‘o ‘0'... ..a'l .02. It!!!) .3 L :03 .1 v- ......I.. \ -I'I‘l‘.’ 11o)..." . .vl 5‘0]. V Ir .. y. .ule ‘v .32.? . . A «Lintwlxlt l1 . n 1 - 2 , 1.2.5:-.. .. .... . z: .. S. 3.2.: mg... 3.93%.qu : gagsféf JEE...§%§§§. Egg? .. .e ‘l 9..., Wi\llll§llilll\l\\\ This is to certify that the dissertation entitled PHYSIOLOGICAL RESPONSES OF PRUNUS CERASUS TO WHOLE-PLANT SOURCE MANIPULATION presented by DESMOND R . LAYNE has been accepted towards fulfillment of the requirements for Ph. D. degreein HORTICULTURE Major professor Date August 19, 1992 MSU i: an Affirmative Action/Equal Opportunity Institution 0-12771 L I LIBRARY W'Chlgan State Unlversity i l l PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE 3U“: '2; 100K “J” U fif—T—fil MSU Is An Affirmative Action/Equal Opportunity Indltution PHYSIOLOGICAL RESPONSES OF mm ME TO WHOLE-PLANT SOURCE MANIPULATION by Desmond Richard Layne A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1992 ABSTRACT PHYSIOLOGICAL RESPONSES OF m m TO WHOLE-PLANT SOURCE MANIPULATION by Desmond Richard Layne The source-sink ratio of one-year-old, potted sour cherry trees (Bums mm L.) was altered by whole—plant partial defoliation or continuous illumination to determine if trees were primarily sink limited and to elucidate the mechanism whereby photosynthetic enhancement or inhibition occurred. Partial defoliation resulted in a photosynthetic enhancement within one day that was attributed to higher stomatal conductance (g), estimated photochemical (o) and carboxylation (k) efficiencies and ribulose-l,S-bisphosphate (RuBP) regeneration rates. The increase in net CO, assimilation (A) was associated with reduced carbon partitioning to starch and increased partitioning to 'sucrose and sorbitol. This altered partitioning suggested that as sink strength increased following defoliation, 3-phosphoglycerate (3-PGA) accumulated in the cytosol which inhibited fructose-2,6—bisphosphate production. As a result, fructose-1,6-bisphosphatase activity probably increased, leading to accumulation of glucose-6-phosphate which activated sucrose-phosphate-synthase and enhanced sucrose synthesis. Photosynthetic enhancement was maintained over 32 days and senescence was delayed in partially defoliated plants. This may have been due to the production of new photosynthetic machinery, increased availability of root- derived cytokinins or both. Within one day, continuous illumination resulted in an inhibition of photosynthesis that was attributed to lower stomatal conductance, estimated photochemical and carboxylation efficiencies and MP regeneration rates. This decrease in A was associated with a decrease in variable and maximal fluorescence and an increase in instantaneous fluorescence indicating that leaves were photoinhibited and that irreversible damage had occurred to photosystem II. In addition, leaves of continuously illuminated plants partitioned 80% more carbon to starch and significantly less to sucrose and sorbitol. This altered partitioning indicated that the sink limitation was aggravated by continuous illumination leading to an insufficient utilization of sucrose from the leaf. This then may have reduced orthophosphate availability for exchange with triose—phosphates from the chloroplast. Accumulation of 3-PGA in the chloroplast probably activated ADP-glucose pyrophyosphorylase and enhanced starch biosynthesis. Whether photochemical and biochemical events occurred simultaneously and/or to the same degree to lead to the observed responses is uncertain. Evidence is provided in support of a sink limitation in young, potted sour cherry trees. D-ICATION This thesis is dedicated to the memory of my grandparents Claude and Ossie Layne. Their genuine love of plants and the enjoyment they projected in the cultivation and care thereof was influentialtomeatayoung age. lwishthey couldbeheretoseemy childrengrow with the same appreciation and love for plants that they shared with me. ACKNOWLEDGEMENTS I would like to express my sincere thanks to my major professor and mentor Dr. J .A. Flore. His invaluable enthusiasm, interest, advice, encouragement, support, guidance, and above all, his friendship provided a foundation upon which my graduate experience here at M.S.U. was able to develop and grow. His example as advisor, teacher, scientist, administrator, and person isonethatlhopeto emulateinmy own way. I am also grateful to the members of my guidance committee: R.M. Beaudry, M.J. Bukovac, K.L. Poff, and J. Preiss. Their interest, suggestions, criticisms, and guidance helped balance both my curriculum and research efforts so that I would be equipped to begin a career as a scientist. Valuable discussions regarding the thesis research with the following scientists is acknowledged with appreciation: R. Dickson, R. Gee, A.N. Lakso, J .W. Moon, and M.N. Sivak. I would also like to express thanks to M. Kwantes and J .D. Everard for their advice and assistance in developing the carbohydrate analysis protocols. The following people are thanked for help with plant material: K. Gordon, A. Herner, M. Hubbard, and M. Lloyd. .1 would like to thank LE. Sage and J. Jollay for laboratory assistance. A. Herner, J. Jollay, M. Lloyd, J. Masabni and M. Sakin are thanked for their help with data collection. I would also like to thank J. Jollay and M. Lloyd for acquiring reading materials for me at the library. J .W. Moon provided me the opportunity to study rubisco and along with R.G. Jensen, P. Adams, and G. Zhu, his interest and expertise provided a valuable learning opportunity at the University of Arizona in Tucson for which I am thankful. I am grateful for the friendship, support, and comradery of Rod Eckert, John Erwin, Pete Petracek, Tom Fernandez, Mike McClean, Jim Faust, and Mark Yelanich among others in the department. . My sincere gratitude goes to my family. My parents have always believed in me and encouraged me to work hard and aim high. My father’s advice and example has been both inspirational and invaluable. The unceasing support and love from my wife Cheryl has helped make the ordeal of graduate school both bearable and worthwhile. My sons Stephen and Michael make my life worth living. TABLE OF CONTENTS LIST OF TABLES ............................................. Vl LIST OF FIGURES ........................................... vn LIST OF SYMBOLS AND ABBREVIATIONS .......................... xnl INTRODUCTION ............................................ 1 SECTION I: RESPONSE OF SOUR CHERRY m CERASUS L. CV. ’MONTMORENCY’) TO WHOLE-PLANT SOURCE MANIPULATION ..... 3 Abstract .............................................. 4 Materials and Methods ..................................... 7 Statistical design .................................... 9 Gas exchange measurements ............................ 9 Results ............................................... 10 Gas exchange over time ............................... 10 Relationship between g, and A ........................... ll - Discussion ............................................ 11 Literature cited .......................................... 14 ii SECTION II: PHYSIOLOGICAL RESPONSES OF m CERASUS L. TO WHOLE-PLANT SOURCE MANIPULATION. I. PARTIAL DEFOLIATION AND PHOTOSYNTHETIC ENHANCEMENT ..................... 23 ABSTRACT ........................................... 25 MATERIALS AND METHODS ............................... 28 Gas exchange measurements ............................ 29 Diurnal gas exchange ................................. 29 Long-term effects ................................... 30 ' ‘Chl determination ................................... 30 Response to CO2 and low 02 ............................ 31 Gas exchange measurements for changing environmental conditions . . . . 31 Respiration and carbon partitioning ........................ 32 Soluble sugar and starch determination ...................... 33 RESULTS ............................................ 34 Diurnal gas exchange ................................. 34 Response to PPFD .................................. 35 Response to CO, and O2 ............................... 35 Respiration and carbon partitioning ........................ 36 Long-term effects ................................... 37 DISCUSSION .......................................... 37 ACKNOWLEDGEMENTS .................................. 42 LITERATURE CITED ..................................... 42 iii SECTION III: PHYSIOLOGICAL RESPONSES OF m CERASUS, L. TO WHOLE-PLANT SOURCE MANIPULATION. II. CONTINUOUS ILLUMINATION AND PHOTOSYNTHETIC INHIBITION ............. 59 ABSTRACT ........................................... 61 MATERIALS AND METHODS ............................... 64 Gas exchange measurements ............................ 65 Diurnal gas exchange ................................. 66 Gas exchange response to CO2 and O2 ...................... 66 Time to full photosynthetic recovery ....................... 67 Chi fluorescence .................................... 68 Chi determination ................................... 68 RESULTS ............................................ 69 Diurnal gas exchange ................................. 69 Response to CO, and O, ............................... 69 Time to full photosynthetic recovery ....................... 70 Chi fluorescence .................................... 70 Chi content ....................................... 71 DISCUSSION .......................................... 71 ACKNOWLEDGEMENTS .................................. 74 LITERATURE CITED ..................................... 74 iv SECTION IV: PHYSIOLOGICAL RESPONSES OF m CERASUS L. TO WHOLE-PLANT SOURCE MANIPULATION. III. GAS EXCHANGE, CHLOROPHYLL FLUORESCENCE, WATER RELATIONS AND CARBON PART'I'TIONING ........................................ 87 ABSTRACT ........................................... 89 MATERIALS AND METHODS ............................... 92 Gas exchange measurements ............................ 93 Chi fluorescence measurement ........................... 94 Leaf xylem water potential ............................. 94 Soluble sugar and starch determination ...................... 95 RESULTS ............................................ 96 Gas exchange over time ............................... 96 Leaf water status ................................... 97 Chi fluorescence .................................... 97 Carbon partitioning .................................. 97 DISCUSSION .......................................... 98 ACKNOWLEDGEMENTS .................................. 101 LITERATURE CITED ..................................... 102 SUMMARY AND CONCLUSIONS ................................. 114 LIST OF TABLES SECTION II Table I. The cfl'cct ofpartial defoliation (defol) on r, k, A”, c, ,,,, A... I, and R, of expanded leaves of one-year-old, potted sour cherry trees with time. ........ 49 Table II. The eflEct of whole plant partial defoliation on leaf R, of emanded leaves of one-year-old, potted sour cherry trees over time. ..................... 50 Table III. The efi'ect of partial defoliation on leaf soluble carbohydrate content of apanded leaves of one-year-old, potted sour cherry trees over time. ........ 51 Table IV. Long-term eflbcts of whole plant partial defoliation on A, g, chl a, chl b, P chl, SLD of expanded leaves of one-year-old, potted sour cherry trees over SECTION 111 Table I. The efl‘ect of continuous illlanination on P, k, Am, C, 3,0, A... and I, of expanded leaves of one-year-old, potted sour cherry trees with time. ........ 81 Table II. The efl'ect of continuous illumination (C.L.) for 1, 2, or 3 days on leaf chl a, b, and total chl content of expanded leaves of one-year-old, potted sour cherry vi SECTION IV Table I. The efl'ea of partial defoliation (dejle or continuous illumination (C.L.) on leaf xylem water potential of expanded leaves of one-year-old, potted sour cherry LIST OF FIGURE SECTION I Figure 1. Effects of partial defoliation (defol.) or continuous illumination (24 h) on diurnal (A) net CO2 assimilation and (B) stomatal conductance over time for one- year-old, potted sour cherry trees. Treatments: Control - nondefoliated, 14 h photoperiod (O); Defoliated - 14 h photoperiod (O); 24 h Light - nondefoliated, 24 h photopuiod (I); and 24 h/Defol. - defoliated, 24 h photoperiod (Cl). Gas exchange was measured at 12:00 for 0 and 288 hours following treatment and at 10:00 and 15:00 daily for days 1 through 11. Control plants were defoliated on day 7 after the 15:00 measurement (note arrow). Each point represents the average (iSE) of 16 leaves. .......... - l ....................... Figure 2. Effects of partial defoliation or continuous illumination on diurnal net CO2 assimilation (A) to internal CO2 concentration (C,) ratio over time for one-year- old, potted sour cherry trees. Treatments: Control - nondefoliated, 14 h photoperiod (O); Defoliated -. 14 h photoperiod (0);.24 h Light - nondefoliated, 24 h photoperiod (I); and 24 h/Defol. - defoliated, 24 h photoperiod (Cl). Gas exchange was measured at 12:00 0 and 288 hours following treatment and at vii 20 10:00 and 15:00 daily between days 0 and 11. Control plants were defoliated on day 7 after the 15:00 measurement (note arrow). Each point represents the average (iSE) of 16 leaves. ................................. 21 Figure 3. The relationship between stomatal conductance and net CO2 assimilation over time for one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated, 14 h photoperiod (a); Defoliated - 14' h photoperiod (0); 24 h Light - nondefoliated, 24 h photoperiod (I); and 24 h/Defol. - defoliated, 24 h photoperiod (C1). Linear regressions appear about the raw data. Data for (A) and (C) are for days 1 through 7 following treatment. Data for (B) and (D) are for days 1 through 12 following treatment. ........................ 22 SECTION II Figure 1. Time course for the diurnal response of (A) A, (B) R,' (units as for A), and (C) g. to whole plant partial defoliation in one-year-old, potted sour cherry trees. Treatrnents: Control - nondefoliated (O) and Defoliated - partially defoliated plant (0). Gas exchange was measured hourly from 08:00 to 20:00 and R. was measured at 21:30 daily. Each point represents the average (+, -, or 18E) of Figure 2. The relationship between g. and A over time for one-year-old, potted sour cherry tress Treatments: (A) Control - nondefoliated (O) and (B) Defoliated - partially defoliated plant (0). Points are from hourly gas exchange measurements (08:00 to 20:00) for day-l (day before treatment) through day 7 (7 days after treatment). Raw data appears about the regression lines. ....... 54 viii Figure 3. Effect of partial defoliation on the response of A to PPFD 9 days after treatment for one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated (O) and Defoliated - partially defoliated plant (0). Regression lines occur about the data. Data points which represent the average (:1: SE) of 8 leaf measurements appear about the regression lines. .................. 55 Figure 4. Effect of partial defoliation on the respome over time of A to C, of one-year- old,pottedsourcherrytrees. Eachfigure(A-F)comparestheday0 pretreatment response (0) with the treatment response for the subsequent day (0). Treatments: day 0 - before partial defoliation; days 1 (A), 2 (B), 3 (C), 4 (D), S (E), and 6 (F) correspond to l, 2, 3, 4, 5, and 6 days following defoliation, respectively. Regression lines occur about the data. Arrows point to the data collected at ambient C0, partial pressure (35 :1; 2.5 Pa). Data points - which represent the average (18E) of 4 leaf measurements appear about the regression lines. ......................................... 56 Figure 5. Effects of partial defoliation on (A) A, (B) R., and (C) leaf starch content over time for one-year-old, potted sour cherry trees. A was measured daily at 10:00. Daily R. was measured at 22:00, 02:00, and 06:00. The arrow indicates the time at which plants were partially defoliated. Each point represents the average (iSE) of at least 4 leaf measurements. ........................... 57 figure 6. The relationship between R, and leaf starch content for leaves ‘of partially defoliated one-year-old, potted sour cherry trees. Data points which are single leaf measurements appear about the regression line. ................... 58 SECTION III figure 1. Effects of continuous illumination (C.L.) on diurnal (A) A and (B) 8. over time for one-year-old, potted sour cherry trees: Gas exchange was measured at 09:00, 12:00, 03:00, and 20:00 for each day. Treatmmts: day 0 (0-24 h) before C.L.; day 1 (25-48 b), day 2 (49-72h), and day 3 (73-96h) represent 1, 2, and 3 days following C.L., respectively; day 4 (97-120h), day 5 (121-144h), and day 6 (145-168h) represent recovery 1, 2, and 3 days following return to 14 h photoperiod, respectively. Arrows point to the time at which plants were returned to 14 h photoperiod. Each point represents the average (18E) of 4 leaf figure 2. Effect of continuous illumination (C.L.) on the response over time of A to Q of one-year-old, potted sour cherry trees. Each figure (A - F) compares the day 0 pretreatment response (0) with the treatment respome for the subsequent day (0). Treatments: day 0 - before C.L.; days 1 (A), 2 (B), and 3 (C) represent 1, 2, and 3 days following C.L., respectively; days 4 (D), 5 (E), and 6 (F) represent recovery 1, 2, and 3 days following return to 14 h photoperiod, respectively. Regression lines occur about the data. Arrows point to file data collected at ambient C02 partial pressure (3512.5 Pa). Data points which represent the average (:1: SE) of 4 leaf measurements appear about the regression figure 3. Time course for the response of (A) A and (B) 2. to continuous illumination (C.L.) in one-year-old, potted sour cherry. trees. Treatments: 1, 2, and 3 days of CL. (C, O, I), respectively. Gas exchange was measured daily at 10:00. Each point represents the average (:1; SE) of 20 leaf measurements. ......... figure 4. Time course for the response of (A) Fv/Fm, (B) Fv, and (C) F0 to contimlous illumination (C.L.) in one-year-old, potted sour cherry trees. Treatments: 1, 2, and 3 days of CL. (0, O, I), respectively. Chl fluorescence was measured daily at 15:00. Each point represents the average (iSE) of 20 leaf SECTION IV figure 1. Effects of partial defoliation or continuous illumination on (A) A and (B) g, over time for one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated, 14 h photoperiod (O); Defoliated - 14 h photoperiod (O); 24 h Light - 24 h photoperiod (I). Gas exchange was measured beginning at 10:00 daily between days -1 and 7. Control plants were defoliated on day 0 after the 10:00 measurement. Each point represents the average (iSE) of at least 8 leaf figure 2. Effects of partial defoliation or continuous illumination on (A) E or (B) C, over time for one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated, 14 h photoperiod (O); Defoliated - 14 h photOperiod (O); 24 h Light - 24 h photoperiod (I). Gas exchange was measured beginning at 10:00 daily between days -1 and 7. Control plants were defoliated on day 0 after the 10:00 measurement. Each point represents the average (:I: SE) of at least 8 leaf figure 3. Effects of partial defoliation or continuous illumination on (A) Fv/Fm, (B) Fv, and (C) Fo over time for one-year-old, potted sour cherry trees. Treatments: xi Control - nondefoliated, 14 h photoperiod (O); Defoliated - 14 h photoperiod (O); 24 h Light - 24 h photoperiod (I). Chl fluorescence was measured at 15:00. Control plants were defoliated on day 0 at 17:00. Each point represents the average (iSE) of at least 8 leaf measurements. ................... 