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"' 1659:}; ‘17-‘55”: ! “”3“.“ _ ?\wlq@b’7 MICHIGAN STATE II II III III IIIIIIII IIIIIIIIIIIIIIIIL " ' ‘ 300548 0284 ~L:BRARY Michigan State University This is to certify that the dissertation entitled THE EFFECT OF FRUIT REMOVAL ON LEAF PHOTOSYNTHESIS, WATER RELATIONS, AND CARBOHYDRATE PARTITIONING IN SOUR CHERRY AND PLUM. presented by Riccardo Gucci has been accepted towards fulfillment of the requirements for Ph. D. degree“, Horticulture : 5 Major professor Date May 15, 1988 MSU is an Affirmative Action/Equal Opportunity Institution 0- 12771 M._.._ _ MSU LlBRARlES ‘ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. THE EFFECT OF FRUIT REMOVAL ON LEAF PHOTOSYNTHESIS, WATER RELATIONS, AND CARBOHYDRATE PARTITIONING IN SOUR CHERRY AND PLUM BY Riccardo Gucci A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1988 gaaflazo ABSTRACT THE EFFECT OF FRUIT REMOVAL ON LEAF PHOTOSYNTHESIS, WATER RELATIONS, AND CARBOHYDRATE PARTITIONING IN SOUR CHERRY AND PLUM BY Riccardo Gucci The effect of fruit removal on photosynthesis and gas exchange parameters of leaves from field-grown, mature sour cherry (Prunus cerasus L. 'Montmorency') and plum (arugus domestic; L. 'Stanley') trees was studied with respect to environmental conditions, and carbon and water status of the leaf. Removal of fruits at the end of stage III caused an immediate (within 48 h) decrease of net photosynthesis (Pn) equal on average to 41% in 1985, 26% in 1986, and 27% in 1987 for sour cherry, 25% in 1986 and 0% in 1987 for plum. Removal of plum fruits during stage II reduced Pn 32% in 1986 and 16% in 1987. Pn inhibition after fruit removal persisted 2-4 wks in sour cherry, but only 7-10 days in plum when fruits were removed during stage II. In both species Pn inhibition was greatest in the afternoon, lower or negligible in the morning. Pn decrease of defruited trees was paralleled by similar, simultaneous changes in stomatal conductance (gs), whereas calculated levels of intercellular C02 (Ci) did not change significantly after fruit removal. Defruiting did not significantly affect chlorophyll content or leaf water potential, but defruited trees had lower osmotic potentials. Stomatal closure and lower osmotic potentials of defruited trees did not appear to be related to loss of leaf turgor. Non-stomatal contribution to the post-harvest decrease of Pn calculated from field and laboratory measurements of assimilation-internal COZ-response curves ranged from 55% to 76%. Photorespiration rates were similar for fruiting and defruited plum trees, while in sour cherry Pn inhibition due to fruit removal decreased from 27% of the control at 21% 02 to 14% at 2.5% 02. Levels of soluble sugars (fructose, glucose, inositol, sorbitol, and sucrose) in the leaf were not significantly different before and after fruit removal. Leaf starch content increased 2-3-fold within 24 h from fruit removal. Analysis of transmission electron micrographs of leaf mesophyll cells of sour cherry did not reveal disruption of thylakoid membranes due to enlargement of starch grains in the chloroplast. The results are interpreted as evidence for sink- induced inhibition of leaf Pn after fruit removal and support the hypothesis of feedback regulation of Pn by levels of non-structural carbohydrates in the leaf. ACKNOWLEDGEMENTS I am grateful to my major professor Dr. J. A. Flore for his direction, support, and criticism during the course of my graduate studies. I would also like to thank Drs. M.J. Bukovac, A.C. Cameron, F.G. Dennis, D.I. Dickmann, K.L. Poff, and A.J.M. Smucker for serving on my guidance committee, Mr. P.D. Petracek for helping me with the Transmission Electron Microscope, and Ms. L.E. Teichman for her general assistance in the laboratory. ii Guidance Committee: The journal paper format was chosen for this dissertation in accordance with departmental and university regulations. The dissertation is divided into four sections and an appendix. Section one and two are intended for publication in The Journal of the American Society for Horticultural Science, sections three and four are intended for publication in Physiologia Plantarum. TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . vii LIST OF FIGURES . . . . . . . . . . . . . ix LIST OF ABBREVIATIONS . . . . . . . . . . . xv INTRODUCTION . . . . . . . . . . . . . . 1 Section I The Effect of Fruit Removal on Photosynthesis and Water Relations of Sour Cherry . . . . . . . . 3 Abstract. . . . . . . . . . . . . . 4 Introduction . . . . . . . . . . . . 6 Materials and Methods . . . . . . . . . 8 Results . . . . . . . . . . . . . . 16 Discussion . . . . . . . . . . . . . 41 Literature Cited . . . . . . . . . . . 47 AbbrEViations O O O O O O O O O O O O 51 Section II Non-Stomatal Limitations, Photorespiration, and Carbohydrate Partitioning during Inhibition of Photosynthesis following Fruit Removal in Sour Cherry 52 Abstract. . . . . . . . . . . . . . 53 Introduction . . . . . . . . . . . . 54 Materials and Methods . . . . . . . . . 55 Results 0 O O O O O O O O O O O O O 63 iv Discussion . . . . . . . . . . . Literature Cited . . . . . . . . . Abbreviations . . . . . . . . . . Section III Diurnal and Seasonal Changes in Leaf Photosynthesis following Fruit Removal in Plum. The Influence of Stage of Fruit Development Environmental Conditions . . . . . . . . Abstract. . . . . . . . . . . . IntrOduction O O O O O O O O O 0 Materials and Methods . . . . . . . Results 0 O O O O O O O O O O 0 Discussion . . . . . . . . . . . Literature Cited . . . . . . . . . Abbreviations . . . . . . . . . . Section IV Diurnal and Seasonal Changes in' Leaf Photosynthesis following Fruit Removal in Plum. Net II. Water Relations, Photorespiration, and Partitioning of Non-Structural Carbohydrates in the Leaf . Abstract . . . . . . . . . . . . Introduction. . . . . . . . . . . Materials and Methods. . . . . . . . Results . . . . . . . . . . . . Discussion . . . . . . . . . . . Literature Cited . . . . . . . . . Abbreviations . . . . . . . . . . Page 67 79 84 85 86 88 90 93 111 117 120 121 122 124 126 131 157 163 168 Page SUMMARY AND CONCLUSIONS. . . . . . . . . . . 169 REFERENCES 0 O O O O O O O O O O O O O O 17 3 Appendix. 0 o o o o o o o o o o o o o o 184 vi Table LIST OF TABLES Section I Angles of mature leaves from fruiting and defruited sour cherry trees measured 7 days after removal of fruits at the HRC, East Lansing, in 1985. O O O O O O O O O O O O O O 0 Inhibition of photosynthesis in leaves from spurs or terminal shoots of sour cherry following harvest of mature fruits, HRC, East Lansing, 1985 Changes in net photosynthesis (Pn), stomatal conductance (g ), and intercellular C02 (Ci) of fruiting and deIruited sour cherry trees located at .the HRC, East Lansing, in 1986 . . . . . Chlorophyll content in fruiting and defruited sour cherry trees, HRC, East Lansing, in 1987. . Leaf water potential, osmotic potential, and turgor pressure of fruiting and defruited sour cherry trees, HRC, East Lansing, in 1987 . Section II Coefficients of estimated demand (a) and supply (b) gas exchange curves and relative coefficients of determination for fruiting and defruited treatment, HRC, 1987 . . . . . . . . . . Photorespiration of fruiting and defruited sour cherry shoots measured in the laboratory and in the field at HRC, East Lansing in 1987 . . . . Section III Increase in shoot elongation and cross sectional areas of limbs from fruiting and defruited plum trees. Date of harvest: June 15, 1986 . . . . Page 25 26 30 37 38 61 66 99 Table Total sink strength of plum fruits and amount of Pn inhibition following partial, localized harvests at fruit maturity . . . . . . . . Correlation coefficients (r) between changes in Pn and gS calculated from time course and diurnal measurements in plum . . . . . . . . . . Section IV C02 assimilation rates of fruiting and defruited plum trees measured at ambient (21%) or low (2.5% or 0.5%) 02 concentration in the field and in the laboratory . . . . . . . . . . . . . Appendix Changes in chlorophyll content of leaves from fruiting and defruited plum trees at HRC, East Lansing. Fruits removed on June 16, 1987 . . . Diurnal changes in chlorophyll content in leaves from fruiting and defruited plum trees 1 day after fruit removal. Fruits removed on June 16, 1987 O O O O O O O O O O O O O O 0 viii Page 112 113 144 186 187 LIST OF FIGURES Figure Section I Schematic representation of leaves and fruits on excised shoots from fruiting and defruited sour cherry trees used for 4C pulsing. . . . . . Growth curve of sour cherry fruits in 1987. Date of full bloom: April 26, 1987. Date of fruit harvest: July 7, 1987 (indicated by arrow). Vertical bars indicate 1 SE (n=25) . . . . . Minimum and maximum temperatures during the course of field gas exchange measurements in 1985 (a), 1986 (b), 1987 (c). Arrows indicate the date of harvest. Needle bars indicate daily rainfall, squares at the top of the graph indicate cloud cover ( lcloudy: npartially cloudy; casunny). The number shown at the right of each graph denotes monthly precipitation in mm Changes in net photosynthesis (a), stomatal conductance (b), and intercellular CO (c) of fruiting and defruited sour cherry trees in 1985. Date of harvest: July 3, 1985. Vertical bars are i SE (n=12-20). Measurements started between 1230 and 1430 hr . . . . . . . . . . . The effect of different leaf/fruit ratio on net photosynthesis of fruiting and defruited sour cherry trees in 1985. Symbols are means of 6-10 leaves. Vertical bars indicate Least Significant Differences at the 5% level. . . . . . . . Changes in net photosynthesis (a), stomatal conductance (b), and intercellular C02 (c) of fruiting and defruited sour cherry trees located at NWHES, Traverse City, in 1986. Date of fruit harvest: July 15, 1986 (indicated by arrow). Vertical bars indicate i SE (n=18-20). Pre- harvest measurements started at 1400 hr; post- harvest measurements started for each date at 1300, 900, 1500, and 900 hr respectively . . . ix Page 15 18 20 23 28 33 Figure 7. Morning (a, c, e) and afternoon (b, d, f) changes on net photosynthesis (a,b), stomatal conductance (c,d), and intercellular C02 (e, f) of leaves from fruiting and defruited sour cherry trees in 1987. Date of harvest: July 7. (indicated by arrow). Morning measurements started between 930 and 1130 hr: afternoon measurements started between 1300 and 1500 hr. Vertical bars are 1 SE (n=16). . . . . . . . . . . . Pattern of carbon translocation in shoots excised from defruited (A, B, C, D) and fruiting (E4 F, G, H) mature sour cherry trees in 1987. C pulsing performed 3, 4, 10, and 11 days after harvest. (A, B, E, G) represent patterns in the upper half of the shoot, (C, D, G, H) in the lower half. Pulsed leaves indicated by arrows (see also Figure 1) . . . . . . . . . . Relationship between leaf net photosynthesis (Pn) and stomatal conductance (gs) for fruiting and defruited sour cherry trees in the morning (a, c) and afternoon (b, d) of 1986 (a, b) and 1987 (c, d). Each symbol is the mean of 6-10 observations. 1986 measurements were made at NWHES, Traverse City, Michigan; 1987 measurements were made at HRC, East Lansing, Michigan . . . . . . . Section II Schematic diagram of gas-exchange equipment used to measure C02 response curves and photorespiration in the field. Abbreviations: ADC, Analytical Development Co, Hoddesdon, England; IRGA, infrared gas analyzer. . . . . C02 response curves of fruiting and defruited sour cherry trees measured with an open gas exchange system (Sams and Flore 1982). Measurements made in the laboratory 9 days after fruits were harvested. Date of fruit harvest: July 7, 1987. Environmental conditions: leaf T 28-29°C, dew point of ingoing air 0.6°C, ambient C93 £58m 340 pl 1—1 to 50 pl 1- , PAR=1000 pmols m s . Equation at top of the graph refers to the model used to fit demand curves for gas exchange. Gas supply curves were fitted by linear regression using g values of fruiting and defruited treatment at 343 pl 1' . . . . . . Page 35 4O 44 58 65 Figure 3. Changes in leaf content of soluble sugars after fruit harvest in sour cherry: a) 1986, NWHES, Traverse City, Michigan; b) 1987, HRC, East Lansing. Arrows indicate date of harvest. Soluble sugars were: fructose, glucose, inositol, sorbitol, and sucrose. Vertical bars i SE . . . Changes in leaf starch content after fruit harvest in sour cherry: a) 1986, NWHES, Traverse City, Michigan; b) 1987, HRC, East Lansing, Michigan. Arrows indicate date of harvest. Vertical bars): SE. . . . . . . . . . . Transmission electron micrographs of palisade (A, B) and spongy (C, D) mesophyll cells of leaves collected from sour cherry trees 1 h before (A, C) and 24 h after (B, D) fruit removal. Legend: c. chloroplast: s. starch grain; w. cell wall. Magnification: A. 5800; B. 4100; C. 11800; D. 6300 . . . . . . . . . . . . . . . Transmission electron micrograph of an enlarged starch grain in the chloroplast of a spongy mesophyll cell from leaves of sour cherry 24 h after fruit removal. Sampling at 1400 hr. Legend: c. chloroplast; p. plastoglobule: s. starch grain; w. cell wall. .Magnification: 57700 . . Section III Fresh and dry weights of plum fruits in 1986 and 1987. Full bloom dates: April 27, 1986: April 29, 1987. Arrows indicate time of fruit removal. Vertical bars indicate 1 SE (n=20-25) . . . . (a, c) Minimum and maximum temperatures, precipitation, and cloud cover during the first period (June) of fruit removal in 1986 and 1987. Arrows indicate date of harvest. Needle bars indicate daily rainfall, squares on top of the graph cloud cover ( l cloudy; apartially cloudy: D clear). Thick bars separate months; the number shown at the end of the month denotes monthly precipitation. (b, d) Inhibition of photosynthesis after fruit removal at stage II of development. Vertical bars indicate 1 SE. . . . . . . . xi Page 69 71 73 75 96 98 Figure 3. (a, c) Environmental variables during the second period (September) of fruit removal in 1986 and 1987. Symbols are the same as in Figure 2a. (b,d) Changes of photosynthesis after removal of mature fruits in 1986 and 1987. Vertical bars indicate 1 SE 0 O O O O O O I O O O O O O O 0 Changes in photosynthetic rate (Pn), stomatal conductance (gs), and intercellular CO (Ci) following defruiting during stage II in the morning and afternoon. Vertical bars i SE (n=18-20). . . . . . . . . . . . . . Changes in photosynthetic rate (Pn), stomatal conductance (gs), and intercellular C0 (Ci) following defruiting during stage II in the morning and afternoon. Gas exchange parameters expressed as percentage of the mean of preharvest values . . . . . . . . . . . . . . Diurnal changes in net photosynthesis (Pn), stomatal conductance (gs), and intercellular C02 (Ci) of leaves from fruiting, defruited, or girdled shoots of field-grown plum trees. Branches defruited on June 15, 1986. Different letters indicate significant differences between treatments at each single date. . . . . . . Section IV Changes in photosynthetic rate (Pn), transpiration (Tr), and water use efficiency (WUE) following removal of plum fruits at stage II during the morning and afternoon in 1987. Arrows indicate date of fruit removal. Vertical bars 1 SE (n=16-20) . . . . . . . . . . Diurnal changes in net photosynthesis (Pn), transpiration (Tr), and water use efficiency (WUE) of leaves from fruiting and defruited plum .trees 1 day after fruit removal. Fruits were harvested during stage II of development (June 15, 1986). Verical bars i SE (n=6-8) . . . . Effect of defruiting on leaf water potential (solid line), osmotic potential (dashed line), and turgor pressure (TP) of fruiting and defruited plum trees. Fruits removed on June 17, 1987. Arrows indicate date of fruit removal. Vertical bars + SE (n=4-9) . . . . . . . . xii Page 102 105 107 109 134 136 138 10. Diurnal changes in leaf water potential (solid line), osmotic potential (dashed line), and turgor pressure of fruiting and defruited plum trees 1 day after removal. Fruits removed on June 17, 1987. Arrows indicate date of fruit removal. Vertical bars i SE (n=4-9) . . . . . . . . Changes in net photosynthesis (Pn)(circles) and stomatal conductance (gs)(triangles) in relation to calculated levels of intercellular C0 (Ci) for leaves of fruiting (open symbols? and defruited (solid symbols) plum trees 1 day after fruit removal in 1987. Measurements made in the field at ambient C02 ranging from 100 pl 1"1 to 345 pl 1'1, and 02 concentration equal to 0.5% . Gas chromatographic analysis of soluble sugars from fully expanded leaves of field-grown plum trees and -phenyl-D-glucopyranoside (internal standard) using trimethylsilyl derivatives. a. fructose. b. sorbitol. c. glucose. d. inositol. e. E-phenyl-D-glucopyranoside. f. sucrose. . . Content of hexose (fructose, glucose), translocatable (sorbitol, sucrose) and total soluble sugars of leaves from fruiting (open circles) and defruited (solid circles) plum trees during the morning and afternoon. Fruits removed on June 16, 1987 (date indicated by arrow). Vertical bars 1 SE . . . . . . . . Diurnal changes in soluble sugar content of leaves from fruiting (open circles) and defruited (solid circles) plum trees 1 day before (a) and 1 day after (b) fruit removal. Fruits were removed on June 16, 1987. Hexose sugars are fructose and glucose, translocatable are sorbitol and sucrose: total sugars also include inositol and raffinose. Vertical bars 1 SE. . . . . . . . . . . Changes in leaf starch content of fruiting and defruited plum trees in the morning and afternoon. Arrows indicate date of fruit removal. Vertical bars 3 SE (n=3-6) . . . . . . . . Diurnal changes in leaf starch content of fruiting and defruited plum trees 1 day before (a) and 1 day after (b) fruit removal. Fruits removed on June 16, 1987. Vertical bars i,SE . . xiii 140 142 146 148 150 152 154 Figure 11. Diurnal changes in starch content of leaves from fruiting, defruited, and girdled fruiting shoots of field-grown plum trees 4 days after fruit removal. Fruits