBiSCo and other soluble proteins (Givnish, 1988). In black locust, the slopes of the light response curves for PanI the field grown plants were steeper than those of the greenhouse grown plants. The light response for Gstr other species showed light saturations of Gsoccurred at less than 50 percent of full sunlight and seem to coincide with the light saturation of Pn(Ten.gt gt., 1977; Ku gt gt., 1977; Lapido gt gt., 1984). Leaf conductances in Engelmann spruce (Ptggg engelmannii Parry ex Engelm.), subalpine fir (Abies lasiocarpa/Hook./Nutt.), lodgepole pine (Piggg contorta var. latifolia Engelm.), and aspen (Populus tremuloides Michx.) increased with PAR up to full sunlight (Kaufmann, 1982). This observation is in agreement with the model of Givnish model (1986) which predicts an increase in Gs with PAR in conditions where water is not limiting. These species must have light saturation of Pn close to full sunlight, otherwise their water use efficiency will be low because of loss of water without accompanying gain in carbon. Gsin black locust increased with PAR like P“. The response of Pn to light could be limited by stomatal control of gas exchange (Jurik gt g;., 1984). Gs had the same pattern as Pa in response to PAR in Douglas fir (Meinzer 1984), larch (Anderson, 1982), and Schima superba 27 Gardn. and Champ. (Sun and Ehleringer, 1986). In this study, (% of black locust had different patterns and did not parallelimapatterns, suggesting a weaker relationship between Gsand Pn.The correlation analysis supports this conclusion. On the other hand,<3nhad the same patterns as PM in addition to the strong correlation between Gnland Pn indicating that.Fh is limited by an.Seemann gt gt. (1988) suggested that the rate of’Fmbin response to increased PAR is often limited by the rate at which the activity of RuBiSCO increases, which by itself is determined by the rate of activation of the catalytic sites and degradation of the RuBiSCO inhibitor, 2-carboxyarabinitol 1-phosphate. The response of Pn to TL observed for black locust is similar to those reported for other C3temperate woody plants (Chabot and Lewis, 1976; Anderson, 1982; Jurik gt g;., 1984; Moon gt _;., 1987). The decline in Pnen: temperatures above optimum was associated with reduction in stomatal aperture (Moon gt gt., 1987). This suggestion is supported by observations of Gspmtterns similar to Pn (Anderson, 1982; Lawrence and Oechel, 1983). In black locust, Gs did not have much role in the decline of Pn at TL above optimum. The correlation between Gs and Pn were non- significant at all T1, The analysis of variance also showed non significant differences in Gsand T among the different TL in the greenhouse grown plants. Gm was only strongly correlated with.Pm in field grown plants. 28 If the decline in Pn at TL above optimum is not associated with the limitations to gas exchange between the atmosphere and the chloroplasts, the decline inim1at higher temperatures could be attributed to temperature effects on the electron transport system, photophosphorylation, or the photosynthetic carbon reduction cycle. Stidham gt gt. (1982) have studied the effect of temperature on electron transport and photophosphorylation in isolated chloroplast membranes. They observed reduced proton uptake at temperatures above 35°C and suggested that Pn in whole leaves at 35-40°C temperatures may be limited by changes in stromal environment and /or a decrease in ATP production due to thermal uncoupling of electron transport. The optimum temperature for the grass species they studied was 25°C, which indicates the involvement of other factor(s) for the decline of Pn between 25 and 35°C. Marcus gt _t. (1981) observed reduced ribulose bisphosphate carboxylase (RuBPcase) activity at higher temperatures and suggested that RuBPcase plays an important role in plants' response to temperature. The reduction in RuBPcase activity could be due to either decreased solubility of COzat higher temperature (Ku and Edwards, 1977) and /or increased photorespiration at higher temperatures (Stidham gt _t., 1982). Increased Prcould reduce RuBPcase activity by limiting the regeneration of ribulose bisphosphate, the COzacceptor and substrate for RuBPcase. On the other hand, Downtown and Slatyer (1972) 29 suggested that the temperature response in cotton was not mediated by RuBPcase activity. Monson gt gt. (1982) observed a shift upwards in the temperature optima when the ambient C02 concentration was raised to 800 ppm and suggested that the decline of Pn at above-optimal temperatures in their study not accountable by Ozinhibition was due to the decreased solubility of C02 at higher TL. A shift in the optimum temperature of Pn in saturating C02 levels have also been reported by Jurik gt gt. (1984). The dependence ofIH1on both growth and measurement temperatures was observed in Atriplex lentiformis, a C4 evergreen shrub (Pearcy, 1977) . The Pn of plants grown at higher temperatures was lower at temperatures below the temperature optima than those plants grown at low temperatures, but the temperature optima for the plants grown at higher temperatures were 10°C higher. The Pn in cotton peaked at temperatures corresponding to the growth temperatures (Downtown and Slatyer, 1972). Pearson and Hunt (1972) also suggested that a lower temperature optima for Pn results from lower temperatures during the growing period. The temperature optima for field grown black locust plants were lower by about 5%:than the greenhouse grown plants . The Pn rates below the temperature optima were higher in the field grown plants. If black locust plants behave the same as Atripleg lentiformis, cotton, and alfalfa, the response curves suggest that the field grown plants were growing at a lower temperature than the greenhouse grown plants. The 30 weather data showed that in fact the daily minimum temperatures were lower in the field than the greenhouse. Minimum temperatures close to §%:were recorded. An alternative explanation for the observed responses of Pntx> 11,1s related to senescence or aging. Ledig and Clark (1977) observed a shift in the optimum temperatures from 25° to 30°C in 38-day-old seedlings to 15° to 25°C in 88-day-old seedlings. Similar shifts in optimum temperatures of Populus grandidentata Michx. were observed between leaves 1., 1984). In measured in July and September (Jurik gt. both studies, the Pnretes at the temperature optima were lower in the old seedlings than those measured in September. In black locust, the H1at temperature optima in the field plants were slightly higher than the greenhouse plants, so senescence or aging could be ruled out as the reason for the observed responses. Rd could also contribute to the decline of Pn at above- optimal temperatures. The temperature response of dark respiration for some species indicate that Rd could triple or quadruple at temperatures 10-1§%2above optimum (Luukkanen and Kozlowski, 1972; Fock gt gt., 1979). The decline in PD at above-optimal temperatures is a result of reductions in activity at all three partial processes of photosynthesis as well as increased mitochondrial respiration. However, the contribution from each process could vary depending on growth conditions, species, and experimental conditions. 31 The response of’Pm to varying levels of C02 concentrations of black locust (Figure 3) was somewhat different from poplars (Luukkanen and Kozlowski, 1972: Jurik gt gt., 1984) and sour cherry (Sams and Flore, 1982). The difference was that the increase in H1with increasing C02 above 350 ppm was reduced in black locust. This could have resulted from either limitation by the electron transport system, decreases in G5 and Gm, and/or measurement at above optimum temperatures. Since light saturation of Pn in black locust did not occur at 1900 umolJans'gt 350 ppm C02 (Figure 1), it is reasonable to assume that electron transport and ATP production could be limiting at higher C02 concentrations. The two families which showed constant Pn above 500 ppm were measured at about 330C5fi;- Since Pn of C3 plants at ambient C02 concentration is always limited by COzavailability at the carboxylation site, higher C02 concentrations will increase P" by removing the limitation of carboxylation by reduced C02. Higher C02 concentrations could also increaseifilby decreasing ribulose bisphosphate utilization by Prbecause of increased carboxylase/oxygenase ratio (Ku gt gt., 1977). The non-significant family X PAR, family X1T1” and family X Cchencentration interactions in this study suggest that studies on gas exchange of black locust can be done at any PAR, TL, and C02 level as long as they are kept constant for all plants. REFERENCES Anderson, J.E. 1982. Factors controlling transpiration and photosynthesis in Tamarix chinensis Lour Ecology 63:48- 56. Bjorkman, 0., N.K. Boardman, J.M. Anderson, S.W. Thorne, D.J. Goodchild, and A.N. Pyliotis. 