112 figure 4. Effects of partial defoliation or continuous illumination on leaf (A) starch, (B) sorbitol, 'and (C) sucrose content over time for one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated, 14 h photoperiod (O); Defoliated - 14 h photoperiod (O); 24 h Light — 24 h photoperiod (I). Control plants were defoliated on day 0 at 17:00. Each point represents the average (18E) of at least xii AC, CA—l-P chl a chl b C1350 F-2,6-BP FBPase Fm Fo Fv Fv/Fm GA Glc-6-P 1PM LAR PAR P chl 3-PGA rubisco RuBP SLD SLW SPS triose-P VPD LIST OF SYMBOLS AND ABBREVIATIONS net CO, assimilation net CO2 assimilation rate at 35 Pa C02 curve describing the relationship between A and C, net C02 assimilation rate at 90 Pa (900 M) C02 2-carboxyarabinitol-1—phosphate chlorophyll a chlorophyll b internal C02 concentration internal CO2 concentration at 35 Pa CO2 light compensation point transpiration rate fructose-2,6-bisphosphate fructose-1,6-bisphosphatase maximal fluorescence instantaneous fluorescence variable fluorescence . photochemical efficiency gibberellin glucose-6-phosphate stomatal conductance rate integrated pest management estimated carboxylation efficiency leaf area reduction stomatal limitation to A photosynthetically active radiation protochlorophyll 3-phosphoglycerate orthophosphate dark respiration rate estimated photorespiration rate ribulose-1,5-bisphosphate carboxylase/oxygenase ribulose—l ,5-bisphosphate specific leaf density specific leaf weight sucrose phosphate syntbase triose phosphate vapor pressure deficit CO2 compensation point estimated photochemical efficiency or quantum yield leaf xylem water potential xiii Guidance Committee: The journal-article format was adopted for this dissertation in accordance with departmental and university requirements. Each section was prepared as a self-standing manuscript so there is some duplication in the Materials and Methods portion of each section of the dissertation. Section I was prepared and styled for publication in Journal of the American Society for Horticultural Science. Sections 11, Ill, and IV were prepared and styled for publication in Plant Physiology. xiv INTRODUCTION Plant growth and yields are limited by both the supply, and demand for photoassimilates. Net C0, assimilation (A) of plants is limited primarily by the activity of sink tissues (fruits, shoots, and roots) or the activity of source tissues (expanded leaves). A sink limited condition can be demonstrated by the enhancement of source leaf A brought about by reducing plant leaf area, shading part of the canopy, or continuously illuminating plants. The source limited condition can be demonstrated when an increase in atmospheric CO, brings about a lasting increase in source leaf A. Whether a given plant is source or sink limited also depends on its stage of development. In the spring, as buds are breaking, and mobilized reserve carbohydrates are being depleted, trees are source limited because a canopy of foliage has yet to fully develop. In the summer, as fruit are maturing and root and shoot growth may have slowed or stopped, if crop load is low (ie. due to a spring frost), trees may be sink limited. Since crop loads in mature sour cherry orchards are unpredictable from year to year (eg. frost, etc.), and due to the complications associated with measuring gas exchange in the field (eg. weather conditions) we opted to develop a model vegetative plant system to examine sink limitations to A. Using greenhouse grown potted trees allowed us to control the growing environment and conditions under which data was collected. By controlling environmental factors we could then focus on the responses of young trees to wholeplant source manipulation. Detailed studies were conducted in growth chambers to document the response of one- year-old, potted sour cherry trees (2mm m L., cv. ’Montmorency’ on Mahaleb rootstock) 2 to whole-plant partial defoliation or continuous illumination. Chlorophyll fluorescence was utilized to examine treatment effects on precarboxylation phenomena. Gas exchange was measured to determine C02 uptake during carboxylation and C0, efflux during photorespiration and dark respiration. Changes in leaf chlorophyll and specific leaf weight were monitored to document changes in photosynthetic machinery. Leaf carbohydrate content was measured to determine the flux of carbon between starch in the chloroplast and sucrose and sorbitol in the cytosol. Leaf water potential was measured to assess plant water status. The main objectives of this research were to: i. determine if young, potted sour cherry trees are sink limited; ii. document changes in chlorophyll fluorescence, gas exchange, carbon partitioning, photosynthetic machinery, and leaf water relations in response to source manipulation; iii. provide an explanation for the responses that occur following source manipulation; iv. suggest the regulatory sites in the carbon flux cycle that are involved so that photosynthetic enhancement or inhibition may occur following source manipulation; and v. Outline potential areas of further investigation. SECTION I: RESPONSE OF SOUR CHERRY m CERASUS L. CV. ’MON'TMORENCY’) 'TO WHOLE-PLANT SOURCE MANIPULA'TION Desmond R. Layne1 and LA. Flore‘. talisman 0 9011271.; U.'?fl _:!.' ' ans .~l‘l' til 13,. Received for publication . Acknowledgment is made to the Michigan Agricultural Experiment Station for their support of this research. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulation, this paper must be hereby marked Went solely to indicate this fact. This research was supported in part by USDA grant No. 88-34132-3380. ‘ Present Address: Forestry Sciences Laboratory, 5985 Hwy K, P.O. Box 898, Rhinelander, WI 54501 . 2 Professor Cellular and Plant Physiology Response of Sour Cherry (Bums mg L. cv. ’Montmorency’) to Whole-Plant Source Manipulation Addmgnaljndemm, photosynthesis, stomatal conductance, internal C02 concentration, RuBP, source/sink manipulation, limitation, photoinhibition, photooxidation, feedback inhibition, light compensation point, rubisco, carboxylation efficiency, photochemical efficiency, F—2,6-BP, FBPase, starch, sucrose, sorbitol, defoliation, continuous light . AW A, net CO2 assimilation; Ci, internal CO2 concentration; F—2,6-BP, fructose-2,6- bisphosphate; FBPase, fructose-1,6-bisphosphatase; g,, stomatal conductance; PAR, photosynthetically active radiation; Pi, orthophosphate; VPD, vapor pressure deficit; RuBP, ribulose-1,5-bisphosphate; rubisco, ribulose-l,5-bisphosphate carboxylase/oxygenase. Ahm The source-sink ratio of one-year-old, potted sour cherry trees was manipulated by whole-plant partial defoliation or continuous illumination to investigate the mechanism whereby photosynthetic enhancement or inhibition occurs and to determine if trees were sink limited. Partial defoliation resulted in a significant enhancement of net C0, assimilation (A) and stomatal conductance (g,) within one day. Continuous illumination resulted in a significant reduction of A and g, within one day. Partially defoliated plants that were continuously illuminated were not photosynthetically inhibited. Two days after partial defoliation or following two days in continuous light, A and g, had stabilized. Nondefoliated (control) plants had a diurnal variation 5 in which A and g, were higher at 10:00 than 15:00 each day. Partial defoliation of continuously illuminated plants resulted in full photosynthetic recovery within 5 days when plants remained continuously illuminated. g. and A were related for each treatment, but partially defoliated and continuously illuminated plants had the highest and lowest A per unit g,, respectively. The data indicate that trees were sink limited and possible mechanisms to explain the above responses are discussed. Herold (1980) described a source (expanded leaf) as the provider of photosynthetic carbon compounds and a sink (fruit, shoot, root) as the consumer thereof. He noted that sink activity included all metabolic processes associated with sustaining life, growth and development. Wardlaw (1990) stated that limitations to net C0, assimilation (A) are primarily related to the demand of sink tissues and the activity of source tissues. A source limited condition occurs when production of photoassimilate limits growth and productivity, whereas, when utilization of photoassimilate limits growth and productivity, a sink limited condition occurs (Baysdorfer and Bassham, 1985). Whole-plant manipulation to alter the source/sink ratio is a technique that can be utilized to investigate the source or sink limited condition. In a previous study (Layne and Flore, 1992), we demonstrated that whole plant partial defoliation of one-year-old, potted sour cherry trees over a five month growing period reduced shoot growth and leaf number but plant dry weight was not significantly reduced until 30% of the plant leaf area was removed. The photosynthetic enhancement that has been observed following whole-plant partial defoliation in diverse crops (Aoki, 1981; Baysdorfer and Bassham, 1985; Heichel and Tamer, 1983; Hodgkinson, 1974; Satoh et al., 1977; Tschaplinski and Blake, 1989a; von Caemmerer and Farquhar, 1984; Wareing et al., 1968; Williams and Farrar, 1988; 6 Wolk et al., 1983) is probably due to the relief of a sink limited condition. As a result of decreasing the source/sink ratio, it is possible that stomatal conductance (g_) (de Jong, 1986), ribulose-1,5-bisphosphate carboxylaseloxygenase (rubisco) activity (von Caemmerer and Farquhar, 1984; Warning et al., 1968) and ribulose-l,5-bisphosphate (RuBP) regeneration rate (von Caemmerer and Farquhar, 1984) were enhanced. This enhancement of A indicates that the source leaves are often operating below their maximum photosynthetic potential (Tschaplinski and Blake, 1989a). Continuous illumination extends the period over which photosynthesis can occur and as a result, growth enhancement has been observed in different crops (Bonsi et al., 1992; Darrow, 1933; Smith, 1933; Wheeler et al., 1986). Continuous illumination may increase source capacity more than sink capacity and lead to accumulation of carbohydrates in leaves (BOhning, 1949; Sawada et a1, 1989). Sharkey (1985) suggested that under conditions where carbohydrates accumulate, utilization of photoassimilate is the process most sensitive to that condition. The accumulation of carbohydrates in source leaves may lead to a feedback inhibition of A (Foyer, 1988). Starch accumulation in the chloroplast may impair light interception by the thylakoid membrane (Wildman, 1967) or interfere with intercellular C0, transport (Nafziger and Koller, 1976). Sucrose accumulation in the cytosol may cause a direct feedback inhibition of sucrose synthesis (Herold, 1980) or reduce the availability of orthophosphate (Pi) for exchange with triose-phosphates from the chlor0plast. 1f the latter occurs, fructose-2,6-bisphosphate (F-2,6-BP) biosynthesis in the cytosol increases (Cseke and Buchanan, 1983), directly reducing fructose-1,6- bisphosphatase (FBPase) activity and sucrose synthesis (Stitt et al., 1987). In addition to the enhanced growth of some species under continuous illumination, leaf injury has been observed in tomato (Hillman, 1956) and potato (Tibbits et al., 1990). The short- term reduction in A that often occurs following exposure to strong light (photoinhibition) precedes 7 the photodestruction of photosynthetic pigments (photooxidation) that may occur following longer- term exposure (Powles, 1984). Tibbits et al. (1990) found that temperature cycling (warmer day, cooler night) could alleviate the detrimental effects that continuous illumination at a constant temperature had on potato growth and development. Wheeler et a1. (1991) suggested that feedback inhibition of A may have occurred under conditions of continuous high light and high C02 because poor tuber initiation led to a sink limited condition. Whether the detrimental affects of continuous illumination on particular plant species were due to photoinhibition, photooxidation, or feedback inhibition remains to be fully elucidated. Many studies have been conducted to evaluate the whole-plant effects of source manipulation by continuous illumination or partial defoliation but there is a paucity of information for deciduous fruit trees and, more particularly, plant species with . polyol metabolism (Loescher, 1987). We chose to develop a vegetative, model plant system to investigate sink limitations to A. The objectives of this study were to: 1) document the gas exchange responses to partial defoliation and/or continuous illumination; 2) establish if there is a diurnal response of A during the day for plants grown in a growth chamber; 3) examine how the relationship between g, and A is affected by whole-plant manipulation; and 4) determine how long it takes for full photosynthetic recovery following whole-plant manipulation. Finally, we suggest how this model system may be utilized to: i. establish whether the sink limited condition exists; and ii: elucidate how source leaf A is regulated in one-year-old, potted sour cherry trees (Emails m L.). Materials and Methods Forty dormant one-year-old sour cherry trees (cv. ’Montmorency’ on Mahaleb rootstock) were planted in 11 liter plastic pots with 9.5 liters of sterilized greenhouse soil mix (5 sandy loam 8 :3sphagnumpeat:2torpedosand(byvolume),p1-I = 7.0). Alltreeswerecuttoanactivebud (0 to 10 cm above the bud union) and placed in an environmentally controlled greenhouse (day and night average temperatures were 28 and 23C, respectively). Trees were trained to a single ' shoot from which all laterals were removed as they appeared. Peter’s soluble 20N-20P-20K fertilizer .(500 ppm) was applied every 3 weeks and trees were watered every 3 days. Pesticides [5-O-demethylavermectin (abamectin, Avid), cyano(4-fluoro-3-phenoxyphenyl)methyl-3(2,2- dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate (cyfluthrin, Decathlon), and d-(2- chlorophenyl)-d-(4-chlorophenyl)—5pyrimidine-methanol (fenarimol, Rubigan) were applied as necessary according to label rates. Following 9 weeks of active growth in the greenhouse, leaf number and total leaf area was determined for each plant. Each plant had approximately 20 to 25 fully expanded leaves at this time and leaves were still emerging. Gas exchange was measured on the four most recently expanded leaves per plant using an ADC LCA2 portable photosynthesis system (Analytical Development Company, Hoddesdon, UK) under the following conditions (flow rate = 400 ml min", leaf temperature = 30 :1; 2C, vapor pressure deficit (VPD) = 3 kPa, ambient C0, ’= 350 j: 10 pl 1", and photosynthetically active radiation (PAR) 2 1000 umol ln'2 s“). The sixteen most uniform plants were selected for this experiment based on total leaf area and initial gas exchange characteristics. Selected plants were moved from the greenhouse to a Conviron PGV36 walk-in plant growth chamber (Conviron Systems of America, Pembina, N .D.). The growth chamber was programmed to the following settings (07:00: air temperature = 28C, relative humidity = 50%, PAR = 800 umol in2 s" -> which is saturating for leaf A of sour cherry; 21:00: air temperature = 23C, relative humidity = 50%, PAR = 0 mol m" 3"). Plants were randomly assigned to each of the following whole-plant source manipulation treatments: i. Control - nondefoliated, 14 h photoperiod; ii. Defoliated - partially defoliated, 14 9 h photoperiod; iii. 24 h Light - nondefoliated, 24 h photoperiod; and iv. 24 h/Defol. - partially defoliated, 24 h photoperiod. For the whole-plant partial defoliation treatment, approximately 70-75% of the total leaf area was removed by excising all expanded leaves below the fourth fully expanded leaf from the apex. Continuous illumination was accomplished by reprogramming the growth chamber so that the lights were always on. Plants that received a 14 h photoperiod were moved to the other side of the growth chamber behind a light screen (which reduced PAR to $2 umol m" s"; the light compensation point was 14 limo] m" s") at 21:00 and then returned to the illuminated side at 07:00 each day. Immediately following the last measurement 7 days following treatment, the Control and 24 h Light plants were partially defoliated as described above. Wm Plants were arranged in a completely randomized design. There were four replicate plants per treatment and four leaves were measured per plant (16 measurements per ' treatment). Analysis of variance was conducted for treatment comparisons at each date. Linear regression analysis was used to determine the relationship between g, and A. The PC version of SAS (SAS Institute Inc, Cary, NC) was used for all statistical analysis. ' W The four most recent fully expanded leaves on each plant were tagged and leaf area was determined according to Kappes (1985) where LA = (length x width x 0.671). Gas exchange was measured in the growth chamber with the portable ADC unit daily on each of the four leaves per plant under the following conditions (flow rate = 400 ml min“, leaftemperature = 32 :1: 2C, VPD = 4kPa, ambientCO2 = 350 j; 10p.l l“, andPAR = 800 umol m" s“). On days 0 and 12 following treatment, gas exchange was measured beginning at 12:00. On days 1 through 11, gas exchange was measured at beginning at 10:00 and then again beginning at 15:00 for each of the four leaves per plant. Gas exchange parameters were calculated using the BASIC computer program of Moon and Flore (1986). 10 Results W The effects of partial defoliation or continuous illumination on diurnal A and g, over time are presented in Figure 1. One day following manipulation, A was 27% higher and 48% lower than control for defoliated and 24 h light plants, respectively. g, was 80% higher and 35% lower for defoliated and 24 h light plants, respectively. By 2 days following manipulation, A was 47% higher and 57% lower than control for defoliated and 24 h light plants, respectively. Between 2 and 7 days after treatment, leaves from defoliated and 24 h/defol. plants had significantly higher A and g. rates than controls but 24 h light plants had significantly lower A and g, rates. The photosynthetic inhibition following continuous illumination was maintained as long as plants were continuously illuminated. A and g, of control plants fluctuated diurnally and were higher at 10:00 than at 15:00. After 7 days, control and 24 h light plants were defoliated. A and g, steadily increased for these plants during the 5 day recovery period. Twelve days following the initiation of the experiment, A was not significantly different among the treatments. The photosynthetic inhibition from continuous illumination was completely ameliorated 5 days after partial defoliation. Following the initial manipulation, internal CO, concentration (CD was always significantly higher in 24 h light plants than in control plants (data not shown). The effects of partial defoliation or continuous illumination on the diurnal A:C, ratio over time is presented in Figure 2. The ratio of these two parameters is an estimate of the photosynthetic efficiency of the leaf (CO, assimilation + CO2 available at the site of carboxylation). Since A of 24 h light leaves was significantly lower and Q was significantly higher than in control leaves, the efficiency of these leaves was greatly reduced. On the other hand, leaves of defoliated plants had higher A but comparable C, to control resulting in a higher 11 efficiency. The efficiency of control leaves was comparable to leaves 24 hldefol. plants. In Figure 1, A of 24 h light leaves that were defoliated after 7 days increased to the same level as control in 5 more days but the efficiency of these leaves was lower due to the significantly higher C, levels. . W The relationship between g, and A during the ’response period’ is presented in Figure 3. Since plants from both control and 24 h light treatments were defoliated by day 8, onlythedatafor days 1 through7 wereusedfortheregressionanalysis. Ontheother hand, since plants from both the defoliated and 24 hldefol. treatments were manipulated on day 1, the data for days 1-12 were used for the regression analysis. Note that the relationship between g, and A over time was significant (at P=0.05) for all treatments except the 24 h light treatment. The strongest relationship was for leaves of nonmanipulated control plants. The highest slope for the linear regression function was observed for the leaves of defoliated plants indicating that these leaves had the highest A per unit g,. Dismission partial defoliation reduced the source-sink ratio and probably relieved a sink limited condition. Stomatal behavior may have accounted for part of the photosynthetic enhancement that was observed following defoliation. Tschaplinski and Blake (1989a) observed a similar increase A and g, in decapitated may; trees. de long (1986) concluded that the higher photosynthetic rateoffruitingpeachtrees versusthosethathadbeendefruited early intheseason was dueto enhanced stomatal conductance. Hudson et al. (1992) suggested that stomatal function was independent of total leaf rubisco activity though. Partial defoliation of bean (von Caemmerer and Farquhar, 1984) and soybean (Wareing et al., 1968) resulted in an increase in rubisco activity 12 forbothcropsandanincreaseinRuBPregenerationrateforbean. lnapreviousstudy(Layne and Flore, 1992), we found that the photosynthetic enhancement of individual leaves to leaf area reduction was due to higher estimated photochemical and carboxylation efficiencies and RuBP regeneration rate and we suggest that a similar short-term (over days) mechanism is involved in leaves of partially defoliated plants noted here. Further experimentation, utilizing conventional carbohydrate analysis, “CO, pulse/chase to follow changes in photosynthetic metabolites, followed by isolation of regulatory enzymes to determine any changes in activity should help to shed light on the short-term mechanism whereby photosynthetic enhancement occurs. In the longer tam (weeks), the sustentation of higher A may have been due to the production of new photosynthetic machinery (Hodgkimon, 1974; Ness and Woolhouse, 1980; Satoh et al., 1977) and delayed senescence due to increased availability to root-derived cytokinins (Neuman and Stein, 1984; Satoh et al., 1977; Wareing et al., 1968). The decline in A in the afternoon for control plants was associated with a similar decline ing,. Thismay havebeendueto amild waterstress (WeberandGates, 1990) leadingtoan accumulation of ABA resulting in lower g,. Leaf xylem water potential was not affected by partial defoliation or continuous illumination (section IV) but ABA content of leaves was not determined. IsuspectthatthedeclineinAintheaflernoonwasnotduetoawater stress condition. The absence of a relationship between g, and A for continuously illuminated trees is curious and indicates that under conditions where photoinhibition may occur, the coupling of g, to A breaks down. It remains