were removed on June 15, 1986. Different letters indicate significant differences between treatments at each single date (P=0005) O O O O O O O O O O I 0 Appendix Gas chromatographic analysis of soluble sugars from fully expanded leaves of field-grown sour cherry trees and p-phenyl-Dglucopyranoside (internal standard) using trimethylsilyl derivatives. Bar indicates retention time . . . Diurnal changes of Pn and g8 of leaves from fruiting and defruited shoots of field-grown plum trees 1 day after fruit removal. Branches defruited on June 16, 1987. Vertical bars 1 SE. Sectors of the canopy defrrited and measured for photosynthesis during Experiment 1 of partial defruiting of plum trees in 1986 (schematic) . . Sectors of the canopy defruited and measured for photosynthesis during Experiment 2 of partial defruiting of plum trees in 1986 and 1987 (values shown refer to percentages in 1986; schematic). Changes in Pn, Tr, and WUE following removal of plum fruits during stage II in the morning and afternoon in 1987 expressed as percentage of pre- harvest values. Arrows indicate date of fruit removal . . . . . . . . . . . . . . Diurnal changes in Tr, and WUE of leaves from fruiting and defruited plum trees 4 days after fruit removal. Fruits removed on June 15, 1986 . Gas chromatographic analysis of soluble sugars of standard solutions prepared as described in Section IV (Materials and Methods). Bar indicates retention time . . . . . . . . . . . . xiv Page 156 185 189 191 193 195 197 199 A/Ci Ca CE Ci CSA GC gs IC L/F Lg J OP PAR Pi Pn R RH RuBP Rubisco T TEM TP Tr VPD WP WUE LIST OF ABBREVIATIONS CO assimilation response curve to internal CO2 a ient CO carboxylation efficiency intercellular C02 cross sectional area stomatal contribution to a change in Pn stomatal conductance cloudiness index leaf to fruit ratio stomatal limitation to Pn molar concentration of cell sap leaf osmotic potential photosynthetic active radiation inorganic phosphate net photosynthesis gas constant relative humidity ribulose bisphosphate ribulose bisphosphate carboxylase/oxygenase air temperature transmission electron microscopy turgor pressure transpiration vapor pressure deficit leaf water potential water use efficiency XV INTRODUCTION In order to maximize yields and resistance to various stresses of fruit trees it is necessary a better understanding of the relationship between sources and sinks for assimilates. Compensatory changes of Pn in response to source-sink manipulations and environmental conditions have been shown to occur in several species. Evidence in favor or against changes of leaf net photosynthesis after removal of the reproductive sink has been reported for many. Erunus species. However, data reported in the literature are seldom comparable because of differences in plant material (species, age, cultural practices), experimental methods, and/or environmental conditions. In addition, in only a few cases a clear distinction has been made between the effect on photosynthesis due to the elimination of the reproductive sink (blossoms, flowers, or fruitlets at an early stage), and changes of photosynthesis caused by removal of fruits when they are strong sinks for assimilates. These two situations' may have a different impact on gas exchange parameters of the tree. In fact, if compensatory changes of Pn occur in response to altered sink strength, then the stage at which fruits are removed is critical to detect any effect of the fruit on gas exchange variables. Determining the physiological response of fruit trees to removal of fruits in the field is important not only to optimize cultural practices like pruning, fruit thinning, and irrigation, but also to understand phenomena like June drop and alternate bearing. For this reason, gas exchange parameters, water relations, and carbohydrate partitioning were measured when developing or mature fruits were removed from sour cherry or plum trees in the field. Sour cherry and plum were chosen because of their different rate of fruit growth and period of development that allowed to study the "fruit effect on Pn" under different climatic conditions during the growing season. The objective of this study was to characterize the physiological and environmental conditions under which the "fruit effect" can be detected in sour cherry and plum and to determine the mechanism of regulation of photosynthesis by the fruit. Section I THE EFFECT OF FRUIT REMOVAL ON PHOTOSYNTHESIS AND WATER RELATIONS IN SOUR CHERRY. ABSTRACT Removal of mature fruits from field-grown sour cherry trees (Prunus cerasus L. cv. 'Montmorency') caused an immediate (within 24-48 h) decrease in leaf net photosynthesis (Pn) over 3 growing seasons. Pn inhibition averaged to 41% in 1985, 0% and 51% in 1986, and 27% in 1987 (afternoon measurements). The decrease of Pn appeared as early as 24 h after harvest and was particularly evident in the afternoon, or when air temperatures were above 28 C and light intensity high (PAR 1500-2000 Pmols m‘2 s'l). Pn rates of harvested trees remained significantly lower than fruiting ones for 2-3 weeks after removal of fruits. The decline of Pn after fruit removal was similar in spur and terminal leaves and was not associated with changes in leaf chlorophyll content. Pn rates were higher in both treatments when the source was limited with respect to sink strength (leaf/fruit ratio < 2). Despite symptoms of apparent wilting of the foliage after fruit harvest, there was no significant decrease in turgor due to treatment. Changes in Pn rate were paralleled by similar, simultaneous changes in stomatal conductance (gs). Stomatal conductance of trees with fruits removed also diminished sharply in the afternoon. Calculated levels of intercellular C02 (Ci) remained approximately constant (190-220 pl 1'1) for both treatments during the course of the experiment. In addition, Ci of defruited trees was usually equal to or higher than that of fruiting trees. Therefore, both stomatal and non stomatal factors seem to be responsible for the observed inhibition of leaf photosynthesis following fruit removal. INTRODUCTION The leaves of sour cherry trees appear cupped after fruits are removed, as if the tree is under water stress (Flore and Gucci 1986). Fruit removal has been reported to cause a decrease of photosynthesis in many annual (Hall and Milthorpe 1975: Kriedemann et al. 1976: Lenz 1979; Plant and Mayoral 1984) and perennial species (Downton et al. 1987; Fujii and Kennedy 1985; Lenz 1979; Schaffer et al. 1987). On the other hand, removal of flowers or fruitlets (stage I) only slightly reduced leaf photosynthesis of peach (DeJong 1986), sweet cherry (Roper et al. 1988), and sour cherry (Sams and Flore 1983). Other physiological responses to fruit removal include decreased stomatal conductance and increased leaf water potential (DeJong 1986; Downton et al. 1987; Kriedemann et al. 1976; Schaffer et al. 1987), altered levels of phytohormones (Hoad et al. 1977: Kriedemann et a1. 1976: Setter et al. 1980), and altered carbohydrate partitioning within the leaf and among different plant organs (Hall and Milthorpe 1978; Roper et al. 1988; Schaffer et al. 1987). The decrease in stomatal conductance and the change in Pn appear to be simultaneous, but it is still not clear whether the decrease in Pn is caused by stomatal closure or also by nonstomatal factors. Further, is stomatal closure driven directly by substances translocated to or from the fruit or by changes in carbon metabolism? Since ABA accumulation in detached grapevine leaves may be triggered by loss of turgor (Loveys and During 1984), extended periods of water stress may account for ABA-induced stomatal closure and the cupped appearance of sour cherry leaves after harvest. On the other hand, Pn regulation by carbon metabolism would be achieved either because high levels of intercellular C02 cause stomatal closure, or because of direct feedback inhibition in the photosynthetic reduction cycle to regenerate ribulose-1,5-bisphosphate. Field studies on the effect of fruit removal on Pn of woody species are often inconclusive because experimental conditions are not well defined. Yet, environmental and experimental conditions may limit Pn rates of woody species more than internal mechanisms (Nelson 1984). Pn rates of sour cherry, under controlled or natural conditions, may be limited by both environmental and internal factors (Flore and Sams 1986; Sams and Flore 1983; Roper et al.1988). In this study I report on the effect of fruit removal on gas exchange of sour cherry, with particular emphasis on the interaction between changes in photosynthetic response and environmental parameters, and differences in source to sink (leaf/fruit) relationships. Further, I have attempted to determine if the wilted appearance of sour cherry foliage after fruit harvest was due to water stress. Fruit removal was performed at maturity, when the fruit still presented a relatively strong sink. To assess environmental variability, fruit removal was repeated over 3 growing seasons . MATERIALS AND METHODS Plant material. Three pairs of adjacent sour cherry trees (Prunus cerasus L. cv. Montmorency on Mahaleb rootstock), planted in 1979, were selected similar in size, vigor and crop load in an orchard at the Horticultural Research Center (HRC) of Michigan State University, East Lansing (latitude 43°N, altitude 288 m) in spring 1985. Rows were oriented north-south, trees were spaced at 8.2 x 3.0 m, and soil type was a Miami loam of pH = 5.5-6.0. Six trees were used in 1985, four in 1986 and 1987. In 1986 additional measurements were taken on four trees, spaced 2.0 x 6.0 m, planted in 1981 on an Emmet sandy loam (pH=6.2) at the Northwestern Horticultural Experiment Station (NWHES), Traverse City (latitude 45°N, altitude 245 m). At both locations, trees were randomized in blocks, each containing 2 trees. Pesticides were applied according to commercial recommendations (Mich. Ext. Bul. E154, Fruit Pesticide Handbook). Pruning, fertilization, and other cultural practices were performed according to standard practices. Leaffifruit ratioI fruit growth and removal. Leaf/fruit ratios for each tree were estimated yearly by counting leaves and fruits on 4-5 limbs on the east and west sides of the canopy of each tree after fruit set (mid stage II of fruit development). Leaves from these same shoots were later measured for gas exchange, water relations and chlorophyll content. Shoots for gas exchange measurements were chosen based on position, girth, length, and crop load. Leaf lamella angles were measured with a protractor 7 days after fruit removal, considering the leaf midvein as the origin of the angle, and the 2 half blades as the sides. Fruits (n=20-25) were collected weekly from about 35 days after full bloom until fruit maturity for growth measurements. Estimated dates of full bloom at the HRC location were: April 24, 1985; April 26, 1986; April 26, 1987. Fruits were transported, sealed in a plastic bag under ice, to the laboratory where fresh weights were measured with a Mettler AB 163 analytical balance (Mettler Instruments AG, Greifensee, Switzerland). Longitudinal, cheek and suture diameters were measured with a precision caliper. Dry weights were determined after drying the fruits to constant weight at 105°C. During this study 2 trees were completely harvested at fruit maturity each year. Dates of fruit harvest were: July 3, 1985: June 30, 1986; July 7, 1987. One additional tree was defruited on July 14, 1985. Trees were harvested by hand from 1700 to 2100 hr. Two trees were also harvested at NWHES on July 16, 1986. Yields per tree amounted to 25 and 36 kg in 1985, 13 and 7 kg in 1986, and 44 and 30 kg in 1987 (HRC, East Lansing). Envirdnmental sparameters. Values of minimum/maximum temperatures, rainfall and degree of cloudiness were taken from daily weather records from each station. 10 figs exchange parameters. Leaf net photosynthesis (Pn), stomatal conductance (gs), PAR, leaf temperature, and air relative humidity (RH) were measured with a portable open gas exchange system equipped with a Parkinson broad leaf chamber (model ADC LCA-2, Analytical Development Co., Hoddesdon, England) operated at the following conditions: saturating light intensity (PAR > 1000 pmols 111'2 5'1), ambient C02 of 330-340 pl 1'1, flow rate 0.4 l/min, inlet RH 6-11%, unless otherwise stated. The cuvette was clamped onto the leaves and readings were taken after differential C02 had equilibrated (within 30-50 seconds). Relative humidity of the outgoing air and cuvette temperature were also monitored for calculation of gs. One spur leaf and one leaf apical to the fruit on 1-year-old wood were measured on 3-5 shoots per tree for a total of 12-20 leaves per treatment at each determination. Leaves were tagged at the beginning of the experiment and subsequent measurements were made on the same leaves. Adjacent leaves were chosen in those cases in which physical damage had occurred. Pn measurements started between 1200 and 1600 hr in 1985. In 1986 and 1987, weather permitting, Pn was measured on opposite sides of the canopy twice a day (morning and afternoon). Leaf photosynthesis, conductances and internal C02 were calculated as previously described (Moon and Flore, 1986). The experimental component of changes in gas-exchange parameters was calculated by subtracting the decrease (or summing the increase) of the 11 control value for that specific variable from the change observed for the defruited treatment. The experimental design was a randomized complete block. Treatment comparisons were made at each date by ANOVA, where appropriate, and means were separated by LSD or SE. ea chloro h . Chlorophyll content was determined according to the method of Moran (1982). Five to ten leaves per tree were collected at each time of Pn measurement, and immediately frozen in dry ice. Three l-cm diameter discs per sample were punched from the lamella, weighed, and suspended in N,N-dimethyl-formamide (0.5-0.6% w/v). The remaining tissue was stored at -17°C until the osmotic potential was determined. Analyses were repeated 3 times per tree. Leaf discs were recovered after extraction and their weights recorded after drying to constant weight at 105° C. Results were calculated on the basis of both weight and area. Leaf potentials. Leaf water potential of 3 to 4 leaves per tree was determined with a portable pressure bomb (PMS Instrument Company, Corvallis, Oregon) at each time of Pn measurement. Leaves were similar in size, position on the spur _or shoot, distance from fruits, apparent turgor, and exposure to light to those used to measure photosynthesis. Two procedures for water potential measurement were employed. That of Turner and Long (1980) was used in 1986 12 and 1987, whereby the leaf was enclosed in a zip-seal plastic bag prior to collection. In 1985 the leaf was harvested without using the zip-seal plastic bag. The time between detachment of the leaf and placing it‘ into the pressure chamber never exceeded 30 seconds. Comparisons between water potential of harvested and fruiting trees were not affected by the change in method. Leaf osmotic potential was measured on frozen tissue leftover from chlorophyll analysis. Major veins were excised, the thawed tissue was placed in a 5 cc syringe, and 10 ul of the cell sap was forced out with the plunger and used for solute determination with a vapor pressure osmometer (5500 M2448 Wescor Inc, Logan, Utah) at 37° C. Solute concentration (mmol/kg) was converted into osmotic potential using the Van't Hoff equation (OP= R x T x J), where R= gas constant, T= temperature at which the measurement was made, and J= molar concentration of cell sap. Osmotic potential determinations were made in triplicate for each tree at each date. Turgor pressure was calculated as the difference between water and osmotic potentials. 14C studies. Shoots from defruited and control trees were excised at the base between 800 and 900 hr and immediately transported with bases in water to the laboratory (< 10 min.), where they were recut under water to a maximum length of 45 cm and allowed to equilibrate in a stainless steel 13 ventilated hood for 4 hours (light intensity = 600-700 pmols '2 s‘l; T = 25°C; ambient coz= 350-370 p1 1’1). Light was m provided by four 400 W high pressure sodium vapor lamps (General Electric, LU 400/40). Three leaves from the median portion of the shoot were chosen so to have 3-4 leaves apical and 4-5 basal to them. Fruiting shoots bore 5 to 10 fruits in the lower part of the shoot (Figure 1). 