1972. Effect of light intensity during growth of Atriptex patula on the capacity of photosynthetic reactions, chloroplast components and structure. Carnegie Inst. Wash. Year Book 71:115-135. Brix, H. 1971. Effects of nitrogen fertilization on photosynthesis and respiration in Douglas-fir. Forest Sci. 17:407-414. Chabot, B.F. and A.R. Lewis. 1976. Thermal acclimation of photosynthesis in northern red oak. Photosynthetica 10:130-135. DeLucia, E.H. and W.K. Smith. 1987. Air and soil temperature limitations on photosynthesis in Engelmann spruce during summer. Can.J.For.Res.17:527-533. Downtown, J. and R.O. Slatyer. 1972. Temperature dependence of photosynthesis in cotton. Plant Physiol.50:518-522. Fock, H., K. Klug and D.T. Canvin. 1979. Effect of carbon dioxide and temperature on photosynthetic C02 uptake and photorespiratory CQzevolution in sunflower leaves. Planta 145:219-223. Givnish, T.J. 1986. Optimal stomatal conductance, allocation of energy between leaves and roots, and the marginal cost of transpiration. In: On the Economy of Plant Form and Function. T.J. Givnish, ed. Cambridge Univ. Press, Cambridge MA. pp. 171-213. . 1988. Adaptation to sun and shade: a whole plant perspective. Aust. J. Plant Physiol. 15:63-92. 32 33 Jurik, T.W., J.A. Weber and D.M. Gates. 1984. Short-term effects of C02 on gas exchange of leaves of bigtooth aspen (Populus qrandidentata) in the feild. Plant Physiol.75:1022-1026. Kaufmann, M.R. 1982. Leaf conductance as a function of photosynthetic photon flux density and absolute humidity difference from leaf to air. Plant Physiol. 69:1018-1022. Koike, T. 1987. Photosynthesis and expansion in leaves of early, mid, and late successional tree species, birch, ash, and maple. Photosynthetica 21:503-508. Kramer, P.J. and J.P. Decker. 1944. Relation between light intensity and rate of photosynthesis of loblolly pine and certain hardwoods. Plant Physiol. 19:350-358. Ku, 8.8. and G.E. Edwards. 1977. Oxygen inhibition of photosynthesis. I. temperature dependence and relation to Oz/COgsolubility ratio. Plant Physiol.59:986-990. Ku, 8.8., G.E. Edwards, and C.B. Tanner. 1977. Effects of light, carbon dioxide, and temperature on photosynthesis, oxygen inhibition of photosynthesis, and transpiration in Solanum tuberosum. Plant Physiol.59:868-872. Lapido, D.O., J. Grace, A.P. Sandford, and R.R.B. Leakey. 1984. Clonal variation in photosynthetic and respiration rates and diffusion resistances in the tropical hardwood Triplochiton scleroxylon K. Schum. Photosynthetica 18:20-27. Lawrence, W.T. and W.C. Oechel. 1983. Effects of soil temperature on carbon exchange of taiga seedlings. II. Photosynthesis, respiration and conductance. Can.J.For.Res.13:850-859. Ledig, F.T. and J.G. Clark. 1977. Photosynthesis in a half- sib family experiment in pitch pine. Can.J.For.Res.7:510-514. Luukkanen, O. and T.T. Kozlowski. 1972. Gas exchange in six Populus clones. Silvae Genetica 21:220-229. Marcus, V., S. Lurie, M.A. Stevens and J. Rudich. 1979. High temperature effects on RuBP carboxylase and carbonic anhydrase activity in two tomato cultivars. Physiol.Plant.53:407-412. McMillen, G.G., and J.H. McClendon. 1983. Dependance of photosynthetic rates on leaf density thickness in deciduous woody plants grown in sun and shade. Plant Physiol. 72:674-678. 34 Mebrahtu, T., and J.W. Hanover. 1989. Heritability and expected gain estimates for traits of black locust in Michigan. Silvae Genetica (in press). Meinzer, F.C. 1982. The effect of light on stomatal control of gas exchange in Douglas fir (Pgeudotguga menziesii) seedlings. Oecologia 54:270-274. Moon, J.W. and J.A. Flore. 1986. A basic computer program for calculation of photosynthesis, stomatal conductance, and related parameters in an open gas exchange system. Photosyn.Res.7:269-279. Moon, J.W.Jr., J.F. Hancock,Jr., A.D. Draper and J.A. Flore. 1987. Genotypic differences in the effect of temperature on COzassimilation and water use efficiency in blueberry. J.Amer.Soc.Hort.Sci.112:170- 173. Monson, R.K., M.A. Stidham, G.J. Williams III, G.E. Edwards, and E.G. Uribe. 1982. Temperature dependence of photosynthesis in Agropyron smithti Rydb. I. factors affecting net COzuptake in intact leaves and contribution from ribulose-1,5-bisphosphate carboxylase measured in vivo and in vitro. Plant Physiol. 69:921- 928. Nelson, N.D. and P. Ehlers. 1984. Comparative carbon dioxide exchange for two Populus clones grown in growth room, greenhouse, and field environments. Can.J.For.Res. 14:924-932. Nobel, P.S., L.J. Zaragota, and W.K. Smith. 1975. Relation between mesophyll surface area, photosynthetic rate, and illumination level during development for leaves of Plectranthug parviflorus Hencke). Plant Physiol. 55:1067-1070. Patterson, D.T., J.A. Bunce, R.S. Alberte, and E.V. Volkenburgh. 1977. Photosynthesis in relation to leaf characteristics of cotton from controlled and field environments. Plant Physiol. 59:384-387. Pearcy, R.W. 1977. Acclimation of photosynthetic and respiratory carbon dioxide exchange to growth temperature in Atriplex lentiformis (Torr.) Wats. Plant Physiol. 59:795-799. Pearson, C.J., and L.A. Hunt. 1972. Effects of pretreatment temperature on carbon dioxide exchange in alfalfa. Can. J. Bot. 50:1925-1930. Pereira, J.P., W.E. Splittstoesser, and W.L. Ogren. 1986. Photosynthesis in detached leaves of cassava. Photosynthetica 20:286-292. 35 Salisbury, F.B. and C.W. Ross. 1985. Plant physiology. Third ed., Wadsworth Pub.Co., California. p540. Sams, C.E. and J.A. Flore. 1982. The influence of age, position and environmental variables on net photosynthetic rate of sour cherry leaves. J.Amer.Soc.Hort.Sci. 107:339-344. Schulze, E.-D., O.L. Lange, M. Evenari, L.Kappan, and U. Buschbom. 1974. The role of air humidity and leaf temperature in controlling stomatal resistance of Prunus armeniaca L. under desert conditions. Oecologia 17:159-170. Seemann, J.R., M.U.F. Kirschbaum, T.D. Sharkey, and R.W. Pearcy. 1988. Regulation of ribulose-1:5 bisphosphate carboxylase activity in Alocasia macrorrhiza in response to step changes in irradiance. Plant Physiol. 88:148-152. Stidham, M.A., E.G. Uribe, and G.J. Williams III. 1982. Temperature dependence of photosynthesis in Agropyron smithii Rydb. II.contribution from electron transport and photophosphorylation. Plant Physiol. 69:929-934. Tan, C.S., T.A. Black, and J.U. Nryamah. 1977. Characteristics of stomatal diffusion resistance in a Douglas-fir forest exposed to soil water deficits. Can. J. For. Res. 7:595-604. CHAPTER 2 EFFECT OF LEAF TEMPERATURE ON DARK RESPIRATION AND PHOTORESPIRATION OF BLACK LOCUST 36 ABSTRACT Rd and Pr were studied at leaf temperatures ranging from 10° to 40°C in black locust families. Rd increased with TL in all families and the percent reduction of Pn by Rd reached as high as 35% at 40°C. Pr peaked at 10-15°C and followed the same pattern as Pn (21% O2) and true Pn (1.8% 02) . The temperature response curves had lower temperature optima than observed before (Chapter 1). The lower temperature optima in this study were attributed to chilling night temperatures before measurements. Plotting Tm,against the sum of true Pu and Rd at each measurement TL to account for losses of CO2 fixed indicated that Rd and P, are not the causes of the decline in2m1of black locust at leaf temperatures above optimum. 