unclear at this point why the uncoupling occurs, physiologically. _ - _ The accumulation of carbohydrates in the leaf toward the end of the photoperiod is probably a more likely explanation for the afternoon decline in A (Foyer, 1988). Continuous illumination may have aggravated the sink limited condition that already existed for control plants. The inhibition of A upon continuous illumination was related to a 13 lower g, as observed for defruited peach trees (de long, 1986) but since C. was actually higher in these leaves, relative to the control plants, we suggest that lower g, wu of no physiological significance. In addition, the decrease in A may have been due to feedback inhibition (Foyer, 1988), photoinhibition, photooxidation (Powles, 1984) or a combination thereof. If leaves were photoinhibited, the inhibition of A may have been the result of peroxidation of lipids in the thylakoid mmbrane of the chloroplast (Mishra and Singhal, 1992) and removal of the Q. protein of P811 which impairs electron transport (Kyle, 1987). Since no leaf chlorosis or necrosis was observed during 7 days of continuous light, we suspect that photooxidation did not occur. The full photosynthetic recovery following defoliation of 24 h light plants may have been due to relief of feedback inhibition by depletion of accumulated starch (Sasek et al., 1985), reduced competition for root derived cytokinim (Neuman and Stein, 1984; Satoh et al., 1977; Wareing et al., 1968) and production of new photosynthetic machinery (Hodgkinson, 1974; Ness and Woolhouse, 1980; Satoh et al., 1977). Until a more detailed short-term evaluation is conducted, utilizing chlorophyll fluorescence, carbohydrate analysis, and measuring changes in leaf chlorophyll content, the involvement and relative significance of these phenomena, individually, or in combination with each other, remains equivocal. Continuous illumination of partially defoliated plants did not lead to photosynthetic inhibition. Defoliation may have increased the sink demand so that the export rate (Tschaplinski and Blake, 1989b) was high enough to prevent carbohydrates from accumulating even though plants were in contimlous light. Since photosynthetic inhibition was not exhibited for 24 hldefol. plants, this raises a very interesting question regarding the role of carbohydrates in the phenomenon of photoinhibition. Is there a direct interaction between starch grains and the thylakoid membrane which affects electron transport or is the effect indirect due to starch grains shading the thylakoid membrane (W ildman et al., 1967)? Further experimentation is necessary 14 to distinguish the relative importance and possible synergism between feedback inhibition and photoinhibition under conditions where they might be occurring simultaneously. This model plant system provides an ideal opportunity to investigate sink limitatiom to A and to study the mechanisms whereby photosynthetic enhancement and inhibition occur. Goals of our future efforts include utilization of this system to elucidate possible mechanisms regulating source leaf A of sour cherry and to study polyol biosynthesis. Literature cited Aoki, S. 1981 . Effects of plucking of young tea plants on their photosynthetic capacities in the mature and overwintered leaves. Japan. lour. Crop Sci. 50:445-451. Baysdorfer, C. and l.A. Bassham. 1985. Photosynthate supply and utilization in alfalfa. A developmental shift from a source to a sink limitation of photosynthesis. Plant Physiol. 77:313-317. Bdhning, R.H. 1949. Time course of photosynthesis in apple leaves exposed to continuous illumination. Plant Physiol. 24:222-240. Bonsi, C.l{., P.A. Loretan, W.A. Hill, and D.G. Mortley. 1992. Response of sweet potatoes to continuous light. HortScience 27:471. Cseke, C and B. Buchanan. 1983. An enzyme synthesizing fructose-2,6-bisphosphate occurs in leaves and is regulated by metabolite effectors. Fed. Europ. Biochem. Soc. Lett. 155:139-142. Darrow, G.M. 1933. Tomatoes, berries and other crops under continuous light in alaska. Science 78:370. de long, T.M. 1986. Fruit effects on photosynthesis in Emma mica. Physiol. Plant. 15 66: 149-153. Foyer, G.H. 1988. Feedback inhibition of photosynthesis through source-sink regulation in leaves. Plant Physiol. Biochem. 26:483-492. Heichel, G.H. and N.C. Turner. 1983. CO, assimilation of primary and regrowth foliage of red maple (Am mhnnn L.) and red oak (Gyms mm L.): response to defoliation. Oecologia 57:14-19. Herold, A. 1980. Regulation of photosynthesis by sink activity - the missing link. New Phytol. 86:131-144. Hillman, W.S. 1956. Injury of tomato plants by continuous light and unfavorable photoperiodic cycles. Am. J. Bot. 43:89-96. Hodgkinson, K.C. 1974. Influence of partial defoliation on photosynthesis, photorespiration and transpiration by lucerne leaves of different ages. Aust. J. Plant Physiol. 1:561-578. Hudson, G.S., J.R. Evans, S. von Caemmerer, Y.B.C. Arvidsson, and TJ. Andrews. 1992. Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase content by antisense RNA reduces photosynthesis in transgenic tobacco plants. Plant Physiol. 98:294-302. Kappes, EM. 1985. Carbohydrate production, balance, and transpiration in leaves, shoots and fruits of ’Montmorency’ sour cherry. Ph.D. Diss., Mich. State Univ., E. Lansing (Diss. Abstr. 86-13300). Kyle, DJ. 1987. The biochemical basis for photoinhibition of photosystem II, p. 197-226. In: Kyle, D.l., C.B. Osmond, and CJ. Arntzen (eds.). Photoinhibition. Elsevier Sci. Pub. B.V. Amsterdam. Layne, D.R. and l .A. Flore. 1992. Photosynthetic compensation to partial leaf area reduction in sour cherry. J. Amer. Soc. Hort. Sci. 117:279-286. Loescher, W.H. 1987. Physiology and metabolism of sugar alcohols in higher plants. Physiol. 16 Plant. 70:553-557. Mishra, R.K. and GS. Singhal. 1992. Function of photosynthetic apparatus of intact wheat leaves under high light and heat stress and its relationship with peroxidation of thylakoid lipids. Plant Physiol. 98:1-6. Moon, J.W. and l.A. Flore. 1986. A BASIC computer program for calculation of photosynthesis, stomatal conductance, and related parameters in an open gas exchange system. Photosynthesis Res. 7 :269-279. Nafziger, ED. and HR. Koller. 1976. Influence of leaf starch concentration on C0, assimilation in soybean. Plant Physiol. 57:560-563. Ness, PJ. and H.W. Woolhouse. 1980. RNA synthesis in M chloroplasts. II: Ribonucleic acid synthesis in chloroplasts from developing and senescing leaves. I . Exp. Bot. 31:235-245. Neumann, P.M. and Z. Stein. 1984. Relative rates of delivery of xylem solute to shoot tissues: Possible relationship to sequential leaf senescence. Physiol. Plant. 62:390-397. Powles, SB. 1984. Photoinhibition of photosynthesis induced by visible light. Ann. Rev. Plant Physiol. 35:15-44. Sasek, T.W., B.V. DeLucia, and B.R. Strain. 1985. Reversibility of photosynthetic inhibition in cotton after long-term exposure to elevated CO2 concentrations. Plant Physiol. 78:619-622. Satoh, M., P.E. Kriedemann, and B.R. Loveys. 1977. Changes in photosynthetic activity and related processes following decapitation in mulberry trees. Physiol. Plant. 41:203-210. Sawada, S., Y. Hasegawa, M. Kasai, and M. Sasaki. 1989. Photosynthetic electron transportand carbon metabolism during altered source/sink balance in single-rooted soybean leaves. Plant Cell Physiol. 30:691-698. l7 Sharkey, TD. 1985. Photosynthesis in intact leaves of C, plants: Physics, physiology and rate limitations. Bot. Rev. 51:53-105. Smith, F. 1933. Researches on the influence of natural and artificial light on plants. I. On the influence of the length of day - preliminary researches. Meld. Norg. Landbukshoiskole 13:1-228. Stitt, M., S. Huber, and P. Kerr. 1987. Control of photosynthetic sucrose formation, p. 327-409. In: M.D. Hatch and N.K. Beardman (eds.). The biochemistry of plants, vol.10. Academic Press, New York. Tibbits, T.W., S.M. Bennett, and W. Can. 1990. Control of continuous irradiation on potatoes with daily temperature cycling. Plant Physiol. 93:409-411. Tschaplinski, T.l . and TJ . Blake. 1989a. Photosynthetic reinvigoration of leaves following shoot decapitation and accelerated growth of cappice shoots. Physiol. Plant. 75: 157-165. Tschaplinski, TJ. and TJ. Blake. 1989b. The role of sink demand in carbon partitioning and photosynthetic reinvigoration following shoot decapitation. Physiol. Plant. 75:166-173. von Caemmerer, S. and GD. Farquhar. 1984. Effects of partial defoliation, changes 'of irradiance during growth, short-term water stress and growth at enhanced p(CO¢) on the photosynthetic capacity of leaves of W mm L. Planta 160:320-329. Wardlaw, I.F. 1990. The control of carbon partitioning in plants. New Phytol. 116:341-381. Wareing, P.F., M.M. Khalifa, and K.l. Treharne. 1968. Rate-limiting processes in photosynthesis at saturating light intensities. Nature 220:453-457. "Weber, l.A. and D.M. Gates. 1990. Gas exchange in gums mm (northern red oak) during a drought: analysis of relations among photosynthesis, transpiration, and leaf conductance. Tree Physiol. 7 :215-225. Wheeler, R.M. and T.W. Tibbits. 1986. Utilization of potatoes for life support systems in space. 18 I. Cultivar-photoperiod interactions. Am. Potato 1. 63:315-323. Wheeler, R.M., T.W. Tibbits, and A.I-I. Fitzpatrick. 1991. Carbon dioxide effects on potato growth under different photoperiods and irradiances. Crop Sci. 31:1209-1213. Wildman, S.G. 1967. The organization of gram-containing chloroplasts in relation to location of some enzymatic systems concerned with photosynthesis, protein synthesis, and ribonucleic acid synthesis, p. 295-319. ln:Biochemistry of chloroplasts, vol.2. W.T. Goodwin, (ed.). Academic Press, New York. Williams, l.H.H. and l.F. Farrar. 1988. Endogenous control of photosynthesis in leaf blades of barley. Plant Physiol. Biochem. 26:503-509. Wolk, l.O., D.W. Kretchman, and D.G. Ortega, Jr. 1983. Response of tomato to defoliation. J. Amer. Soc. Hort. Sci. 108:536-540. 19 Captions for figures figure 1. Effects of partial defoliation (defol.) or continuous illumination (24 h) on diurnal (A) net CO2 assimilation and (B) stomatal conductance over time for one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated, 14 h photoperiod (O); Defoliated - 14 h photoperiod (O); 24 h Light - nondefoliated, 24 h photoperiod (I); and 24 h/Defol. - defoliated, 24 h photoperiod (El). Gas exchange was measured at 12:00 for 0 and 288 hours following treatment and at 10:00 and 15:00 daily for days 1 through 11. Control plants were defoliated on day 7 after the 15:00 measurement (note arrow). Each point represents the average (18E) of 16 leaves. figure 2. Effects of partial defoliation or continuous illumination on diurnal net CO, assimilation (A) to internal CO, concentration (C,) ratio over time for one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated, 14 h photoperiod (O); Defoliated - 14 h photoperiod (O); 24 h Light - nondefoliated, 24 h photoperiod (I); and 24 h/Defol. - defoliated, 24 h photoperiod (in). Gas exchange was measured at 12:00 0 and 288 hours following treatment and at 10:00 and 15:00 daily between days 0 and 11. Control plants were defoliated on day 7 after the 15:00 measurement (note arrow). Each point represents the average (:1: SE) of 16 leaves. figure 3. The relationship between stomatal conductance and net CO2 assimilation over time for one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated, 14 h photoperiod (O); Defoliated - 14 h photoperiod (O); 24 h Light - nondefoliated, 24 h photoperiod (I); and 24 thefol. - defoliated, 24 h photoperiod (Cl). Linear regressions appear about the raw data. Data for (A) and (C) are for days 1 through 7 following treatment. Data for (B) and (D) are for days 1 through 12 following treatment. (umol m'2 s") STOMATA‘L CONDUCTANCE NET (:02 ASSIMILATION (mmol m"2 s") 20 Control Defoliated - 24 h Light . 24 h/Defol. 1 . l 95 ‘ 144 192 A 240 288 TIME FOLLOWING TREATMENT (hours) AzC. RATIO (omol m”2 s“) 80 60 20 21 N i\+’ .r’ \ N '- +M- Control Defoliated ‘ H 24 h Light ‘ l a L a 1 I L ii 24 h/DefOI. 48 95 144 192 240 288 TIME FOLLOWING TREATMENT (hours) ISTOMATAL CONDUCTANCE (mmol m“2 s“) 22 ' l ' l l I ' l ' l ' l T l ' l A e Control . B o Defoliated . Y = —-15 + 11X (r=.76) Y = —45 + 15X (r=.65) 160t- 80 "‘ 120r o — 80- - 40— e O i l ' l i l ' r . l ' l . l i l C I 4 h Light - D D 24 h/Defol. ‘ Y = 39 + 4x (r=.14) Y = —-3 + 10x (r=.42) 160- ‘7 ‘ B ago a o o 120- -‘ ‘ a 80 4O NET CO2 ASSIMILATION (mmol nn‘2 8“) SECTION II: PHYSIOLOGICAL RESPONSE OF ms CERASUS L. TO WHOLE- PLAN’T SOURCE MANIPULA‘TION. I. PARTIAL DEFOLIA'TION AND PHOTOSYN'TIIE'TIC ENHANCEMENT“ DesmondRichardIayne’andJamesAfiol-e . O I O I . en hunt-Hello 9'|!*11_'u.|‘l’-I I I en‘s .-‘i I! u' Received for publication Accepted for publication 23 24 ‘ Supported by the Michigan Agricultural Experiment Station and by USDA grant No. 88-34132- 3380. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulation, this paper must be hereby marked Mensa; solely to indicate this fact. 2 Present address: Forestry Sciences Laboratory, 5985 Hwy K, P.0. Box 898, Rhinelander, WI 54501. 3W A, net C02 assimilation rate; RuBP, ribulose-1,5-bisphosphate; FBPase, fructose-1,6-bisphosphatase; F-2,6-BP, fructose-2,6-bisphosphate; Glc-6-P, glucose-6-phosphate; SLD, specific leaf density; VPD, vapor pressure deficit; LAR, leaf area reduction; R4, dark respiration rate; chl a, chlorophyll a; chl b, chlorophyll b; P chl, protochlorophyll; R., estimated photorespiration rate; C., internal C02 concentration; AC,, curve describing the relationship between A and c.; P, CO, compensation point; k, estimated carboxylation efficiency; 1,, stomatal limitation; A_,, net C0, assimilation rate at 90 Pa (900 llL/L) C0,; cp, light compensation point; e, estimated photochemical efficiency or quantum yield; g,, stomatal conductance to C0,; A3,, net C0, assimilation rate at 35 Pa C0,; Cu”, internal CO, concentration at 35 Pa C0,; CA-l-P, 2—carboxyarabinitol-1-phosphate; 1PM, integrated pest management. 25 ABSTRACT The source-sink ratio of one-year-old, potted sour cherry trees (2mm m L.) was reduced by whole-plant partial defoliation to determine if trees were sink limited and to elucidate the basis of photosynthetic enhancement. In the short-term, within 24 h, net co, assimilation (A) of the most recently expanded source leaves of partially defoliated plants was significantly higher than nondefoliated (control) plants throuwout the photoperiod. Between two and seven days following defoliation, A was 30 to 50% higher and g, was 50 to 100% higher than in controls. Carboxylation efficiency (k) and RuBP regeneration rate had significantly increased within two days and remained consistently higher for the nine days followed. Carbon partitioning to starch decreased while partitioning to sorbitol and sucrose increased over time following defoliation. Dark respiration (R) decreased as starch content decreased over time following defoliation. 4’ was significantly higher nine days following defoliation. The diurnal decline in A was not due to starch accumulation but probably feedback inhibition by soluble carbohydrates in the cytosol. In the long-term, leaf senescence was delayed as indicated by higher A and g, in combination with higher chl content up to 32 days following defoliation. The enhanced sink strength and photosynthetic enhancement of source leaves following whole-plant partial defoliation indicated that trees were primarily sink limited. Photosynthetic enhancement following whole-plant partial defoliation has been observed in diverse crops including soybean (W areing et al., 1968), lucerne (Hodgkinson, 1974), mulberry (Satoh et al., 1977), tea (Aoki, 1981), tomato (Wolk et al., 1983), maple (Heichel and Turner, 1983), bean (von Caemmerer and Farquhar, 1984), alfalfa (Baysdorfer and Bassham, 1985), barley (Williams and Farrar, 1988), and 291111115 (Tschaplinski and Blake, 1989a). A threshold level of defoliation is reached when further defoliation cannot be compensated for and growth and yield are reduced. By determining damage thresholds for defoliation from insects, mites and diseases, one can predict if a particular level of infestation will be detrimental. Damage thresholds depend on the stage of crop development and the amount of crop present. Damage thresholds are an important component in IPM’. By choosing pesticides that maximize the effect on the target organism and minimize the effect on beneficial organisms, and by applying them only when a threshold is reached, more effective pest control is achieved and pesticide load into the environment is reduced. The time from defoliation to photosynthetic enhancement varies among species and depends on the extent and degree of defoliation. By partially defoliating a plant, the source-sink ratio is reduced and hence sink strength is increased. Sink strength is the product of sink size and sink activity (Warren Wilson, 1972). Ho (1988) suggested that "the import rate of assimilate, measured as the sum of the net carbon gain and respiratory carbon loss I by a sink organ, should give an appropriate estimate of the actual sink strength". In the short-term, the photosynthetic enhancement from increased sink strength is partly due to increased g, (delong, 1986) and enhanced k and RuBP regeneration rates of the remaining source leaves (W arcing et al., 1968; von Caemmerer and Farquhar, 1984). Photosynthetic enhancement may continue for weeks or months and leaf senescence is delayed in many crops (Gifford and Marshall, 1973; Hodgkinson, 1974; Satoh et al., 1977; Ness and WoolhOuse, 1980). 27 This is in part due to increased production of chloroplasts (Woolhouse, 1967), plastid proteins (W arcing et a1, 1968), chlorophyll and other photosynthetic components (Ness and Woolhouse, 1980). The competition for root-derived cytokinin (Wareing et al., 1968; Satoh et al., 1977; Neumann and Stein, 1984) is also reduced which may delay senescence and sustain the photosynthetic enhancement of the remaining leaves. Thedecline ofA observed inmany crops inthe afternoon may bethe resultoffeedback inhibition (Foyer, 1988). Herold (1980) suggested that there may be a direct inhibition of sucrose synthesis by sucrose accumulation in the cytosol. There may also be a reduction in fructose-1,6-bisphosphatase (FBPase) activity in the cytosol due to the production of F-2,6-BP (Stitt et al., 1987). Accumulation of starch grains in the chloroplast may affect the photosynthesis by shading the light reaching the thylakoid membrane (Warren Wilson, 1966), increasing the diffusive path lengths or interfering with intercellular C02 transport (Nafziger and Koller, 1976) thereby reducing A. In contrast, Potter and Breen (1980) demonstrated that leaf starch content was not related to A in soybean or sunflower. ‘ Many studies have been conducted on the responses of annual field crops and vegetables to partial defoliation but few have been conducted on perennial deciduous fruit trees. Sour cherry is of additional interest since in addition to sucrose, it produces a similar quantity of the sugar alcohol sorbitol during its photosynthetic metabolism (Loescher, 1987). The objectives of this study were use of one-year-old, sour cherry trees (2mm mm L.) as a model system to create a situation whereby they were sink limited. In addition we wanted to elucidate the factors regulating the responses to whole-plant source manipulation by partial defoliation by documenting: 1) short-term changes in: i.diurnal gas exchange; ii. response to PPFD, C0,, and 0,; iii. R, and carbon partitioning; and 2) long-term changes in gas exchange, chlorophyll content and SLD. 28 MA'I'ERIAIS AND METHODS Dormant one-year-old sour cherry trees (Bums m L., cv. ’Montmorencyi on Mahaleb rootstock) were planted in 11 L plastic pots with 9.5 L of sterilized greenhouse soil mix [5 sandy loam : 3 spth peat :2 torpedo sand (by volume), pH = 7.0]. All trees were cut to anactivebud (Oto 10cm abovethebudunion)andplaced inanenvironmentally controlled greenhouse (day and night means 28 and 23°C, respectively). Trees were trained to a single shoot from which all laterals were removed as they appeared. Peter’s soluble 20N-20P-20K fertilizer (500 gig) was applied every three weeks and trees were watered every three days. Pesticides [5-O-demethylavermectin (abamectin; Avid), cyano(4-fluoro-3-phenoxyphenyl)methyl- 3(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate (cyfluthrin, Decathlon), and d-(2- chlorophenyl)-d-(4-chlorophenyl)-5pyrimidine-methanol (fenarimol, Rubigan) were applied as Following nine weeks of active growth in the greenhouse, leaf number and total leaf area was determined for each plant. Each plant had 25 fully expanded leaves on average at this time and leaf emergence was still occurring. Gas exchange was measured on the four most recently expanded leaves per plant using an ADC LCA2 portable photosynthesis system (Analytical Development Company, Hoddesdon, UK) under the following conditions: flow rate = 400 mL/min, leaftemperature = 30 j: 2°C, ambientCO2 = 35 j: lPa, VPD = 3kPa, andPAR 2 1000 umol ln'2 s" PPFD. The 16 most uniform plants were selected for the first experiment based on total leaf area and initial gas exchange characteristics. Selected plants were moved from the greenhouse to a Conviron PGV36 walk-in plant growth chamber (Conviron Systems of America, Pembina, ND). The growth chamber was programmed to a 16 h photoperiod at the following semngs,(07:00: air temperature = 28°C, relative humidity = 50%, PAR = 800 umol . 