14C02 pulsing was performed as described by Kappes (1985) with the following modifications: a) three adjacent leaves were enclosed in bags of approximately 1.5 liter capacity containing ambient air. Bags were prepared from sheets of Mylar 30 (polyethylene terephthalate polyvinylidene chloride coated) (DuPont, Wilmington, Delaware). This material was chosen for its low permeability to C02; b) l4C02 was released by reacting 0.7 ml of 14C-sodium bicarbonate diluted to 10 ‘PCi/ml (56 mCi/mmol, in sterile aqueous solution obtained from ICN, Irvine, California) and 1 ml lactic acid (20% v/v in deionized water). Leaves were exposed to 14C02 for 30 minutes, then bags were removed. C02 uptake estimated from radioactivity incorporated into the pulsed leaves ranged from 85% to 95%. Translocation patterns were determined by autoradiography. Shoots were sampled 3 hrs after pulsing, mounted on paper and dried for 48 hours at 75 C in a forced draft oven. The dry samples were exposed to X-Omat AR film (Eastman Kodak Co., Rochester, NY) for 10 days and then developed according to manufacturer guidelines. Levels of detection by autoradiography and 14 Figure 1. Schematic representation of leaves and fruits on excised shoots from fruiting and defruited sour cherry trees used for 14C pulsing. Figure 1 O 00 14C-pulsed leaves 10 cm OO 00 OO O O O fruits ooO 16 time-course study (0.5-24 hours) of transport after pulsing were determined using a Biological Oxidizer - OX400 (R. J. Harvey Instrument Corporation, Hillsdale, New Jersey) operated at 02 and N2 flows of 0.3 l/min. Leaf discs (30-60 mg dry tissue), and shoot and fruit sections (70-130 mg dry tissue) were combusted at 2 and 4 minute cycles respectively, the products trapped in 15 ml of Carbon-14- Cocktail (R.J. Harvey Instrument Company, Hillsdale, New Jersey), and the radioactivity counted in a liquid scintillation counter (1211 Rackbeta, LKB-Wallac, Turku, Finland). Combined efficiency of combustion and counting estimated by carbon-14 standards (CFR.101, Amersham International plc, UK) ranged from 65 to 80%. RESULTS Fruits were removed at the end of stage III in all 3 years of the study. At this stage cherry fruits were still relatively strong sinks for assimilates because of their size and relative activity. Changes in both diameter and dry weight in 1987 are illustrated in Figure 2. Fgeit effect in 1985 During the first 2 weeks of July, average maximum and minimum air temperatures (T) were 26.5° and 12.1° C respectively for the 10 days preceding harvest. Maximum and minimum T for the month were 27.5°C and 13.9°C (figure 3a), while average leaf temperature at time of measurement was 17 Figure 2. Growth curve of sour cherry fruits in 1987. Date of full bloom: April 26, 1987; Date of fruit harvest: July 7, 1987 (indicated by arrow). Vertical bars indicate i SE (n=25). DRY WEIGHT (mg) 650 18 Figure 2 600- ssoI 500- 450- 400- 350- 300- 250- 200- 150- 100- 50- 0 dry weight "",gr_..—-é. his diameter .zr”‘_--_-ér. T I MEAN DIAMETER (mm) I “-0 r T r l l r r T r i 30 35 40 .45 50 55 50 65 7O 75 80 85 DAYS AFTER FULL BLOOM 19 Figure 3. Minimum and maximum temperatures during the course of field gas exchange measurements in 1985 (a), 1986 (b), 1987 (c). Arrows indicate the date of harvest. Needle bars indicate daily rainfall, squares at the top of the graph indicate cloud cover ( I cloudy; a partially cloudy; D«clear). The number shown at the right of each graph denotes monthly precipitation in mm. 20 56.5 4323. A85 4.2.123. e55 4.2.125 2 3 2 .. a . m... m. m... U... 1 a fix- Io 0 Cl \. 0 3 I”. a \U. .\ .\\ 7 I 0\ till). 0.. o F A 3...... U at: c I!) A III: .1 v I. \\ 5 if}..- .c A n 2 luv. .15. I. : .llli C .I .‘I r A .k ,u o: :0: LI... LR 1|". \|8 .\ 4n“|\ 0 our 9.. 2 lot (it o I I.I "II . F 3 \u ‘U‘ I "' bx \\\III I I u .. I y.-. a. s l 1 " m Q: TV-.. 1.01:: U \\I \V 1 U o,» U a). J A. .s.. g \.\ J ‘l.\‘\ 'U" ."' owl .\ '10.- "n‘ ' II II. ‘ ||I|‘ w bx.N 0\\\e \iL Vii ennui 0:1: 1.‘ so a”. Ola”. 4 I? '11\ Fl '5'-" di" [5 A. u I... In . .. a x b A. I. c C .11 R15 - I.Ppb—-_-I-—I.I-_-—-b—-p_.bbbhFP-_-I. ‘4‘H‘ “‘Ud‘4d.d“J‘-4“1I‘1‘41.‘1‘11“-JI“‘4~‘+“-“Iq‘II‘-.‘I‘I‘-I“I#quqlll 0 3 0 7 4 1 8 5 2 9 6 3 6 O 4 8 2 6 6 0 4 8 2 6 0 3 3 2 2 2 l l l J .J 2 l I. 3 3 2 cl cl 81 $23.32“: :2 CV $25.33: «2 § $25.25.. «a 21 31.3°C, within the optimal range for Pn in sour cherry (Sams and Flore 1983). Most days were cloudless and PAR was 2 s'1 at the time of Pn usually > 1500 pmols m- measurements, well above the light saturation level for Pn (22-30" c at 1200 pmols m'2 s'1 and VPD=O-6.6 kPa: Sams and Flore 1982). Precipitation totalled 20 mm in the first 15 days of July, and 32 mm in the second 15 days. Rains were uniformly distributed in July (Figure 3a) and usually occurred at night. Fruit removal caused a 34% decrease in Pn rates (from 11.8 to 7.8 pmols 111"2 5'1) within 24 h from treatment, and a 49% decrease after 8 days (Figure 4a). Such differences persisted for the next 15 days, then diminished. By the 4th week after harvest differences were no longer significant (P=0.05) and Pn rates of harvested trees approached pre- harvest values (Figure 4a). Leaf Pn did not decrease when fruits were removed on July 17, 1985 (data not shown). Average Pn rates of fruiting trees were 11.7 Pmols m-2 s'l, comparable with those reported in the literature for cherry (Sams and Flore 1983; Roper and Kennedy 1986). Stomatal conductance (gs) was also reduced by defruiting, and decreased from 96 to 36 mmols m"2 s'1 24 h after harvest. The experimental component of gs decrease was 2 -1 estimated to be about 20 mmols m' s and was particularly evident 1 and 9 days after harvest. Values of gS for 2 -1 fruiting trees ranged from 70 to 120 mmols m' s over the duration of the study (Figure 4b). Calculated values of 22 Figure 4. Changes in net photosynthesis (a), stomatal conductance (b) and intercellular CO (c) of fruiting and defruited sour cherry trees in 1985. ate of harvest: July 3, 1985. Vertical bars are 1 SE (n=12-20). Measurements started between 1230 and 1430 hr. c o. :0 "'2 Q E .5 23 Figure 4 13— fail—I .5‘ W/\ a 9" \ 8/ ‘5 +1 1 3: ~ \ I _ ‘9 l— \ \I 5"- - at O—OHorveeted T O-OContr-ol '1 filri'l'l'l‘irrr 1 9 13 17 21 23 29 33 IT I Y I fir' I r I rs'u I35- H Harvested - 'L H Control . . V—r fifi F r I ‘ I '—r . I r I v 1 . 5 9 13 17 21 25 29 33 r I— "[ Y r v ‘ r1 I v I r I f I r 225 — .. 200 - .. 175 r-_- .. 1:0 - l _ / I 125 - .. .. t H Harvested o—o Control r I ' l ' I h . r T , . I r 9 13 17 21 25 29 33 DAYS AFTER HARVEST 24 intercellular C02 (Ci) fluctuated from about 190 pl 1"1 to 135 P1 1'1, mainly due to the large decrease of gS on day 1, 9, and 13 after harvest (Figure 4b-4c). Leaf water potentials (WP) were about -2.0 MPa for both treatments at the beginning of the experiment, remained approximately constant from day 5 to day 9, then decreased as low as -4.0 MPa (day 21-26) (data not shown). Nevertheless, no WP differences between harvested and fruiting trees were found. Osmotic potentials were not recorded in 1985. The apparent cupping of leaves on harvested trees was confirmed by measurements of leaf angles 7 days after harvest. Mean angles were significantly narrower in defruited than in fruiting trees (P=0.05). Differences were larger for spur than for terminal leaves (Table 1). No differences in Pn inhibition due to leaf type, spur or terminal were found. Pn of terminal leaves was 66, 75, 72, and 74% of that of control 1, 9, l4, and 21 days after harvest respectively. Percentages for spur leaves were 80, 66, 70, and 89% at those same dates (Table 2). Pn rates were consistently higher in trees which had a low leaf/fruit (L/F) ratio (Figure 5). Inhibition of Pn after harvest was similar at the different L/F ratios , except at 14 and 21 days after harvest, when Pn decrease was greater for trees with a low source/sink ratio. 25 Table l. Angles of mature leaves from fruiting and harvested sour cherry trees measured 7 days after removal of fruits at the HRC, East Lansing, 1985. Leaf position Defruited Control LSDz Spur 96 a 113 b 9.3 Terminal 82 a 86 a 10.1 Mean 89 a 98 b 7.7 2 Each number is the average of 30 leaves per treatment. Letters indicate differences within rows at the 0.05 level using LSD mean separation test. Measurement taken with a protractor on July 7, 1985. 26 Table 2. Inhibition of photosynthesis in leaves from spurs and terminal shoots of sour cherry following harvest of mature fruits, HRC, East Lansing in 1985. N e t P h o t o s y n t h e s i 52 Days after (pmols 111'2 5'1) harvest --------------------------------------------- Terminal shoot Spur Defruited Control Defruited Control - 0.5 12.3 13.2 11.4 12.1 1 7.5a 11.4b 8.1a 10.1b 9 8.7a 11.6b 7.8a 11.8b 14 8.9a 12.3b 8.8a 12.5b 21 7.8a 10.5b 8.6a 9.7ab 2 Each number represents the average of 6-10 leaves/ treatment. Letters indicate statistical differences within rows at the 0.05 level using Duncan's Multiple Range Test. Absence of letters within a row indicates lack of significant differences. 27 Figure 5. The effect of different leaf/fruit ratio on net photosynthesis of fruiting and defruited sour cherry trees in 1985. Values are means of 6-10 leaves. Vertical bars indicate Least Significant Differences at the 5% level. NET PHOTOSYNTHESIS (pmols m" s“) o—O u—A A 28 Figure 5 6 0 Control 0 Harvested p — L/F>2 Q / ‘0 --. L/F< 2 31 n \ 1\ , ’2’ ' \ a 5 L‘ . \G— —-— ‘I’ 3 1; -—G—' 0" / \ , —-— ’ —. \ \ ’ "' ’ % 7- 4.- II I II I I I ,1. TI I rt UTTrUIIII ”13 15 20 25 30 36 DAYS AFTER HARVEST IUTTIUUIIII‘UfrrUII 4O 29 Erui§_sffe2§_in_12§§ In 1986 fruit load was only 10 kg/tree and L/F ratio between 5 and 10 for all four trees. Environmental conditions were often sub-optimal for Pn during the experimental period (Figure 3b). Average maximum and minimum T during the 10 days before harvest were respectively 24.4’ and 13°C, during the 10 days after harvest 27.2°C and 13.9’ C. Cloudy conditions occurred on 10 out of 13 days (Figure 3b) and under such conditions PAR was usually about 100- 300 pmols m"2 '1 s . Total rainfall in July was 70 mm. No significant differences in Pn were found between control and defruited trees following treatment (Table 3). A similar experiment was conducted at NWHES two weeks later when environmental conditions were similar to those recorded in East Lansing in 1985, cherry fruits were at the same stage as those harvested at the beginning of July in 1985, and L/F ratios were less than 5. The experiment was conducted for only four days. Mean leaf temperatures were 28.63 27.6: 33.53 36.63 35‘C respectively at the times of measurement. VPD inside the cuvette varied from 2.3 kPa on day 0 to 4.2 kPa on day 3 (afternoon). Pn was reduced 14 to 51% during the 3 days following fruit removal (Figure 6a). Pn rates of fruiting trees, comparable with those recorded in 1985, increased from 11.3 to 13.2 Pmols m"2 s'1 after the first 24 h, but declined to 2 -1 8.9 pmols m' s on day 2. The experimental component of the Pn change was therefore approximately 22%. Values of 30 Table 3. Changes defruited July 2, 1986. 1300 and 1700 hr. in net photosynthesis conductance (gs) and intercellular C02 (Pn), stomatal (Ci) of fruiting and sour cherry trees located at the Horticultural Research Center, East Lansing, in 1986. Fruits removed on Measurements made in the afternoon between Days after fruit removal Gas exchange parameterz Fruits -12 -9 -1 +1 +7 Pn + 16.1a 14.2 17.3 16.8 11.3 (pmols m’z s'l) - 14.6b 15.0 18.1 16.3 11.0 Gs + 130 116 102 98 117a (mmols m’2 s'l) - 154 128 114 127 l4lb Ci _ + 216a 216 175 179a 231 (pl 1 1) - 246b 221 186 221b 251 2 Different letters indicate significant differences (P=0.05) between treatments within parameters and dates. No letter indicates means not statistically different. Each value is the mean of 10 replications. 31 stomatal conductance were low for both treatments (50-100 mmols 111'2 s'l), but gS was significantly higher in fruiting trees (Figure 6b). Ci did not differ significantly between the 2 treatments Figure 6c). F it e ct in 987 In 1987 Pn did not decrease significantly until 3 days after harvest. The decrease amounted to 27% in the 2 5'1, plus an afternoon (from 9.8 to 8.2 pmols m" experimental component equal to 10%) but was not apparent in the morning. The 2-day delay for the appearance of the fruit effect can be explained by the limited light and sub- optimal temperatures prevalent on the date of harvest and immediately thereafter (for this reason, fruit removal was postponed to July 7th: Figure 3c). The inhibition was considerably lower than that observed in 1985 (20% vs 40%) (note that the same trees were used in both years). The effect of fruit removal was lower in the morning than in the afternoon (Figure 7a, 7b). No differences were found in the morning (measurements starting between 900 and 1100 hr), but Pn rates dropped 27% in the afternoon (measurements started from 1300 to 1600 hr) (Figure 7b). The effect remained significant in the afternoon for 18 days after harvest, when the measurements were terminated. Pn was not measured in both morning and afternoon on every day because variable environmental conditions prevented replication of measurements at a different time of the day. Average Pn 32 Figure 6. Changes in net photosynthesis (a), stomatal conductance (b), and intercellular C02 (c) of fruiting and defruited sour cherry trees located at the Northwestern Horticultural Experimental Station, Traverse City, in 1986. Date of fruit harvest: July 15, 1986 (indicated by arrow). Vertical bars indicate 3 SE (n=18-20). Pre-harvest measurements started at 1400 hr; post-harvest measurements started for each date at 1300, 900, 1500, and 900 hr respectively. 33 Figure 6 120— 30- ‘- N-w 230 '— e 210 L- V 170- Ml") 150 -—1 o-II‘ 1 i DAYS AFTER HARVEST 34 Figure 7. Morning (a,c,e) and afternoon (b,d,f) changes in net photosynthesis (a,b), stomatal conductance (c, d), and intercellular C02 (e, f) of leaves from fruiting and defruited sour cherry trees in 1987. Date of harvest: July 7, 1987. (indicated by arrow). Morning measurements started between 930 and 1130 hr; afternoon measurements started between 1300 and 1500 hr. Vertical bars are 1 SE (n=16). 35 7 Figure ._.m w>zma mo.o map um mmocmummuwo HMOwumwuoum muoowocfl muwuumq .mo can m3 coozumn mocmnmmufic on» mm omumHooaoo whammmum Mommas .Hmfiusouom owuosmo can nouns you muouommm mum mUCMOHuflcmflm Hooflumflumum mo msomwummeou .Amov ucoeummuu\mw>ooa oIv no Adzv usofiuoou9\mo>oma wIw mo omnum>o map ma Hones: zoom mn.o om.o mz mw.m I mo.m I mz hw.a I mw.a I ma >H=b mw.o m>.o mz mh.~ I m¢.m I o~.o nhm.a I ooh.a I ma hash mH.H mm.a omgu nmm.m I on.N I wo.o an.H I oom.a I m haah 6H.H mo.a mz om.~ I o¢.m I mz ~¢.H I mv.a I N wash Houusoo .pfizuuma 0mg Houucoo .ufisuwma 0mg Honusoo .uflsuumo 25 25 ---..------mma ........... 33 musmmmum uomuse Hoflucmuom DauOEmo Hoflucmuom Hmaoz .hmma .h >H5b "Ho>oamu uwsuu mo upon mcflmcoq umom .mwmuu pmuflzumoo can vcfluflsuu mo ousmmmum uomusp can Howpswuom ofluoamo .Hofiucmuom nmuoz mama .m manna 39 Figure 8. Patterns of carbon translocation in shoots excised from defruited (A, B, C, D) and fruiting (E, F, G, H), mature sour cherry trees in 1987. C 4 pulsing performed 3, 4, 10, and 11 days after harvest. (A, B, E, G) represent patterns in the upper half of the shoot, (C, D, G, H) in the lower half. Pulsed leaves indicated by arrows (see also Figure 1). Details of experimental procedure are reported in Materials and Methods and Figure 1. 41 water stress or water loss. Turgor was always maintained (minimum of 0.7 MPA on July 12 and 18) and there were no apparent differences between the treatments. 14C studies showed that ripe cherry fruits continued to import assimilates from fully expanded leaves (Figure 8). Translocation from the leaves on the median portion of the shoot was directed acropetally in shoots from defruited trees, and/or basipetally (towards the fruit) in fruiting shoots Figure 8). This pattern of translocation occurred even when shoots were pulsed 2 weeks after the date of harvest). DISCUSSION Leaf Pn of sour cherry trees decreased up to 51% of pre-harvest values 48 h after fruit removal under field conditions. The decline of Pn was not always evident in the 3 years of study. For example, defruiting did not reduce Pn rates of defruited trees at the East Lansing location in 1986, but had a marked effect on the same trees in 1985 and 1987. Environmental variables were different between 1985 and 1986. Climatic conditions intermediate between 1985 and 1986 occurred in 1987, when Pn inhibition became evident only three days after harvest (after two cloudless days). Pn inhibition of defruited trees was particularly evident in the afternoon, and was not associated with changes in chlorophyll content. Fruiting and defruited trees showed similar changes of Pn per unit variation of gS 42 (slopes of the regression lines) both in the morning and the afternoon of 1986 (Figure 9a, 9b); Pn increase per unit Gs of defruited trees was greater than fruiting ones in 1987 (Figure 9c, 9d). Since positive correlation between Pn and g5 was lower in the morning than in the afternoon and average gS values were lower in the afternoon than in the morning for both treatments in both years, Pn was probably limited by stomatal aperture in the afternoon. Decrease of gS associated with fruit removal (Downton et al. 1987; Kriedemann et al. 1976; Schaffer et al. 1987) or midday depression of photosynthesis (Raschke and Resemann 1986: Tenhunen et al. 1981) have also been reported for several woody species. Midday stomatal closure of Arbutus and Quercus has been related to increasing VPD in the afternoon in natural environments (Tenhunen et al. 1980). Under simulated natural conditions gs of hazelnut leaves was as low as 25 mmols m'2 s'1 at a VPD of 3.0 kPa and a WP of -1.9 MPa (Schulze and Kuppers 1979). In hazelnut gS progressively decreased with long-term water stress, but not with short- term water stress. Stomatal closure during short-term water stress seemed to be entirely dependent on air humidity and temperature (Schulze and Kuppers 1979). Because of the type of experiment conducted in this study I do not have conclusive evidence as to whether stomatal closure in sour cherry in the afternoon was regulated by environmental parameters alone. Further studies will have to be conducted in a controlled environment. Although transient periods of 43 Figure 9. Relationship between leaf net photosynthesis (Pn) and stomatal conductance (gs) for fruiting and defruited sour cherry trees in the morning (a, c) and afternoon (b, d) of 1986 (a, b) and 1987 (c, d). Each symbol is the mean of 6-10 observations. Measurements were made at the Northwestern Horticultural Station, Traverse City, Michigan, in 1986, and at the Horticultural Research Center, East Lansing, Michigan, in 1987. 44 Figure 9 9% TE w_oEEv on com m: cm. nm— 00— mp on .nN ATm 7.: £955 on cow n: on. mm. co. mm on mw ii _ . n - _ _ n lli . . . - _ p .3 oo o 7 L1 .0530 o L- 3125.: o. . plate: e I. #l? .O‘ . d o .m o .m e .\o\ u :2. . s. u G... é. a \I o n o .e.oo a... E “w e om 336.9221... .m u a . .. . . e " oo<¥\e \ \ . . ‘\ 4 m .\m -2 w. o .E LVMVF @ O S o o 0” b\1\\ . a ewe e co. .2 z o o . win-IVo\ E. .N. o. 2.: . e. ... n -oéHubJ. . m a 2:... . .21... s \o\\o\ . o o. o c . .E ... o a a : a a E ( I o e\ o o O N -w— 0 we HNQ. O o Q~.o n 3.. .m: _o o A: O .m— A_-m TE m_oEEv on 9% TE 2955 mm mm_ om. mm. on... mm 00.,“ mm mm. om. nm_ 0...: mm . 