37 INTRODUCTION The Pn of plants decreases at higher TL. The decline of Pn above optimum TL probably result from both the limitation of diffussion of gases from outside the leaf to the site of carboxylation (Ku gt gt.,1977: Moon gt gt., 1987) and internal factors. The internal factors limiting Pn at higher TL are increased Rd (Grahm, 1980) , deceased electron transport and photophosphorylation (Stidham gt gt., 1982), decreased C02 fixation by the photosynthetic carbon reduction cycle (Monson gt gt., 1982) , and increased P,- (Hew gt gt. , 1969) . Increases in Rd were observed for many 1., 1969; Hofstra and Hesketh, 1969: species (Hew gt Pearcy, 1977: Lawrence and Oechel, 1983: Koike and Sakagami, 1985) . In black locust, Rd at 25°C in both field and greenhouse grown plants were 5-14% of Pn at 1900 umol.m'2.s'l PAR (Chapter 1) . The responses of Pr to TL in plants studied so far have shown two distinct patterns. In the first group of plants, the optimum temperatures for P, are the same as the optimum temperatures for Pn (Hofstra and Hesketh, 1969: Ku gt gt. , 1977) , while the temperature optima for P, in other plants are different from Pn and are usually 10-15°C higher than the temperature optima for Pn (Jolliffe and 38 39 Tregunna, 1968: Pearson and Hunt, 1972). The main goal of this study was to evaluate whether Rd and P,- are the causes for the decline in Pn of black locust at higher temperatures. MATERIALS AND METHODS Plant material The plant material and the growth conditions are described in Chapter 1, in the materials and methods section for field grown plants. Gas exchange Measurements of the response of Rd to TL were made on 24 seedlings from 6 half-sib families. ngwas increased in steps up from 10° to 39° C. P, was determined as the difference between Pn at 1.8% 02 (true Pu) and Pn at 21% 02 at four TL: 10, 20, 30, and 40°C. The PAR level was maintained 2.s'lThe gas exchange system and calculations at 1900 umol.m' used to determine the gas exchange variables are described in detail elsewhere (Chapter 1) RESULTS Dark respirationtion (Rd) Rd increased with TL in all families (Figure 1) . The percent reduction of Pa by Rd also increased with TL (Figure 1) . The percent reduction by Rd was calculated as Rd/(Pn+ Rd) at each TL. The Pn was taken from the temperature response 40 Rd (mg C02.dm'2.hr") M (a) b 01 O) ‘1 T 4O 35 30* 25- 20- 15" 10— 96 REDUCTION 140 -— anoa 120i -+-FAM308 10° _ 1? FAM 385 4} nmaae so ~ + FAM 408 -0- FAM 418 60 — (mmol.m'2.s'1) 40- 20- O ' ' 1 l I '10 0 1° 20 3O 40 50 LEAF TEMPERATURE (DEGREE C) Figure 1. Effect of T on Rd, % reduction of Pn by R , and Gs in the dark of field grown black locust half-51b families measured at 350 ppm C02. The values are averages of four seedlings. 41 data of these families done a week earlier (Chapter 1). Both Rd and percent reduction of Pa by Rd were very small at the 2.hr'l optimum temperature range, and reached upto 6 mg C02.dm' and 35%, respectively, at 40°Cfm2.Significant variation was observed among the families in Rd(}01 probability level). Family x Tm,interaction was significant (.01 probability level). (% peaked at 15°C in all families, declined sharply to about 20 mmol.nf2.s'5t 20°C in some families and at 25°C in the rest of the families and then remained constant to the highest TL (Figure 1) . Gs at the peak was about 120 mmol.m'2.s" l in 4 families. T generally increased with Ti,in all families, but the course to the highest T was variable (data not shown). There were no significant variations among families in (k‘while significant variations were observed in T and CL Family x TL interactions were non-Significant in G5, T, and Ci. Gs was positively and significantly correlated with T. Photorespiration (Pa P.- decreased with increasing TL in 4 families (Figure 2) . The peak P,- in 2 families was at 15°C TL. The pattern of true Pn (Pn at 1.8% 02) for each family was similar to the pattern of Pr except in 2 families where the Peak Pn was at a TL 10°C lower or higher than Pr (Figure 2) . Response of PD (21% 02) to TL was the same as true Pn except in one family (Figure 2) . The percent reduction of Pa by P, was the highest 42 at 40°C TL and the lowest at the optimum TL in all families (Figure 3). Significant variations (.01 probability level) were observed among families in Pr, true Pu, and Pn while family x TL interactions were nonsignificant (.05 probability level) in Pr, true Pu, and P“. The performance of each family relative to others remained constant when the families were ranked by Pr, true P“, and Pn except families 308 "and 416 switched positions in Pr. The difference in P,- between families 308 and 416 was only 0.2 mg CO2.dm'2.hr'l, however. The correlation between P; and true Pn was significant (Table 1), supporting the results of the ranking. Gs at both 1.8% and 21% 02 decreased with increasing TL in all families except in one in which the peak Gs was at 20°C (data not shown). Gs at 1.8% 02 was higher than Gs at 21% 02. Gm at 1.8% 02 peaked at 20°C in all families except one and the values were much more higher than Gm at 21% 02 (data not shown). At 21% 02, Gm also peaked at 20°C in 4 families and at 10°C in 2 families (data not shown). T generally increased with TL in both 1.8% and 21% 02 and the values were more or less similar (data not shown). Significant family variations were observed in Gs, Gm, and T in both 1.8% and 21% 02. Family x TL were non- significant in all variables. The correlation coefficients among Pr, true P“, P“, Gs, Gm, and T are presented in Table 1. 43 Pr d .5 N 0 0| 0 I I (mg C02.dm'2.hr'1) 01 60 50 - 40- 30* TRUE P“ 20- (mg C02.dm'2.hr") 35" 30 _ —-— FAM 208 -I- FAM 308 25 --.)... FAM 385 -8— FAM 388 2° 2*- PAM 408 0 15_‘. ' FAM 410, 10" P (mg coz.dm'2.hr") 0 I l 1 l 1 '10 0 1O 20 30 4O 50 LEAF TEMPERATURE (DEGREE C) Figure 2. Effect of TL on Pr, true P", and Pn of field grown black locust half-sib families measured at a PAR of 900 umol.m'2.s'1. The values are averages of four plants. 44 O) O no: 9,. (mg C02.dm'z.hr'1) (A) «A 0| 0 O O I I I M O r cub O I 5.. >— L—- >— )— 70 40F 30- 96 REDUCTION 20 ~ A A —‘— PAM 208 ‘1'" PAM 308 ‘9)“ PAM 388 10 — ~8- PAM 888 -><- PAM 408 ~<>- PAM 418 o 1 i i l l l -10 0 1O 2O 30 40 50 60 LEAF TEMPERATURE (DEGREE C) Figure 3. Effect of TL on % reduction of Pn by Pr and photosynthesis of field grown black locust half-sib families. The photosynthesis values were calculated after removing the effects of Rd and Pr. The values are averages of four seedlings. 45 Table 1. Correlation coefficient values among Pn,true Pn, Gs, Gm, and T at 1.8% 02 for black locust half-sib families. Pf Pn Gs Gm T Pr - Pn .90 - Gs .60 .73 - Gm 082 091 048 '- T .16 .14 .00 .20 - 46 DISCUSSION The Pn of C3 plants decreases at high TL. The decline in Pa is attributed to decreased G5 and Gm (Ku _e_t_ gt., 1977) , increased Pr(Mcnson gt gt., 1982), increased electron transport (Stidham gt gt., 1982), and decreased C02fixation by the photosynthetic carbon reduction cycle (Monson gt gt., 1982). Since the solubility of C02decreases more than the solubility of 02at high temperatures (Ku and Edwards 1977), the carboxylase activity of RuBiSCO decreases more than the oxygenase activity at higher temperatures (Marcus gt gt., 1981). The resulting change in carboxylase/oxygenase ratio is expected to increase Pr(Ku and Edwards 1977). Monson gt gt. (1982) suggested that the decline in P“ at temperatures above optimum is largely due to Pp.The data of Keys gt gt. (1977) and Jolliffe and Tregunna (1968), both on wheat, suggest the reduction inlmaby Prat higher temperatures. The reduction of Pn by 02 is due to two effects: (1) stimulation of Pn,and (2) inhibition of true Pn(1mdwig and Canvin 1971). On the other hand, Fock gt gt. (1979) observed no correlation between Prand the solubility ratios of Ozand CO2 with increasing temperatures. Data suggesting P,- was not the cause of the decline in Pn at higher temperatures were reported by Ku gt _t. (1977), Drake and Raschke (1974), and Hofstra and Hesketh (1969). These contradictory conclusions about the effects of P, on the decline of Pa at temperatures above optimum are based on different sets of data. The conclusion that Pris 47 responsible for the decline of Pn at higher temperatures is based on the difference in the optimum temperatures for Pn in temperature response curves at lower 02 (usually 2%) and 21% 02. For example, Jolliffe and Tregunna (1968) observed temperature optima between 20 and 26°C at 21% 02 and temperature optima between 30 and 36°C at 3% 02. Therefore, the decrease in Pn at temperatures up to 30-36°C at 21% 02 must have been due to 02 inhibition (Pr) . The conclusion that P; is not resposible for the decline of Pn at higher temperatures is based on data showing the same patterns and optimum temperatures for Pn at 2% and 21% O2 (Ku gt gt., 1977: Drake and Raschke, 1974) . In black locust, Pn at both 1.8% and 21% 02 had the same patterns and temperature optima and I suggest that P,- was not the cause for the decline of Pn at temperatures above optima in black locust. Lower Pn at lower 02 levels than 21% 02 was observed at low temperatures (Jolliffe and Treguna, 1973: Cornic and Louason, 1980) and high CO2 partial pressures and light intensities (McVetty and Canvin, 1981). Some of the explanations for this phenomenon are: (1) Photoinhibition by high PAR (Cornic and Louason, 1980), (2) reduced ribulose- 1,5-bisphosphate regeneration at low 02 (McVetty and Canvin, 1981), and (3) low phosphate concentration in the chloroplast stroma (Sharkey, 1985). The percent reduction in Pa caused by P,- in black locust increased from as low as 27.5% at 10°C to 58% at 40°C. Increased percent reduction of Pa by P.