29 m" s"; 21:00: air temperature = 23°C, relative humidity = 50%, PAR = 0 umol m” s"). Whole-plant partial defoliation was accomplished by removing all expanded leaves below the fourth fully expanded leaf from the apex (corresponding to a 70% whole-plant LAR). Gas exchange measurements Whole-leaf gas exchange was measured using the open gas exchange system 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 the leaf chambers; (b) air flow entering the chambers was regulated using the following Matheson equipment (Matheson Instruments, Horsham, PA): 8100 series flow meters and 8200 series mass flow controllers connected to a model 8219 multichannel Dyna-Blender. Ambient C02 concentrations were measured using a portable ADC LCA2 infrared gas analyzer and 02 concentration was determined with 3 0-260 Beckman oxygen analyzer (Beckman Instruments, Inc., Irvine, CA). Trees and assimilation chambers were placed in a different but identical Conviron PGV36 walk-in plant growth chamber (set to the same conditions as described above). Unless otherwise indicated, gas exchange measurements were conducted under the following conditions (air temperature of 28°C, 50% relative humidity, PAR = 300 umol m" s“). Four leaf chambers were used simultaneously in which the following conditions were maintained, unless otherwise indicated: inlet C02 and 0,, 35 :1: 1.5 Pa and 21 kPa, respectively; leaf temp. 29 :l; 1°C; VPD 1.0 :1: 0.2 kPa; inlet flow rate 3 lein. Diurnal gas exdlange On three separate dates (three replicates over time) four plants were selected and two treatments were imposed on two plants each: i. nondefoliated control; and ii. partial defoliation 30 (70% LAR). The fourth fully expanded leaf of each plant was inserted into an individual leaf chamber (where it remained for the duration of the experiment). The day before defoliation was designated as day zero. On day one, the plants were partially defoliated at 08:00. Gas exchange wasmeasuredhourlyduringthephotoperiodfrom08:00t020:00andthenngasmeasuredat 21:30 following a 30 min exposure to darkness on days zero through eight. On day nine, a response to PPFD was measured (as described below). Plants were then returned to the walk-in growth chamber that was described first above. Long-term effects After the measurements of diurnal gas exchange and response to PPFD for the three replicates over time were completed, gas exchange was measured on the same four leaves of all 12 plants with the portable ADC LCA2 unit. Since the experiment was replicated three times, the plants from each replicate had three different recovery durations. Leaf areas were measured for all leaves. For each plant, leaves one through four were sampled for chlorophyll (as described below) and then leaves were dried in an oven at 80°C for seven days so that SLD - could be determined (dry weight of leaf + by leaf area). Chl determination Four discs (0.324 cm’) were punched from the lamina of each leaf using a paper holepunch. Fresh weight of discs was recorded and then chlorophyll was extracted in 7 mL N ,N- dimethylformamide in darkness at 5°C for 36 hours. Absorbance of extracts was read at 664, 647, and 625 nm on a Hitachi U-3110 UVlVis spectrOphotometer (Hitachi Ltd., Tokyo). Calculations for chl a, chl b and P chl were made according to Moran (1982). 31 Response to CO, and low 0, From the same population of 16 plants, the four remaining plants from one block were brought to the growth chamber in the laboratory. Gas exchange measurements were made on days zero through six, and day nine following whole-plant partial defoliation as described above. CO, and low 02 response were measured daily between 09:30 and 14:30 (as described below). All four plants were defoliated immediately alter the last gas exchange measurement on day zero. Gas exchange measurements for changing environmental conditions Response to PPFD was determined by decreasing PPFD in a stepwise manner (15 min acclimation at each step) to the following levels 1800, 1400, 980, 530, 380, 265, 150, 110, 70, 45, and 0 umol m" s”. PAR to supplement that in the growth chamber was provided by GE 400 W metal halide lamps. PPFD levels were produced using a combination of neutral density filters. PAR was measured with a recently calibrated LI-COR 190 PAR sensor (LI-COR, Inc. Lincoln, NE). R. was estimated by comparing A at ambient 02 (21 kPa) with that at reduced 02 (1.5 kPa). Response to CO2 was determined by increasing the C02 concentration stepwise to the following levels: 7, 10, 15, 20, 25, 30, 35, 40, 50, 70, and 90 Pa. Leaves were allowed to acclimate for 20 min at each C02 level. Gas exchange parameters were calculated using the BASIC computer program of Moon and Flore (1986). Responses of A to PPFD and A to CS were analyzed for each treatment by nonlinear regression. Curve fitting was performed using the Marquardt compromise method of successive approximatiom. The best fit curve, evaluated by analysis of residuals and r’ was the monomolecular asymptotic function (Hunt, 1980) of the type: Y =~B(l) * [1.0 - 3(2) (5(3) ’X] 32 where B(1), B(2), and B(3) are the asymptotic value, minimum value and rate constant, respectively. This polynomial was selected because by inspection it provided direct estimates of specific physiological processes and it exhibited curvilinear features that represented the data. Individual leaf AC, and A versus PPFD response curves were developed using this polynomial. From the AC, response curve, I‘ was extrapolated as the C, at which A was zero. k was calculated from the raw data as the slope in the linear portion of the Ac. curve. 1, was calculated according to the differential method of Jones (1985). A.I was determined as the A at 90 Pa C0,. Limitations due to RuBP regeneration were expressed by a reduced A at saturating CO, concentrations (Farquhar and Sharkey, 1982). From the A versus PPFD curve, Rd was calculated from the raw data at 0 umol in2 s" PPFD. cp was extrapolated from the curve at the PPFD level in which A was wo. ¢ was calculated from the raw data as the slope of the A versus PPFD curve in the linear portion between 0 and 150 mo! in2 s" PPFD. Respirationanrl carbon partitioning A similar set of plants to that described above were used for this experiment. The 15 most uniform plants were selected from the greenhouse and moved to the walk-in growth chamber in the laboratory. One day before partial defoliation (designated as day -1), whole leaf gas exchange was measured on the four most recently expanded leaves of all 15 plants proceeding from 10:00 until 12:00. R,I was measured at 22:00 (alter 1 h of darkness), 02:00 (after 5 h of darkness) and 06:00 (after 9 h of darkness; 1 h before the lights were turned on again). On day zero, all 15 plants were partially defoliated between 21:00 and 22:00. The fifth and sixth fully expanded leaves from the apex were immediately frozen in liquid N2 and stored at -80°C for carbohydrate analysis to provide a day zero leaf carbohydrate status for each plant. On days zero through four, gas exchange was measured beginning at 10:00. One plant was destructively 33 harvestedateachnightmeasurementondayszerothroughfour. Thefourharvestedleavesper plant were pooled as one sample, frozen in liquid N, and immediately stored at -80°C. Soluble sugar and starch determination Stored leaf samples (-80°C) were lyophilized, weighed and ground in a Wiley mill to pass through a 40-mesh screen. 100-mg subsamples were extracted three times each (20 min) with 3.5 mL of 80% ethanol. The homogenates were centrifuged at 1500 x g for 5 min alter each extraction, supernatants were transferred into 50 mL polypropylene centrifuge tubes, and 5 mL of ultrapure water and 5 mL of reagent grade chloroform was added to partition the chi from the sample extract. The tubes were capped, shaken vigorously and then centrifuged at 470 x g for 3 min. The upper, clear aqueous phase containing the soluble sugars was transferred to a test tube and evaporated to dryness using a Savant Speedvac SC200 equipped with a refrigerated condensation trap RT4104 at -103°C (Savant Instruments, Inc., Farmingdale, NY). The pellet was also evaporated to dryness and it, along with the soluble sugar Samples, was stored in a glass desiccator over an anhydrous desiccant. Sorbitol, Glc, Pm, and Sue were separated and quantified by HPLC. A Dionex series 40001 system was used which was equipped with a gradient pump, CarboPac PAl column, autosampler and pulsed amperometric detector connected to a model 4270 integrator (Dionex Corp., Sunnyvale, CA). The column was eluted with ultrapure water and 200 mM NaOH which had been sonicated for 30 min, degassed with helium for 1 hour and then continuously degassed during the analysis. A combination of gradient and isocratic elution was used by programming the gradient pump as follows: i. prior to sample injection, the column was eluted with 65% water and 35% NaOH; ii. at time = 0 min, the sample was injected and the gradient then was adjusted to 75% water and 25% NaOH by time = 0.1 min; the elution remained at this ratio until time 34 = 1.5min; in fromtime =1.5minto9.0minthegradientwasadjustedtoendupat0% water and 100% NaOI-I; iv. From time = 9 min to 28 min, the column was isocratically eluted with 100% NaOH. The flow rate of eluent was constant at 1 mL/min. The column was maintained at room temperature. The sample was prepared for HPLC analysis by adding 1 mL of ultrapure water, vortexing 3X - each followed by at least 5 min to stand. The sample was then transferred to a 1.5mLEppendorftubeandspunat10000ngor10minonaBeckmanmicrofuge(Beckman Instruments, Inc., Irvine, CA) to pellet any particulate matter that remained in the sample. The sample was then diluted 1:100 with ultrapure water and a 0.5 mL aliquot was loaded into a sample vial. Each sample was analyzed three times. Sugars were identified and quantified on the basis of retention time and peak heights of sugar standards. Starch in the pellet was measured using the method of Roper et al. (1988) modified as follows: samples were incubated at 55°C for 16 h with amyloglucosidase (Boehringer Mannheim 208 469) and then assayed colorimetrically using glucose oxidase (Sigma Chemical Co.). Absorbance at 440 nm was read with a Hitachi U-3110 UV/Vis spectrophotometer (Hitachi Ltd., Tokyo). Each sample was assayed three times. RESULTS Diurnal gas exchange A pronounced diurnal response of net CO, assimilation rate (A) and g, was observed for both control and partially defoliated plants in which A typically reached a maximum value at 11:00 and then declined in the afternoon (Fig. 1A,C). One day (or 24 h) after treatment, A and g, were significantly higher in the defoliated plants at most times during the day. Between two 35 and seven days following defoliation, A was 30 to 50% higher and g. was so to 100% higher in leaves of defoliated plants than in leaves of nondefoliated, control plants. R. was significantly lower in leaves of partially defoliated plants from two days afier defoliation (Fig. 113). Based on linear regression analysis, there was a significant correlation between g. and A duringthephotoperiodover theninedays ofmeasurement (Fig. 2). Thetightestfitofthedata to the regressionfunctionwas from lowerto mediumA and itbeganto breakdown atthehigher A. The y-intercept and slope were lower and higher, respectively, for leaves of partially defoliated plants indicating greater A per unit g, than in the control. Respollse to PPFD A was significantly higher at each light intensity for partially defoliated plants 9 days after treument (Fig. 3). R. was -0.7 versus -0.3 umol m“2 s" for the control and partially defoliated plants, respectively. cp was 14 versus 6 umol ln'2 s" for the control and partially defoliated plants, respectively. d was 67% higher in leaves of partially defoliated plants than in control plants (.0226 versus .0135 mol CO2 fixed per mol of PPFD absorbed). Light saturation occurred at about 800 umol in2 s" PPFD. Response to CO, and O, A highly significant relationship was observed between A and Q for each date of memurement (r2 = .96, on average) (Fig. 4). The response of leaves 9 days following defoliation was almost identical to that observed 6 days following defoliation (Fig. 4F) and was not included in this figure. A was significantly higher at all C0, levels (except 7. Pa) on each day following whole plant partial defoliation. 1‘ and R, were not affected by partial defoliation (Table 1). Two days following defoliation, k was 89% higher than it was before defoliation and 36 it remained significantly higher on each subsetplent day of measurement. 2 and 4 days following partial defoliation, A,” was 26 and 53% higher, respectively, than it was before defoliation. By 2 and 3 days following defoliation, Q 3,, was reduced in comparison with the predefoliation values but the differences wae not significant by day 4. A at saturating C02 concentratiom and A.“ were significantly higher at each date following defoliation. A- reached a value of 56% higher than it was before defoliation by 6 days following defoliation. The stomata presented a slightly increased limitation to A between 1 and 3 days following defoliation, but this was ameliorated thereafter. Respiration and carbon partitioning Leaf A rate increased by 30% one day following partial defoliation (Fig. 5A). By 3 or 4 days following treatment, the A rate had almost doubled from the predefoliation value. R. was higher before partial defoliation and it progressively decreased following treatment (Fig. 5B). R. fluctuated during the dark period but it was not consistently higher or lower at any given time during the night following defoliation. Leaf starch content decreased over time following partial defoliation (Fig. 5C). Starch content was reduced by almost 40% four days following treatment. Starch content was typically highest and lowest at the beginning and the end of the dark period, respectively. By 06:00, leaf starch was usually less than 0.1% of the leaf dry weight. There was a good correlation between leaf starch content and respiration rate (Fig. 6). As leaf starch content decreased, dark respiration rate also decreased and m ma. The effect of whole-plant partial defoliation on R. was consistently observed in four experimental replicates (Table 11). Each day following defoliation, R,l was significantly lower than it was before treatment. By 6 days following treatment, the average R.I rate of leaves was only 7% of that observed for the same leaves before defoliation. 37 Leaf carbon partitioning to sorbitol and Suc increued over time following defoliation (Table III). Sorbitol and Suc content was always highest and lowest at the beginning and end of the dark period, respectively. On average, soluble carbohydrates comprised about 20% of the total leaf dry weight. The relative proportions thereof for sorbitol, Glc, Pm and Sec were 45, 5, 4, and 46%, respectively. Defoliation did not significantly alter the relative partitioning among these four major soluble carbohydrates. Sorbitol is an acyclic sugar alcohol, the reduction product of Glc-6-P, which is a major phloem-translocated carbohydrate in ms species (Loescher, 1987). Long-term effects A and g. remained significantly higher in leaves of partially defoliated plants up to 32 days following defoliation (Table IV). Chl a, chl b, and P chl were not significantly higher for leaves of partially defoliated plants until 32 days after defoliation. SLD was only 7% higher in leaves of defoliated plants 32 days after defoliation. DISCUSSION Partial defoliation altered the source-sink balance in sour cherry trees and probably relieved a sink-limiting condition (Sawada et al., 1989) resulting in an enhancement of leaf photosynthesis. The type and extent of defoliation, and the plant species under investigation affect whether photosynthetic enhancement occurs and the time before such enhancement is observed. Tschaplinski and Blake (1989b) suggested that carbohydrate reserves in the stems of decapitated mum trees played an important role in buffering short-term responses to altered sink demand. They further noted that until carbohydrate reserves were severely depleted by sink 38 activity, an enhancement of source A would not occur. Decapitated mulberry trees took 6 days before photosynthetic enhancement was observed (Satoh et al. 1977). In partially defoliated bean (von Caemmerer and Farquhar, 1984) and soybean plants (Wareing et al., 1968), significant enhancementsoonccurred within2to3days. Insourcherrytrees, Awasenhancedwithin 24 h of partial defoliation (figure 1). If the argument of Tschaplinski and Blake (1989b) applies to these sour cherry trees, then the reserve carbohydrate supply in their stems was small and was rapidly depleted within 24 h. The photosynthetic enhancement was due, in part, to the combined influences. of increased g., e, k, and RuBP regeneration rate. deiong (1986) found that the enhanced A of fruiting versus defruited peach trees was primarily related to stomatal behavior. von Caemmerer and Farquhar (1984) noted increases in RuBP regeneration for bean and, like Wareing et al. (1968) using soybean, also observed enhanced rubisco activity in source leaves of partially defoliated plants. By 9 days following treatment, 4: was significantly higher in partially defoliated sour cherry trees. Since daily light responses were not measured, we cannot be sure exactly when it increased, but since it did increase, it indicates that light capture and electron transport processes underlying RuBP regeneration were enhanced following defoliation. The enhancement of A was not due to a reduced oxygenase activity of rubisco since R. was unaffected by defoliation. In spite of the enhanced sink demand following partial defoliation, A decreased in the afternoon for partially defoliated sour cherry trees. The relationship between A and g. was quite strong throughout the diurnal photoperiod over time indicating that the decline in A was due, in part, to a similar decline in g,. Tschaplinski and Blake (1989a) noted similar diurnal responses of A and relationships between A and g, for rejuvenated stump leaves of decapitated Romans plants. Loveys et al. (1987) found in apricot trees (2mm mm L.), which are closely related to sour cherry, that ABA was relatively unimportant in the control of g,. Since ABA 39 concentration and leafwater potential was not measured in these leaves, the possibility of a mild water stress induced stomatal closure in the afternoon cannot be completely ruled out. The trees wereadequatelywateredandexhibitednovisualsymptomsofwaterstress. Itiscurrentlynot known whether plants of the genus 2mm contain a tight-binding inhibitor of rubisco (Seemann et al. 1985) that may lead to partial rubisco deactivation in the afternoon. But since gas exchange measurements were made at a saturating light intensity, the deactivation of rubisco by CA-l-P appears unlikely (Berry et al., 1987). Foyer (1988) suggested that towards the end of the photoperiod, the loss in photosynthetic capacity was partly due to a reduced capacity of sink tissues to import and utilize photoassimilate. One mechanism whereby the decline in A in the afternoon could be direct feedback inhibition of sucrose synthesis by sucrOse (Herold, 1980). A reduced cycling of Pi to the chloroplast and an increased synthesis of F-2,6—BP in the cytosol could decrease the activity of cytosolic FBPase and result in decreased rates of sucrose synthesis .