9...... .5130 o I.—. .3139 o .. poi->5: o 0‘. 1I.v $3230: o A)? O ooeeoo\§\,oo\oo .o d an... .2 o o o o 6 :2. .2. o\o%o o u 3 :66 . n: . s. e .a . ml 0 O o o\o\ a mu 3...: o 2 _ u E. . m 00 w 00 \DO 00 Q— o .\\. \C o .91 Q a o u- o. o o .2 .\o. co m i I o o . . o on o\ a: o O I: z . O. 0 $6 4... s. o\ o o ,E A: (U 3 ~26 . 8.. . .5 w o o :5 4.. .. a— o .5 G a. m a as... . :3 . s. .: -Dm .c. (,-s .33: 31017.1?!) ud niua $7011: (I-5 2- "' 45 mild water stress may have occurred especially during the hottest part of day, there was no evidence of symptoms of long-term water stress. Therefore, lack of turgor is probably not responsible for the decrease of gS after fruit removal in sour cherry. Diurnal stomatal movements have also been reported to be regulated by levels of ABA. Stomatal closure following accumulation of ABA in the leaf occurs in soybean (Setter et al. 1980), grapevine (Loveys 1984), and apricot (Loveys et al. 1987). In grapevines grown in a semiarid environment (South Australia), ABA seemed to account for midday stomatal closure, whereas air temperature and humidity appeared responsible for stomatal behavior later in the day (Loveys and During 1984). In both grapevine and apricot grown in a similar environment ABA accumulation was not the result of loss of turgor (Loveys 1984; Loveys et al. 1987). Since afternoon stomatal closure was particularly evident only for defruited sour cherry trees grown in a much more humid environment (Michigan), further studies on the effect of fruit removal will have to consider changes of ABA levels in the leaf in response to fruit harvest. It has also been hypothesized that growth regulators other than ABA may regulate leaf photosynthesis and the flow of assimilates to the fruit by stimulating fruit growth and sink strength (Chalmers et al. 1976; Herold 1980: Hoad et al. 1977; Neales and Incoll 1968). From the data presented thus far it is not possible to determine whether the change 46 of Pn rate after removal of cherry fruits was caused by direct action of phytohormones or was the consequence of feedback inhibition due to carbohydrate accumulation (for a review of both hypotheses and their implications see Neales and Incoll 1968, or Herold 1980). Calculated Ci levels of defruited sour cherry trees equalled or exceeded Ci levels of fruiting trees, which suggests that rates of Pn and g8 may regulate Ci/Ca ratio and maintain approximately constant. Evidence for Pn regulation by endogenous factors after fruit removal also derives from different Pn rates when L/F ratios are above a certain threshold (Figure 5 and Table 3). In 1986 all trees had a high L/F ratio (range 5-10) and bore a small crop (about 10 kg/tree), which could have resulted in compensatory changes of Pn in response to sink strength modifications similar to those described in bean, pepper, cucumber,and apple (Borchers-Zampini et al. 1980 :Kriedemann et al. 1976: Hansen 1969: Plaut and Mayoral 1984). Since vegetative growth was vigorous, assimilate supply and demand might have been influenced more by the growth of shoots, stems, and leaves in those relatively young trees. It is also interesting that, in sour cherry, the flow of assimilates from leaves located in the central portion of the shoot was reoriented towards the vegetative apex after fruit removal even when fruits were beyond the stage of full maturity (Figure 8). The presence of 14C-activity in overripe fruits indicated that even at this late stage of 47 development the fruit was still a relatively active sink for assimilation products. Analogous redistribution of assimilates has been previously reported for apple and grapevine at times of rapid fruit growth and import of assimilates into the fruit (Calo' and Iannini 1975; Hansen 1970). The effect of L/F ratio, high levels of Ci for defruited trees, and changes in carbon allocation due to the presence of mature fruit seem to indicate that nonstomatal components and carbon partitioning are affected by fruit removal and may be the primary determinants of post-harvest Pn inhibition. LITERATURE CITED 1) Borchers-Zampini, C., A. Bailey-Glaumm, J. Hoddinott and C.A. Swanson. 1980. Alterations in source-sink patterns by modifications of sink strength. Plant Physiol. 65:1116-1120. 2) Calo', A. and B. Iannini. 1975. Indagini sulla migrazione dei prodoEXi di assimilazione della vite mediante l'uso del . Rivista di Viticoltura ed Enologia. Episode II. 7:278-301. 3) Chalmers, D.J., B. van den Ende and P.H. Jerie. 1976. The effect of (2-chloroethyl)phosphonic acid on the ' sink strength of developing peach (Prunus persica L.) fruit. Planta 1311:203-205. 4) Cowan, I.R. and G.D. Farquhar. 1977. Stomatal function in relation to leaf metabolism and environment. Symp. Soc. Exp. Biol. 31:471-505. 5) DeJong, T.M. 1983. C02 assimilation characteristics of five Prunus tree fruit species. J. Amer. Soc. Hort. 801. 108:303-307. 6) Downton, W.J.S., W.J.R. Grant and B.R. Loveys. 1987. Diurnal changes in the photosynthesis of field-grown grape vines. The New Phytol. 105:71-80. 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 48 Flore, J.A. and R. Gucci. 1986. The influence of harvest on photosynthesis and water relations of cherry and peach. HortScience 21(3):895 (Abstr.). Flore, J. A. and C.E. Sams. 1986. Does photosynthesis limit yield of sour cherry (Prunus cerasus) 'Montmorency'. pp 105-110. In: Regulation of Photosynthesis in Fruit Trees (A.N. Lakso, F. Lenz, eds). Symp. Proc. Publ., NY State Agr. Exp. Sta., Geneva, NY. Fujii, J.A. and R.A. Kennedy. 1985. Seasonal changes in the photosynthetic rate in apple trees. A comparison between fruiting and non-fruiting trees. Plant Physiol. 78:519-524. Hall, A.J. and F.L. Milthorpe. 1978. Assimilate source- sink relationships in Capsicum annuum L. III. The effect of fruit excision on photosynthesis and leaf and stem carbohydrates. Aust. J. Plant Physiol. 5:1-13. Hansen, P. 1969. 14C-studies on apple trees. IV. Photosynthate consumption in fruits in relation to the leaf-fruit ratio and to the leaf-fruit position. Physiol. Plant. 22:186-198. Hansen, P. 1970. 14C-studies on apple trees. VI. The influence of the fruit on the photosynthesis of the leaves and relative photosynthetic yields of fruits and leaves. Physiol. Plant. 23:805-810. Herold, A. 1980. Regulation of photosynthesis by sink activity - The missing link. The New Phytol. 86:131-144. Hoad, G.V., B.R. Loveys and K.G.M. Skene. 1977. The effect of fruit-removal on cytokinins and gibberellin- like substances in grape leaves. Planta 136:25-30. Kappes, E.M. 1985. Carbohydrate production, balance and translocation in leaves, shoots and fruits of 'Montmorency' sour cherry. Ph.D. Dissertation, Michigan State University, East Lansing, Mich. Kriedemann, P.E., B.R. Loveys, J.V. Possingham and M. Satoh. 1976. Sink effects on stomatal physiology and photosynthesis. pp 401-414. In: Transport and Transfer Processes in Plants (Wardlaw, J.F. and J. B. Passioura, eds.). Academic Press, London. 17) 18) 19) 20) 21) 22) 23) 24) 25) 26) 49 Kirschbaum, M.U.F. 1987. Water stress in Eugaliptus pauciflora: a comparison of effects on stomatal conductance with effects of the mesophyll capacity for photosynthesis, and investigation of a possible involvement of photoinhibition. Planta. 171:466-473. Lenz, F. 1979. Fruit effects on photosynthesis, light- and dark-respiration. pp161-164. In: Photosynthesis and Plant Development (R. Marcelle, H. Clijsters, and M. Van Poucke, eds), Dr. W. Junk, The Hague. ISBN 9-06- 193594-6. Loveys, B.R. 1984. Diurnal changes in water relations and abscisic acid in field-grown Vitis vinifgra cultivars. III. The influence of xylem-derived abscisic acid on leaf gas exchange. The New Phytol. 98:563-573. Loveys, B.R. and H. During. 1984. Diurnal changes in water relations and abscisic acid in field-grown Vitis vinifera cultivars. II. Abscisic acid changes under semi-arid conditions. The New Phytologist. 97:37-47. Loveys, B.R., S.P. Robinson and W.J.S. Downton. 1987. Seasonal and diurnal changes in abscisic acid and water relations of apricot leaves (Prunus armeniaca L.). The New Phytol. 107:15-27. 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. Moran, R. 1982. Formulae for determination of chlorophyllous pigments extracted with N,N- dimethylformamide. Plant Physiol. 69:1376-1381. Neales, T.F. and L.D. Incoll. 1968. The control of leaf photosynthesis rate by the level of assimilate concentration in the leaf: a review of the hypothesis. Bot. Rev. 2:107-125. Nelson, N.D. 1984. Woody plants are not inherently low in photosynthetic capacity. Photosynthetica. 18:600- 605. Plaut, Z. and M.L. Mayoral. 1984. Control of photosynthesis in whole plants by source-sink interrelationships. pp161-164. In: Advances in Photosynthesis Research, vol.IV (Sybesnea, C. ed.), Martinus Nijhoff/ Dr. W. Junk Publishers, The Hague. ISBN 90-247-2945-9. 27) 28) 29) 30) 31) 32) 33) 34) 35) 50 Raschke, K. and A. Resemann. 1986. The midday depression of C02 assimilation in leaves of Arbutus unedo L.: diurnal changes in photosynthetic capacity related to changes in temperature and humidity. Planta 168:546-558. Roper, T.R. and R.A. Kennedy. 1986. Photosynthetic characteristics during leaf development in 'Bing' sweet cherry. J. Amer. Soc. Hort. Sci. 111:938-941. Roper, T.R., Keller, J. D., Loescher, W.H. and C.R. Rom. 1988. Photosynthesis and carbohydrate partitioning in sweet cherry: fruiting rffects. Physiol. Plant. 72:42-47. 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. Sams, C.E. and J.A. Flore. 1983. Net photosynthetic rate of sour cherry (Prunus cerasus 'Montmorency' L.) during the growing season with particular reference to fruiting. Photosyn. Res. 4:307-316. Schaffer, B., L. Ramos and S.P. Lara. 1987. Effect of fruit removal on net gas exchange of avocado leaves. HortScience 22:925-927. Schulze, E.D. and M. Kuppers. 1979. Short-term and long-term effects of plant water deficits on stomatal response to humidity in Corylus avellana L. Planta 146:319-326. Setter, T.L., W.A. Brun and M.L. Brenner. 1980. Effect of obstructed translocation on leaf abscisic acid, and associated stomatal closure and photosynthesis decline. Plant Physiol. 65:1111-1115. Tenhunen, J.D., 0.L. Lange and M. Braun. 1981. My and B.R. Strain. 1981. Effect of carbon dioxide enrichment on chlorophyll content, starch content and starch grain structure in Trifolium subterraneum leaves. Physiol. Plant. 51:171-174. 51 KEYWORDS: Prunus cerasus, inhibition of photosynthesis, stomatal conductance, intercellular C02, environment, fruit removal. ABBREVIATIONS: Pn, net photosynthesis: gs, stomatal conductance; Ci, internal C02: VPD, vapor pressure deficit; L/F, leaf/fruit ratio: T, air temperature; PAR, photosynthetic active radiation; WP, water potential; OP, osmotic potential; TP, turgor pressure; R, gas constant: J, molar concentration of cell sap. Section II NON-STOMATAL LIMITATIONS, PHOTORESPIRATION, AND CARBOHYDRATE PARTITIONING DURING INHIBITION OF PHOTOSYNTHESIS FOLLOWING FRUIT HARVEST IN SOUR CHERRY 52 53 ABSTRACT Nonstomatal limitations and leaf carbohydrate partitioning during inhibition of net photosynthesis (Pn) occurring after fruit harvest were studied in sour cherry (Prunus cerasus L.) over two growing seasons. Stomatal contribution to the decrease of Pn ranged from 32 to 40% in a controlled environment and from 25 to 45% under field conditions. Under laboratory conditions, reducing the 02 concentration from 21% (air) to 2.5% increased Pn 73% in defruited trees, but only 47% in controls. Similar results were obtained under field conditions at 02 concentrations of 21% vs 0.5%. Both harvested and control treatments showed evidence of 02-sensitivity of Pn. The levels of soluble sugars (fructose, glucose, inositol, sorbitol, and sucrose) in leaves were not significantly affected by defruiting. In contrast, starch levels in the leaf increased about 3-fold within 48 h after fruit harvest in both years (from 0.8 to 2.9% dry wt. in 1986 and from 0.6 to 1.9% dry wt. in 1987), but dropped to pre-harvest levels 3 days after harvest in 1987. The large non-stomatal contribution to Pn decrease after harvest and the changes in starch levels support the hypothesis that defruiting results in end- product inhibition of photosynthesis. 54 INTRODUCTION I have shown in the previous section that stomatal behavior does not appear to account completely for the inhibition of Pn caused by fruit harvest in sour cherry. Nonstomatal limitations are reportedly the most relevant component in the afternooon depression of Pn in grapevine (Downton et al 1987) and Qggrgus (Tenhunen et al. 1984). However, stomatal limitations have been claimed to be totally responsible for changes in Pn in other systems or for other types of manipulations (Downton et al. 1988; Kriedemann et al. 1976; Setter et al. 1980). Obstructed translocation by chilling or girdling, fruit removal, or continuous illumination have all been shown to decrease Pn rate in different species (Azcon-Bieto 1983; Claussen et al. 1985; Mayoral et a1. 1985; Setter et a1. 1980). Photorespiration has been implicated in Pn decrease after fruit removal in citrus (Lenz 1979), but not in other systems (Peet and Kramer 1980). Accumulation of non- structural carbohydrates due to a reduction in sink strength or excessive assimilate supply has been hypothesized to regulate Pn rates via end—product inhibition at the source level (Azcon-Bieto 1983; Neales and Incoll 1968). There is evidence for changes both at the level of sucrose synthesizing enzymes affecting the rate of carbohydrate export out of the leaf (Claussen et a1. 1985; Ho 1979; Pharr et al. 1985; Rufty and Huber 1983) and Pn inhibition caused physically by accumulation of starch 55 (Milford and Pearman 1975; Nafziger and Koller 1976; Schaffer et al. 1986). The objectives of this study were to quantify the non- stomatal limitations to Pn after fruit harvest in field- grown 'Montmorency' sour cherry trees, to determine if photorespiration contributed to changes in Pn, and to measure changes in non-structural carbohydrates in the leaf mesophyll. MATERIALS AND METHODS Elant material. Four 8-year-old sour cherry trees (Prunus cerasus L. cv. Montmorency) were chosen in an orchard at the Horticultural Research Center (HRC) of Michigan State University, East Lansing, Michigan, in 1987, and four 5-year old sour cherry trees at the Northwestern Horticultural Experiment Station (NWHES), Traverse City, Michigan, in 1986. Trees were spaced 8.2 x 3.0 m at the HRC and 6.0 x 2.0 m at the NWHES. Fruits were removed on July 16, 1986 at NWHES, and July 7, 1987 at HRC. Pesticides were applied according to commercial recommendations (Mich. Ext. Bul. E154, Fruit Pesticide Handbook). Pruning, fertilization and other cultural practices were performed according to standard practices. ‘Other details about plant material, location, and experimental conditions are as reported in Section I. 56 gas exchange measurements. All measurements in the field were made in an open system using an ADC LCA2 portable infrared gas analyzer equipped with a Parkinson broad leaf chamber (Analytical Development Company, Hoddesdon, UK) at 2 '1) and saturating light intensity (PAR > 1000 pmols m' 5 air temperatures between 24 and 32 C. Field measurements of photorespiratory rates and COZ-response curves were taken using a gas tank containing approximately 2000 pl 1'1 C02 in nitrogen and an ADC GD600 gas diluter (Analytical Development Company, Hoddesdon, UK) connected in series to an ADC air supply unit, chamber and analyzer (Figure 1). Photorespiration was estimated by comparing Pn rates at ambient C02 (325-340 p1 1'1) and 02 (21%) with rates at low 02 (0.5%). cog-response curves were obtained by decreasing the C02 concentration of ingoing air from 330 Pl 1'1 to about 50 pl 1'1 with the gas diluter. Levels of C02 in the chamber were monitored with an ADC LCAZ portable infrared analyzer. Flow rate was 0.6 l/min, and pressure in the gas diluter was maintained above 0.1 MPa. Measurements were taken on four leaves of those regularly measured for gas exchange (see also Section I) from one tree/treatment at PAR 2 5'1, cuvette temperature 31-35° C, air > 1500 Pmols m- relative humidity 60-70%, air temperature 28-32° C, and relative humidity of incoming air 15-25%. Measurements started at 1200 hr and were run on 2 consecutive days (9-10 days after defruiting). Photorespiration and C02 response curves were measured in the laboratory using the open system 57 Figure 1. Schematic diagram of gas-exchange equipment used to measure C0 curves and estimate photorespiration under field conditions. Abbreviations: ADC, Analytical Development Company, Hoddesdon, England; IRGA, infra-red gas analyzer. C02 tank (LIE;:__. Flowmeter .Q 58 Figure 1 leaf Parkinson broad-leaf chamber.