- with increasing 48 temperatures were reported by Pearson and Hunt (1972), Jolliffe and Tregunna (1968), and Ku gt gt. (1977). The higher percentage of reduction of Pa by P, at higher temperatures were due to more decrease inimlat 21% than 1.8% O2,which could have resulted from increased affinity of RuBiSCO for 02 than C02 (Lainge e_t gt., 1974) . The similarity in the patterns of Pa and P; in response to Tiwas well as the similarity in the ranking of the families forimqand Prindicate an association between these two processes. Linear relationships between Pu and P, were observed for Populus clones (Luukkanen and Kozlowski, 1972). On the other hand, Frazer and Bidwell (1972) suggested that these two processes are independently controlled by different factors during leaf ontogeny, or if they are controlled by the same factor, they are regulated in a different manner. Obviously,2fi1and Prin black locust seem to be regulated by the same factors. 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Ann... 0. ...0 ml. .5. .00. :22... cm .00.0 00.0 .000 .0.0h 0&0..0& >00 .00: >00 80.0 30m .00 00.0l0.a 000.0: ..0000) m. .0 .0000. 300.0 00 0..0.. .00. 000 0.30.0 000 00:.0> 000: .0 0.00% 68 reciprocal of Trincreased with drought (Priera gt gt., 1986). Pn was very weakly correlated with the growth traits: height, foliage dry weight, stem dry weight, root dry weight (Table 3). Similar observations were reported by Ledig and Botkin (1974), Ceulmans gt gl. (1980), and Lapido gt gt. (1984). Significant and positive correlations were also reported by Mahon gt gt. (1977), Ceulmans and Impens (1983), and Nelson and Ehlers (1984) . When the Pn of whole plants (Pn X leaf area) was correlated with height, stem dry weight, and total dry weight, the correlation coefficients were higher and positive (Table 3). Since studies have shown that Pn decreases with leaf age (Dickmann, 1971: Reich, 1984; Nelson, 1985), my estimate of whole plant Pn could be much higher than the actual Fm. But the relationship with height, stem dry weight, or total dry weight will not be affected. The lack of strong correlations between plant Pu and total dry weight in this study could be due to: 1) variations in Ejamong families; 2) variations in the magnitude of self shading among the plants: and 3) variations among families in Pa rate during a course of a day and over the age of a leaf. Lapido gt gt. (1984) showed that consideration of Kjimproved the relationship between Pn and productivity. 69 Leaf traits There was no significant difference in SLW among the families. Significant genetic variation in SLW was found among genotypes for a grass species (Dictonia spicata), and broad sense heritability was very low (0.07) (Schreiner gt gt., 1984). SLW was weakly correlated with the gas exchange traits in black locust. Significant and positive correlations between SLW and Pn were observed for Dictonia spicata (Schrenier gt gt., 1984), poplar (Nelson and Ehlers, 1984), Lgttg spp (Oren gt gt., 1986), and Eitgg,gt;g§ (Oren gt gt., 1986). Hobbs and Mahon (1985) suggested that SLW might influence Chl content and RuBiSCO carboxylase activity. SLW increases with leaf age (Oren gt gt., 1986) and is affected by light conditions (Nelson and Ehlers, 1985; Oren gt _t., 1986). Significant variation in Chl b and total Chl content was observed, but weak correlations were found between Chl and other gas exchange or growth traits. A positive correlation between leaf biomass and Chl content was reported by Gratani and Fiorentino (1986) for Quercus itgg. Significant genetic variation in Chl content and a significant correlation with Pn was observed for maize (Bear and Schrader, 1985a) and pea (Hobbs and Mahon, 1985). Since chlorophylls are involved in energy trapping and transduction rather than as a substrate for chemical reactions, they are usually not limiting factors in photosynthesis (Baer and Schrader, 1985b). 70 Significant variation was observed in leaf area and it was highly correlated with total dry weight and stem dry weight. Previously, Okafo and Hanover (1978) found significant correlation between leaf area and plant dry weight in trembling and bigtooth aspens. Growth traits The mean performance of the families in growth traits is presented in Table 4. Black locust grew at a rate of at least 1 cm and 3 cm a day in the slow and fast growing families, respectively, beginning 3 weeks after germination. Black locust grew at a faster rate (4-6 cm a day) when grown in 18.5 litre pots compared with 7.5 litre pots (unpublished data). Highly significant (.01 probability level) differences in height growth were found among families after 10 weeks of growth and at the end of the study. Both the variation and the relative performance of each family were similar to their performance in a field progeny test (Mebrahtu and Hanover, in press). Height was not strongly correlated with total dry weight (Table 3), and that is why any attempt to relate and h shoot growth fails to show the casual relationship betweenimmand total dry weight production. Height was only weakly correlated with diameter at root collar. There was significant variation (.01 probability level) in above ground:below ground dry weight ratio among families. No significant variation was found in root dry 71 weight or foliage dry weight but stem and total dry weight production did vary significantly. Mahon gt gt., 1977) reported significant correlation between Pu and total plant weight and weak correlation betweenlnnand shoot dry weight in cassava. They suggested that total plant weight should be used instead of shoot dry weight in studies involving gas exchange. The influence of carbon partitioning on the relationship betweenimmand productivity was demonstrated by Pearcy and Ustin (1984). They investigated the photosynthetic potential and plant growth rate of three different species: the C4grass (Spgttina foliosa), the C3 sedge (Scirpus robustgg) and the Q3stem-succelent shrub (Salicornia virginica) . Pn was highest in the C4 grass and lowest in the shrub, the sedge being intermediate. The relative growth rate showed the reverse: the shrub having the highest and the grass the lowest. This reversal was attributed to greater photoassimilate allocation to shoots in the shrub which compensated for lower'Fh. The apparent adaptation strategies for these different species might also pertain to individual plants within a variable species. Despite significant variation among families in above ground:below ground dry weight ratio, the correlation between stem dry weight and total dry weight was very high (Table 3), mainly due to non-significant differences in root and foliage dry weights. It seems that in black locust stem dry weight can be used instead of total dry weight in studies where total weight determinations are difficult. REFERENCES Baer, G.R. and L.E. Schrader. 1985a. Relationships between C02 exchange rates and activities of pyruvate, pi dikinase and ribulose bisphosphate carboxylase, chlorophyll concentration, and cell volume in maize leaves. Plant Physiol. 77:612-616. . 1985b. Inheritance of DNA concentration, and cellular contents of soluble protein, chlorophyll, ribulose bisphosphate carboxylase, and pyruvate, pi dikinase activity in maize leaves. Crop Sci. 25:916-923. Bahari, Z.A., S.G. Pallardy, and W.C. Parker. 1985. Photosynthesis, water relations, and drought adaptation in six woody species of oak-hickory forests in central Missouri. For. Sci. 31:557-569. Boltz, B.A., B.C. Bongarten and R.O. Ryugo. 1986. Seasonal patterns of net photosynthesis of loblolly pine from diverse origins. Can. J. For. Res. 16:1063-1068. Carpenter, S.B. and J.W. Hanover. 1974. Comparative growth and photosynthesis of black walnut and honeylocust seedlings. For. Sci. 20:317-324. Ceulmans, R. and I. Impens. 1980. Leaf gas exchange processes and related characteristics of seven poplar clones under laboratory conditions. Can. J. For. Res. 10:429-435. . 1983. Net C02 exchange rate and shoot growth of young poplar (Populus) clones. J. Exp. Bot. 34:866-870. Ceulmans, R., I. Impens and V. Syeenackers. 1987. Variations in photosynthetic, anatomical, and enzymatic leaf traits and correlations with growth in recently selected Populus hybrids. Can. J. For. Res. 17:273-283. Comstock, J. and J. Ehleringer. 1984. Photosynthetic responses to slowly decreasing leaf water potentials in Bncelia frutescens. Oecologia (Berlin) 61:241-248. 72 73 DeJong, M., A. Tombesi and K. Ryugo. 1984. Photosynthetic efficiency of kiwi (Actinidig ghinensis Planch.) in response to nitrogen deficiency. Photosynthetica 18:139-145. Dickmann, D.I. 1971. Photosynthesis and respiration by developing leaves Of cottonwood Populus deltoides Bartr.). Bot.Gaz. 132:253-259. Farguhar,G.D. and T.D. Sharkey. 1982. Stomatal conductance and photosynthesis. Ann.Rev.Plant Physiol. 33:317-345. Gifford, R.M., J.H. Thorne, W.D. Hitz and R.T. Giaquinta. 1984. Crop productivity and photoassimilate partitioning. Sci. 225:801-807. Gratani, L. and E. Fiorentino. 