(Stitt et al., 1987). In any case, the decline is clearly not due to starch accumulation in the chloroplast since starch content was actually reduced over time yet the magnitude of the decrease in A from 11:00 to 20:00 on a given day hardly changed over time. Both soluble and storage leaf carbohydrates were depleted in the leaf during the night as was observed in barley (Farrar and Farrar, 1985). Leaf R, in the night was significantly reduced over time following partial defoliation in sour cherry trees and there was a good correlation between R. and leaf starch content. The variability of R, during the dark Mod though indicates that factors in addition to starch content were also involved. Avery et al. (1979) noted similar effects of whole plant partial defoliation on potted apple trees. They found that A and R. were related to substrate levels and that A increased while R. decreased when transport from the leaf increased. They suggested that photosynthesis (source activity) was modulated by removal of carbohydrates from the leaf (sink demand) and that there was a negative feedback via the phloem- 4o transport system. Fondy and Geiger (1982) suggested that starch accuimilation and degradation in source leaves of m m L. was controlled exogenously by the leaf sucrose level and endogenously by the photoperiod of photosynthetic duration. Mullen and Koller (1988) noted that the rate of assimilate export during the night for soybean leaves was closely associated with the rate of starch mobilization but not closely related with either leaf sucrose content or respiratory CO2 loss. They further noted that starch stopped accumulating before the end ofthe light period and that there was a 75 min delay before starch was mobilized in the dark. Since sink strength was incremed by partial defoliation in sour cherry trees, partitioning to starch as a reserve may have occurred at the expense of supplying sink demand, and hence was reduced. It is also possible that some of the depletion in leaf starch over time was due to mobilization thereof in the light. Starch mobilization in the light has been observed in cottonwood, spinach and pea, respectively (Dickson and Larson, 1975; Pongratz and Beck, 1978; Kruger et al., 1983). Senescence delay of the remaining foliage following defoliation has been observed in divase crops. Ness and Woolhouse (1980) observed that when bean plants were decapitated, both RNA and chlorophyll synthesis in the chloroplast increased dramatically within about 4 days. Reduced chlorophyll degradation, or enhanced synthesis could partly account for the observed enhancement of «it of partially defoliated sour cherry trees. The thickness of the leaf palisade layer may have increased which could increase leaf volume and hence, photosynthetic machinery, as was observed in lucerne, mulberry, and bean, respectively, following defoliation (Hodgkinson, 1974; Satoh et al., 1977; Ness and Woolhouse, 1980). The long-term, sustained enhancement of A following partial defoliation in sour cherry trees may in part be due to the increased production of chloroplasts (W oolhouse, 1967) and plastid protein mntent (W arcing et al. 1968). Since there were fewer leaves on the defoliated trees, the competition for root—derived cytokinin 41 should have been reduced which may have influenced senescence delay (Wareing et al., 1968; Satoh et al., 1977; Neumann and Stein, 1984). Whole plant partial defoliation of actively growing sour cherry trees increased sink demand and resulted in photosynthetic enhancement. This indicated that photosynthesis was not operating at maximum capacity and that nondefoliated plants were indeed sink limited. Increasing sink demand clearly influenced the photosynthetic efficiency and partitioning of carbon in sour cherry trees. Given the compensatory ability of the remaining leaves following partial defoliation, the extent to which whole plant leaf area can be reduced before there is a significant affect on growth and yield remains uncertain. In a previous report, Layne and Flore (1992) demonstrated that whole plant dry weight accunnilation of potted trees was not significantly reduced until the leaf area of the tree was reduced by 30% but there was a significant downward trend as the level of defoliation increased. This level of defoliation is a threshold which may change with tree age, fruit load and environmental predisposition. Thresholds for tree defoliation are a necessary input for making 1PM decisions regarding pest control in fruit orchards.- Clearly, plants can be exposed to different types and levels of defoliation without detrimental effects on growth and productivity. With the increasing concerns about the impact of pesticides on the environment, knowledge of damage thresholds and the incorporation thereof into an [FM strategy may ultimately lead to decreased pesticide application. Achieving this goal is paramount to the success of IPM ad0ption. With lower operation costs for the producer and reduced environmental contamination while preserving beneficial predator and parasite insect and mite species which provide biological control gratis, [PM is a management strategy that provides numerous benefits to agriculture. 42 ACKNOWLEDGEMENTS The authors gratefully acknowledge the assistance and expertise provided by Michael Kwantes and Dr. John D. Everard in developing and conducting the soluble sugar and starch analyses. We would also liketothankDrs. Mirta N. Sivak, Jack Preiss, and Alan N. Laksofor invaluable input and advice. LITERATURECITED 1. Add S (1981) Effects of plucking of young tea plants on their photosynthetic capacities in the mature and overwintered leaves. Japan Jour Crop Sci 50:445-451 2. Avery D J, Preistley C A, TH-eharne K J (1979) Integration of assimilation and carbohydrate utilization in apple. In: R Marcelle, H Clijsters, M Van Poucke, eds, Photosynthesis and Plant Development. Dr W. Junk bv Publishers, The Hague, pp 221-231 3. Baysdorfer C, Basshain J A (1985) Photosynthate supply and utilization in alfalfa. A developmental shift from a source to a sink limitation of photosynthesis. Plant Physiol 77:313-317 4. 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Academic Press, New York, pp 7-30 Warren Wilson J (1966) An analysis of plant growth and its control in Arctic environments. Ann Bot 30:383-402 Williams J H H, Farrar J F (1988) Endogenous control of photosynthesis in leaf blades of barley. Plant Physiol Biochem 26:503-509 Wolk J O, Kretcliman D W, Ortega D G Jr (1983) Response of tomato to defoliation. J Amer Soc Hort Sci 108:536-540 Woolhouse H W (1967) The nature of senescence in plants. In: Symp Soc Exp Biol, Camb. Univ. Press, 21:179-213 47 Captions for figures Figure 1. Timecoursefor thediurnal responseof(A) A, (B) R, (unitsasfor A), and (C) g, to whole plant partial defoliation in one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated (e) and Defoliated - partially defoliated plant (0).. Gas exchange was measured hourly from 08:00 to 20:00 and R. was measured at 21:30 daily. Each point represents the average (+, -, or :|:SE) of 6 leaf measurements. figure 2. The relationship between g, and A over time for one-year-old, potted sour cherry trees. Treatments: (A) Control - nondefoliated (O); and (B) Defoliated - partially defoliated plant (0). Points are from hourly gas exchange measurements (08:00 to 20:00) for day -1 (day before treatment) through day 7 (7 days after treatment). Raw data appears about the regression lines. Figure 3. Effect of partial defoliation on the response of A to PPFD 9 days after treatment for one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated (O) and Defoliated - partially defoliated plant (0). Regression lines occur about the data. Data points which represent the average (iSE) of 8 leaf measurements-“appear about the regression lines. Figure 4. Effect of partial defoliation on the response over time of A to C, of one-year-old, potted sour cherry trees. Each figure (A - F) compares the day 0 pretreatment response (0) with the treatment response for the subsequent day (0). Treatments: day 0 - before partial defoliation; days 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), and 6 (F) correspond to 1, 2, 3, 4, 5, and 6 days following defoliation, respectively. Regression lines occur about the data. Arrows point 48 to the data collected at ambient CO, partial pressure (35 i- 2.5 Pa). Data points which represent the average (1:815) of 4 leaf measurements appear about the regression lines. Figure 5. Effects of partial defoliation on (A) A, (B) R,, and (C) leaf starch content over time for one-year-old, potted sour cherry trees. A was measured daily at 10:00. Daily R‘ was measured at 22:00, 02:00, and 06:00. The arrow indicates the time at which plants were partially defoliated. Each point represents the average (1: SE) of at least 4 leaf measurements. Figure 6. The relationship between R. and leaf starch content for leaves of partially defoliated one-year-old, potted sour cherry trees. Data points which are single leaf measurements appear about the regression line. 49 Tam. meeaofpaniatdefvuaaoudefwonr. m... cu... A... smear W leaves of one-year-old, potted sour cherry trees with time. Means for each day are compared relative to Day 0 by orthogonal contrasts in each column. Means are averages of four leaf measurements. NS, ', ", “"3 Nonsignificant or significant at P = 0.05, 0.01, or 0.001 respectively. Absence of NS or asterisks in a column indicates a lack of significant differences among days. Days 1‘ k A360 Cissn A—a lg R1 afiei' (11.11101 (mol CO, (umol C0, (pmol C0, (umol C0, (%) (%) defol CO2 in" s") in" s“) mol“) in" 3") mol") 0 68.5 0.044 8.98 279.4 17.93 20.6 30.2 1 69.5 0.042 NS 7.71 NS 270.3 NS 21.82 * 35.0 *" 27.3 2 60.5 0.083 ""‘ 11.35 *" 241.7 "* 24.85 *“ 36.2 *" 27.7 3 66.0 0.075 *** 12.83 m 260.9 * 27.84 m 27.1 .. 25.3 4 66.8 0.080 "" 13.77 *“ 285.4 NS 24.44 ""‘ 19.4 NS 25.5 5 69.0 0.080 ““ 13.46 "" 274.7 NS 25.96 "* 22.7 NS 33.1 6 68.8 0.084 “* 13.76 *“ 267.0 NS 28.02 "" 25.2 * 31.4 9 67.3 0.083 "* 13.48 *" 269.7 NS 25.90 “* 23.7 NS 25.8 50 Table II. The efectofwholeplantpartialdefoliationon lequ, ofcxpandedleaws of one-year-old. ported sour cheny trees over time. R. is expressed as the percent change from the day 0 pretreatment value for the same leaf. Means for experimental replicates 1, 2 and 3 are the average of 2 determinations. Means for experimental replicate 4 are the average of 4 determinations. The overall mean and SE are based on the pooled data for all experimental replicates. The absence of data in a given column indicates that data was not collected at this date. Expuimental . Time following treatment (days) replicate 0 1 2 3 4 5 6 1 100 81 80 66 32 19 5 2 100 81 71 55 34 19 3 3 100 54 58 41 59 37 13 4 100 65 62 57 43 - - Mean 100 70 68 55 42 25 7 (:1; SE) (4.8) (4.8) (4.8) (4.8) (5.8) (5 .8) 51 Table III. The eject of whole plant partial defoliation on soluble carbohydrate content of amanded (saws of one-year-old, potted sour cherry trees over time. Data are means ofthree subsamples per sample. SE apply to the means for each day, respectively. Soluble carbohydrate content (percent dry weight) Day Time Sorbitol Glc Fru Suc Total 0 22:00 6.7 1.1 1.1 8.4 17.3 1 22:00 8.4 1.0 0.7' f 10.3 20.4 1 02:00 6.8 1.0 0.9 8.8 17.5 1 06:00 6.4 0.9 0.6 7.7 15.6 SE Day 1 0.2 0.06 0.05 q 0.84 0.91 2 22:00 10.7 1.2 1.2 10.2 23.3 2 02:00 10.7 0.8 0.7 9.0 21.2 2 06:00 7.6 0.7 0.7 7.3 16.3 ' SE Day2 0.4 0.01 0.05 0.51 0.33 3 22:00 11.6 1.2 0.2 12.0 25.0 3 02:00 8.4 1.4 0.2 8.6 18.6 3 06:00 8.3 0.6 0.6 6.0 15.5 SE Day 3 0.4 0.01 0.01 0.11 0.16 4 22:00 11.4 1.2 1.0 13.1 . 26.7 4 02:00 10.0 1.2 0.6 10.6 22.4 4 06:00 9.3 1.2 0.8 8.1 19.4 SE Day 4 0.3 0.04 0.10 0.37 0.44 52 Table IV. Long-term cflicts ofwholeplantpartialdcfoliationonA, g,. chla, chlb, P chl, ED, an amended leaves of one-year-old. potted sour diary trees. Means are averages of four replicates. NS, ‘, "'3 and “‘3 Nonsignificant or significant at P = 0.05, 0.01, or 0.001, respectively. Treatment A g. chla chlb Pchl SLD (1411101 C02 (mmol C02 048 can") 04: an") 048 cm”) (ms cm") in” s") in" s") 11 WWW W defoliation Control 10.4 75.3 43.9 18.9 9.9 9.8 Defoliated ‘ 13.5 94.0 44.7 19.4 10.0 8.7 Significance * * NS NS NS NS 23 WWW PM“ 440%" Control 10.1 68.3 49.5 21.6 10.2 8.8 Defoliated 14.1 137.0 50.0 21.1 10.3 9.0 Significance *" *“ NS NS NS NS 32 days foUoivlng partial defoliation Control 7.9 48.9 28.9 14.8 9.1 8.9 Defoliated 13.3 91.3 44.7 19.7 9.9 9.5 Significance as: an: alias as: as: NS 53 1 A 0 Control } o Defoliated i 16 14 12 1O 8 Arm etc: .083 < 6 0.0 -O.5 - .Bt G. when”... i. saw...” new... we... ennui? towns. ..e U (I -1.0 24 48 72 96 120 144 168 0 Time following treatment (h) gS (mmol m"2 s“) 54 300 _A to Co'ntrol1 I l I I I ' I Y = -O.63 + 8.7X (r=.57) 200“ ' 3": ° 2. 3:? ~- 100 - L O . . 300 _B o Delfolioted I I o L °°° I Y = —39.7 + 12.7X (r=.59) o° 0 g " O 200 - 100 - Q r l A (,umol rn‘2 s") 55 ' T ' I l r r i l . a Control " o 1 4 r O Defoliated _ 12 — O 10 :_ 0 ‘ L- -4 8 — 0 r 5 _ _ CD 3+ 4 __ 0 _. - «t . 2 ._," _ 0‘, —————————————————————— __ _1 l l L l r I r l 1 0 300 600 900 1200 1500 1800 PPFD (,umol 'nn’2 S“) A (,umol m‘2 s“) 56 (D '8' l ' l 12. .r r , e DAY 0 " 0 DAY 0 0 . . 0 ‘DAY 5‘ - J 0 ‘DAY 6, o 100 200 300 +400 500 600 0 1E1 foo 300 400 500 600 700 Ci (nmol mol") 57 J a 1e a a . _ . _ . . . .14 q a _, . q 4 _ a all _ . ellldl q a . a 1 o . . w ..- n r .llfllla 1 T01 t 1'1 r I 1 Tel. .lel. I’ll; ll. LIT l TIT i r 1.1 l f i 41.1. .I..l. 41.1.. .l.l. a r rlOTi 1 rL’l i r A 1.1 r t t w ... v ... 1 1.1 r TIOII 12‘24‘36 4s 60‘72‘84‘96 rel Tel. .9 7 ‘I. v a T ‘1. Ill. All Tl"; A f B . . . rC g » Fl~ » _ _ p _ rlL _ _ _rl.ts. » . p _ _1t r e _ t _ s p L 9 7 5 3 4| 9 5 7 9 4| 3 5 7 5 O 5 O 5 O O 1 1 1 1 1 o. o. 0 1 1. 1. 1. 5 3. 2 2 1. 1. o. _ . _ _ _ _ _ 0 O 0 0 0 O 0 v 95063 >6 ecoouoov 9% TE 683 < Aim TE 6&5 m “coecoo £806 .63 108 -24 —12 0 ' Time following treatment (h) 58 -0.4 —0.6 - —0.8 - —1.0— —1.2- —1.4_ Rd (,umol nn'2 3") -1.6— Y = —0.59 -— 1.64X (r = —-o.632) 1 I n 0.05 L l l . 0.15 0.25 0.35 0.45 Leof storch content (percent dry weight) SECTION III: PHYSIOLOGICAL REPONSES OF m CERASUS L. TO WHOLE- PLANT SOURCE MANIPULATION. II. CONTINUOUS ILLUMINATION AND PHOTOSYNTIIEI'IC INHIBITION1 DunnodRichardlayne’nndJmAFlore helium-m ' conmy' UJ't?’ .11; I. emu .-‘1 .102!‘ u' “31 Received for publication Accepted for publication 59 ‘ Supported by the Michigan Agriculmral Experiment Station and by USDA grant No. 88-34132- 3380. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulation, this paper must be hereby marked advertisement solely to indicate this fact. ’ 3 Present address: Forestry Sciences Laboratory, 5985 Hwy K, P.0. Box 898, Rhinelander, WI 54501. 3W A, net C0, assimilation rate; RuBP, ribulose—1,5—bisphosphate; VPD, vapor pressure deficit; chl a, chlorophyll a; chl b, chlorophyll b; P chl, protochlorophyll; 12,, estimated photorespiration rate; Q, internal C02 concentration; ACi, curve describing the relationship between A and Ci; P, CO, compensation point; It, estimated carboxylation efficiency; 1,, stomatal limitation; A.” net C02 assimilation rate at 90 Pa (900 uL/L) C0,; cp, light compensation point; g., stomatal conductance; A,” net C0, assimilation rate at 35 -Pa C0,; Q 3,, internal CO, concentration at 35 Pa C0,; Fo, instantaneous fluorescence; Fv, variable fluorescence; Fm, maximal fluorescence; Fv/Fm, photochemical efficiency. 61 The source-sink ratio of one-year-old, potted sour cherry trees (2mm m L.) was altered by whole-plant contimrous illumination to determine if trees were primarily source or sink limited and to elucidate the means by which photosynthetic inhibition and subsequent recovery occur. Within one day of continuous illumination, A, k, Fv, Fo, Fv/Fm, and RuBP regeneration of the most recently expanded source leaves were significantly reduced. During the exposure to continuous illumination, A,” was reduced two to three-fold and k was reduced up to four-fold. g, was slightly reduced by contimrous illumination but C, was not significantly affected suggesting that the physical limitation to A was small. The decrease in Fv, and Fv/Fm indicated that leaves were photoinhibited within one day. The decrease in F0 following continuous illumination indicated that there was a reversible regulatory mechanism whereby the damage to PSII centers could be repaired. Since leaf chl was not altered by 1,_ 2, or 3 days of exposure to continuous illumination, photooxidation probably did not occur. The time to full photosynthetic recovery from continuous illumination increased as the duration of exposure increased. It is evident that continuous illumination enhanced a sink limited condition resulting in photosynthetic inhibition where both photochemical and biochemical processes were affected. End product inhibition is also a possible contributor to the observed decline in A. Whether biochemical or photochemical events were affected first, or occurred simultaneously to result in the observed photosynthetic inhibition and the magnitude of the response that can be ascribed to each remains to be determined. 62 Plant growth enhancement in continuous illumination has been documented for at least two and a half centuries. Smith (1933) reported the observations of Linneaus, in 1739, where grain was sown and harvested in only 58 days during the short summer in Lulea, Lapmarck in the Arctic. Darrow (1933) noted that tomato plants could be set in the field and develop a good cropinonlyéweeksduringthesummerinAlaska. Attemptsweremadetorecreatethe continuous illumination due to natural light in the Arctic, using artificial light in combination with natural light in greenhouses in temperate climates. Arthur (1936) reported that tomato plants developed foliar injury within five to seven days of continuous illumination in a greenhouse. He noted that effects of both light quality and intensity produced characteristic changes in chlorophyll and leaf structure. BOhning (1949) documented the effect of continuous illumination over time on the photosynthesis of apple leaves. He noted that the decrease in photosynthesis over days for ”shade" leaves may have been due to photooxidation of one of the photosynthetic enzymes. He suggested that the decrease in photosynthesis over weeks for "sun" leaves was probably due to chlorophyll bleaching but end product accumulation may have also been involved. Hillman (1956) found that foliar injury of tomato under continuous illumination was related to photoperiod, a shorter photoperiod was noninjurious. He also noted that the sensitivity to photoperiod and subsequent damage took place in young developing leaves but was diminished or absent in mature leaves. Photoinhibition, the reduction of photosynthetic capacity of a plant following exposure to light intermities in excess of that required to saturate photosynthesis, is a short-term phenomena that precedes the longer-term exposure of plants to strong light that can result in photodestruction of photosynthetic pigments (photooxidation) (Powles, 1984). Under most conditions, damage to PSII can be repaired as long as the cellular repair mechanisms can 63 matchtherateofdamage (Kyle, 1987). Itisnotuntiltherateofdamageexceedstherateof repair that a net loss in photosynthetic capacity (photoinhibition) occurs (Kyle, 1987). Currently, the potential for growing crops in Controlled Ecological Life Support Systems (CBLSS) in space is being evaluated. To maximize yields over a set time interval, potato (Wheeler et al., 1986) and sweetpotato (Bonsi et al., 1992) were grown under continuous (24 h) lighting to extend the duration of photosynthesis. Sweetpotato plants grown under 24 h light had storage root fresh weight yields that were from 18 to 29 times higher than plants grown in 12 h light (Bonsi et al., 1992). Tibbits et al. (1990) noted that potatoes grown under 24 h light at a constant temperature (18°C) were stunted, had chlorotic leaves that abscised and had minimal tuber formation. They found that when grown under 24 h light with fluctuating temperatures (22°C day/ 14°C night), plants developed normally and produced abundant growth, including tubers. Wheeler et al. (1991) noted that under 24 h lighting, the absence of a photoperiodic stirrmlation for tuber development remlted a sink limited condition where plants produced more shoot growth and essentially no tubers. They suggested that under conditions of 24 h light, high PPFD, high C0,, and lagging tuber initiation, feedback inhibition due to the buildup of carbohydrates in the leaf may have occurred. Many studies have been conducted on the responses of annual field crops and vegetables to continuous illumination but few have been conducted on perennial deciduous fruit trees. Sour cherry is of additional interest since in addition to sucrose, it produces a similar quantity of the sugar alcohol sorbitol during its photosynthetic metabolism (Loescher, 1987). In addition to the . -. benefits of continuous illumination for growth and productivity of some plants under certain conditions, it can be used as technique to study sink limitations, feedback inhibition, and in cases where leaf damage results, both photoinhibition and photooxidation. The objectives of this study were to document the following responses of source leaves of one-year-old, potted sour cherry 64 (2mm m L.) trees to whole-plant source manipulation by continuous illumination: 1. diurnalgasexchange; 2. responsestoCO,and0,;and3. changesinchlcontentandchl fluorescence. An additional objective was to determine the time required for full photosynthetic recovery following a continuous illumination treatment. MATERIALS AND METHODS Dormant one-year-old sour cherry trees (Bums m cv.’Montmorency’ on Mahaleb rootstock) were planted in 11 L plastic pots with 9.5 L of sterilized greenhouse soil mix [5 sandy loam: 3 sphagnumpeat:2torpedosand(byvolume), pH = 7.0]. Alltrees werecuttoan active bud (0 to 10 cm above the graft union) and placed in an environmentally controlled greenhouse (day and night means 28 and 23 °C, respectively). Trees were trained to a single shoot from which all laterals were removed as they appeared. Peter’s soluble 20N-20P-20K fertilizer (500 ug/g) was applied every three weeks and trees were watered every three days. Pesticides [5-0-demethylavermectin (abamectin, Avid), cyano(4-fluoro-3-phenoxyphenyl)methyl- 3(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate (cyfluthrin, Decathlon), and d-(2- chlorophenyl)-d-(4-chlorophenyl)—5-pyrimidine-methanol (fenarimol, Rubigan) were applied as necessary. Following nine weeks of active growth in the greenhouse, leaf number and total leaf area was determined for each plant. Each plant had 25 fully expanded leaves on average at this time and was still actively growing. Gas exchange was measured on the four most recently expanded leaves per plant using an ADC LCA2 portable photosynthesis system (Analytical Development Company, Hoddesdon, UK) under the following conditions: flow rate = 400 mL/min, leaf temperature = 30 j: 2°C, ambientCO, = 35 j; lPa, VPD’ = 3kPa, andPAR 21000umol 65 in" s". The four mom uniform plants were selected for the first experiment based on the combined characteristics ofleafnumber, total leefarea, and initial gas exchange characteristics. Selected plants were moved from the greenhouse to a Conviron PGV36 walk-in plant growth chamber (Conviron Systems of America, Pembina, ND). The growth chamber was programmed to a 16-h photoperiod at the following settings (07 :00: air temperature = 28°C, relative humidity = 50%, PAR = 800 umol m’2 s"; 21:00: air temerature = 23°C, relative humidity = 50%, PAR = 0 umol in2 s“). Whole-plant continuous illumination was accomplished by reprogramming the growth chamber so that the lights were always on. Previous studies (Section I) documented that A of the most recently expanded leaves of untreated (control) sour cherry trees, identical to those described here, was constant for at least two weeks when trees were at this stage of development. Based on this condition, we decided to make comparisons of the effect of contimious illumination on the response observed for the same leaf prior to treatment rather than compare with separate untreated (control) plants. This permitted more replications for statistically evaluating the continuous illumination effects on the same leaves over time. Gas exchange measurements Whole-leaf gas exchange was measured in the laboratory using the open gas exchange system 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 C0, concentrations at the inlet and outlet of the leaf chambers; (b) air flow entering the chambers was regulated using the following Matheson equipment (Matheson Instruments, Horsham, PA): 8100 series flow meters and 8200 series mass flow controllers connected to a model 8219 multichannel Dyna-Blender. Ambient C0, concentrations were measured using a 66 portableADCLCAZinfraredgasanalyzerand0,concemrationwasdeterminedwitha0-260 Beckman oxygen analyzer (Beckman Instruments, Inc., Irvine, CA). Trees and assimilation chambers were placed in a differem but identical Conviron PGV36 walk-in plant growth chamber (set to the same conditions as described above). Unless otherwise indicated, gas exchange measurements were conducted under the following conditions: air temperature of 28°C, 50% relative humidity, PAR = 800 umol m" s“. Four leaf chambers were used simultaneously in which the following conditions were maintained, unless otherwise indicated: inlet C0, and 0, 35 1: 1.5 Paand 21 kPa, respectively; leaftemp. 