___ L_____. outlet to atmosphere ADC ' Portable IRGA f T‘ We ADC Air Supply Unit - ADC - A ———p Gas diluter ' ' J air 59 described by Sams and Flore (1982) and modified as follows: a) An ADC 225 Mk3 infrared Gas Analyzer (Analytical Development Company, Hoddesdon, UK) was used to measure differential C02 concentrations at the inlet and outlet of leaf chambers; b) air flow entering the chambers was regulated with Matheson 8100 series mass flowmeters and Matheson 8200 series mass flow controllers connected to a Matheson multichannel Dyna-Blender 8219 (Matheson, Instruments, Horsham, Pennsylvania). Four shoots were excised, using the precautions recommended by Lakso (1980), from fruiting and defruited trees at 900 hr and taken to the laboratory where they were left to acclimate for 3 hours at air temperature of 23°C and 1000 pmols m-2 s'1 PAR. Light was provided by GE 400 W multivapor lamps (metal halide, General Electric). Once acclimated, one leaf (4th or 5th from apex) per shoot was inserted into a leaf chamber. Four chambers were used at a time. C02 response curves were determined by decreasing the C02 concentration to the compensation point. C02 concentrations at the chambers exits were monitored with an ADC LCA2 portable infrared analyzer, 02 concentrations with a 0-260 Beckman oxygen analyzer (Beckman Instruments Inc., Irvine, California). Measured leaves were maintained at 1000 Pmols m"2 s'1 PAR, 27.5-28.5° C leaf temperature, and dew point of 1-1.5° C equivalent to a VPD of about 2.85 kPa. Flow rates to the chambers were 2.3 i 0.1 l/min depending on leaf size and photosynthesis rate. Measured leaves were harvested and 60 their area determined with a LICOR LI 3000 leaf area meter (LI-COR Inc, Lincoln, Nebraska). Calgalations. statistical analysis, and curve fitting Gas exchange parameters were calculated according to the equations by vonCaemmerer and Farquhar (1981) as previously described (Moon and Flore, 1985). Stomatal contributions to the change of Pn after fruit harvest were calculated according to the methods proposed by Farquhar and Sharkey (1982) and Jones (1985). The following equations were used: a) Lg = Pno - Pn / Pno (Farquhar and Sharkey 1982) where Lg is the stomatal limitation to Pn, Pn is the assimilation rate that actually occurs, and Pno the assimilation rate that would occur if resistance to C02 diffusion was zero; b) Gc= 1 { Pn + Pn } rg -rg (Jones 1985) where CC is the stomatal contribution to the change in occurring from condition 1 to 2, r9 the stomatal resistance, and rm the residual resistance to C02 diffusion. Photosynthesis, and stomatal and mesophyll resistances used in the equations refer to means of values at 330 ‘pl 1'1 ambient C02. Curve fitting was done by computer using the Marquardt method of successive approximations. The best fit for demand curves (A/Ci), evaluated by R2 and analysis of residuals, was given by a nonlinear model of the type: Pn = 8(1) x exp {8(2) Ci} + 8(3)} 61 Table 1 . Coefficients of estimated demand (a) and supply (b) gas exchange curves and relative coefficients of determination for fruiting and defruited treatment, HRC East Lansing, 1987. T r e a t m e n t Coefficientz ---------------§ --------------------------- Defruited R Fruiting R2 8(1) 589.79 0.81 - 28.14 0.77 8(2) 1.13 x 10’4 1.81 x 10'2 8(3) - 591.61 14.13 C(1) 22.14 0.99 27.69 0.98 C(2) - 0.065 - 0.082 2 Coefficients refer to the following equations: (a) Pn = 8(1) x exp {8(2) x Ci) + 8(3) (b) Pn = C(1) + C(2) x Ci wgere Pn is net photosynthesis, Ci is intercellular C02, and R is the coefficient of determination of the regression curves. Measurements made in the laboratory on excised shoots 9 days after fruit removal. 62 The estimated coefficients of demand curves for fruiting and harvested trees are reported in Table 1. Carboxylation efficiencies at each concentration of Ci were calculated from derivatives of the demand curves at the point of the chosen Ci concentration, as follows: d(Pn)/d(Ci) = 8(1) x exp{8(2) x Ci} x 8(2) Gas supply curves for each treatment were calculated by the equation Pn=(Ca-Ci) x gs (Farquhar and Sharkey 1982) using gS values at Ca= 330 P1 1'1. Gas exchange measurements in the field were made according to a randomized complete block design (2 blocks, each consisting of 2 trees; 6-10 replications per tree). The design for gas exchange in the lab was completely randomized with leaves as replications. Means were separated by LSD (P < 0.05). Comparisons were made only between treatments at each single date. Carbohydrate aaalysis . Analyses of soluble sugars (glucose, fructose, sucrose, sorbitol, and inositol) were performed by gas chromatography, analysis of starch by enzymatic degradation and color reaction. Details about the methods used are reported in Section IV. Iransmissign electrgn migrosgapy. Samples for TEM were collected from 3 leaves per treatment at 1400 hr immediately before and 24 h after fruits were removed. Samples were fixed in 4% glutaraldehyde on ice and postfixed in 2% 0504 at room temperature. Samples were dehydrated with ethanol 63 and embedded in Mollenhauer mixture resin (Mollenhauer 1964). Sections were stained with uranyl acetate and Reynolds lead (Reynolds 1963). RESULTS Calculated stomatal contribution to the post-harvest decrease of photosynthesis ranged from 32% to 40% in a controlled environment and from 25% to 45% in the field. Carboxylation efficiency (CE) of fruiting sour cherry trees was about 20% higher than that of defruited ones at 100 P1 1'1 Ci, corresponding to an ambient C02 (Ca) of 180-220 pl 1'1 (Figure 2). Photorespiration estimated from Pn rates at 21% 02 and 2.5% 02 in the laboratory and in the field (0.5%) are reported in Table 2. The ratio of Pn values from harvested and control trees measured in the laboratory was slightly higher at 2.5% 02 (86%) than at 21% 02 (73%). The difference of Pn at 2.5% 02 and 21% 02 expressed as a percentage of values at 21% 02 was 73% for harvested and 47% for control treatment when measured in the laboratory, 71% and 42% when measured in the field. Leaf content of soluble sugars was not significantly affected by defruiting in 1986 and 1987 (Figure 3). Sorbitol was the most abundant among the soluble sugars in the leaf (about 56%), followed by sucrose (27%), glucose (9%), and fructose (7%). Inositol was only found in traces (< 0.5%). There was no clear trend 64 Figure 2. C02 response curves of fruiting and defruited sour cherry trees measured with an open gas exchange system (Sams and Flore 1982). Measurements were made in the laboratory 9 days after fruits were harvested on July 7, 1987. Environmental conditions: leaf T 28-29°C; dew point of air going in o the cuvette 0.6‘95 ambient C02 from 340 pl 1'1 to 50 pl 1 ; PAR 1000 pmols m s . Equation at top of the graph refers to the model used to fit demand curves for gas exchange. Gas supply curves for each treatment were fitted by linear regression using values of stomatal lconductance of fruiting or defruited treatment at 340 pl 1' . 65 Figure 2 18 t." 0 Control E5 2 O ‘ - g 12‘ o O s , - ' m E}- 9_ DEMAND CURVE O . I § 0 . a ‘ ‘ -‘ >.. - 4 8 6 a. O / l- " o A, g Q E 3.. . . SUPPLY CURVE Z .. o , Ofi'r-rltII'I'III‘IIIIHI'FIF 0 30 60 90 120 150 180 210 240 270 INTERNAL 002 (11.1 1-1) 66 .8890 «83 mo .838 can 035 05 pm 5m 8953 5088 93 8. 3&3 PS 6.2 8n u 93 33 i mmmuommu 8 6.56m u a “Tm use 395 83 A mam ”50¢ 05 5 855680 358555 6%. mm.~ u 93 3.5 1 8?on u N8 609mm Imém u .H. “Hum ~13 magi oooa n mg 29058023 0:» 5 9035500 $855 .55“ 05 5 .8 83 B on: 8.0 .33885 65 5 83 8. 633 .85 88 mpg; .83 .m 35. so 80.832 B5B“ 283 .3 98 3 35. co no 888:8 38 .35888 m 8.8 8:15 mo momma .88 38 m m 3 K E 3.8 S 2. at 6:8 86 3~.~ Anna 36 8S 3:8 mm 3.3 8mg... m6 8 3.3 2.3 3v m.~ 9 3S 3..” am me 3.3 $6 3 3 GS 388 as 85.58 Q 858 a. 85386 E Amy E Ame 03 o o\a o ruin «IE 381v Egg umz Aalm NIB macaw: 385g #02 camfim >Hououonoq .33 5 6523 ummm Gm: up 303 05 5 can #35333 05 5 38cm 3.86 .58 68.558 Ba 65530 no 83588808 80508 En 3855885 . m 035 67 in levels of soluble sugars over time due to fruit harvest (Figure 3). Starch levels increased on average 350% times within 48 h after harvest in 1986 (Figure 4). In 1987 leaf starch levels of harvested trees increased from 0.6% to 1.95% 24 h after harvest, but decreased to pre-harvest value 3 days after harvest (0.66% dry wt.). Starch levels in control leaves also increased 24 h after harvest (from 0.60% to 1.46% dry wt.), but dropped to 0.33 and 0.37% 3 and 5 days after harvest respectively (58% of pre-harvest values). The differences between control and defruited trees were significant at all times of sampling after fruit harvest in 1986 (Figure 4a), and 4 out of 5 times in 1987 (Figure 4b). Increase in leaf starch was confirmed by enlargement of starch grains in chloroplasts of mesophyll cells (Figure 5). Analysis of about 45 cells (spongy and mesophyll) from TEM micrographs revealed a 3-fold increase in the area of the chloroplast occupied by starch grains after fruit removal. However, no breakage of chloroplasts or disruption of membranes could be observed due to the enlargement of such grains (Figure 6). DISCUSSION The decrease in leaf Pn following removal of fruits in sour cherry appeared to be regulated by several factors. 68 Figure 3 . Changes in leaf content of soluble sugars after fruit harvest in sour cherry: a) 1986, NWHES, Traverse City, Michigan; b), HRC, East Lansing. Soluble sugars were fructose, glucose, inositol, sorbitol, and sucrose. Arrows indicate date of harvest. Vertical bars t SE. LEAF SOLUBLE SUCARS (% dry wt) LEAF SOLUBLE SUGARS (7. dry wt) 69 Figure 3 10.0- o—o Control H Harvested I I I 0 l ' 2 DAYS AFTER FRUIT REMOVAL 32.0! 28.0: 24.0: 20J3: 1613: 1213: 8.0-: 450- 0i) o—o Control H Harvested P l TWII I I I I 4).—1 01 -:6 -1 0 1 2 3 DAYS AFTER FRUIT REMOVAL 70 Figure 4 . Changes in leaf starch content after fruit harvest in sour cherry: a) 1986, NWHES, Traverse City, Michigan; b) 1987, HRC, East Lansing. Arrows indicate date of harvest. Vertical bars 1 SE. LEAF STARCH (7. dry wt) LEAF STARCH (7. dry wt) 71 Figure 4 o—o Control o—e Harvested DAYS AFTER FRUIT REMOVAL 2.0- 1.85 1.6d 1.4- 1.2- 1.0- 0.8- 0.6- 0.4- 0.2- 0.0 G—D Control H Harvested DAYS AFTER FRUIT REMOVAL 72 Figure 5 . Transmission electron micrographs of palisade (A, 8) and spongy (C, D) mesophyll cells of leaves collected from sour cherry trees 1 h before (A, C) and 24 h after (B, D) fruit removal at HRC in 1987. Magnification: A. 5800x; B. 4100x; C. 11800x; D. 6300x. Legend: 0) chloroplast; s) starch grain; w) cell wall. 73 Figure 5 2pm — e '0; c fly!- 1 c 1} 5 a ‘ 'W f W 1" V (Ir . r" r . 4 o “‘ "VF-W ° ‘ ‘ . fl . 09. _ ‘ .‘ '7’. s c w .' c 3 1 In. s . I w ‘ - . a ’N -._r,:: - .2, Hi . . 74 Figure 6 . Transmission electron micrograph of an enlarged starch grain in the chloroplast of a spongy mesophyll cell from leaves of sour cherry 24 h after fruit removal. Note the apparently undamaged arrangement of the thylakoid membranes in the chloroplast. Sampling done at 1400 hr. Legend: c) chloroplast; p) plastoglobule; s) starch grain; w) cell wall. Magnification: 57700x. Figure 6 76 Nonstomatal components contributed more than stomatal ones. Analysis of A/Ci curves for harvested and control treatment seemed to indicate that major limitations to Pn were in the region of RuBP regeneration rate (Farquhar and Sharkey 1982; Jones 1973), whereas lower CE of leaves from harvested trees only occurred at Ci < 150 pl 1’1 (Figure 2). 02-sensitivity of Pn was not lost after fruit removal (Table 2). Since percentages of Pn ratios of harvested and control treatment decreased from 27% at 21% 02 to 14% at 2.6% 02, post-harvest Pn inhibition was slightly relieved at low 02. This also seemed to be confirmed by percentages of photosynthetic enhancement at low 02 (72% of Pn at high 02 for harvested trees, 45% for control trees) (Table 2) and the slight increase of the C02 compensation point after harvest (Figure 2). Limitations of RuBP regeneration capacity, RuBP carboxylation activity, and increase in C02 compensation point have also been reported during midday depression of Ph in Quercus (Tenhunen et al. 1984). This may indicate that the post-harvest decline of Pn is not a phenomenon specifically induced by fruit removal, but it may represent a general type of response to environmental conditions or lack of sink strength. The involvement of photorespiration in the Pn response to fruit removal has also been reported in citrus (Lenz 1979) and it is probably effective through a release of competitive inhibition of 02 for Rubisco (Ogren and Bowes 1971). Involvement of photorespiration is also in 77 agreement with the hypothesis of RuBP limitations, since the RuBP oxygenase also uses RuBP and, if RuBP is in limited supply , the presence of 02 can rapidly reduce the rate of carboxylation. Increases in levels of non-structural carbohydrates after fruit removal have been reported for citrus (Schaffer et al. 1986), plum (Section III), pepper (Hall and Milthorpe 1978), eggplant (Claussen et al. 1985), and cucumber (Barrett and Hamling 1978), but not for sweet cherry (Roper et al. 1988). In sour cherry, only starch increased after harvest, whereas soluble sugars showed no clear trend (Figures 3 and 4). Changes in leaf starch content not accompanied by changes of soluble sugars have also been reported for many species in response to various source-sink manipulations (Maidsen 1968; Milford and Pearman 1975; Nafziger and Koller 1976; Ho 1979). Such a trend could be due to the rapid metabolism of soluble sugars and their conversion into starch. Such changes have also been related to changes in the activity of sucrose or starch synthesizing enzymes (Claussen et al. 1985; Pharr et al. 1983; Rufty and Huber 1985) and/or reduced rates of translocation and export of assimilates from the leaf (Ho 1979; Fondy and Geiger 1980). Stitt (1986) has recently shown that leaves may have an excess capacity for electron transport so that Pn rate can be enhanced if the rates of RuBP regeneration and carboxylation are increased. He also suggested that the 78 rate of sucrose synthesis may be the ultimate limitation for photosynthesis since it may prevent the recycling of Pi (Stitt 1986). The discussion of the hypothesis of Pi limitations on Pn is beyond the scope of this paper (see the following references for details: Sharkey 1985; Sharkey et al. 1986; Stitt 1986; Sivak and Walker 1986). However, limitations in the utilization of triose phosphate have also been confirmed during Pn inhibition by ABA feeding or exposure to dry air in Arbutus (Loske and Raschke 1988). Since in this study we did not attempt to determine the activity of enzymes involved in carbon photosynthetic metabolism or the role of metabolites other than the major non-structural carbohydrates, it is impossible to say whether Pn was inhibited by synthesis of translocatable sugars or by utilization of triose phosphates. 0n the other hand, we did investigate whether the post; harvest increase in starch might have resulted in physical damage to the chloroplasts of mesophyll cells. Analysis of TEM micrographs showed enlargement of starch grains consistent with the increase of starch content determined by enzymatic analysis, but no disruption of chloroplast or thylakoid membranes was apparent (Figures 5 and 6). Physical damage to the chloroplast by starch grains has been suggested as a possible mechanism of Pn inhibition and cause for leaf chlorosis in response to various source-sink manipulations (Cave et al. 1981; Nafziger and Koller 1976; 79 Schaffer et al. 1986), but there is still contradictory evidence about the regulatory role of starch on leaf Pn (Little and Loach 1973; Potter and Breen 1980). Because of the low starch content of sour cherry leaves and the absence of chloroplast breakage, starch probably plays no major role in the post-harvest photosynthetic decline, but may be an additional factor playing on the overall depression of photosynthesis. In conclusion, although the data presented here are not conclusive, A/Ci curves, photorespiration, C02 compensation point, and increase in leaf starch content all seem to point towards limitations in the rate of RuBP regeneration rate as a likely mechanism of inhibition. Nevertheless, further experiments on the effect of elevated concentrations of C02 ( > 350 pl 1'1) on the stomatal and non-stomatal response of sour cherry leaves and the activity of enzymes of carboxylation and regeneration of RuBP are needed to elucidate the mechanism of Pn inhibition after fruit harvest. LITERATURE CITED 1) Azcon-Bieto, J. 1983. Inhibition of photosynthesis by carbohydrates in wheat leaves. Plant Physiol 73:681- 686. 2) Barrett, J.E. and H.J. Hamling. 1978. Effects of developing fruits on production and translocation of 14C labeled assimilates in cucumber. HortScience. 13:545-547. 3) .4) 5) 6) 7) 3) 9) 10) 11) 12) 80 Cave G., L.C. Tolley and B.R. Strain. 1981. Effect of carbon dioxide enrichment on chlorophyll content, starch content and starch grain structure in Trifolium subterraaeum leaves. Physiol. Plant. 51:171-174. Claussen, W., J.S. Hawker and B.R. Loveys. 1985, Sucrose synthase activity, net photosynthetic rates and carbohydrate content of detached leaves of eggplant as affected by attached stems and shoots (sinks). J. Plant Physiol. 119:123-131. Downton, W.J.S., B.R. Loveys and W.J.R. Grant. 1988. Stomatal closure fully accounts for the inhibition of photosynthesis by abscisic acid. The New Phytol. 108:263-266. Farquhar, G.D. and T.D. Sharkey .1982. Stomatal conductance and photosynthesis. Ann. Rev. Plant. Physiol. 33:317-345. Fondy, B.R. and D.R. Geiger. 1980. Effect of rapid changes in sink-source ratio on export and distribution of products of photosynthesis in leaves of Beta vulgaris L and Phaseolus vulgaris L. Plant Physiol. 66:945-949. Ho, L.C. 1979. Partitioning of 14C-assimilate within individual tomato leaves in relation to the rate of export. In: Photosynthesis and Plant Development (Marcelle, R., Clijsters, H., Van Poucke, H. eds). Dr. J.Junk Publ., The Hague. Jones, H.G. 1973. Limiting factors in photosynthesis. The New Phytol. 72:1081-1094. Jones, H.G. 1985. Partitioning stomatal and non- stomatal limitations to photosynthesis. Plant, Cell and Environm. 8:95-104. Kriedemann, P.E., Loveys, B.R., Possingham, J.V. and M. Satoh. 1976. Sink effects on stomatal physiology and photosynthesis. In: Transport and Transfer Processes in Plants (Wardlaw, J.F., J.B. Passioura, eds), pp 401- 414, Academic Press, London. Lakso, A.N. 1982. Precautions in the use of excised shoots for photosynthesis and water relations measurements of apple and grape leaves. HortScience 17:368-370. 13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 81 Lenz, F. 1979. Fruit effects on photosynthesis, light- and dark-respiration ppl61-164 .In: Photosynthesis and Plant Development (R Marcelle, H Clijsters, M Van Poucke eds). Dr W Junk , The Hague ISBN 9-06-193594-6. Little, C.H.A. and K. Loach. 1973. Effects of changes in carbohydrate concentration on the rate of net photosynthesis in mature leaves of Abies balsamea. Can. J. Bot. 51:751-758. Loske, D. and K. Raschke. 