1986. Relationship between chlorophyll content and leaf biomass for Quercus ilex in a Mediterranean maquis stand. Photosynthetica 20:267-273. Hobbs, S.L.A. and J.D. Mahon. 1985. Inheritance of chlorophyll-deficient mutants of common wheat. Crop Sci. 25:1031-1034. Knudson, L.L., T.W. Tibbitts and G.E. Edwards. 1977. Measurement of ozone injury by determination of leaf chlorophyll concentration. Plant Physiol. 60:606-608. Lambers, H. 1987. Does variation in photosynthetic rate explain variation in growth rate and yield. Netherlands J. of Agri.Sci. 35:505-519. Lapido, D.O., J. Grace, A.P. Sandford and R.R.B. Leakey. 1984. Clonal variation in photosynthetic and respiration rates and diffusion resistances in the tropical hardwood Triplochiton sclerxylon K. Schum. Photosynthetica 18:20-27. Ledig, F.T. 1969. A growth model for tree seedlings based on the rate of photosynthesis and the distribution of photosynthate. Photosynthetica 3:263-275. Ledig, R.T. and D.E. Botkin. 1974. Photosynthetic C02-uptake and the distribution of photosynthate as related to growth of larch and sycamore progenies. Silvae Gen. 23:188-192. Ledig, F.T. and J.G. Clark. 1977. Photosynthesis in a half- sib family experiment in pitch pine. Can. J. For. Res. 7:510-514. Luukkanen, O. and T.T. Kozlowski. 1972. Gas exchange in six Populus clones. Silvae Gen. 21:220-229. 74 Mahon, J.D., S.B. Lowe and L.A. Hunt. 1977. Variation in the rate of photosynthetic C02 uptake in cassava cultivars and related species of Manihg . Photosynthetica 11:131-138. Mebrahtu, T. and J.W. Hanover. 1989. Heritability and expected gain estimates for traits of black locust in Michigan. Silvae Gen. Moon, J.W. and J.A. Flore. 1986. A basic computer program for calculation of photosynthesis, stomatal conductance, and related parameters in an open gas exchange system. Photosyn. Res. 7:269-279. Nelson, N.D. 1985. Photosynthetic life span of attached poplar leaves under favorable controlled environmental conditions. For. Sci. 31:700-705. Nelson, N.D. and D. Michael. 1982. Photosynthesis, leaf conductance, and specific leaf weight in long and short shoots of Populus ‘Tristis #1‘ grown under intensive culture. For. Sci. 28:737-744. Nelson, N.D. and P. Ehlers. 1984. Comparative carbon dioxide exchange for two Populus clones grown in growth room, greenhouse, and field environments. Okafo, O.A. and J.W. Hanover. 1978. Comparative photosynthesis and respiration of trembling and bigtooth aspens in relation to growth and development. For. Sci. 24:103-109. Oren, R., E.-D. Schulze, R. Matyssek and R. Zimmermann. 1986. Estimating photosynthetic rate and annual carbon gain in conifers from specific leaf weight and leaf biomass. Oecologia (Berlin) 70:187-193. Pearcy, R.W. and S.L. Ustin. 1984. Effects of salinity on growth and photosynthesis of three California tidal marsh species. Oecologia (Berlin) 62:68-73. Pereira, J.S., J.D. Tenhunen, O.L. Lange, W. Beyschlag, A. Meyer and M.M. David. 1986. Seasonal and diurnal patterns in leaf gas exchange of Eucalyptus globulus trees growing in Portugal. Can. J. For. Res. 16:177- 184. Pezeshki, S.R. and J.L. Chambers. 1986. Stomatal and photosynthetic response of drought-stressed cherrybark oak (Quetcus falcata Var. pagedaefolia) and sweet gum (Liggidambar styraciflua). Can.J.For.Res. 16:841-846. 75 Pezeshki, S.R., R.D. DeLaune and W.H. Patrick, Jr. 1986. Gas exchange characteristics of bald cypress (Taxodium distichum L.): evaluation of responses to leaf aging, flooding, and salinity. Can. J. For. Res. 16:1394-1397. Reich, P.B. 1984. Relationships between leaf age, irradiance, leaf conductance, C02 exchange, and water use efficiency in hybrid poplar. Photosynthetica 18:445-453. Sawada, S. and T. Hayakawa. 1984. Effect of Bordeaux mixture on net photosynthetic rate and stomatal and intercellular resistances in apple leaves. Photosynthetica 18:69-73. Scarascia-Mugnozza, G., T.M. Hinckely and R.F. Stettler. 1986. Evidence for nonstomatal inhibition of net photosynthesis in rapidly dehydrated shoots of Populus. Can. J. For. Res. 16:1371-1375. Scheiner, S.M., J. Gurevitch and J.A. Teeri. 1984. A genetic analysis of the photosynthetic properties of populations of Danthonig spicgtg that have different growth responses to light level. Oecologia (Berlin) 64:74-77. Sharkey, T.D. and M.R. Badger. 1982. Effects of water stress on photosynthetic electron transport, photophosphorilation, and metabolite levels of Xanthium strumarium mesophyll cells. Planta 156:199-206. Sorensen, F.C. and W.K. Ferrell. 1973. Photosynthesis and growth of Douglas-fir seedlings when grown in different environments. Can.J.Bot. 51:1689-1698. Tenhunen, J.D., O.L. Lange, J. Gebel, W. Beyschlag and J.A. Weber. 1984. Changes in photosynthetic capacity, carboxylation efficiency, and C02 compensation point associated with midday stomatal closure and midday depression of net C02 exchange of leaves of Quercus suber. Planta 162:193-203. Tenhunen, J.D., O.L. Lange, M. Braun, A. Meyer, R. Losch and J.S. Pereira. 1980. Midday stomatal closure in Arbutus unedo leaves in a natural macchia and under simulated habitat conditions in an environmental chamber. Oecologia (Berlin) 147:365-367. Wintermans, J.F.G.M. and A. DeMots. 1965. Spectrophotometric characteristics of chlorophylls a and b and their phenophytins in ethanol. E.B.A. 109:448-453. Wong, S.G., J.R. Cowan and G.D. Farquhar. 1979. Stomatal conductance correlates with photosynthetic capacity. Nature 282:424.426. » CHAPTER 4 DIURNAL PATTERNS OF GAS EXCHANGE OF BLACK LOCUST (Robinia pseudoacacia) 76 ABSTRACT The diurnal patterns of gas exchange of black locust were studied using greeenhouse and field grown seedlings. In all experiments, Gm was highly correlated with Pn while the correlation with Gswas variable. Attempts were made to determine the factors responsible for causing variation in (k and an In the greenhouse, light intensity was the limiting factor and the pattern of Pn paralleled changes in the light intensity during the day. Midday depression in Pa of well watered black locust plants occurred consistently in plants treated with night light. Pa in field grown plants peaked early morning and declined from the peak even though PAR and temperature were increasing to optimum levels. The depression in Pn was accompanied by depressions in G5, Gm, and T. Under water stress conditions, Pm in black locust was limited by the availability of water in the pots, but black locust maintained high T at TL exceeding 35°C and a VPD close to 5.6 kPa. Black locust does not seem to regulate stomatal aperture to moisture stress conditions. In greenhouse grown plants, family differences inifiawere observed only in the afternoon, and there were no family X time interactions. 77 INTRODUCTION The Pn of plants varies during the course of a single day, because environmental and internal conditions change during the day. This phenomenon is very important to fully understand the vast literature on photosynthesis. Attempts to relate P" with growth have ended in disappointing results (Chapter 1; Ledig and Botkin, 1974: Lapido gt gt., 1984). The common practice in most studies was to measure the Pa of plants in a given set of conditions in a greenhouse, growth chamber, or in the field and correlate such measurements with growth parameters (height, relative growth rate, dry weight, etc.). The absence of significant relationship between Pu and growth can be attributed to many factors including failure to consider the variation in the diurnal patterns of Pn among genotypes or species. The relative performance of genotypes may vary within a single day. For example, some genotypes maintain similar’Em for greater part of the day, while others decrease or increase their rates at different times of a day. Therefore, a single or even several measurements ofimaat selected hours or conditions in a day may not reveal the real relationship between growth and Pn unless no diurnal variation occurs. The same problem 78 79 is apparent in studies done to determine the factors that limit.Pm, Since the external and internal factors limiting Pn shift in importance during the day (Kramer and Kozlowski, 1979 ), data from measurements at a given time of a day will only provide information on those factors that limit Pnan: that given time. The main objective of this study was to measure the diurnal pattern ofin1of different black locust families. Elucidation of the factors limiting Pn was also attempted. MATERIALS AND METHODS A. GREENHOUSE Plant material: Seedlings from 9 half-sib families selected for contrasting growth performance were grown from seed in 7.4 litre pots in a greenhouse. The potting mix was peat moss, perlite, and vermiculite (3:1:1). The seedlings were watered adequately and fertilized with Peters slow release fertilizer (20,20,20). Micronutrients were