29 i1°C;VPD1.0 i 0.2 kPa; inletflow rate 3 lein. Dlmlalgesexehange 'l‘hefourthfiillyexpandedlenfofeachplantwasinsertedintoanindividualleafchamber (where it remained for the duration of the experiment). The day before continuous illumination was designated as day zero. Plants remained in continuous illumination from 07:00 on day zero until 21:00 on day four. For days five through seven, plants were returned to a 16 h photoperiod. Gas exchange was measured daily at 09:00, 12:30, 15:00 and 20:00. C0, and 0, responses were measured daily between 09:30 and 14:30 (as described below). Gas exchange response to C0, and 0, Response to CO, was determined by increasing the CO, concentration stepwise to the following levels: 7, 10, 15, 20, 25, 30, 35, 40, 50, 70, and 90 Pa. Leaves were allowed to acclimate for 20 minutes at each C0, level. Gas exchange parameters were calculated using the BASIC computer program of Moon and Flore (1986). Response of A to C, was analyzed by nonlinear regression. Curve fitting was performed using the Marquardt compromise method of - -—./, ,a 67 successive approximations. The bea fit curve, evaluated by analysis of residuals and r" was the monomolecular asymptotic function (Hunt, 1980) of the type: Y = B(l) * [1.0 - 3(2) {0(3) ' x] where B(1), B(2), and B(3) are the asympmtic value, minimum value and rate constant, respectively. This polynomial was selected because it provided direct estimates of specific physiological processes and it exhibited curvilinear features that represented the data. Individual leaf AC, response curves were developed using this polynomial. From the AC, curve, 1‘ was extrapolatedastheC,atwhichAwaszero. kwascalculatedfromtherawdataastheslopein the linear portion of the AC, curve. 1, was calculated according to the differential method of Iona (1985). A... was taken as the A at 90 Pa C0,. Limitations due to RuBP regeneration were expressed by reduced A at saturating C0, concentrations (Farquhar and Sharkey, 1982). R, was estimated by comparing A at ambient C0, (21 kPa) with that at reduced 0, (1.5 kPa). Time to full photosynthetic recovery A similar set of 30 plants were trained, watered, and fertilized as described above. Plants were grown in an identical growth chamber to that described previously under a 16 h photoperiod (PAR = 800 umol ln'2 s", R.H. 50%, day and night temperatures 26 and 22°C, respectively). Following nine weeks of active growth, whole plant leaf area was determined and gas exchange was measured using a portable ADC LCA2 photosynthesis measurement system. The most uniform 15 plants were selected based on leaf number, total leaf area and initial gas exchange characteristics. The remaining nonselected 15 plants were removed from the growth chamber. Five plants each were randomly selected and subjected to either one, two, or three days of continuous illumination. Recovery was followed for at least six days after the treatment. 68 Continuous illumination was accomplished by reprogramming the growth chamber so that the lights were always on. Once a given group of five plants had received the appropriate continuous lighttreatrnent, they were movedtotheother sideofthegrowthchamberbehindalightscreen (which reduced PAR to s 2umol m" s"; cp was 14 umol m" s") at 21:00 and then returned to the illuminated side at 07:00 each day. Once all plants had received their continuous light neeunengtllechamberwasreprogrammedth 16-hphotoperiod. Oneachday forthedurationoftheexperiment, gasexchangewas measuredbeginning at 10:00 with the portable ADC LCA2 unit and chl fluorescence was measured beginning at 15:00 (as described below). All measurements were made on the four most recently fully expanded leaves per plant. Each day following the chlorophyll fluorescence measurement, one leaf disc from each of the four leaves per plant was harvested for chl determination (as described below). Chl fluormcence A Morgan CF-1000 chl fluorescence measurement system (P.K. Morgan Instruments, Inc., Andover, MA) was used. Leaves were dark acclimated for 15 min using dark acclimation cuvettes. Leaves were irradiated with 1000 urnol nt-‘is-l PPFD actinic light and chl fluorescence kinetics were recorded over a 60 s sample time. Chl deter-urination One disc was punched from the lamina of each leaf using a paper holepunch (punch area = 0.324 cm’). The four discs per plant were pooled as a sample. Chl was extracted in 7 mL N,N-dimethylformamide in darkness at 5 °C for 36 hours. Absorbance of extracts was read at 69 664, 647, and 625 nm on a Hitachi U-3110 UVIVis spectrophotometer (Hitachi Ltd., Tokyo). Calculations for chl a, chl b and p chl were made according to Moran (1982). REULTS Diurnal gas exchange A decreased more than two-fold alter one day of continuous illumination (Fig. 1A). The normal diurnal response ofhigher A and g, (Fig. 1B) in the morning and lower A and g, in the afiernoon disappeared under contimlous illumination. Returning plants to a 14-h photoperiod for three days was not long enough to overcome the inhibition of A resulting from three days of continuous illumination. A significant linear relationship was observed between g. and A on each day for the combined diurnal data (data not shown). Response to C0, and 0, A was significantly lower at each C0, level (except 7 Pa) each day in continuous light (Figs. 2A-C) and during each of the three days following return to a 14-h photoperiod (Figs. 2D- F). The relationship between A and C, was not as strong (based on r2 values for the regression functions) following continuous illumination. 1‘ was not altered by continuous illumination (Table I). k was reduced by almost four-fold after one day in continuous light. It remained significantly lower (by two-fold) even three days following return to a 14h photoperiod. A,” was reduced by continuous illumination. C, m was higher one day following continuous illumination but then not different from the pretreatment value on each subsequent day. A at samrating CO, was dramatically reduced when trees were continuously illumimted (Figs. 2A—C). A...x was not significantly reduced by continuous illumination. Two and three days following continuous 70 illumination, the stomatabegantopresentanincreased limitationto Abutthiswasrelieved following return to a 14-h photoperiod. R. averaged about 35% for leaves and no significant treatrnentdifferenceswerenoted(datanotshown). Time to full photosynthetic recovery After one day of continuous illumination, A was significantly reduced in all trees (Fig. 3A). When plants were continuously illuminated for one day, it took two days before the A rate of those same leaves recovered to the rate which was observed before treatment. When leaves were continuously illuminated for two or three days, it took five days before the A rate of those same leaves recovered to that rate which was observed before treatment. By eight days following one day of continuous illumination, plants actually had higher A and g, (Fig. 3B) rates than before treatment. Chl fluorescence Fv/Fm was inhibited by continuous illumination (Fig. 4A). Continuous illumination for one day inhibited Fv/Fm significantly but full recovery occurred by the next day after return to a 14h photoperiod. Continuous illumination for two or three days resulted in an inhibition of Fv/Fm that took four to five days for full recovery. Both Fv and F0 were inhibited by continuous illumination (Figs. 4B,C). By nine days following C.L. exposure, Fv and F0 were still significantly reduced relative to the pretreatment levels. The values for Fv/Fm for plants before continuous illumination (0.760, on average) are in close agreement with the values determined for other C, species (ijrkman and Demmig, 1987). 71 Chl content Continuous illumination for one, two, or three days did not significantly change leaf chl content, orthe chl ato b ratio (data notshown) (Table II). Chl contentdid increase slightlyover time following continuous illumination. P chl content was not altered by continuous illumination and was 1.0 pg cm”, on average (data not shown). DISCUSSION Continuous illumination altered the source/sink balance in sour cherry trees and probably enhanced an existing sink limited condition and an inhibition of leaf photosynthesis resulted. Photosynthetic inhibition from contimlous illumination has been observed in diverse crops including apple (BOhning, 1949), 93m (Mooney and Billings, 1961), wheat (King et al. (1967), sunflower (Potter and Breen, 1980), and soybean (Kerr et al., 1985; Sawada et al., 1989). One explanationfor thedecline inA isthatcarbohydrates builtup in the leafcausingan endproduct inhibition of photosynthesis as suggested by BOhning (1949). This could be the result of excess starch accumulation in the chloroplast (Potter and Breen, 1980; Kerr et al., 1985, Sawada et al., 1986) or sugar accumulation in the cytosol (Mooney and Billings, 1961; King et al.,, 1967; Sawada et al., 1986). Accumulation of starch or sugar would indicate that utilization of photosynthate (sink strength), rather than assimilation thereof, was the process most sensitive to continuous illumination (Sharkey, 1985). The absence of a consistent diurnal variation in A and g, while continuously illuminated suggested that leaves may have been continuously feedback inhibited during this time. It is possible that amount of starch mobilized in the light (Kruger et al., 1983)andtheamountsofsucroseandsorbitolexportedtothesinkswasatsuchalowrate that photosynthate accumulated in the leaf and a feedback inhibition of A resulted. 72 Light trapping, electron transport and rubisco activity, as indicated by the reduced Fv/Fm, estimated RuBP regeneration rate and k, also contributed to the limitation to A brought on by continuous illumination. Reduced photochemical efficiency suggested that there was an impaired production or consumption of ATPINADPH in photosynthesis. If production of NADPH was reduced, sorbitol biosynthesis would be affected more than sucrose biosynthesis (Loescher, 1987). Herold (1980) noted that soluble sugar accunnllation may reduce the rate of RuBP regeneration by decreasing the available stromal Pi. A phosphate limitation in the chloroplast can reduce both photOphosphorylation and electron transport. This is likely due to a decreased ATP/ADP ratio and depression of 3-PGA reduction (Robinson and Walker, 1981). Harris et al. (1983) noted a progressive decline in the photosynthetic capacity of spinach leaf discs when mannose (which sequesters Pi) was added. They found that A was inhibited by lowering 0, to 2 kPa suggesting that a suppression of P-glycolate synthesis could exacerbate this Pi limitation. The Pi requirement of chloroplasts varies with 0, pressure (U soda and Edwards, 1982) and increasing oxygenation (by increasing 0, pressure) should increase A by making Pi more available (Sharkey, 1985). In contrast, we did not see an inhibition of A upon exposure to low 0, but rather an enhancement (Warburg effect) suggesting that the leaves were not Pi limited in the chloroplast. The increased lI suggested that, in addition to the mesophyll, the stomata presented a slightly increased physical limitation to A as the duration of exposure to continuous light increased. Since c. was not significantly reduced and stomatal function has been found to be independent of total leaf rubisco activity (Hudson et al., 1992), the physiological consequence of reduced g, appeared to be minor in contrast to that noted by de long (1986). Photoinhibition precedes chlorophyll bleaching (photooxidation) (Powles, 1984). Karukstis (1991) stated that photoinhibition of photosynthesis is manifested at the leaf, thylakoid, and P811 level by losses in C0, fixation, electron transport capacity, and Fv, respectively. 73 Indeed, according to the above conditions, cherry leaves exposed to continuous illumination were photoinhibited. Since leaf chlorophyll content did mt decrease under contimlous illumination, we assume that photooxidation did not occur. Like William and Farrar (1988) using barley, we noted that as the time in continuous light increased, the amount of reduction of Fv increased. Photoinhibition dannge may be a combination of peroxidation of the lipids in the thylakoid membrane (Mishra and Singhal, 1992) and removal of die D1 protein (previously referred to as the ’0, protein’ by Kyle, (1987)) after sustained effects on the oxidizing side of PSII have occurred (Demmig-Adams, 1990). Since the observed photoinhibition resulted in a decrease in F0 over time, fllis indicated that there was a reversible regulatory mechanism whereby the damage to PSII centers could be repaired (Krause, 1988). The restoration of FvIFm following return to a 16 h photoperiod in cherry leaves indicated that recovery from photoinhibition had indeed occurred (Krause, 1988). The amount of time before full photosynthetic recovery was observed increased as the duration of continuous illumination increased. Repair to PSI] involves removal of damaged Q. protein by a highly efficient and specific membrane-bound protease (Kyle, 1987). Sincethereisno Q.proteinturn‘over inthedark, itappearsthattheremustbea vacancy created by removal of damaged protein before synthesis and insertion of the new protein can occur (Kyle, 1987). Demmig-Adams (1990) suggested that the carotenoid, zeaxanthin, also plays an important role in preventing or reducing damage to PSII. She noted that the photoan facilitated by zeaxanthin is from de-excitation of the excited singlet state of chlorophyll in the chlorophyll pigment bed under excess light. Since detailed determinations of gas exchange, chlorophyll fluorescence, and carbon partitioning were not determined over the first 24 hours following continuous illumination, it is not possible to say whether biochemistry or photochemistry was affected first. Accumulation of starch grains in the chloroplast affected the photosynthetic apparatus (Warren Wilson, 1966; 74 Wildman, 1967) and may have interfered with intercellular C0, transport (Nafziger and Koller, 1976), thereby reducing A. It is indeed possible that carbohydrate buildup and reduced ability to dissipate light energy occurred simultaneously leading to the observed phalomena. Clearly, though, precarboxylation events and carboxylation were both affected and photosynthetic inhibition resulted. At this point, the magnitude of the inhibition that can be ascribed to these . respectiveprocesses, andthemechanismfordamagerepairremainsequivocal. ACKNOWLEDGMENTS We would like to thank Dr. Mirta N. Sivak for invaluable input and advice. LITERATURE CITED 1. Arthur J M (1936) Plant growth in continuous illumination. In: B M Duggar, ed, Biological Effects of Radiation. McGraw-Hill, New York, pp 715-725 2. leirkman 0, Dernmig B (1987) Photon yield of 0, evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170:489-504 3. Bilhning R H (1949) Time course of photosynthesis in apple leaves exposed to continuous illumination. Plant Physiol 24:222-240 4. BonsiCK,MretanPA,HillWA,MortleyDG(1992)Responseofsweetpotatoes to continuous light. HortScience 27:471 ' 5. Dale J (1985) Carbohydrate partitioning and metabolism in crops. Hort Rev 7:69-108 10. ll. 12. 13. 14. 15. 75 Darrow G M (1933) Tomatoes, berries and other crops under continuous light in > Alaska. Science 782370 Wong T M (1986) Fruit effects on photosynthesis in Emma mica. Physiol Plant 66:149-153 . Demmig-Adarm B (1990) Carotenoids and photoprotection in plants: A role for the xanthophyll zeaxanthin. Biochim Biophys Acta 1020: 1-24 ‘ Farquhar G D, Snrkey T D (1982) Stomatal conductance and photosynthesis. Ann Rev Plant Physiol 33:317-345 HarrisGC,CheesbrouthK,WalkerDA(l983) MeasurementofC0,andH,0 vapor exchange in spinach leaf discs. Effects of orthophosphate. Plant Physiol 71:102-107 Harold A (1980) Regulation of photosynthesis by sink activity - the missing link. New Phytol 86:131-144 Hillman W S (1956) Injury of tomato plants by continuous light and unfavorable photoperiodic cycles. Am J Bot 43:89-96 HudaonGS,EvansJR, vonCaemmererS,Arvidsson YB C,Andrews TJ(1992) Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase content by antisense RNA reduces photosynthesis in transgenic tobacco plants. Plant Physiol 982294-302 Hunt R (1980) Asymptotic functions. In: Plant Growth Curves. Univ Park Press, Baltimore, pp 121-146 Jones H G (1985) PartitiOning stomatal and non-stomatal limitations to photosynthesis. Plant Cell Environ 8:95-104 16. 17. 18. 19. 20. 21. 22. 23. 24. 76 Karukstis K K (1991) Chlorophyll fluorescence as a physiological probe to the photosynthetic apparatus. In: H Sheer, ed, Chlorophylls. CRC Press Inc, Boca Raton, pp 769-795 KerrPS,RultyTW,HuberSC(1985)Endogenousrhythmsinphotosynthesis, sucrose phosphate synthase activity, and stomatal resistance in leaves of soybean (Glycine m [L.] Merr.). Plant Physiol 77:275-280 ‘King RW, Wardth F, Evans LT (1967) Effectof assimilate utilization on photosynthetic rate in wheat. Planta 77:261-276 Krause G H (1988) Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms. Physiol Plant 74:566-574 Kruger N J, Bulpin P V, ap Rees T (1983) The extent of starch degradation in the light in pea leaves. Planta 157:271-273 Kyle D J (1987) The biochemical basis for photoinhibition of photosystem II. In: D J Kyle, C B Osmond, C I Arntzen, eds, Photoinhibition. Elsevier Sci Pub B V Amsterdam, pp 197-226 Inescher W H (1987) Physiology and metabolism of sugar alcohols in higher plants. Physiol Plant 70:553-557 Mishra R K, Singhal G S (1992) Function of photosynthetic apparatus of intact wheat leaves under high light and heat stress and its relationship with peroxidation of thylakoid lipids. Plant Physiol 98:1-6 Moon 1 W Jr, Flore J A (1986) A BASIC computer program for calculation of photosynthesis, stomatal conductance, and related parameters in an open gas exchange system. Photosynthesis Res 7 :269-279 26. 27. 28. 29. 30. 3.1. 32. 33. 