1988. Determination of carbon-reduction-cycle intermediates in leaves of Arbutus unedo L. suffering depressions in photosynthesis after application of abscisic acid or exposure to dry air. Planta 173:275-281. Madsen, E. 1968. Effect of C02 concentration on the accumulation of starch and sugar in tomato leaves. Physiol. Plant. 21:168-175. Mayoral, M.L., Z. Plaut and E. Reinhold. 1985. Effect of sink-source manipulation on the photosynthetic rate and carbohydrate content of cucumber cotyledons. J. Exper. Bot. 171:1551-1558. Milford, G.F.J. and I. Pearman. 1975. The relationship between photosynthesis and the concentrations of carbohydrates in the leaves of sugar beet. Photosynthetica. 9:78-83. , Mollenhauer, H.H. 1964. Plastic embedding mixtures for use in electron microscopy. J. Biophys. Biochem. Cytol. 4:191-194. 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. Photosynt. Res. 7:269-279. Nafziger, E.D. and H.R. Koller. 1976. Influence of leaf starch concentration on C02 assimilation in soybean. Plant Physiol. 57:560-563. Neales, T.F. and L.D. Incoll. 1968. The control of leaf photosynthesis rate by the level of assimilate concentration in the leaf: a review of the hypothesis. Bot. Rev. 2:107-125. Ogren, W.L. and G. Bowes. 1971. Ribulose diphosphate carboxylase regulates soybean photorespiration. Nature New Biol. 230:159-160. 24) 25) 26) 27) 28) 29) 30) 31) 32) 33) 34) 82 Peet, M.M. and P.J. Kramer. 1980. Effects of decreasing source-sink ratio in soybean on photosynthesis, photorespiration, transpiration and yield. Plant, Cell and Environ. 3:201-206. Pharr, D.M., S.C. Huber and H.N. Sox. 1985. Leaf carbohydrate status and enzymes of translocate synthesis in fruiting and vegetative plants of Cucumis sativus L. Plant Physiol. 77:104-108. Potter J.R. and P.J. Breen. 1980. Maintenance of high photosynthetic rates during the accumulation of high leaf starch levels in sunflower and soybean. Plant Physiol. 66:528-531. Reynolds, 8.3. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17:208-212. Roper, T. R., Keller, J.D., Loescher, W.H. and C. R. Rom. 1988. Photosynthesis and carbohydrate partitioning in sweet cherry: fruiting effects. Physiol. Plant. 72:42-47. Rufty, T.W., Jr. and S.C. Huber. 1983. Changes in starch formation and activities of sucrose phosphate synthase and cytoplasmic fructose-1,6-bisphosphatase in response to source-sink alterations. Plant Physiol. 72:474-480. 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. Sams, C.E. and J.A. Flore. 1983. Net photosynthetic rate of sour cherry (Prunus cerasus 'Montmorency' L.) during the growing season with particular reference to fruiting. Photosynt. Res. 4:307-316. Schaffer, A.A., Liu, K., Goldschmidt, E.E., Boyer, C.D. and R.Goren. 1986. Citrus leaf chlorosis induced by sink removal: starch, nitrogen and chloroplast ultrastructure. J. Plant Physiol. 124:111-121. Setter, T.L., W.A. Brun and M.L. Brenner. 1980. Effect of obstructed translocation on leaf abscisic acid, and associated stomatal closure and photosynthesis decline. Plant Physiol. 65:1111-1115. Sharkey, T.D. 1985. Photosynthesis in intact leaves of C3 plants: physics, physiology and rate limitations. Bot. Rev. 51:53-105. 35) 36) 37) 38) 39) 83 Sharkey, T.D., M. Stitt, D. Heineke, K. Raschke and H.W. Heldt. 1986. Limitation of photosynthesis by carbon metabolism II. 02-insensitive C0 uptake results from limitation of triose phosphate uti ization. Plant Physiol. 81:1123-1129. Sivak, M.N. and D.A. Walker. 1986. Photosynthesis 1a vivo can be limited by phosphate supply. The New Phytol. 102:499-512. Stitt, M. 1986. Limitation of photosynthesis by carbon metabolism. I. Evidence for excess electron transport capacity in leaves carrying out photosynthesis in saturating light and C02. Plant Physiol. 81:1115-1122. Tenhunen. J.D., 0.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 C0 exchange of leaves of Quercas suber. Planta 162:193- 03. von Caemmerer, S. and G.D. Farquhar. 1981. Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153:376-387. 84 KEYWORDS: Stomatal limitations, Eraaas cerasus , soluble sugars, starch. ABBREVIATIONS: Pn, net photosynthesis; g , stomatal conductance; Ci, intercellular C02; Ca, ambienE C02; PAR, photosynthetic active radiation; T, leaf temperature; A/Ci, response curve of CO assimilation to internal C02; Lg, stomatal limitations; c, stomatal contribution to a change in Pn; TEM, transmission electron microscopy; CE, carboxylation efficiency; RuBP, ribulose bisphosphate; Rubisco, ribulose bisphosphate carboxylase/oxygenase; Pi, inorganic phosphate. Section III DIURNAL AND SEASONAL CHANGES IN LEAF NET PHOTOSYNTHESIS FOLLOWING FRUIT REMOVAL IN PLUM. I. THE INFLUENCE OF STAGE OF FRUIT DEVELOPMENT AND ENVIRONMENTAL CONDITIONS 85 86 ABSTRACT The effect of fruit removal on net photosynthesis (Pn), stomatal conductance (gs), internal C02 (Ci), and chlorophyll content of leaves from mature plum trees (Prunus domestica L. cv. Stanley) was determined in the field at two stages of fruit development over 2 consecutive growing seasons. Gas exchange parameters were measured with a portable infrared open system. When fruits were removed during stage II (mid-June), Pn decreased from 12.6 to 8.5 pmols m"2 s"1 24 h after harvest in 1986, and from 12.1 to 10.2 umols m"2 s'1 in 1987. Pn inhibition was greatest in the early afternoon (30-45% of pre-harvest values), less (15-25%) or absent in the morning. Lower Pn rates in defruited trees persisted for 6-8 days in both years. Recovery of Pn in harvested trees coincided with vegetative growth 6 times more vigorous than that of fruiting trees in the first 6 weeks after fruit removal. Leaf chlorophyll content was similar (2.2 pg mg'1 fresh wt.) in defruited and fruiting trees. Fruit harvest at the end of stage III reduced Pn 25% after 24 h in 1986, but had no effect in 1987. The difference between years was probably related to both sink activity and environmental factors The decline of Pn following fruit harvest appeared to be confined to the defruited sector of the canopy. Midday depression of photosynthesis was observed only in harvested trees. Calculated levels of intercellular CO2 (Ci) were not significantly affected by defruiting. Water relations, 87 feedback inhibition by assimilation products, and direct action of phytohormones are suggested as mechanisms responsible for reduced Pn after fruit removal in plum. 88 INTRODUCTION The presence of the fruit reportedly enhances leaf net photosynthesis in eggplant (Lenz 1979), pepper (Hall & Brady 1977), soybean (Mondal et al. 1978), strawberry (Choma et al. 1982), citrus (Lenz 1979), and grapevine (Downton et al. 1987). On the contrary, no cpnsistent effect has been shown in sour cherry (Sams & Flore 1983) or sweet cherry (Roper et al. 1988). In most crops the "fruit effect" on photosynthesis seems to be largely dependent on the particular stage of fruit development at which gas exchange measurements are taken. Higher photosynthetic rates have been correlated with the presence of fruits during periods of maximum fruit growth rate and consequent demand for assimilates in tomato (Starck et al. 1979) and peach (Chalmers et al. 1975; DeJong 1986). Fruiting reduces above- and/or below-ground vegetative growth in tomato (Starck et a1. 1979), pepper (Hall and Milthorpe 1978), strawberry (Forney and Breen 1985; Schaffer et al. 1985), peach (Crews et al. 1975; DeJong 1986), and apple (Hansen 1967; Maggs 1963). Certain conditions may be necessary before a positive sink effect on photosynthesis is observed. Data reported in the literature are seldom comparable because of plant material, environmental differences during growth, and methods used to measure gas exchange (Roper et al. 1988). Furthermore, the physiological mechanism of sink-dependent regulation of Pn is still unclear (Guinn and Mauney 1981). 89 Plum is a suitable and convenient system for studying the effect of fruit removal on photosynthesis of fruit trees, because of its long period of fruit development and potential for heavy cropping. Photosynthetic characteristics of plum trees have been described to be very similar to those of peach (DeJong 1983), which have been extensively characterized (Crews et al. 1975; DeJong 1983), but few studies have been reported for plum. In peach the presence of the fruit reportedly stimulates photosynthesis even to the point of overcoming light limitations for leaves in the inner part of the canopy (Chalmers et al. 1975). The same authors found a significant decline in photosynthesis in those leaves following harvest of mature fruits. On the contrary, DeJong (1986) noticed lower rates of photosynthesis in nonfruiting peach trees only during periods of high fruit growth. However, differences between fruiting and non-fruiting trees were small. ‘ There are no known studies on the effect of fruit removal on photosynthesis and gas exchange of plum. An understanding of the dynamics of vegetative and reproductive development is particularly important in view of the alternate bearing habit of some plum varieties. It is also important to determine the interaction between fruit removal and with environmental and physiological parameters. For the above reasons, I measured gas-exchange parameters following removal of fruits at pit hardening or at maturity over two years. 90 The purpose of the present study was to characterize the short- and long-term photosynthetic responses to fruit removal in plum, with particular emphasis on the environmental and physiological conditions under which such responses occur in the field. MATERIALS AND METHODS Plant material. Three pairs of plum trees (Ergaa§__ggma§rlga L. cv. Stanley) planted in 1976 on a Miami loam at the Horticultural Research Center of Michigan State University, East Lansing (latitude 43° N, altitude 288 m) were selected based on similarity in vigor, crop load, leaf/fruit ratio and pre-harvest Pn rates in spring 1986. Trees were spaced 3.3 x 8.2 m, aligned in 4 rows oriented north-south, and trained to an open center. The same trees were utilized in 1987. The six trees were randomly assigned to treatments according to a randomized block design. Fertilizers and pesticides were applied according to commercial recommendations. Fruit growth and harvest. A sample of 20-25 fruits were collected weekly from 2 weeks after full bloom to maturity for growth measurements. Fruits were immediately weighed, their length, suture and cheek diameters measured with a precision caliper, and then oven dried at 105°C to constant weight and dry weights recorded. Full bloom dates were estimated to be April 27, 1986 and April 29, 1987. In both 91 years fruits were removed by hand in stage II and at the end of stage III. Dates of harvest weré: June 15, 1986; June 16, 1987; September 2, 1986 and 1987. Fruit densities were established by counting all the fruits on each scaffold and removing the selected percentage. Experiments on partial harvest were performed only at fruit maturity in both years. Percentages of harvest were 50, 75, and 100% in 1986, and 60, 75, 90, and 100% in 1987. Average yield per tree was 17 kg in June 1986, 21 kg in June 1987, and 24 kg in September 1987 (yield data not available for September 1986). Fruiting shoots of 2 additional plum trees on the same site were girdled with a knife by removing 0.5 cm of bark at approximately 50 cm below apex. Girdling was performed on June 10, 1986. Fruits were not removed from girdled shoots. Leaflfruit ratio and vegetative growth. A leaf/fruit ratio equal to 4.1 for each tree was estimated by counting leaves and fruits on 4-5 limbs on the east and west sides of the canopy. Vegetative growth of fruiting and harvested trees was estimated from cross-sectional areas and elongation of at least 3 differently oriented scaffold-limbs per tree. Cross-sectional area of the limb was calculated from 2 diameters measured 50 cm distal to the insertion of each scaffold on the trunk. Vegetative elongation was calculated by summing the terminal growth of all 1-year-old shoots longer than 10 cm present on the same limb. 92 Environmental parameters. Daily records of minimum and maximum temperatures, rainfall, and degree of cloudiness were taken from the records of the local weather station. The cloudiness index was calculated as the sum of the number of cloudy days divided by the number of days in the period of study. The number of cloudy days was calculated using the following scale: clear= 0; partially cloudy = 0.5; cloudy = 1. Gas axchange parameters. Net photosynthesis, stomatal conductance, PAR, leaf T, and air relative humidity were measured with an ADC LCA-2 portable open gas exchange system equipped with a Parkinson broad leaf chamber (Analytical Development Company, Hoddesdon, UK) on 6-8 leaves distributed on 3-4 limbs (shoots) per tree at each time of measurement. Two fully expanded leaves were used on each shoot, one on a fruit-bearing spur, the other apical to the spur. Measurements were always taken on the same leaves, unless leaves were inadvertently damaged during one of the measurements (damaged leaves amounted to less than 5% for each time course study). Leaves measured in September were not the same as those measured in June of the same year. In the partial harvest experiment, 2 trees were used as controls and 2 were totally harvested, but only one tree was used for intermediate treatments. Diurnal variation of gas exchange parameters was measured twice in June 1986 (1 and 4 days after harvest, from 600 to 1900 hr), and once in June 93 1987 (1 day after harvest from 800 to 1800 hr). In 1987 gas exchange was always measured twice a day (morning and afternoon). In order to investigate how localized the effect of fruit removal on Pn was, opposite sides of the canopy were partially harvested in 1986 and Pn measured on the west side to allow afternoon measurements. In 1987 Pn was measured only on the defruited side. Time of measurement for experiment 1 (trees defruited on the east side) was between 1215 and 1430 hr, for experiment 2 (trees defruited on the west side) between 1430 and 1630 hr. Unless otherwise noted the Pn unit was operated as follows: flow rate= 0.4 l/min; ambient 002 = 325-345 pl 1’1; PAR > 1000 pmols m"2 s'l. Gas exchange parameters were calculated as previously described (Moon & Flore 1986). Data were analyzed statistically by ANOVA within dates according to a randomized complete block design. Means were separated by LSD (P=0.05) unless otherwise indicated. Chlorophyll. Chlorophyll content was determined in 1987 using the method of Moran (1982) as described in Section I. Data were blocked by date . RESULTS The effect of fruit removal on gas exchange of plum was studied at two different times of the year. Fruits were removed on June 15-16, or September 2, these dates 94 corresponding respectively to stage II and the end of stage III on the double sigmoid curve typical of stonefruits (Lilleland 1932) (Figure 1). The warmest part of the year begins in Michigan in mid-June (Figure 2a). Average minimum and maximum temperatures were respectively 12.1’and 25.6 °C in June 1986, 16.1" and 28°C in July 1986, 14° and 28°C in June 1987, 17° and 29.8°C in July 1987 (figure 2a). Rainfall was relatively abundant in these months averaging 112 mm in June and 65 mm in July, sufficient for growth and production of fruit trees without irrigation. Fruit removal during stage II reduced Pn rates 35% within 24 h in 1986 (from 12.6 to 8.5 pmols m'2 s'l) (Figure 2b), 20% within 48 h in 1987 (Figure 2d). Significant differences (P=0.05) between Pn rates of defruited and fruiting trees remained apparent for approximately 7-9 days. The decline of Pn was due to neither early senescence of leaves from defruited trees nor changes in chlorophyll content. Leaf chlorophyll content was about 2.27 pg mg'l fresh wt (5.61 mg dm'z), and the chlorophyll [a/b] ratio approximately 3.5 at each date of sampling for each treatment. Chlorophyll content did not vary diurnally (data not shown). In both years, Pn rates of defruited trees returned to pre-harvest values 9-10 days after harvest (Figure 2b). Recovery was associated with vigorous growth of the canopy of defruited trees (Table 1). Total shoot length per cm2 CSA of defruited trees was 6 times greater than that of fruiting trees when measured on Aug. 1, 95 Figure 1. Fresh and dry weights of plum fruits in 1986 and 1987. Full bloom dates: April 27, 1986; April 29, 1987. Arrows indicate times of fruit removal. Vertical bars indicate t SE (n= 20-25). FRESH WEIGHT (9) 96 Figure 1 45- 404 l 30- zo- 10- 1986 1987 , 14$. DRY WEIGHT (9) DAYS AFTER FULL BLOOM fii'l'l'lfTI'IfTrr‘r' 20 30 40 50 60 70 80 90100110l2013 2 0 0 J. c: 97 Figure 2. (a, c) Minimum and maximum temperatures, precipitation, and cloud cover during the first period (June) of fruit removal in 1986 and 1987. Arrows indicate the date of harvest. Needle bars indicate daily rainfall. Squares on top of the graph cloud cover ( I cloudy; a partially cloudy; Clclear). Thick bars separate months; the number shown at the end of the month denotes monthly precipitation. (b, d) Inhibition of photosynthesis after fruit removal during stage II of development. Vertical bars indicate j SE. 98 Figure 2 '0' i U m or n En PH ' “' \. E"; ‘- v .. 2' +4 5. Z N ~EN >- C‘O— . x O O Q) "' ‘3 n 2 O n o _ 2 |_ H N N :> — p a . LL. 0 a fit ti 0 . 0 LL. P3 . < n U) - c” > <— 0 \a ._ C I: t 0.6!. d— O Q a 9L. ‘ I . . . fl 0 O I I 0 - a) ‘0 N a: - _ H‘ (w: o--- 3°12”) (,5 .41: new!) I‘d, 114 “"”T"““” @NDlwwmm . Q L: In E - :2 ......____.'_o I .I 1""? Lo L” i J l— '9) —° I “'9 N T a h. _ .In:—n '2 l- ‘- _ n H: --; .- u n 61 ‘ .