also added. The greenhouse was maintained at 27°C and 17°C day and night temperatures, respectively. The plants were grown for 9 weeks under a 20 hr photoperiod before the first measurement. Gas exchange: Gas exchange measurements were done on leaflets of the 9u‘leaves (from the youngest fully expanded leaf) of each 8O plant using a portable open gas exchange system (ADC model LCA-Z) under the following four different conditions. i. seedlings well watered, measurment done inside a greenhouse (May 28,1988) ii. seedlings well watered, but measurements done outdoors (May 29, 1988) iii. seedlings kept under light (700 umicromol.m'2.s’l) overnight before the measurement day. Plants were well watered and measurement done outdoors (May 30, 1988) iv. seedlings were not watered for 2 days before measurement. Measurements done outdoors (June 18, 1988). On all days the maximum ambient temperature was 35°C and there were no clouds. The parameters recorded during measurement and the calculations used for Pn:(%: GuU'T, Ch and VPD are according to Moon and Flore (1986) as described in Chapter 3. B. FIELD Plant material Seedlings from 6 half-sib families were grown from seed in 7.4 pots in a greenhouse in conditions as described in Chapter 1. Three weeks after germination, the seedlings were transferred to the open field. The seedlings were watered daily. 81 Gas exchange The seedlings were grown for 70 days in the field before the gas exchange measurements. Determinations of Pn (k, and Ciwere done on matured leaves using a closed gas exchange system, LI-6200 Portable Photosynthesis System (LI- CoR Inc, Lincoln, NE). Measurements were made on three separate days on 18 seedlings. As a precaution against cloud overcasts, the instrument was set to take 3 sample readings every 5 seconds. Leaf areas of the leaves in the leaf chamber were measured with a delta T leaf area meter ( delta T Devices Ltd.). Calculations of Pn,(%, and Ciwere done using the program provided with the LI-6200 instrument. RESULTS A. GREENHOUSE GROWN PLANTS i. Well watered, in greenhouse Em_increased to a maximum at about 14:00 and then decreased with the declining PAR level (Figures 1). The pattern was similar in all families and paralleled changes in PAR (Figure 2). The diurnal response of Gnland T followed the same pattern as Pn (Figures 1 & 2), while that of G5 was different in different families (Figure 2). In some families, Gsdecreased at about 11:00 and increased to a peak by 14:00 and declined. In other families, asshowed the same pattern as Pn,(hn,and T with the maximum reached at 14:00. 11,and VPD peaked earlier in the day (10:45) than from Ph (10:45) (Figure 2). 82 No significant familly differences in Pn were observed among families during the morning hours, while there were significant differences among families in Pm in the afternoon. Family X measurement time interaction was non- significant. The correlation coefficients between Pn and G5 were non-significant in the morning hours and significant in the afternoon hours. In all measurement hours, Pn was strongly and positively correlated with Gn,(<0.01 probability level). ii. Well watered, outdoors This experiment was designed to test if the Pn of greenhouse grown plants is different between measurements made outside and inside a greenhouse. The diurnal pattern of lhlat this measurement day was different from that of May zaflkimqpeaked at 10:00 in almost all families, and then either decreased gradually, or depressed the hour after, or remained constant for few hours before they were depressed (Figure 3) . In the morning hours, Pn did not parallel changes in PAR (Figure 4), while Haseem to parallel changes in PAR in the afternoon. The pattern of diurnal responses of G5, Gm, and T was similar to that of Pn (Figures 3 & 4) . The patterns of Tl,and VPD are shown in figure 4. Variations for Pn among families were non-significant in the morning and significant in the afternoon. Family X measurement time interaction was non-significant. Both Gs and Gnywere strongly and significantly correlated with P". 83 iii. Light pretreatment, well watered, outdoors At this measurement day which was done to determine if the accumulation of photosynthetic end products inhibits P", the diurnal pattern ofimawas different from those of May ”HF>n increased to a maximum at about 10:00, 28u‘and 29 followed by a depression at about 14:00and then increased again at 15:00 before it deceased at 18:30 (Figure 5). The pattern ofim1did not follow the PAR pattern (Figure 6). The diurnal response of Pn was not paralleled by G5 and T (Figures 5 & 6). On the other hand, Gnlshowed similar pattern to Pn (Figure 5) . The values of P“, Gs, Gm, and T were th . The low compared to those observed on May 28u‘and 29 patterns of Ti,and VPD are presented in figure 6. There was no significant variation in Pa among families in the morning and the afternoon, but differences in Pa were significant among measurement hours. Correlations between Pu and G5 and Pu and Gm were relatively lower and significant. iv. Unwatered, outdoors The diurnal response of Pn was different from the previous experiments.1fi1peaked at 11:00 and then kept decreasing for 5 hours and finally reached a smaller second peak at 16:00 in some families (Figure 7). The patterns of Gs and Gm were similar to Pn (Figures 7) . The values for Pn, G3,(hn,and T were lower than the previous days. Some seedlings were wilting at the end of the day. The patterns of T, PAR, and T1 and VPD are presented in Figure 8. 84 A 25 'u _ .C.‘ 20 2‘5 15 " “13 0:» 10 r U o 5" E 0 160 A 140 '— ‘m 120 T" “q: ' 100 ~ 0 5: 80 ~ H o 60 i" E 40 - V 20 - o l 1 l l l 60 50 ” 'm 40— $ 0 0. j 30 _. O ch g 20 '— r. 'r v —°— tam 012 "i— tam 051 .. 10 5 -B- fani 329 4"- !am 386 ‘0' tom '40:“ o 1 l l l 1 7:00 8:45 10:45 13:45 15:45 19:15 22:00 TIME OF DAY Figure 1. Diurnal patterns of Pn ,G , and Gs of greenhouse grown black locust half-sib families measured inside a greenhouse. The values are averages of two plants. 85 ' Q o- .-- s o - a b ‘ o - (mmol.m'2.s'1) ” —‘— tam 012 —+— lam 051 4‘" tam 20\8 ‘9‘ {am 329 + Mm 386 ‘9“ 18m 408 35 4 l 2.5 I 20 11 (0CD ‘*‘TEMP -+-vpo «15 .i. (eax) GdA I 10 '1 _a .5 O N o o o o I 1 PAR (umol.mi.s o 1 l l l 1 7:00 8:45 10:45 13:45 15:45 19:15 22:00 TTNHE<- 111m 386 , '0' I 10 l l l l l l 0 7:00 8:45 10:00 11:45 13:45 15:45 17:00 19:00 20:30 22:00 TIME OF DAY Figure 3. Diurnal patterns of P , Gs, and (Gm) of greenhouse grown black locust half-Sig families measured outdoors. The values are averages of two plants. 87 .“ _- \ I \ I I \ \ I , \ \ ’ 1 \ \ I ’ \ ‘ I I = u. \ \ ’ ’ I : F'. ' x x ‘I I - , I ’ \ \ I \ I ’ ’ ‘ "..., \ , ‘ I l \ ’ \ ’ ’ ‘0 o ‘ I I \ I I . c . ,, I * am 051 -+— 15m 063 4‘" 15m 20 1" —8— 1am 255 -><- 13m 330‘ -€>- 13m 403 "0 Va (mmol.m’2.s") o l l l L l l l l 40 4 30F .13 —*-TEMP -+-vpo '1': <° C) IN) 0 l M (can) adA 10r , -* n: ~1 .1112 -b '0 0| 0 O O O O l I .5 O O O T l l l l l J l l o 7:00 3:45 10:00 11:45 13:45 15:45 17:00 19:00 20:3022:00 11NH5(DF'EM¥( Figure 4. Diurnal patterns of T, PAR, VPD, and T of greenhouse grown black locust half-sib families measured outdoors. The values are averages of two plants. 88 J .5 01 f - 1 1 1 1 1 1 1 I \ 1 b I I I I I I l I z I I l ' . ,, I I l , . I I . 1 I p 1 I 1 1 I \ l \ 1 1 I 1 § \ ‘ 1 1 1 1 1 1 PI'I (mg coz.dm'2.hr“) 8 1 ) .5 o O 1 —‘— 13m 012 —+— tam 051 '1“ 1am 063 80* —B- 1am 055 -‘- 1am 355 --<>~ 11m 386 \, I“ N \ \ . 4/ \ . .. \ \ 20 ’— --------------------- .44 o l l 1 1 1 7:00 8:30 10:15 14:15 17:15 18:30 20:00 TIME OF DAY Figure 5. Diurnal Patterns of P , Gs, and Gm of greenhouse grown black locust half-Sig families measured outdoors. The plants were kept under 700 1.1mol.111'2.s’1 PAR overnight. The values are averages of two plants. 89 —‘— tam 012 “+— 13111 051 "1‘” tam 063 “9* fam 255 "*" tam 385 '9" 13m 385+ (mmol.m°2.s'1) 4o 4 20" ..2 TI. (0 C) "“‘TEMP -—h-vpo 10“ -1 3000 FA 2500 I. 2000: -2 I 1500 I 1000 500 _ pan (umol.m o 1 1 1 1 1 7:00 8:30 10:15 14:15 17:15 18:30 20:00 11hflEICN:IDAN' Figure 6. Diurnal patterns of T, PAR, VPD, and.T' of greenhouse grown black locust half-sib fami ies measured outdoors. The plants were kept under 700 umol.m'2.s'1 PAR overnight. The values are averages of two plants. (can) 055 90 14 7" 12 1'" L1 43. 10 ~ ‘1‘ “as 8” c;~ er 0 4 U‘ E v 2 - o 1 L 1 1 1 L 1 120 100 r ”K 'm 80* N. Ufa. 60»- :-1 E 40* V 20— O L 1 1 1 1 1 1 40 “*— tam 012 "’1— 13111 051 '*‘ 13m 255 ‘9‘ (am 3 9 + 13111 365 "9" tam 408 A 30- . "_,-.-“ I I ,, H '7 -- ' ------- a". m *’ N. v ....... 9 ------- n 0' 5: 20 - ‘ " H O ‘9 E , 10 * ° 0 1 1 1 J 1 1 1 8:00 9:15 10:45 12:00 14:00 15:30 17:00 18:15 20:00 TIME OF DAY Figure 7. Diurnal patterns of P", G3, and G"I of greenhouse grown black locust half-ib families measured outdoors. The plants were not watered for two days before measurement. The values are averages of two plants. 