77 Mooney H A, Billing W D (1961) Comparative physiological ecology of arctic and alpine populations of m djgyna. Ecol Monogr 31: 1-29 Moran R (1982) Fornnllae for determination of chlorophyllous pigments extracted with n,n-dimethylformamide. Plant Physioli69: 1376-1381 Nafziger E D, Keller H R (1976) Influence of leaf starch concentration on C0, assimilation in soybean. Plant Physiol 57:560-563 Potter J a, Breen P J (1980) Maintenance of high photosynthetic rates during the accumulation of high leaf starch levels in sunflower and soybean. Plant Physiol 66:528-531 Powles S B (1984) Photoinhibition of photosynthesis induced by visible light. Ann Rev Plant Physiol 35:15-44 Robinson S P, Walker D A (1981) Photosynthetic carbon reduction cycle. In: J Preiss, ed, The Biochemistry of Plants, Vol 8. Academic Press Inc., New York, pp 193-236 Sams C E, Flore J A (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 Sawada s, Hayakawa r, ruktrsln K, Kasai M (1986) Influence of carbohydrates on photosynthesis in single, rooted soybean leaves used as a source-sink model. Plant Cell Physiol 27 :591-600 Sawada S, Ibsegawa Y, Kasai M, Sasaki M (1989) Photosynthetic electron transport and carbon metabolism during altered source/sink balance in single-rooted soybean leaves. Plant Cell Physiol 30:691-698 Sharkey T D (1985) Photosynthesis in intact leaves of C, plants: Physics, physiology and rate limitations. Bot Rev 51:53-105 35. 37. 38. 39. 41. 42. 78 Smith F (1933) Researches on the influence of natural and artificial light on plants. 1. 0n the influence of the length of day - preliminary researches. Meld Norg ' Landbukshoiskole 13:1-223 nbbitr r w, Bennett 8 M, Geo W (1990) Control ofcontinuous irradiation on potatoes with daily temperature cycling. Plant Physiol 932409-411 Usuda H, Edwards G E (1982) Influence of varying C0, and orthophosphate concentrations on rates of photosynthesis, synthesis of glycolate and dihydroxyacetone phowhate by wheat chloroplasts. Plant Physiol 692469-473 Warren Wilson J (1966) An analysis of plant growth and its control in Arctic environments. Ann Bot 30:383-402 Wheeler RM, Steffen K L, Tibbits TW, PaltaJP (1986) Utilizationofpotatoesfor life support systems. II. The effects of ternperature under 24-h and 12-h photoperiods. Am Potato I 63:639-647 Wheeler R M, Tibbits T .W, Fitzpatrick A H (1991) Carbon dioxide effects on potato growth under different photoperiods and irradiances. Crop Sci 31:1209-1213 Wildman S G (1967) The organization of gram-containing chloroplasts in relation to location of some enzymatic systems concerned with photosynthesis, protein synthesis, and ribonucleic acid synthesis. In: W T Goodwin, ed, Biochemistry of Chloroplasts, Vol 2. Academic Press, New York, pp 295-319 Williams J H H, Farrar J F (1988) Endogenous control of photosynthesis in leaf blades of barley. Plant Physiol Biochem 26:503-509 79 Captions for figures Figure 1. Effects of continuous illumination (C.L.) on diurnal (A) A and (B) g, over time for one-year-old, potted sour cherry trees. Gas exchange was measured at 09:00, 12:00, 03:00, and 20:00 for each day. Treatments: day 0 (0-24 h) before C.L.; day 1 (25-48 b), day 2 (49-72h), and day 3 (739%) represent 1, 2, and 3 days following C.L., respectively; day 4 (97-120h), day 5 (121-144h), and day 6 (145-168h) represent recovery 1, 2, and 3 days following return to 14 h photoperiod, respectively. Arrows point to the time at which plants were returned to 14 h photoperiod. Each point represents the average ($813) of 4 leaf measurements. Figure 2. Effect of continuous illumination (C.L.) on the response over time of A to C, of one- year-old, potted sour cherry trees. Each figure (A - F) compares the day 0 pretreatment response (0) with the treatment response for the subsequent day (0). Treatments: day 0 - before C.L.; days 1 (A), 2 (B), and 3 (C) represent 1, 2, and 3 days following C.L., respectively; days 4 (D), 5 (E), and 6 (F) represent recovery 1, 2, and 3 days following return to 14 h photoperiod, respectively. Regression lines occur about the data. Arrows point to the data collected at ambient C0, partial pressure (351:2.5 Pa). Data points which represent the average (iSE) of 4 leaf measurements appear about the regression lines. figure 3. Time course for the response of (A) A and (3) g. to continuous illumination (C.L.) in one-year-old, potted sour cherry trees. Treatments: 1, 2, and 3 days of C.L. (0, O, I), respectively. Gas exchange was measured daily at 10:00. Each point represents the average (1813) of 20 leaf measurements. 80 Figure 4. Time course for the response of (A) Fv/Fm, (B) Fv, and (C) F0 to continuous illumination (C.L.) in one-year-old, potted sour cherry trees. Treatments: 1, 2, and 3 days of C.L. (O, O, I), respectively. Chl fluorescence was measured daily at 15:00. Each point represents the average (18E) of 20 leaf measurements. 81 Table]. Iheefl'bdofcontinuqusillmrinationonl‘, LA”, Cm, A,_, andl, ofexpanded (saws of one-year-old, ported sour chewy trees wldl time. Means for each day are compared relative to Day 0 by orthogonal contrasts in each column. Means are averages of four replicates. NS, ‘, ", "", Nonsignificant or significant at P = 0.05, 0.01, or 0.001 respectively. Absence of NS or asterisks in a column indicates a lack of significant differences among days. Day Dark Light 1‘ k A3,, C, 3,, A.“ l. (h) (h) (#11101 (11301 C02 (MIDI (#11101 (#11101 (5‘) C0, in2 s“) C0, C0, C0, mol“) in2 s") mol") rn‘2 s“) 0 10 14 75 .3 0.088 12.28 246.8 14.96 11.0 1 0 24 79.8 0.019 *" 5.72 *" 296.7 ** 10.32 18.2 NS 2 0 24 75.3 0.031 *" 3.45 "'" 241.4 NS 12.79 58.1 ““ 3 0 24 79.0 0.026 "* 5.60 " 214.3 NS 8.29 30.5 "‘ 4 10 14 81.0 0.040 *"‘* 7.26 * 242.5 NS 9.80 27.3 NS 5 10 14 74.0 0.039 *“ 7.33 " 261.5 NS 14.94 29.9 NS 6 10 14 72.5 0.043 “* 7.54 NS 242.6 NS 11.43 29.3 NS 82 TableII. 1hr:flbctofcontinuouslllmntnation(€.l..)fi1r1, 2.0r3daysonleqfdlla.b, andtmalddwmemofexpandedleamqfone-year-ddmoaedsourdwnymm time. Meansareaverages of 5 replicates. Leafdiscs were harvested dailyatl62301lnd extractedfor36hbefore absorbsnces were measured. Leastsignificant difference (LSDm) was determined by analysis ofvariance for each day following treatment. C.L. time following tr-eutrnem (days) Duration (Days) 0 l 2 3 4 5 6 7 8 9 all a cement (148 an”) 1 61 61 63 62 65 65 67 69 71 72 2 64 65 63 65 66 67 67 7o 71 72 3 61 61 6o 61 62 63 63 65 66 67 L81), 12 12 10 11 10 10 10 10 10 10 Ch! b content (#8 an") 14 14 15 14 14 15 15 16 17 17 2 15 15 15 15 15 15 15 17 18 18 15 14 14 14 14 15 15 16 16 17 LSD,“ 3.0 3.1 2.9 2.9 2.5 2.7 2.6 2.6 2.2 2.3 Total chl content (#8 an") 75 74 78 77 79 so 82 86 88 89 2 . 79 so 78 so 81 82 82 87 88 89 76 75 75 76 76 7s 77 81 82 83 LSD,” 15 15 13 14 13 13 13 13 12 13 1) A (,umol m'2 3‘ -1) 9S (mmol m‘2 s 83 IIIIIIIIIIIIII 1 l 1 l g l J_ l 1 l L l 1 24 48 72 96 120 144 Time following treatment (h) A (,umol m”2 s") . DAY 0 ‘ 0 DAY 0 0 DAY 51 lo DAY 6‘ o 100 A 200 360 ‘ 400 500 400 500 L 600 660 o 100 260 300 Ci (,umol mol") gS (mmol m“2 S“) H 1 Day C.L.- _ 0—0 2 Days C.L. 2 ‘ H 3 Days C.L. BI l l I. l 1 l l 1 95- 85— 75— 3. .. 25 1 L L l 1 1 1 1 1 O 1 2 3 4 5 6 7 8 9 Time following treatment (days) Fv (relative units) Fo (relative units) Fv/Fm (relative units x 10") 740 720 1300 1150 1000 525 375 325 1 2 3 4 5 6 7 s 9 Time following treatment (days) SECTION IV: PHYSIOLOGICAL REPONSES OF m M L. 'I‘O WHOLE- PLANT SOURCE MANIPULA'I'ION. III. GAS EXCHANGE, CHLOROPHYLL FLUORBCENCE, WATER RELATIONS AND CARBON PARTITIONING‘ DesmondRichardLayne’andJamesAFlol-e o o o o o o .. hat-unaiut Hum! ‘ u .I'=1u.-I..‘ 1. ans .-1‘-1‘u!' Received for publication Accepted for publication 87 ‘ Supported by the Michigan Agricultural Experiment Station and by USDA grant No. 88-34132- 3380. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulation, this paper must be hereby marked W solely to indicate this fact. 3 Present address: Forestry Sciences Laboratory, 5985 Hwy K, P.O. Box 898, Rhinelander, WI 54501. 3W A, net CO2 assimilation rate; RuBP, ribulose-1,5-bisphosphate; FBPase, fructose-1,6-bisphosphatase; F—2,6-BP, fructose-2,6—bisphosphate; Glc-6-P, gluoose-6-phosphate; SLW, specific leaf weight; VPD, vapor pressure deficit; LAR, leaf area reduction; R... dark respiration rate; Q, internal CO, concentration; cp, light compensation point; g., stomatal conductance; ‘1', leaf xylem water potential; GA, gibberellin; E, transpiration rate; Fv, variable fluorescence; Fm, maximal fluorescence; Fo, instantaneous fluorescence; Fv/Fm, photochemical efficiency of PSII; triose-P, triose phosphate; SPS, sucrose phosphate synthase; 3-PGA, 3- phosphoglycerate. The source-sink ratio of one-year-old, potted sour cherry trees (2mm mg L.) was alterw by whole-plant partial defoliation or contimious illumination to determine if trees were primarily sink limited and to elucidate the means whereby photosynthetic enhancement or inhibition occurs. it was not affected by either treatment. A and g, were significantly enhanced or reduced within two days of partial defoliation or contimtous illumination, respectively. The increase in A of partially defoliated plants was associated with reduced carbon partitioning to starch and increased partitioning to sucrose and sorbitol. The decrease in A for continuously illuminated plants was associated with a decrease in Fv, Fv/Fm and an increase in F0 indicating that leaves were photoinhibited and 11m irreversible damage had occurred 10’ P811. In addition, leaves of contimrously illuminated plants partitioned 80% more carbon to starch and significantly less to sucrose and sorbitol. This altered partitioning indicated that the sink limitation was aggravated by contimtous illumination leading to an insufficient utilization of sucrose from the leaf. Whether photochemical and biochemical events occurred simultaneously and/or to the same degree to lead to the observed responses is uncertain. Evidence is provided in support of a sink limitation in young, potted sour cherry trees. Plant growth and yields are limited by both the supply and demand for photosynthate (Wardlaw, 1990). Gifford et al. (1984) suggested that growth and yield increases will be greatest when both carbon source and sink activities are increased simultaneously. A source limitation to A3 occurs when the capacity of the reactions that supply photosynthate is inadequate whereas asinklimitationtvoccurswhattherateatwhichphotosynthateisutilizedisinsufficiem (Baysdorfer and Bassham, 1985). Partial defoliation, shading, and continuous illumination are source manipulation techniques that have been utilized to demonstrate sink limitations in diverse crops. Bassman et al. (1982) found that whole-plant defoliation up to 40% in W had negligible effects on growth. Thorne and Koller (1974) found that shading all parts of soybean plants except for one source leaf, resulted in a curvilinear increase in A, a lO-fold decrease in leaf starch content, and an almost three-fold increase in leaf sucrose content within eight days for that source leaf. g Sawada et al. (1986) continuously illuminated rooted soybean leaves and by five days of exposure found that sucrose and starch levels were almost five and four-fold higher, respectively, while A was reduced by 60% . They noted that A was affected more by the amount of sucrose in the cytosol than by the amount of starch in the chloroplast and that leaves were probably end product inhibited. Contirmous illumination extended the duration of photosynthesis andresultedinagreaterincreaseinsourcecapacitythansinkcapacity,resultinginasink limitation (Sawada et al., 1989). Whether A of a source leaf is regulated by the level of assimilate within itself, by the utilization of assimilate at the sink, or a combination thereof remains equivocal. Neales and Incoll (1968) reviewed the hypothesis that accumulation of assimilates in the leaf is an internal factor which controls A. They noted that root pruning and fruit removal are two sink manipulation techniques that have been utilized in an attempt to demonstrate sink limitations in 91 differentcrops. Ferree(l989)rootprunedyoung,pottedappletreesandfoundthatbothAand shootgrowthwere reducedbutleafcarbohydratelevelswerenotaffected. Richards andRowe (1977) suggested that removal of root tips reduced the supply of cytokinin to the leaves resulting in a reduction of shoot growth. Gucci et al. (1991) found that fruit harvest of mature sweet cherrytrees WWL. cv. Windsor)resultedinasignificantdecreaseinleaannd increaseinleafstarchwithin24h. Althoughminorchloroplastdamage resultedfromstarch accumulation, they suggested that this played only a minor role in the post-harvest photosynthetic inhibition observed. Fruits are not just importers of photoassimilate but also important sources of GA. GA’s produced by the seeds of developing fruits inhibit flower bud development in apple and contribute to the problem of 'biennial bearing’ where a heavy fruit load deveIOps on a tree one year but a light load develops the subsequent year (Road, 1978). Herold (1980) noted that although artificial manipulation of sinks for carbohydrates may result in changes in A, the extent of the adjustment that is a direct outcome of an altered sink requirement is unclear. He suggested that the level and synthesis of phytohormones and nutrients may be altered, thereby indirectly affecting A (eg. hormones altering the permeability of the chloroplast envelope to metabolites). ‘ Wechosetoutilizeavegetativesystemfordlis studyandnottomanipulatesinksas above in order to minimize effects on phytohormone production or utilization. Instead of manipulating sinks, we chose to manipulate source leaves to investigate the sink limited condition of one-year-old, potted sour cherry trees. The major sinks for these vegetative trees are the shoot apex, developing leaves and growing roots. Many studies have been conducted on the responses of annual field crops and vegetables to partial defoliation and continuous illumination but few have been conducted on perennial deciduous fruit trees. Sour cherry is of additional interest since in addition to sucrose, it produces a similar quantity of sorbitol during its photosynthetic 92 metabolism (Loescher, 1987). The objectives of this study were: 1) to document the effect of whole-plant source manipulation by partial defoliation (defoliated) or contimrous (24 h Light) illumination on: i. gas exchange; ii. leaf water relations; iii. chl fluorescence; and iv. carbon partitioning.; and 2) to elucidate factors regulating source leaf A. MATERIALS AND METHODS Fifty dormant one—year-old sour cherry trees (2mm m L. cv.’Montmorency’ on Mahaleb rootstock)wereplantedin11Lplasticpotswith9.5 Lofsterilizedgreenhousesoil mix (5 sandy loam : 3 sphagnum peat : 2 torpedo sand (by volume), pH = 7.0). All trees were cut to an active bud (0 to 10 cm above the graft union) and placed in an environmentally controlled greenhouse (day and night meam 28 and 23°C, respectively). Trees were trained to a single shoot from which all laterals were removed as they appeared. Peter’s soluble 20N-20P-20K fertilizer (500 leg/g) was applied every three weeks and trees were watered every three days. Pesticides [S—O-demethylavermectin (abamectin, Avid), cyano(4-fluoro—3-phenoxyphenyl)methyl- 3(2,2-dichloroethenyl)—2,2-dimethylcyclopropanecarboxylate (cyfluthrin, Decathlon), and d-(2- chlorophenyl)-d—(4-chlorophenyl)-5-pyrimidine—methanol (fenarimol, Rubigan) were applied as necessary. Following nine weeks of active growth in the greenhouse, leaf number and total leaf area was determined for each plant. Each plant had approximately 25 fully expanded leaves at this time and leaf emergence was still occurring. Gas exchange was measured on the four most recently expanded leaves per plant using an ADC LCA2 portable photosynthesis system (Analytical Development Company, Hoddesdon, UK) under the following conditions: flow rate =400mL/min, leaftemperature=3012°C,VPD=3kPa, ambientCO,=35:t lPa,and 93 PAR 2 1000 ml m" s". The 24 most uniform plants were selected for this experiment based onthecombinedcharacteristicsofleafmrmber,totalleflarea,andinitialgasexchange characteristics. Selected plants were moved from the greenhouse to a Conviron PGV36 walk-in plant growth chamber (Conviron System of America, Pembina, N.D.). The growth chamber was programmed to the following settings (07:00: air temperature = 28°C, relative humidity = 50%, PAR = 800 umol m" s"; 21:00: air tunperature = 23°C, relative humidity = 50%, PAR = 0 umol m" s"). After one week of acclimation to the growth chamber, eight plants were randomly assigned to each of the following whole-plant manipulation treatments: i. nondefoliated, 14-11 photoperiod (Control); ii. partially defoliated, l4~h photoperiod (Defoliated); and iii. nondefoliated, 24-h photoperiod (24 h Light). Whole-plant partial defoliation involved removing all expanded leaves below the fourth fully expanded leaf from the apex beginning at 20:00 on day zero (corresponding to a 70% whole-plant LAR). Contimlous illumination commenced when the lights were turned on on day zero, proceeded for the duration of the experiment, and was accomplished by reprogramming the growth chamber so that the lights were always on. By 10:00 on day one when gas exchange was measured, continuously illuminated‘plants had been exposed to 27-h of light whereas defoliated plants had only been defoliated for about 14 h. Plants that received only a 14-h photoperiod were moved to the other side of the growth chamber behind a light screen (which reduced PAR to $2 umol n1°2 s“; cp = 14 ml m" s“) at 21:00 and returned to the illuminated side at 07:00 each day. Gas exchange measurements The four most recently fully expanded leaves on each plant were tagged and leaf area was determined. There were eight replicate plants for each of the three treatments. Gas exchange 94 was measured daily on each of the four leaves per plant under the following conditions: flow rate =400mIJmin,leaftemperan1re=3212°C,VPD=3kPa,ambientCO,=35j:lPa,and PAR = 800umlm"s". Day -1 wasdesignatedastheday beforetheexperimemwas started. Ondays-l drrough7gasexchangewasmeasuredbeginningat10:00foreachofthefourleaves per plant. Gas exchange parameters were calculated using the BASIC computer program of Moon and Flore (1986). Chl fluorescence measurement On days 0, l, 2, 4, and 7 following treatment, leaf chl fluorescence was measured on the same four leaves per plant beginning at 15:00. A Morgan CF-lOOO chl fluorescence measurement system (P.K. Morgan Instruments, Inc., Andover, MA) was used. Leaves were dark acclimated for 15 mimttes using dark acclimation cuvettes and then were irradiated with 1000 umol m" s" PPFD actinic light. Fluorescence kinetics were recorded over a 60 8 sample time. Leaf xylem water potential Immediately following the chl fluorescence measurements (at approximately 17:00), the four most recently expanded leaves were excised and then pressure bombed using a plant moisture stress meter (PMS Instrument Co., Corvallis, OR). Three plants each (one per treatment) were destructively harvested on days 0, l, 2, 4, and 7 following treatment. Immediately following pressure bombing, leaves were frozen in liquid N2 and then stored at - 80°C. 95 Solublesugarandstarchdetermlnatlon Stored leaf samples (-80°C) were lyophilized, weighed and ground in a Wiley mill to pasmmugha40-muhscreen.100-mgmbsamplawueexumedmrwfimeseach(20min) with3.5mLof80% ethanol. Thehomogenates were centrifugedat 1500ngor5 minutes alter each extraction, the supernatants were transferred into 50 mL polypropylene centrifuge tubes and 5mLofultrapurewaterandSmLofreagentgradechloroformwasaddedtopartitionthechl from the sample extract. The tubes were capped, shaken vigorouslyand then centrifuged at 470 x g for 3 minutes. The upper, clear aqueous phase containing the soluble sugars was transferred toatestmbeandevapormedtodrynessusingaSavantSpeedvac SC200equippedwitha refrigerated condensation trap RT4104 at ~103 °C (Savant Instruments, Inc., Farmingdale, NY). The pellet was also evaporated to dryness and then stored along with the soluble sugar samples in a glass desiccator over an anhydrous desiccant. Sorbitol, glc, fru, and suc were separated and quantified using HPLC. A Dionex series 4000i system was used which was equipped with a gradient pump, CarboPac PAl column, autosampler and pulsed amperometric detector connected to a model 4270 integrator (Dionex Corp., Sunnyvale, CA). The column was eluted with ultrapure water and 200 mM NaOH which had been sonicated for 30 min, degassed with helium for 1 hour and then continuously degassed during the analysis. A combination of gradient and isocratic elution was used by programming the gradient pump as follows: i. prior to sample injection, the column was eluted with 65% water and 35% NaOH; ii. at time = 0 min, the sample was injected and the gradient then was adjusted to 75% water and 25% NaOH by time = 0.1 min; the elution remained at this ratio until time = 1.5 min; iii. fromtime =1.5 minto9.0minthegradientwas adjustedto endup at0% water and 100% NaOH; iv. From time = 9 min to 28 min, the column was isocratically eluted with 96 100% NaOH. The flow rate of eluent was constant at 1 mL min". The column was maintained at room temperature. ThesamplewaspreparedforHPLC analysisby adding 1 mLofultrapurewaterandfor each, 3X vortexing followed by at least 5 min to stand. The sample was then transferred to a 1.5mLEppendorftubeandspunat10000ngor lOminonaBeckmanmicrofuge(Beckman Instruments, Inc., Irvine, CA) to pellet any partiallate matter that remained in the sample. The sample was then diluted 1:100 with ultrapure water and a 0.5 mL aliquot was loaded into a sample vial. Each sample was analyzed three times. Sugars were identified and quantified on the basis of retention time and peak heights of sugar standards. Starch in the pellet was measured using the method of Roper et al. (1988) modified as follows: samples were incubated at 55°C for 16 h with