___-_L 5 2 o c n o (wanna-sane: arr 99 Table 1. Increase in shoot elongation and cross sectional diameters of limbs from fruiting and defruited plum trees. Mean shoot Cross sect. Total length Periodz Treatment length area £CSA) /CSA (cm) (cm ) (cm' ) (3) Mid Control 20.5 i 2.1 5.25 i 0.8 54.7 summer Defruited 46.1 t 4.7 7.29 i 1.2 297.0 (1:) , End of Control 37.8 i_3.3 5.52 i 0.9 171.4 growing Defruited 68.4 i 4.3 7.87 i 3.6 400.0 season End-Mid Control 17.3 0.17 116.7 (b-a) Defruited 22.3 0.58 103.0 Z Date of harvest: June 15, 1986. Control trees were harvested on September 30, 1986. Vegetative growth assessed at mid-summer and during winter rest. Values are means t SE. 100 1986. Vegetative growth was the same for both treatments in the second part of the growing season (from Aug. 1, 1986 until leaf abscission) (Table 1). Mean shoot length of harvested trees was more than twice that of fruiting trees in midsummer (46.1 vs 20.5 cm), but only 29% greater in the second part of the growing season (23 vs 17 cm). Diurnal differences in gas exchange parameters were evident because of fruit removal in 1987 (Figure 4). Pn decreased 25% in the afternoon, but no significant differences were apparent in the morning (Figures 4 and 5). Afternoon differences were significant (P=0.05) for 8 days. In 1987 Pn inhibition in the afternoon was about 45% of the pre-harvest value. Pn rates of control trees increased about 20% (from 12.6 to 15.1 pmols m"2 s'l) during the period of rapid fruit growth (between June 15 and July 2) (60 mg dry wt/ day; 158 mg fresh wt/ day). Stomatal conductance (gs) in the morning was not significantly affected by defruiting, but was higher for control trees in the afternoon. Stomatal conductance was higher in the morning than in the afternoon for both treatments until 4 days after harvest, when max T declined from 30-36 to 22-28 C. Gs of control increased about 70% (from 90 to 150 mmols to"2 5'1), showing positive correlation with Pn. When expressed as a percentage of pre-harvest values gs of defruited trees increased about 40%. Values of intercellular C02 (Ci) were similar for both treatments in the morning and afternoon, indicating that both stomatal and mesophyll 101 Figure 3. (a, c) Environmental variables during the second period of fruit removal (September) in 1986 and 1987; (b, d) changes of photosynthesis after removal of mature fruits in 1986 and 1987. Symbols are the same as in Figure 2. Vertical bars indicate : SE. 3 102 Figure HE35: :3.“— mut< m>OZME tam... KHz-.2 m>02um :Dmu «if? m> 1600 pmols m"2 s'l, air temperature 30-34. C, cuvette temperature 34-38 C, ambient relative humidity 48- 50%, ambient C02 345 pl 1'1, flow rate 0.6 ml/min. Photorespiration was estimated by comparison of Pn rates at 129 Excised shoots were used for estimation of photorespiration in the laboratory where environmental conditions could be held constant at 1000 pmols m-2 s"'1 PAR, ambient temperature 24-25’C, cuvette temperature 28.1- 28.5°C, flow rate ranging from 1.7 to 2.2 l/min depending on age and photosynthetic rate of the leaf. Photorespiration was measured by comparing Pn rates at 2.5 vs. rates at 21% 02. C02-response curves were measured by increasing external C02 concentration from 0 to 345 pl 1'1. Field and laboratory measurements were made on material from the same trees on the same day. Stomatal contributions to changes in Pn were estimated from A/Ci curves according to the methods of Jones (1985) and Farquhar & Sharkey (1982) as explained in Section II. Non structural carbohydrates. Six to ten leaves were collected from each tree at each time of measurement, immediately frozen in liquid nitrogen or dry ice, transported frozen to the laboratory, and there lyophilized to constant weight (36-48 hours) in a Virtis 10- 010 automatic freeze-drier (Virtis Inc., Gardiner, New York). Leaves were deveined, and weighed with an analytical balance (Mettler AB 163 Mettler Instruments AG, Greifensee, Switzerland). Leaf area of the dried sample was measured with a portable area meter LI-COR 3000 (LI-COR, Lincoln, Nebraska). Samples were ground in a Wiley mill, passed through a 40 mesh screen, and 50 mg of each sample was 130 extracted 4 times in 2 ml each ‘of 80% ethanol. The homogenates were centrifuged at 1500 rpm (Glc, Sorvall, Connecticut) for 5 minutes after each extraction. The supernatant was used for analysis of soluble sugars, the pellet for starch determination. For determination of the soluble sugars (sorbitol, fructose, glucose, inositol, and sucrose) the supernatants were poured into 50 ml round bottom flasks, evaporated to dryness using a rotary vacuum evaporator in a water bath at 40° C, and the dried samples stored overnight in a desiccator. The samples were converted into oximes ( Roper et al. 1988) and derivatized to tri-methylsilyl ethers (Sweeley et al. 1963). Standards for sorbitol, fructose, glucose, sucrose, and inositol were prepared by dissolving 0.25 g of the first four compounds in 50 ml of 80% ethanol and 0.1 g of inositol in 25 ml of 50% ethanol, then combining the 2 solutions and drying 0.5 ml aliquots in a stream of nitrogen at room temperature. Standards were derivatized in the same way as samples. Analyses were performed using a dual column, temperature programmed Varian 3700 gas chromatograph (Varian Associates Inc, Sunnyvale, California) with flame ionization detectors and 3% OV-17 on 80/100 mesh chromosorb WHP in 2 mm x 2 m glass column. Temperature was programmed from 150 C to 250 C at a rate of 5 C/min. Quantity was calculated using the internal standards with a Spectra Physics SP4100 integrator (Spectra Physics, San Jose', California) and the resulting areas 131 checked at random by cutting the peaks and weighing them with an analytical balance (Mettler Instruments AG, Greifensee, Switzerland). Injections (1 ul) were repeated twice for each sample and results averaged. Soluble sugars were expressed as mg g'1 dry weight. Starch was measured in the pellet remaining after extraction with 80% ethanol using the method of Roper et al. (1988) modified as follows. Samples resulting from the incubation at 55 C for 16 h with amyloglucosidase were diluted with distilled water to 15 ml volumes and three 0.25 ml aliquots from each sample were assayed colorimetrically using glucose oxidase (Sigma Tech. Bull. 510: EC 1.1.3.4). Absorbances were read at 440 nm with a Shimadzu UV-Vis 260 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). RESULTS Transpiration rates of leaves from defruited trees were about 10% and 25% lower than the controls respectively 24 h and 48 h after fruit removal. In the morning there were no consistent differences between the two treatments (Figure 1c). Defruiting did not affect WUE significantly (Figures 1e and 1f). Transpiration rates of fruiting trees were usually higher in the morning than in the afternoon (Figure 1c and 1d). Contrary to what was observed in the time-course response, average diurnal WUE was 37% higher in fruiting trees (Figure 2c). No change of WP occurred after fruit 132 harvest (Figure 3b), but OP decreased, although such decrease was not associated with loss of turgor (Figure 3a). No diurnal differences in WP were found between the two treatments on the dates studied (Figure 4), but defruiting reduced OP starting from about 1130 hr (Figure 4). In order to estimate the stomatal contribution to the decrease of Pn after harvest two methods were used both based on C02-response curves of photosynthesis (Farquhar and Sharkey 1982; Jones 1985) (Figure 5). Stomatal contribution to the decrease in Pn was 31 or 46% when calculated, respectively, by resistance (Farquhar and Sharkey 1982) or sensitivity analysis (Jones 1985) at ambient C02 (330 pl 1'1 corresponding approximately to 180 pl 1""1 Ci). Stomatal conductance did not vary significantly over the range of Ci tested, but was significantly higher (P=0.05) for fruiting 2 5'1: Figure 5). Stomatal trees (150 vs. 112 mmols m' limitations (Lg) to Pn were 53 and 59% respectively for fruiting and defruited trees (Farquhar and Sharkey 1982). (Pn of fruiting trees was 24.2 umols m”2 s"1 , while Pn of defruited trees was 17.3 Pmols m"2 s"1 at 180 pl 1'1 Ci). Calculated Lg overestimated the actual stomatal limitations because they were based on linear A/Ci curves (Farquhar and Sharkey 1982). Photorespiration rates did not differ between defruited and fruiting trees (Table 1), Pn rate was 35% greater at low 02 than at atmospheric 02 concentration for both 133 Figure 1. Changes in photosynthetic rate (Pn), transpiration (Tr), and water use efficiency (WUE) following removal of plum fruits at stage II during the morning and afternoon in 1987. Arrows indicate date of fruit removal. Vertical bars 1 SE (n=l6-20). 134 PP (t3 I-m 61mm!) (,-: ',.u.l clown-u) SlSBHJNASOlOHd EN NOIiVBIdSWHJ. Figure 1 — T -2 ~81 1 § 3 5.... - O o E' E c“ 3 3 ‘6 3E 2 3* {-4021 .> _O 2 "GU—I ,0: <——(> ': .o 'o '4—-°§ L :4 u- 0: I? T ~3L1J 0 at: B— *—< 3'18 -m :44 _Q)... :5 "< mu PO ‘3 _N L ~53 ‘ m o b .s s S‘ s s "a "° 9 :2 ‘5 1 49 ' N <——D ' - 4—0 N o o I‘L 1% 1 «3.1.6.6315? ég$$:‘- J: .51 "cl X (05H 34011-3703 MOLD) ADNBIOL-UB 35h 831VM 135 Figure 2. Diurnal changes in net photosynthesis (Pn), transpiration (Tr), and water use efficiency (WUE) of leaves from fruiting and defruited plum trees 1 day after fruit removal. Fruits were harvested during stage II of development (June 15, 1986). Vertical bars : SE (n=6-8). 136 2 Figure d 4 0—6 Control 24- H Defruited slaw. - J... .1- ..h. . wash-yzw A?» .18 2083 um 1E m_oEE « 10E « «_oEE 981550519. 52 A. . v 8 I . _ ~an V zoséimzé SOLAR TIME 137 Figure 3. Effect of defruiting on leaf water potentials (solid line), osmotic potential (dashed line), and turgor pressure (TP) in plum trees. Arrows indicate date of fruit removal. Vertical bars 1 SE (n = 4-9). TURGOR PRESSURE (MPO) LEAF POTENTIAL (MP0) 138 Figure 3 2.0 1.5- 1.0-1 _ 045- 013 -1.0'- 1 water potential -1.5- / l -2.0- / / \\\ _ .ofi/ \ osmotic potential -2.5- \‘ \\ Defruited H . Control O—O II'I'filr‘T"I"l" 0 3 6 9 12 15 DAYS AFTER FRUIT REMOVAL 139 Figure 4. Diurnal changes in leaf water potential (solid line), osmotic potential (dashed line), and turgor pressure of fruiting and defruited plum trees 1 day after fruit removal. Fruits removed on June 17, 1987. Vertical bars 1 SE (n = 4-9). TURGOR PRESSURE (MPO) LEAF POTENTIAL (MPO) 21) 140 Figure 4 1.5- II)- 045- 01) -11)- 1-1.5- -213- O—O Defruited cr<3 Control water- potential " ,. osmotic ~. .. potential 110 ' 112' 1'41 1'6' 18 ' 20 SOLAR TIME 141 Figure 5. Changes in net photosynthesis (circles) and stomatal conductance (triangles) in response to calculated levels of intercellular CO (Ci) for leaves of fruiting (open symbols) and defruitea (solid symbols) plum trees 1 day after fruit removal in 1987. Measurements made in the field at ambient CO ranging from 100 to 345 pl 1' and 02 concentration equal 50 0.5%. 142 Figure 5 535) (,_s z._uJ S|OUJUJ) OMN CON 00F - u . — b AL 3 .8 653:. CD 004 8 T on T OONI .9550 o 4 635.30 0 4 oa.o "we Pu ~o~.o + om.o~1 ca Nm.o "Na . we mmH.o + Nm.k- u 0 .aummw .Aruq ca j I I I l I -(0 In N OI-CO 3‘) O I I I I (3 PS . S? v- ") CM CM CV (,._s 3111 '03 81011177) Lhfl 143 treatments. Stomatal conductance was different between the treatments but did not vary at low 02 (gS of defruited trees was 115 at 0.5% 02 and 120 mmols m"2 s'1 at 21% 02; 9S of fruiting trees was 131 at 0.5% 02 and 159 mmols m'2 s"1 at 21% 02). The magnitude of post-harvest inhibition (78%) did not change at low 02 (Table 1). Sorbitol was the most abundant soluble sugar in plum leaves, its content ranging from 6 to 12% of dry weight, equal to 50-78% of the total sugar composition of the leaf. Glucose and sucrose were present at about 1-2.5% of dry wt., corresponding to 13-25% of total soluble sugars for sucrose, and to 11-25% for glucose. Sucrose and glucose amounted on average to 15% each, while fructose never exceeded 11% of the total sugar composition. Inositol was only present in traces (less than 0.1%)(Figure 6). Figures 7 and 8 report time-course and diurnal trends of total soluble sugars, translocatable sugars (sorbitol and sucrose), and hexoses (fructose and glucose). Defruiting did not modify the sugar composition of the leaf. Starch content of leaves increased from 0.35% prior to treatment to 0.62% dry wt 24 h after harvest (measurement at 15:35), corresponding to a 79% increase and continued to increase for about 1 week (Figure 9). Since the levels of starch in the fruiting treatment also increased in this time interval (about 28%) the increase of starch content due to fruit removal amounted to about 51%. Average levels of starch in the morning were respectively 0.31% and 0.64% 144 . . 1m 18 maoal coon um50 AU mmlmm nonsuonmmfiou ucofinao “mad man CH mcofluaosoo Housmaconn>cm .mo 30H um musmaunonu zuon non Sausoownnson >no> no: cap moocmuoaosoo nauseoum .«omumq nauncnssn m>numaon nnm “o mnuan "onsumnmasmu ouuo>so 1 1n n1 oenuomn ”moo ucOnn6o A In ~18 mHoaa‘ooeH A Mdm ” pagan on» Ow m:0nufiocoo Houcossonn cm .OCOAHOOAHmon m HO muomum UHMUCMHW GEO mcmwfi mum m05Hw> .ooumd nucm oouNH £003HOQ 060E mucmamhfimmmfi .hwma .oa mash co OO>OEmn muasnm .:0wuonnsoocoo No «m.o no musmaonsmOOE anwm . u mm we we no Awe No son \nmn: m.b >.w m.> m.m oosmnounno no mo.~ + m.oa Hm.H + n.wa Nb ma.m + ¢.n~ on.a + m.oa um.m mm 0H.H + n.m mm.o + m.h Mb mm.N + m.md hm.H + M.HH am a o n o Honusoo pounsnuoo Honucoo oouwsnuwa Awe 11111111111111111111111111111 Awe 11111111111111111111111111111 MAS n\m A In TE ma0£1v n\o A no IE maoalv o c o w u m H m E n m m < «00 c o n u w H m E a m m 4 ~00 w n o u o n o n m A o H 0 a m .hnoumnonoH on» an no pawn“ on» Ca coflumnusoocoo NO Awm.~ no wm.ov 30H no Awamv ucmnnEm no nonsmoofi moon» Esau confisnuoo can ganunsnu no mouon sowuoananmmm Nov .H manna 145 soluble sugars Figure 6. Gas chromatographic analysis of from fully expanded leaves of field-grown plum trees and phenyl-D-glucopyranoside (internal standard) using trimethylsilyl derivatives. a. Fructose. b. Sorbitol. c. Glucose. d. Inositol. e. B-phenyl-D-glucopyranoside. f. Sucrose. 146 Figure 6 147 Figure 7. Content of hexoses (fructose, glucose), translocatable (sorbitol, sucrose), and total soluble sugars of leaves from fruiting (open circles) and defruited (solid circles) plum trees during the morning (a) and afternoon (b). Fruits removed on June 16, 1987 (date indicated by arrow). Vertical bars 3 SE. LEAF SOLUBLE SUGARS (% dry wt) LEAF SOLUBLE SUGARS (z dry-wt) 148 Figure 7 a 0 Control I Defruited 16.0- g ' total 12.0- C2 f 2 =4 l.__,_ V " ¢ \g/ translocatable 8.0- Morning 4.0- é-* - ~ -4»--- _ . 79‘+“‘""=I I"“"-=§l1exose l - .1 . -2 0 2 4 8 1 0 Control I) O Defruited O 16.07 . v “I F=‘+ total 12'0“ ?'¢-:37’4“’7 translocatable 8.0- Afternoon 4.0- /A\\ ,O~A_,_. ‘ 04-41;?“ -" L- "l 'r- :2 hexose I 1 0'0 l l l LU I -2 O 2 4 8 DAYS AFTER FRUIT REMOVAL 149 Figure 8. Diurnal changes in content of soluble sugars in leaves from fruiting (open circles) and defruited (solid circles) plum trees 1 day before (a) and 1 day after (b) fruit removal. Fruits removed on June 16, 1987. Hexose sugars are fructose and glucose, translocatable are sorbitol and sucrose: total sugars also include inositol and raffinose. Vertical bars 3 SE. LEAF SOLUBLE SUGARS (7: dry wt) LEAF SOLUBLE SUGARS (% dry wt) 150 Figure 8 18.0 0—0 Control 16.0- H Dafrmted 14.0... total ' 12.0- fifi—7‘<=:" ¢ ‘1’ 10.0- translocatable 8.0- _ 1 day before fru1t removal 6.0- 4,0- hexosefi - - - -‘- ~ ._ ~ _ 2.- - ~1 ------ - -=¢ ' a 0.0 , 1 I I I 10 12 14 16 18 o—o Control 15-0 ‘ H Defruited ”-0" total 12.0— 10.0- __ .... _- --— / 83" translocatable 6.0- 1 day after fruit removal 4-0' hexose e:::::::£:::::::2::::::2 2.0- b 0‘0 I I I l I 10 12 14 16 18 SOLAR TIME 151 Figure 9 . Changes in leaf starch content of fruiting and defruited plum trees in the morning and afternoon, 1987. Arrows indicate time of fruit removal. Vertical bars 1 SE (n= 3-6). LEAF STARCl-l (% dry wt) 152 Figure 9 1.2- 1.0- 0.8- 0.6- 0.4- 0.2- Morning 1.2- 1.0- 0.8- 0.6- 0.4- 0.2- 0.0 Afternoon o—o Defruited 0—6 Control 0'1 2 ~ 4- I -5 I DAYS AFTER FRUIT REMOVAL 153 Figure 10. Diurnal changes in leaf starch content of fruiting and defruited plum trees 1 day before (a) and 1 day (b) after fruit removal. Fruits removed on June 16, 1987. Vertical bars i SE. LEAF STARCH (7. dry wt) 154 Figure 10 0.8- 0.6- 0.4- 0.2- One doy before fruit removal v 0.8- 0.6- 0.4- 0.2- 0.0 One do;I Ofter fruit removol . 2,21 H Defruited o—o Control IO 1 l I 12 14 15 1% SOLAR TIME 155 Figure 11. Diurnal changes in starch content of leaves from fruiting, defruited, and girdled shoots of field-grown plum trees 4 days after fruit removal. Fruits were removed on June 16, 1986. Different letters indicate significant differences between treatments at each single date (P2 0.05). 156 (ILIBIeM MP 2) HOBVIS 51131 Figure 11 .0 (6 f6 _ao ._ In D .0 r6 .0 CO CD <- _v- .0 .0 (U l“ G 0“? D.” _O ,. .0 u b "'3 13 9.00 Eta _ o c1.b 021:0 U a a I l I r ‘0 O. In. 0. In. 0. SOLAR TIME 157 before and after harvest, in the afternoon 0.34% and 0.78% respectively. The post-harvest increase in starch also showed a clear diurnal trend (Figure 10). The difference in starch levels was particularly evident after 1200 hr. Starch increased from 0.26% to 0.63% in leaves of defruited trees (from 900 to 1815 hr) and from 0.3% to 0.53% in leaves of fruiting shoots (Figure 10). The highest levels of starch were found in leaves of fruiting shoots that had been girdled at the same time the fruit removal treatment had been imposed on other trees (Figure 11). Leaves from fruiting« shoots contained only 0.2-0.7% starch, whereas leaves from girdled shoots contained up to 1.6% at 1800 hr) (Figure 11). Similar diurnal trends were observed by measurements on two other dates. DISCUSSION Calculated stomatal contribution to the post-harvest decrease of Pn did not exceed 46% indicating that non- stomatal components were largely implicated in Pn inhibition. Among possible non-stomatal limitations, photorespiration was not responsible for the decrease of Pn (Table 1). 