91 (mmol.mQ.s4) —‘— 13111 012 _t- tam 051 '1‘“ tam 255 1 7 —B* lam 329 -‘*'* 13m 385 '9' tam 408 o 1 1 1 1 1 J 1 40 6 1 1 (5) b 01 30* 20* .. TL (° C) (Bax) GdA 101- * TEMPERATURE "*— VPD * 2 0 *— p.— p— y— b— p.- p— O 3000 I 2500 I 2000 I 1500 I 1000 500* pm (umol.m'2.s") o 1 J 1 1 1 1 1 8:00 9:15 10:45 12:00 14:00 15:30 17:00 18:15 20:00 TIME OF DAY Figure 8. Diurnal patterns of T, PAR, VPD, and T of greenhouse grown black locust half-sib fami ies measured outdoors. The plants were not watered for two days before measurement. The values are averages of two plants. 92 Variation in Pn among families was significant both in the morning and the afternoon. The correlation between Pn and 35in the morning was significant but lower than between Pn and Gm. B. FIELD GROWN PLANTS i. September 24 Ih,peaked at 10:20 or earlier in one family and then decreased gradually at different rates in different families (Figure 9) . TL and PAR at the peak Pn were about 20°C and 2 1respectively, which are sub optimal Tl,and 1050 umol.nf .s' PAR for black locust (Figure 9). In those families where a recovery of Pnixxlate afternoon was observed, the depressions in Pn were at the highest PAR. (The differences in Pn among the families were highly significant (<0.01 probability level) at each hour of the day (data not shown). (R remained high for the first 2 hours of measurement and then declined sharply before levelling off at about 14:00 (Figure 9). The Gsvalues in the morning hours were very high in 4 of the families. The relative humidity readings in the morning hours were 50-70%, while the relative humidities in the afternoon were close to 30%. Consequently, VPD was higher in the afternoon. This day was a mild one with hazy sunshine and a maximum air temperature about 24°C,.and the plants had ample moisture in the pots all day. The correlation between Pu and Gs increased for the first 2 hours and then stabilized at r about .85. 93 Cidecreased continuously during the course of the day, a difference of about 100 ppm between the first and the last measurement hours (data not shown). The changes in Ciseem to parallel the changes in Gm,and the decline in Ciindicate that more reductions in Gs than Gm. The correlation between Ciand stere non-significant and weak at all measurement hours, while the correlation between Ci and Pa were significant at 3 of the measurement hours (data not shown). ii. October 14 F5 increased to a maximum at about 11:00 in most of the families and later in some families (Figure 10). The decline in.Ph from the peaks were gradual and depressions were observed at 14:45 in those families which showed a recovery of'Fh_in late afternoon. The minimum temperature on the night before this measurement day was below freezing temperature. Theimqvalues on this day were low for black locust. TL and PAR at the peak Pn were only 17.5°C and 1200 2.5-1 umol.m' respectively (Figure 10). Pn was not significantly different (.05 probability level) among the families at all measurement hours. The diurnal responses of Gsfollowed similar patterns as Pn(F&gure 10), and the Gsvalues were about a third of the Gs on September 24. This day was colder particularly in the morning hours with a maximum air temperature of 18°C. The leaves did not stretch to horizontal positions until 11:00. There were strong winds, about 30 kms.hr’lall day and 94 the relative humidity readings were about 30% through out the day. The correlation between Pu and Gs declined during the course of the day (data not shown). Ciremained more or less at a similar level, even though there were multiple peaks and depressions in some of the families (data not shown). The correlation between C1 and Pa were non-significant and weak, but the correlation between Ciand Gswere moderate but non-significant (data not shown). iii. October 15 Pn increased with increasing TL and PAR, and the peak Pn were observed at TLand PAR close to the maxima for the day (Figures 11) . Depressions in P" were observed at different hours in the afternoon in some families. The Pn values were slightly higher than those of October 14, which could have resulted from increased.11,and PAR on this day. The seedlings were also kept indoors the night before the measurement day. The diurnal responses of G5 on this day were marked by multiple peaks and depressions (Figure 11). The depressions were observed at different hours in different families. The maximum air temperature on this day was about 2f%:and relative humidities about 45% and 37%, in the morning and afternoon, respectively. The correlations between Pu and Gs were significant at all measurement hours except the first 2 hours (data not shown). 95 (,35 '2111' Ioum) 8Y8 A 25 .H .- £1 20 N c .3 15 m "I N 10 *" 8 L U‘ 5 E o 1 1 1 1 1 1 1 1 1 700 600 — * “M 208 -*- PAM 303 -+' PAM 335 A _e— “M 33 "" PAM 408 "G“ PAM 413 .0) 500 * . N 0 .E 400 * '3 300 ~ 3 E V 200 * 100 - 0 30 1800 + 3 1600 25 * " 1400 20 * ' " 1200 »~ + U 5 1000 ‘1, 15 ~ ‘4 800 d 5‘ 10 — . - 600 -‘— ERATURE 'F" LIGHT TEMP .. 400 5 ... " 200 o 1 1 1 1 I I 1 1 1 o 8:00 9:00 10:20 11:10 11:40 12:10 18:25 14:05 15:15 16:20 17:00 TIME OF DAY Figure 9. Diurnal patterns of P", Gs , PAR, and TL of field grown black locust half-sib families measured on September 24, 1988. The values are averages of three plants. 96 .5 .5 o N I P (mg COz.dm'2.hr'1) o no a. a 1» I 140 I 120 I '7 100 0 ' 80* 60* 40 * “" AM 208 —+- PAM 303 4:- FM. 335 FAM 386 -><- PAM 403 -<>- p“. 41, I 20 25 2000 20* 1 1500 15* 1 1000 10* 1‘: (° C) —‘— TEMPERATURE ‘1— LIGHT 5,. ‘500 l- ( s 'z-m° tom“) 81d 1 1 I 1 1 1 1 l 1 1 0 0 8:00 9:15 10:15 11:00 11:45 12:4513:3014:05 14:45 15:10 15:3518:00 TIME OF DAY Figure 10. Diurnal patterns of P", Gs, PAR, and TL of field grown black locust half-sib families measured on October 14, 1988. The values are averages of three plants. 97 'u 5 QR S of R N o O m E 180 .01 F," 120 — m a N. 100‘ ‘9 s H 30- 2 5 60 ‘ 4° ” -:- PAM 203 —+—- FAM 303 .11. FAM 335 20 _ -B- PAM 333 -— PAM 403 -<>- FAM 415 0 I 1 I 1 I 1 1 l 1 30 '1300 c1400 25* *1200 a. 20 U 1 “1000 O ‘7 15t *800 cl 5 3600 10* 1 400 5“ —“*TEMPERATURE —+-u0HT 1200 o I I 1 I l l I 1 o 8:00 9:15 10:15 11:00 11:40 12:20 13:00 13:35 14:20 15:00 16:00 TIME OF DAY Figure 11. Diurnal patterns of P", Gs, PAR, and TL of field grown black locust half-sib families measured on October 15, plants. 1988. The values are averages of three (l_s-Z_1n°‘[oum) “a 98 Cidecreased early but remained stable in four of the families the rest of the day (data not shown). The correlations of Ci with Pn and Gs were weak and non— significant (data not shown). DISSCUSION The decrease in Enduring the midday hours, commonly referred to as "midday depression" is attributed to stomatal closure (Mooney g; 31., 1975: Tenhunen et al., 1981; Raschke and Resemann, 1986). The evidence for this conclusion are: (1) similar diurnal response patterns for P“ and G5; (2) strong and significant correlations between Pn and G5 during the course of a day or at least during the noon hours: and (3) maintenance of constant Ciduring the day which reflects the proportional decrease or increase in Pu and Gs (Raschke and Resemann, 1986). These 3 observations are interrelated but one has to present data in support of them, since they are not always related. For example, Cican remain constant by reductions in Pntw'direct temperature effect on electron transport (Stidmah _e_t _a_l., 1982) and reductions in Gs by moisture stress. It would be false to assume in this case that the reductions in Pn were caused by reductions in Gs. The mechanisms by which stomatal closure occurs during the midday hours in those species which show midday depression in Pn,(%, or T are not well understood. Raschke and Resemann (1986) reported that midday depression in P“ and G5 is not caused by temperature (20 to 37°C) if VPD was 99 held constant. They suggested that midday depressions occur if a threshold VPD (20-30 mbar) level is exceeded and there is no correlation between the amount of water loss and reductions in P“. Correlations between VPD and midday depression in Pn were also reported by Sinclair and Allen (1982), but they did not separate the effects of VPD and temperature. In fact, they have shown that the larger VPD were observed on days with higher temperatures. Large VPD will increase water loss from a plant and may cause stomatal closure if there is no moisture in the soil or container to support water loss at the same rate. On May 28th, the light level seems to be the main factor limiting Pn. The peak Pn was observed at the highest light level, which is much lower than the saturation light level for greenhouse