amyloglucosidase (Boehringer Mannheim 208 469) and then assayed colorimetrically using glucose oxidase (Sigma Chemical Co.). Absorbance at 440 nm was read with a Hitachi U-3110 UV/Vis spectrophotometer (Hitachi Ltd., Tokyo). Each sample was assayed three times. RESULTS Gas exchange over time As early as one day following continuous illumination, both A and g, were significantly reduced from that of the control (Figure 1). By two days following partial defoliation, A was significantly higher than control leaves. By seven days following treatment, A of continuously illuminated and partially defoliated leaves were 47% lower and 35% higher, respectively, than control leaves. A similar trend was observed over time for the g, data (relative to that for A) 97 except that the magnitude of the differences were somewhat greater when compared with the control leaves. At each day of continuous illumination, E was significantly lower than in control leaves (Figure 2). E was higher for leaves of partially defoliated plants than controls two, four, and five days following treatment. C, was significantly reduced by continuous illumination after one or twodays. AfiertwodaystherewerenodifferencesinQamongthethreetreatments. Leaf water status Leaf xylem water potential (‘1') was not significantly affected by continuous illumination or whole plant partial defoliation at any date measured (Table I). Chl fluorescence Fv/Fm was considerably reduced for continuously illuminated leaves (Figure 3). Seven days after defoliation, Fv/Fm was significantly higher in leaves of partially defoliated plants than inthe control. Fv was significantly reduced by contimlous illumination relative to control or partially defoliated plants at each date post-treatment. F0 was significantly increased as a result of continuous illumination and this remained higher than the control (or partially defoliated treatment) for the seven days measured. Carbonpartltioning Leaf starch content increased by almost 50% one day following continuous illumination and it remained 20% to 30% higher than the control at each subsequent determination (Figure 4). Starch content of leaves of partially defoliated plants decreased to 50% of the control by four days following defoliation and this remained constant after seven days. For a normal control 98 leaf, starch comprised about 1% of the tissue dry weight. Leaf sorbitol content was significantly reduced at each day following contimlous illumination exposure. By seven days following partial defoliation, sorbitol content was about 14% higher than in control leaves. Sorbitol is an acyclic sugar alcohol, the reduction product of Glc-6-P, which is a major phloem-translocated carbohydrate in 2mm species (Loescher, 1987). Leaf sucrose content was significantly higher one or two days following partial defoliation than in control leaves. Two and three days following contimtous illumination, leaf sucrose content was significantly lower than in the control leaves. By seven days following treatment, there were no significant differences among the treatments for leaf sucrose. There was no significant treatment effect on the relative partitioning of carbon to sorbitol or sucrose in the cytosol (data not shown). Typically, 40% of the total nonstructural carbohydrates in the leaf was sorbitol and 50% was sucrose. DISCUSSION Photosynthetic enhancement following partial defoliation was probably due to the relief of a sink limited condition. By removing source tissue, the sink demand per unit leaf surface increased and hence A of the remaining leaf area increased over time. The photosynthetic enhancement by two days following defoliation may have been due to the combined enhancement of g, (deJong, 1986), rubisco activity (Wareing et al., 1968; von Caemmerer and Farquhar, 1984) and RuBP regeneration rate (von Caemmerer and Farquhar, 1984). We suggest that biochemical processes were affected sooner and/or to a greater extent than either photochemical or physical (g.) processes since although enhancement of A occurred within two days, Fv/Fm was not significantly higher than control plants until seven days following defoliation. Production of new 99 photosynthetic machinery (Hodgkimon, 1974; Satoh et a1, 1977; Ness and Woolhouse, 1980) may account for the enhanced photochemical efficiency alter sevul days. Carbonpartitioningto starch inthechloroplastandsucroseandsorbitolinthe cytosolwas altered within 24 h of defoliation. The reduced partitioning to starch, yet increased partitioning tosucrose, withinonedayindicatedachangeinsinkstrength. Theenhancedsinkdemand following partial defoliation may have resulted in increased exchange of Pi from the cytosol for triose-P from the chloroplast. Accumulation of triose-P in the cytosol inhibits production of F- 2,6-BP at Fru6P,2-kinase (Cséke and Buchanan, 1983). This in turn may have enhanced FBPase activity leading to increased production of Glc-6-P which allosterically activated SPS (Doehlert and Huber, 1983) thereby increasing sucrose synthesis (Rufty and Huber, 1983) and export (Mullen and Koller, 1988). Export rate in the short-term (Grange, 1985) and in the long-term (H6, 1979) is proportional to the photosynthetic rate. Since starch content actually decreased following defoliation, it is possible that starch synthesis was reduced yet utilization was enhanced during the day (Dickson and Larson, 1975; Pongratz and Beck, 1978; Kruger et al., 1983) and during the night as well. In a previous study (Section II) we found that R, of partially defoliated plants was significantly lower than nondefoliated plants four days following defoliation. Since we also found a strong relationship between starch content and 11,, it is likely that within four days, leaf starch was depleted to such an extent that mitochondrial respiration in the night became substrate limited. In fact, in this study, by four days following defoliation starch content was 50% lower and sucrose and sorbitol contents were only marginally affected relative to control plants. Mullen and Koller (1988) found thattherateofassimilateexport(estimatedfromtherateoflossinSLWandtherateofCO, efflux) of soybean leaves at night was associated with the amount of starch reserves and the mobilization thereof but not associated with leaf sucrose concentration. 1m Photosynthetic imtibition following contimlous illumination was probably due to the aggravation of an already sink limited condition. The reduced g, which lowered C, in the first two days, may haveinpartaccountedforthedecreaseinAobserved. Thedecrease inAcoupled with reductiom in FvlFm and Fv indicated that continuously illuminated leaves were photoinhibited (Karukstis, 1991). Since the observed photoinhibition resulted in an increase in Foovertime,misindicatedthatmerewasdamagetothePSIIcentersthatwasnotreadily reversible (Krause, 1988). Obviously, cellular repair mechanisms could not match the rate of damage that was occurring to PS1! and hence photosynthetic capacity was impaired (Kyle, 1987). Under the continuous illumination conditions, extemive peroxidation of thylakoid membrane lipids (Mishra and Singhal, 1992) and damage to the D1 (or ’Q,’) protein (Kyle, 1987) may have occurred. It is also possible that biosynthesis of carotenoids such as zeaxanthin, which provides a photoprotective function by de-exciting the excited singlet state of chl, was reduced leading to chl damage (Demmig—Adams, 1990). Almost 80% more starch accumulated in the chloroplast after one day in continuous light than was present before treatment. Gucci et al. (1991) reported that starch content of sweet cherry leaves increased by 67% within 24 h of fruit harvest. They concluded that although large starch grains accumulated and more damaged chloroplasts resulted, the proportion of the observed post-harvest decline in A due to chloroplast disruption and thylakoid membrane deformation was minor. Wildman (1967) stated that as a chloroplast becomes engorged with starch, its shape is distorted which may tilt the grana away from the direct path of light and ultimately affect electron flow and carbon assimilation. Since PPFD was at nonlimiting levels, I would suggest that it is highly unlikely that starch grain shading of the thylakoid membrane occurred or had any physiological significance. Nafziger and Koller (1976) noted that accumulation of starch grains may interfere with intercellular C0, transport. Since we did not examine chloroplast 101 ultrastructure in this study, it is not possible to comment on the relative integrity of chloroplasts or speculate on the contribution of damage therein to the photosynthetic inhibition we observed. Carbon partitioning was altered in response to continuous illumination such that starch accumulated while levels of sucrose and sorbitol were reduced. This does not rule out a. role of starch in end product inhibition but it strongly indicates that leaves were not feedback inhibited by levels of soluble carbohydrates in the cytosol (Foyer, 1988). The reduced synthesis of sucrose and sorbitol would have made less Pi available to exchange from the cytosol with triose-phosphate from the chloroplast. Accumulation of 3-PGA in the chloroplast probably resulted in activation of ADPeglucose pyrophosphorylase and starch biosynthesis was enhanced (Preiss, 1984). It appeared from this study that photoinhibition and alterations in carbon partitioning were occurring simultaneously in relation to the time at which the continuous light treatment was imposed. Until very short-term studies are conducted over a period of minutes to hours, it remains to be seen if photochemical or biochemical events are affected simultaneously or to the same degree and how these events are related in terms of the photosynthetic inhibition that. results. Whether starch accumulation aggravated the photoinhibition from continuous light remains uncertain. ACKNOWLEDGEMENTS The authors gratefully acknowledge the assistance and expertise provided by Michael Kwantes and Dr. John D. Everard in developing and conducting the soluble sugar and starch analyses. We would also like to thank Drs. Mirta N. Sivak and Jack Preiss for invaluable input and advice. 102 LITERATURE CITE) Basernan J, Myers W,chlrman D, “11th (1982) Effects ofsimulated insectdamage - on early growth of mlrsery-grown hybrid poplars in northern Wisconsin. Can J For Res 12:1-9 Baysdorfer C, Bassham J A (1985) Photosynthate supply and utilization in alfalfa. A developmental shift from a source to a sink limitation of photosynthesis. Plant Physiol 77:313-317 Cease C, Buchanan B (1983) An enzyme synthesizing fructose 2,6 bisphosphateoccurs in leaves and is regulated by metabolite effectors. 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Aust J Plant Physiol 1:561-578 Karulrstls K K (1991) Chlorophyll fluorescence as a physiological probe to the photosynthetic apparatus. In: H Sheer, ed, Chlorophylls. CRC Press Inc, Boca Raton, pp 769-795 ' Krause G H (1988) Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms. Physiol Plant 74:566-574 Kruger N J, Bulpin P V, ap Rees T (1983) The extent of starch degradation in the light in pea leaves. Planta 157:271-273 28 29. 20. 21. 22. 23. 24. 26. 27. 28. 29. 104 Kyle D J (1987) The biochemical basis for photoinhibition of photosystem II. In: D 1 Kyle, C B Osmond, C J Arntzen, eds, Photoinhibition. Elsevier Sci Pub B V Anuterdam, pp 197-226 Inescher W H (1987) Physiology and metabolism of sugar alcohols in higher plants. Physiol Plant 70:553-557 Mishra R K, Slnghal G S (1992) Function of photosynthetic apparatus of intact wheat leaves under high light and heat stress and its relationship with peroxidation of thylakoid lipids. Plant Physiol 98:1-6 Moon J W Jr, Flore J A (1986) A BASIC computer program for calculation of photosynthesis, stomatal conductance, and related parameters in an open gas exchange system. Photosynthesis Res 7:269-279 Mullen J A, Koller H R (1988) Trends in carbohydrate depletion, respiratory carbon loss, and assimilate export from soybean leaves at night. Plant Physiol 86:517-521 Nafziger E D, Koller H R (1976) Influence of leaf starch concentration on CO2 assimilation in soybean. Plant Physiol 57:560-563 . Neales T F, Incoll L D (1968) The control of leaf photosynthesis rate by the level of assimilate concentration in the leaf: a review of the hypothesis. Bot Rev 34: 107-125 Ness P J, Woolhouse H W (1980) RNA synthesis in M chloroplasts. II: Ribonucleic acid synthesis in chloroplasts from developing and senescing leaves. J Exp Bot 31:235-245 Pongratz P, Beck E (1978) Diurnal oscillation of amylolytic activity in spinach chloroplasts. Plant Physiol 62:687-689 Preiss) J (1984) Starch, sucrose biosynthesis and partition of carbon in plants are regulated by orthophosphate and triose-phosphates. Trends Biol Sci 9:24-27 31. 32. 33. 34. 35. 36. 37. 38. 105 RidlardsD,RnweRN(l977)Effectsofrootresn'iction,rootpruningand6- benzylaminopurine on the growth of peach seedlings. Ann Bot 41:729-740 RoperTR,KellerJD,LoescherWH,RomCR(l988)Photosynthesisand carbohydrate partitioning in sweet cherry: Fruiting effects. Physiol Plant 72:42-47 Rufty T W Jr, Huber S C (1983) Changes in starch formation and activities ofsucrose phosphate synthase and cytoplasmic fructose-1,6-bisphosphatase in response to source-sink alterations. Plant Physiol 72:474-480 Satoh M, Kriedemann P E, Loveys B R (1977) Changes in photosynthetic activity and related processes following decapitation in mulberry trees. Physiol Plant 41:203-210 Sawada S, Hayaltawa T, Fultushi K, Kasai M (1986) Influence of carbohydrates on photosynthesis in single, rooted soybean leaves used as a source-sink model. Plant Cell Physiol 27:591-600 Sawada S, Hasegawa Y, Kasai M, Sasalti M (1989) Photosynthetic electron tramport and carbon metabolism during altered source/sink balance in single-rooted soybean leaves. Plant Cell Physi0130:691-698 'I‘horne J H, Koller H R (1974) Influence of assimilate demand on photosynthesis, diffusive resistances, translocation, and carbohydrate levels of soybean leaves. Plant Physiol 54:201-207 von Caemmerer S, Farquhar G D (1984) Effects of partial defoliation, changes of irradiance during growth, short-term water stress and growth at enhanced p(CO,) on the photosynthetic capacity of leaves .of M Maris L. Planta 160:320-329 Wardlaw I F (1990) The control of carbon partitioning in plants. New Phytol 116:341-381 37. 106 WardngPF,KhalifaMM,TheharneKJ(l968)Rate-limitingprocessesin photosynthesis at saturating light intensities. Nature 220:453-457 Wildmn S G (1967) The organization of grana-containing chloroplasts in relation to location of some enzymaic systems concerned with photosynthesis, protein synthesis, and ribonucleic acid synthesis. In: W T Goodwin, ed, Biochemistry of chloroplasts, Vol 2. Acadanic Press, New York, pp 295-319 107 Captions for flgtu'es Figure 1. Effects of partial defoliation or continuous illumination on (A) A and (B) 8. over time for one-year-old, potted sour cherry trees. Trements: Control — nondefoliated, 14 h photoperiod (O); Defoliated - 14 h photoperiod (O); 24 h Light - 24 h photoperiod (I). Gas exchange was measured beginning at 10:00 daily between days -1 and 7. Control plants were defoliated on day 0 after the 10:00 measurement. Each point represents the average (1:81!) of at least 8 leaf measurements. Figure 2. Effects of partial defoliation or continuous illumination on (A) E or (B) Q over time for one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated, 14 h photoperiod (I); Defoliated - 14 h photoperiod (O); 24 h Light - 24 h photoperiod (I). Gas exchange was measured beginning at 10:00 daily between days -1 and 7. Control plants were defoliated on day 0 after the 10:00 measurement. Each point represents the average (1:88) of at least 8 leaf , Figure 3. Effects of partial defoliation or continuous illumination on (A) Fv/Fm, (B) Fv, and (C) Fo over time for one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated, 14 h photoperiod (I); Defoliated - 14 h photoperiod (O); 24 h Light - 24 h photoperiod (I). Chl fluorescence was measured at 15:00. Control plants were defoliated on day 0 at 17:00. Each point represents the average (18E) of at least 8 leaf measurements. 108 Figure 4. Effects of partial defoliation or continuous illumination on leaf (A) starch, (B) sorbitol, and (C) sucrose content over time for one-year-old, potted sour cherry trees. Treatments: Control - nondefoliated, 14 h photoperiod (O); Defoliated - 14 h photoperiod (O); 24 h Light - 24 h photoperiod (I). Control plants were defoliated on day 0 at 17:00. Each point represents the average (18E) of at least 8 leaf measuremwts. 109 Table I. The eject ofparrial defoliation (defol.) or combatants lamination (C.L.) on Ieafxylem Miter potential ofexpanded (saw: ofone-year-old, potted sour cherry tree: over time. _ Leaf water potential (MPa) was determined at 17:00. Means are the average (:85) of at least 8 leaves. Least significant difference (LSDm) was determined by analysis of variance at each date following treatment. Time following treatment (days) Treatment 0 1 2 4 7 Control -l.16 -1.19 -1.11 -1.23 ‘ -l.26 - Defol. -1.15 -1.15 -1.13 -1.10 -1.27 C.L. -1.14 -1.15 -1.13 -l.00 -1.20 LSD m 0.35 0.32 0.31 0.30 0.18 , I I l r l I T l 15 ”A 0 Control _ - 0 Defoliated es -. I 24 h ' h a A 12 _ ng t fl 7 '- 0) r .. E 9 F'~ " .. —o i e — _ < .. 3 _ —l O l- B I I I l I r i q 200 - u _ TU) 0': N 150 — u _ . :l E r- 6 .- E 100..1F - é 64’ i :: ‘ L J J. J. O l J i 1 l l l l 110 Time following treatment (doys) E (mmol m’2 3") Ci (,umol mol") lll I l I l l l l l A a Control 0 Defoliated L— n 24 h Light 290 260 230 i 200 ' 170 l l l l l L l 140 —1 O 1 2 3 4 5 6 7 Time following treotment (days) 'Fv/Fm (relative units x 10") Fv (relative units) Fa (relative units) 112 750 A 700 650 - 600 - 500 ’ a Control 550 -o Defoliated ' - I 24 h Light 1900 1700 - 1500 - 1300 l 1100 900 .B l l l I l r 800 p- l l l l J l 500 0 1 2 3 4 5 6 7 Time following treatment (days) 113 d M t - e g m. a ..._ .m w. h - - 0 e 4. c o 2 \ _ _ . o. - B _ h _ b — b b 3. 1. a 7. . 5. . s s o. . .o o. o. o. o. . o. o. 9592) be “coosmav 9590; be E0803 329m; be Lcoouoav “concoo :0..on .63 ecogcoo 6:98 ”—8.. «coucoo 89.2.5 .63 Time following treatment (days) SUMMARY AND CONCLUSIONS The photosynthetic enhancement that occurred following whole-plant partial defoliation was probably due to the relief of a sink limited condition. As source leaf area was reduced, the sink demand per unit leaf area increased. This photosynthetic enhancement may have occurred because the following factors were enhanced: l) in the short-term: i. stomatal conductance; ii. carboxylation rate; and iii. RuBP regeneration rate; and 2) in the long-term: i. production of new photosynthetic machinery; and ii. availability of root-derived cytokinins. The diurnal decline in net C0, assimilation rate (A) observed in the afternoon may have been due to end-product accumulation causing a feedback inhibition of A or a mild water stress affecting stomatal conductance (g) by a buildup of leaf abscisic acid (ABA). Utilization of “CO, to follow short-term changes in metabolites and measuring changes- in the activity of regulatory enzymes of carbon metabolism should provide insight into the step(s) regulating the observed response. Measurement 'of leaf ABA and water potential over the short-term could distinguish the role of mild water stress in the diurnal decline of A observed. The photosynthetic inhibition that occurred following whole-plant continuous illumination was probably due to the aggravation of an already existing sink limitation. Since sink demand was already low, the ability of source leaves to utilize the additional light energy in photosynthesis became impaired. Once starch accumulated to levels that caused end-product inhibition, it is likely that the excess light energy could no longer be used for productive photochemistry. In turn, as oxygen radicals were produced, thylakoid lipids were probably 114 115 peroxidatively degraded and photosystem 11 (PSI!) was damaged. Carboxylation efficiency and RuBP regeneration rates were also reduced in continuous light. The impairment of A was most likely due to a combination of feedback inhibition and photoinhibition. The recovery of leaves to short-term exposure to continuous light depended upon the exposure duration. Indeed, as long as the rate of damage to PS 11 did not exceed the rate of repair, photoinhibition should not occur. Clearly, two or more days in continuous light was detrimental to leaf A and full photosynthetic recovery was not observed until at least 4 days following return to a normal 14-h photoperiod. In future experiments, it would be desirable to follow changes in A, chlorophyll fluorescence and carbon partitioning over a period of hours following a continuous illumination treatment. In this way it might be possible to determine: i. whether photochemical or biochemical phenomena is affected first; ii. if they are affected simultaneously; and iii. the extent of the photoinhibition that is due to each. In addition, use of “C0, to trace the flow of carbon in the short-term and by determining the activity of the regulatory enzymes in carbon metabolism insight should be gained regarding the step(s) regulating theobserved phenomena. ”Willi/71111111“