02 inhibition in air amounted to about 34% in both fruiting and defruited trees, a value intermediate between that reported by Sharkey (1985) as indicative of RuBP limitation (40%) or RuBP regeneration limitation (30%). No evidence for involvement of photorespiration in Pn changes consequent to source-sink manipulations was found 158 for soybean (Peet and Kramer 1980) and pepper (Hall and Milthorpe 1978). Linearity of A/Ci curves and their estimating from measurements made at 0.5% 02 do not allow to speculate about inhibition of carboxylation parameters. However, limitations of the carboxylation reaction, rather than inhibition within the carbon reduction cycle have been claimed to be responsible for the midday depression of Pn in Arbutus (Loske and Raschke 1987). This phenomenon in many aspects resembles the post-harvest inhibition of Pn in plum, especially with regard to its occurrence and dependence on environmental variables (Section III). Decreased carboxylation efficiency has also been reported for various herbaceous and woody species in response to water stress (Bunce 1977: Bunce 1982 : Briggs et a1. 1986), and has been related to lower translocation rates and diminished pressure flow between sources and sinks that results in accumulation of carbohydrates (Bunce 1982). Sharkey (1984) has also hypothesized that high VPD could decrease water potentials and transpiration rates, which in turn would induce changes in the photosynthetic capacity of leaves. However, in the case of fruit removal in plum, I did not find significant differences in the leaf water status and water potential between fruiting and defruited trees in the two years of study (Figures 3 and 4). Lower transpiration rates of defruited trees appeared essentially regulated by 95 and the higher turgor pressure of defruited trees was probably the result of lower transpiration and osmotic potential. 159 Decreased or constant WUE after fruit removal also seemed to indicate that defruited trees were not experiencing any particular water stress. Lower osmotic potential of defruited trees was evident in the afternoon (Figure 4) and most likely the result of increase in nonosmotic volume due to accumulation of starch rather than to any increase of soluble sugar concentration in the leaf. Osmoregulation resulting from accumulation of starch and/or soluble sugars has been correlated with turgor maintenance or reduced export rates out of the leaf (Acevedo et al 1979: Ackerson 1981). Leaf starch content increased 2-fold within 48 h from fruit removal and was particularly evident in the afternoon (Figures 9 and 10) at a time when Pn inhibition appeared maximum (Section III). Maximum starch accumulation occurred in leaves of girdled fruiting shoots (Figure 11). Starch accumulation following C02 enrichment or shoot girdling and fruit removal produces leaf chlorosis and senescence in soybean and citrus as a result of physical damage to the thylakoid membranes (DeLucia et a1. 1985; Schaffer et al. 1986). It is interesting to note that girdled shoots of plum also senesced early (Section III): although I did not examine chloroplast structure of mesophyll cells it is likely that chlorosis was caused by both starch accumulation and moisture stress (Little and Loach 1973: Schaffer et al. 1986). 160 On the contrary, senescence did not occur early in defruited trees despite the increase in starch (Figure 11). Accumulation of non-structural carbohydrates, occurring in response to source/sink alterations in many species, has been ascribed to Pn inhibition based on correlation analysis (Azcon-Bieto 1983: DeLucia et al 1985: Nafziger and Koller 1976: Peet and Kramer 1980: Schaffer et al. 1986: Thorne and Koller 1974). However, despite the fact that starch may reduce leaf assimilation by enlargement of grains and consequent physical damage to the chloroplast (Schaffer et al. 1986), starch accumulation is probably the result of changes occurring outside the chloroplast. Moreover, if and only if changes of Pn rates are direct responses to modifications of sink demand, fine regulation of Pn is unlikely to be provided by starch, since the hypothesized mechanism of action of starch is merely physical and thus not suitable for fine adjustments. A likely mechanisms of Pn regulation by sink demand is through the supply of Pi. Pn inhibition by utilization of triose phosphates and Pi supply pool has been recently discussed by Sharkey (1985) and Sivak and Walker (1986). According to this hypothesis, in order to maintain a high rate of C02 assimilation, starch and sucrose synthesis must also increase, otherwise triose phosphates will build up and Pi availability will decline (Sharkey 1985). Once the Pi pool is low enough to limit photophosphorylation, C02 assimilation could be regulated by the rate at which starch 161 and sucrose synthesis can metabolize triose phosphates and recycle Pi. Therefore, a critical Pi concentration may exist at which both phosphorylation and triose phosphate utilization are sensitive to changes in the pool size of Pi (Sharkey 1985). Pi availability may also be limited in the cytosol or the chloroplast, even when total cellular Pi is abundant, because of its vacuolar compartmentation. Is the Pi limitation a likely mechanism of Pn inhibition after fruit removal ? Pi limitations have been associated with 02-insensitive photosynthesis (Azcon-Bieto 1983; Sharkey 1985), but 02-insensitivity in the field after fruit removal was not observed. However, I measured responses to low 02 only at temperatures > 25 C, which may have been higher than the threshold of 02-insensitive photosynthesis (Sage and Sharkey 1987). Experiments in a controlled environment are needed to clarify this point. In turn, limitations of Pi availability have been related to reduced rates of translocation due to low sink demand (Grange 1987: Sivak and Walker 1986), and changes in activities of sucrose-synthesizing enzymes (Claussen et al. 1987: Hendrix and Huber 1986). Since the final step of sorbitol synthesis involves a phosphatase the mechanism of Pi sequestration may occur similarly to that seen for sucrose. Pn inhibition due to fruit removal at the pit-hardening stage in plum seems to be a process controlled by several components. There are two pieces of evidence in favor of a 162 major role of nonstomatal limitations: a) the constancy of Ci despite the decrease of Pn and gS (Section III): b) the partitioning of stomatal and non-stomatal components derived from A/Ci curves measured in the field. The documented accumulation of starch is compatible with the hypothesis of end-product inhibition (Azcon-Bieto 1983: Neales and Incoll 1968) and Pi limitations (Sharkey 1985: Sivak and Walker 1986). On the other hand, I did not find changes in levels of soluble sugars, which again points towards a lack of Pi availability (Sivak and Walker 1986). However, since soluble sugars are a much more readily available metabolic currency, variability caused by the large plant system used and environmental parameters may have masked any definite trend. Neither water relations nor photorespiration appeared to be clearly involved in the post-harvest Pn inhibition. The concomitant decrease in gs seemed related to environmental and endogenous parameters (Section III) as described for other species (Raschke 1975; Tenhunen et al. 1984: Tenhunen et al. 1987). However, since we did 'not investigate changes of ABA levels, we cannot rule out u priori the possibility that accumulation of ABA in the leaf regulates stomatal movement after fruit removal (Kriedemann et al. 1975: Raschke 1975). A new perspective on the role of ABA on stomatal regulation was recently presented by Downton et al. (1988). By the use of fluorescence and autoradiography methods they have shown that the nonstomatal 163 inhibition of Pn induced by ABA is an artifact resulting from the techniques used in gas exchange studies and the type of leaf measured (amphistomatous). The determination of the effect of fruit removal on ABA leaf levels and the necessity of further studies in controlled environments are the next two priorities. 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Changes in photosynthetic capacity, carboxylation efficiency, and C02 compensation point associated with midday stomatal closure and midday depression of net CO exchange of leaves of Quazgua auber. Planta 162:193- 03. Tenhunen, J.D., Pearcy, R.W. and 0.L. Lange. 1987. Diurnal variations in leaf conductance and gas exchange in natural environments - In: Stomatal Function (E. Zeiger, G.D. Farquhar & I.R. Cowan), Stanford University Press, Stanford, California. 40) 41) 167 Thorne, J.H. and H.R. Koller 1974. Influence of assimilate demand on photosynthesis, diffusive resistances, translocation and carbohydrate levels of soybean leaves. Plant Physiol. 54:201-207. Turner, N.C. and M.J. Long. 1980. Errors arising from rapid water loss in the measurement of leaf water potential by the pressure chamber technique. Aust. J. Plant Physiol. 7:527-537. 168 KEYWORDS: Photosynthesis inhibition, Egunus uomeatica, water relations, photorespiraton, starch, soluble sugars. ABBREVIATIONS: Pn, net photosynthesis: gs, stomatal conductance: Ci, intercellular C02; Tr, leaf transpiration: WUE, water use efficiency; VPD, vapor pressure deficit: R, gas constant: T, temperature; J, molar concentration; WP, leaf water potential; OP, leaf osmotic potential: TP, leaf turgor pressure; A/Ci, C02 assimilation response curve to intercellular CO : Pi, inorganic phosphate: PAR, photosynthetic active radiation: Lg, stomatal limitation: RuBP, Ribulose-1,5-bisphosphate. SUMMARY AND CONCLUSIONS The optimization of yield per unit of input has always been a major goal in horticulture. Since plant productivity depends largely on carbon assimilation (carbohydrates may represent 90-95% of plant dry weight), studies on the relationship between sources and sinks of assimilates may improve carbon efficiency (yield per unit of leaf area) of fruit crops. This study was an attempt to characterize the relationship between leaves and fruits by measuring changes in gas exchange, water relations, and carbohydrate partitioning following fruit removal in sour cherry and plum under field conditions. Defruiting at the beginning of the summer (June 15 to July 7) reduced Pn rapidly in both species, but the effect persisted longer in sour cherry than in plum, probably because of the different dynamics of development of alternative sinks in the two species. The magnitude of Pn inhibition was similar in both terminal and spur leaves, but greater in the afternoon than in the morning. No apparent change in Pn existed when light and/or temperature were suboptimal for assimilate production or when the leaf/fruit ratio exceeded certain threshold values. The reduction in 169 170 Pn caused by fruit removal was also less evident in plum when fruit growth rate was slower (end of stage III vs. stage II). However, environmental conditions at the two different times of fruit removal were also different and may have influenced the response of the tree to defruiting. Experiments in controlled environments are needed to partition precisely the influence of temperature, light, and ambient humidity on 1the Pn reduction following fruit removal. Since turgor was maintained in leaves of defruited sour cherry trees, the apparent wilting of the foliage after fruit removal does not seem to be related to water stress. Therefore, normal scheduling of irrigation does not need to be modified after fruit harvest. Defruiting also decreased stomatal conductance. Nevertheless, constancy of internal C02 values, lack of strong correlation between Pn and gs, and analysis of A/Ci curves, derived both from field and laboratory measurements, indicate that nonstomatal factors contributed more than stomatal ones to the Pn change. On the contrary, defruiting did not affect photorespiration, chlorophyll content, or concentration of soluble sugars in the leaf. Leaf starch content increased dramatically following fruit removal, but did not cause disruption of chloroplast membranes. Starch accumulation may have been responsible for the decrease of leaf osmotic potential in defruited trees by reducing the nonosmotic 171 volume of the cell. Based on similarities between the results presented in this study and reported cases of Pn inhibition from the literature, feedback inhibition may have limited Pn rate because of reduced carboXylation efficiency, RuBP regeneration rate, or triose phosphate utilization. Evidence and implications for each of these mechanisms in relation to fruit removal have been extensively discussed in this study. Experiments aimed at determining activity of enzymes responsible for synthesis and degradation of translocatable sugars, and Pi availability in the cytosol following fruit removal are necessary to clarify the molecular mechanisms of Pn decline. Another question that needs to be addressed is whether the regulation of stomatal behavior after fruit removal is driven mainly by environmental (VPD, T) or endogenous factors (Ci, ABA). The major limitation of this study is that gas exchange parameters were measured only on 1-year-old shoots, and that water relations and carbohydrate partitioning were analyzed only within the leaf, without considering, for example the rate of carbon export to other tissues and organs. In order to use these results for a model of the whole tree, experiments should be carried out to determine carbon partitioning to stems, trunk, and roots. 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Cowan). Stanford University Press, Stanford, California, USA. Thorne, J.H. and H.R. Koller. 1974. Influence of assimilate demand on photosynthesis, diffusive resistances, translocation and carbohydrate levels of soybean leaves. Plant Physiol. 54:201-207. 105) 106) 107) 183 Turner, N.C. and M.J. Long.1980. Errors arising from rapid water loss in the measurement of leaf water potential by the pressure chamber technique. Aust. J. Plant Physiol. 7:527-537. von Caemmerer, S. and G.D. Farquhar. 1981. Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta. 153:376-387. Wood, B.W. 1988. Fruiting affects photosynthesis and senescence of pecan leaves. J. Am. Soc. Hort. Sci. 113:432-436. APPENDIX 184 Figure 1. Gas chromatographic analysis of soluble sugars from fully expanded leaves of field-grown sour cherry trees and B-phenyl-D-glucopyranoside (internal standard) using trimethylsilyl derivatives. a. Fructose. b. Sorbitol. c. Glucose. d. Inositol. e. B-phenyl-D-glucopyranoside. f. Sucrose. Bar indicates retention time. (Section II) 185 Figure 1 186 Table 1. Changes in chlorophyll content of leaves from fruiting and defruited plum trees at HRC, East Lansing. Fruits removed on June 16, 1987. DAFRz D e f r u i t e d C o n t r o 1 Solar ------------------------------------------ Date time chl pg.chl/ mg Shl'/ chl pg chl/ mg cBl/ [a/b] mg leaf dm [a/b] mg leaf dm fr. wt. LA fr. wt. LA June 1430 -1 4.1 2.28 4.6 3.6 2.20 5.3 15 June 1545 1 3.5 2.25 5.4 3.5 2.01 5.3 17 June 1400 8 3.7 2.72 6.5 3.6 2.75 6.6 26 July 1400 16 3.9 2.22* 5.2 3.9 2.55* 5.9 2 * Values significantly different at 5% level (LSD= 0.32): each value is a mean of 3 replications. Statistical analysis not performed for ratios of chlorophyll [a/b]. DAFR = Days after fruit removal: LA = Leaf area: fr. wt. = Fresh weight; chl. = Chlorophyll. 187 Table 2. Diurnal changes of chlorophyll content in leaves from fruiting and defruited plum trees 1 day after fruit removal. Fruits removed on June 16, 1987. Solar D e f r u i t e d2 C o n t r o 1 time --------------------------------------------- chl. pg.chl/ mg.§hl/ chl. pg.chl./ mg ghl/ [a/b] mg.leaf dm [a/b] mg.leaf dm fr.wt. LA fr.wt. LA 900 3.0 2.12 5.8 3.4 2.13 6.1 1545 3.5 2.25 5.4 3.5 2.01 5.3 1815 3.7 2.19 5.8 3.6 2.06 5.4 z Values are means of 3 replications: no differences are statistically significant. Symbols are same as in Table 1 of Appendix. 188 Figure 2. Diurnal changes of Pn and 98 of leaves fruiting and defruited shoots of field-grown plum trees 1 day after fruit removal. 1987. Vertical bars 1 SE. Branches defruited on June (Section III) from 16, 189 Figure 2 (,.s ,.w sloww) EONVlOnClNOO 'lVlVWOlS 21 CD C) V? 1&3 CD SENS-USPUJ" 43 99" ll ‘ II -S I! ll ~35 II II II ~91 ll +0- // -E // _ // 33 4‘- 1343-116 00 E- 62 0° 1 ‘1 1 *1 *TT ‘1 “3 (15.411810 SISBHINASOIOHOI EN SOLAR TIME 190 Figure 3. Sectors of the canopy defruited and measured for photosynthesis during Experiment 1 of partial defruiting of plum trees in 1986 (schematic). (Section III) 191 Figure 3 Experiment 1 .€%%%%%% vaeacmr?afayafo%%%%%§ clavefoJ?’¢¢f’ifa$fi?§w¢I¢J&J&0¢R¢ ooso...moo.o~ooo 0.. o... a.%%%%%%%%%u%%%%%%%u.ufinuv. ... . . ... ......................... vaaaa¢¢¢nc¢¢a¢aa scar.vacnu.%% ....... ................... ....... . ... . . . . . . . 5...... ........... . . Harvested 50% ...... a... ......... . . . ........... .. . . . o o o o o :33... o... %%&A&J 000...... . o o o o 9 o o.....0.o.¢..:oooo .JVJ&& 0... fl .. .o... .. . onoo... . 3. . . . . ........u.”... ............ ooo...o.¢.. .......... .........._ .... ... ..... .o .3. .~ Pn measurement Harvested 192 Figure 4. Sectors of the canopy defruited and measured for photosynthesis during Experiment 2 of partial defruiting of plum trees in 1986 and 1987 (values shown refer to percentages in 1986; schematic). (Section III). 193 Figure 4 Experiment 2 Control I o%%%%& 0090.. 053%fl3 0000 % Harvested Harvested 194 Figure 5. Changes in Pn, Tr, and WUE following removal of plum fruits during stage II in the morning and afternoon in 1987 expressed as percentage of pre-harvest values. Arrows indicate date of fruit removal. (Section IV) 195 Figure 5 ..._<>O_2mm .EDNE mwt< m><0 m m e N o .~.n.._Io._ ,¢..Iu._ o. m b h » n p n p — “0 bd’ I-N I-O P b bi .8052? h aw 9:522 . \_w -on -0: -on— 4% .3053? J 95:02 % cooEoz< 9:50: (sanleA lSBAJPu-aJd )0 g) 30M 11 "d 196 Figure 6. Diurnal changes in Tr, and WUE of leaves from fruiting and defruited plum trees 4 days after fruit removal. Fruits removed on June 15, 1986. (Section IV) 197 ON a. p - p m— b m2; mfiom .v— . L N. . D— I. b m . I-ID Figure 6 “.3 3:8 0 35:8 0 (.3 ..Lu moww) NOllVUIdSNVBl (o'I-I .-Iow ‘03 Sloww) 3nM 198 Figure 7. Gas chromatographic analysis of soluble sugars of standard solutions prepared as described in Section IV (Materials and Methods). Bar indicates retention time. 199 Figure 7 "ITI’III‘IIIIIIIIIIIIIIIIIIIIIIT