grown blacklocust (Chapter 1). Diurnal patterns ofimqwhich paralleled the PAR pattern were reported by Grant and Ryugo (1984) for kiwifruit and by Pereira gt al. (1986) for Eucalyptus globulus. In both cases, the PAR levels were below 1200 umicromol.m‘2.s'l, indicating a mild day. The diurnal Pn pattern in this study was also paralleled by Gm,<%, and T, but it does not necessarily mean that the changes in Gm,(k, or T caused the observed Pn pattern. Correlation analysis at each measurement hour showed that Qsand T were not significantly correlated with Pn at all measurement hours, while Gm was significantly and highly correlated with Pn (r2>.88) at all times. I have suggested earlier that Pn in black locust may 100 be limited by G“,(Chapter 3) as do other investigators working with other tree species (Pezeshki and Chambers, 1986; Lapido g; 31., 1934) On May 29th, when I measured the greenhouse grown plants out in the open with maximum temperature up to 35°C, I expected the plants to have very low Pn because of the higher temperatures. What I found was that the response of plants was variable depending on stomatal sensitivity and family. Some plants like members of family 51 which have relatively insensitive stomata maintained higher anand Gs beginning early morning. GS was not reduced at higher TL and VPD which can cause stomatal closure (Tenhunen g; al. 1982). Gs and Gm decreased when the available water in the pots decreased with increasing TL and VPD which eventually reduced Pn. Cherrybark oak (W M var W) plants with insensitive stomata similar to these plants have been observed by Pezeshki and Chambers (1986). Plants which had small leaf area maintained a higher Gs for much of the day.1fi,gradually decreased with increasing TL and VPD and finally Gs was reduced to half the maximum at the highest TL at 15:45. Since Gs was highly reduced relative to Gm, lower Ci were observed at this time. Finally, Pn increased with declining temperature before it decreased due to low PAR. (% and Gnlin plants with larger leaf area were reduced early in the day because of the excessiVe water loss when VPD increased to above 3.0 kPa. But Pn, Gs, Gm, and T 101 increased the hours following the depression even though TL and VPD were increasing. The failure of P", Gs, Gm, and T to decrease with increased Tlfland VPD indicates the involvement of another factor such as accumulation of end products. Two-peaked T and Pa diurnal curves were observed by Schulze gt _1. (1974) for Prunus grmeniaca L. under simulated desert conditions. They suggested that the mid-day depression of T andimlobserved were due to stomatal closure which was regulated by the ambient temperature and air humidity. Similar observations were reported under drought conditions for other species (Mooney gt g;., 1975: Tenhunen gt gt., 1984; Benecke gt gt., 1988: Sinclair and Allen, 1982; Tenhunen gt gt., 1981). These two-peaked diurnal curves for T, P", and G5, were not observed under conditions of optimum temperature and no water stress (Tenhunen gt gt., 1982: Sinclair and Allen 1982). The two-peaked curves I observed for black locust in this study (May 3005 could have resulted because of other factors than described above for at least three reasons: 1) the plants were well watered early morning and there was no indication of moisture stress: 2) the:T1,and VPD were similar to the May 29th measurement day; and 3) Pa at this day was lower particularily at the most optimum condition, the first measurement hour. The correlation analysis showed no correlation between Pu and Gm at 8:30 and 15:15, strong correlation at 14:15 (r2=.88), and weaker but statistically significant (:2=.72 and .58 respectively) correlation at 102 10:15 and 18:30. On the other hand, the correlation between Pu and G5 was significant at all hours except it was weak at the first and last hours (r2=.69 and .79 respectively). The results of correlation analyses suggest that the depression in Pn was caused by a factor(s) other than moisture and temperature stresses by regulating Gs and Gm. Even though the highest.11,and VPD were also recorded at the depression, the very low Gs and Gm values observed on this day which had thexcludes the possibility that similar Ti,and VPD to May 29 11,and VPD were the main factors that caused the depression. But they might have been complementary to the factor(s). I suggest that the factor which shaped the diurnal response was the accumulation of photosynthetic end products (carbohydrates). Since the plants were kept under light overnight, the leaves may have already accumulated carbohydrates when gas exchange measurements were begun in early morning. In order for the photosynthetic carbon reduction cycle (PCR cycle) to function well, inorganic phosphate (Pi) has to enter the chloroplasts in exchange for a photosynthetic product, dihydroxyacetone phosphate (DHAP). The Pi is a product of sucrose synthesis in the cytoplasm and starch synthesis in the chloroplast and is used in ATP formation in the process of photophosphorylation. If there is no strong sink to absorb the sucrose formed due to lag in translocation, sucrose synthesis slows, thereby limiting the external Pi pool which will be imported in to the chloroplasts and 103 allocating more triphosphates for starch synthesis. With limited Pi in the stroma of chloroplasts, ATP formation is reduced and eventually ribulose bisphosphate regeneration by the PCR cycle and COzfixation is inhibited. This is what probably happened during the morning hours. A decrease in Pn because of Pi deficiency was reported (Dietz and Foyer, 1986: Foyer and Spencer, 1986: Morison and Batten, 1986).The other effect of limited Pi and thus higher phosphoglycerate (PGA):Pi ratio inside the chloroplasts is the allosteric activation of ADP-glucose pyrophosphorylase, an enzyme involved in starch synthesis within the chloroplasts. Starch synthesis generates Pi at a rate 3 to 4 times lower than maximal COzfixation (Robinson and Walker, 1981). Chatterton (1973) also demonstrated a negative correlation between SLW andlmnduring the course of a day, and suggested the feed-back inhibition of Pn by lag of translocation. A highly significant correlation was demonstrated between starch content of the guard cells and pore width or leaf resistance by Yemm and Willis (1954). Morison and Batten (1986) have observed reductions in Gs because of D-(+)-mannose induced cytoplasmic Pi deficiency. That is probabily why Gsvalues were very low at the depression. Thorne and Koller (1974) have shown that Pu increases with reduction in starch content and suggested that the rise in Pn was the result of an increase in Gm. Gm is the sum of the physical diffusion of C02 in the leaf intercellular spaces to the site of carboxylation and the 104 biochemical incorporation of C02 in the carboxylation reaction (Comstock and Ehleringer, 1984). Thus, Gm can decrease from starch accumulation as a result of decreased C02 fixation due to reduced Pi as described above and increased physical hinderance to C02 diffussion (Thorne and Koller, 1974). During the few hours of low'Pm, the excess sucrose will be utilized in respiration and other metabolic processes to lead to normalization of the Pi/DHAP exchange. The increased Piwill stimulate starch degredation (Preiss gt a1., 1982). Turner gt g1. (1985) showed that gas exchange of Helianthus gggtg is controlled by the availability of water in the soil rather than leaf water potential. Similar observations were reported for the woody species Nerium oleander (Gollan gt _t., 1985). Apparently there is a plant growth factor, that is produced in the roots at some critical soil or root water potential that affects the photosynthetic or stomatal mechanisms. Sinclair and Allen (1982) also suggested the importance of the rate of water supply in regulating Osand the resultant midday depression in Pm.Tmey were able to separate the days with and without midday depression by ranking according to midday VPD. The VPD above which midday depression occurred was 3.0 kPa, but it was even lower in some daYs. In this study of water stressed black locust plants (June 18), I did not observe midday depression despite the fact that the average VPD at midday was close to 4 kPa with,32°Cfn;.Instead the diurnal 105 1%,curves were of two types one with an afternoon recovery from depression late in the afternoon and the second with no afternoon recovery. The decline in Pn with time can be attributed to a decrease in G5 and Gm which could have resulted from decreasing available water and increased TL and VPD. Tenhunen gt g1. (1982) have suggested that water stressed plants do not show an afternoon peak in conductance. Those plants which showed an afternoon recovery had a lower Pn,(%,