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Temperature Dependence of Chlorophyll Fluorescence Induction and Photosynthesis in Tomato as Affected by Temperature and Light Conditions During Growth
J.PlantPbysiol. Vol. 139.pp. 549-554 (1992)
T ernperature Dependence of Chlorophyll Fluorescence Induction and Photosynthesis in T ornato as Affected by T ernperature and Light Conditions During Growth LUUK H. J. JANSSEN, HILLIE
E. W AMS, and PHILIP R.
VAN HASSELT
Department of Plant Biology, University of Groningen, P.O. Box 14, 9750 AA Haren, the Netherlands Received August 12, 1991 . Accepted October 8,1991
Summary The temperature dependence of chlorophyll fluorescence induction and photosynthesis of tomato plants grown at different temperatures and light intensities was studied. Chlorophyll fluorescence induction and photosynthetic activity of leaf discs was determined between 0° and 30°C. Two breakpoints, around 16° and 24 °C, were observed when the maximum of chlorophyll fluorescence induction, Fp , was plotted against temperature. DCMU was used to study the role of photosynthetic electron transport on the breakpoints of Fp. Inhibition of photosynthetic electron transport by DCMU caused the disappearance of the breakpoint at 16°C. The temperature breakpoint at 24 °C remained intact. This result is used to discuss the mechanisms underlying the occurrence of the breakpoints. The low temperature breakpoint at 16°C was ascribed to electron transport limitation between photosystem I and photosystem II at temperatures below 16°C. The high temperature breakpoint was attributed to alterations in light distribution between photosystem I and photosystem II. An increased State II adaptation at temperatures above 24°C is believed to cause a decrease in Fp. Temperatures of both breakpoints were affected by growth conditions of the tomato plants. Compared with optimal growth conditions, suboptimal growth conditions caused a significant decrease in the low temperature breakpoint. Concomitantly, photosynthetic activity and leaf chlorophyll content were decreased. The possibility of using breakpoint temperatures to indicate an adaptation of the thylakoid membrane organisation and functioning to suboptimal growth conditions is discussed.
Key words: Tomato, Lycopersicon esculentum Mill genotype «IVT 1», chilling, cold acclimation, chlorophyll fluorescence induction, growth condition, herbicide, photosynthesis. Abbreviation list: A max = maximal photosynthetic activity; DCMU = 3-(3,4-dichlorophenyl)-I,I-dimethylurea; Fm = maximum fluorescence; Fo = constant fluorescence; Fp = maximum fluorescence induction; QA = primary electron acceptor of photosystem II; qQ = photochemical fluorescence quenching; qN = non photochemical fluorescence quenching.
Introduction Chlorophyll fluorescence measurements have been increasingly used to determine injury in plants due to stress as freezing, chilling, heat, drought and radiation (Havaux and Lannoye, 1985; Oquist and Strand, 1986; Lichtenthaler and Rinderle, 1988). The wide use of chlorophyll fluorescence © 1992 by Gustav Fischer Verlag, Stuttgart
measurements in stress research can be attributed to the advantages that the method provides: it is rapid, non-destructive and can be used on small samples of green plant material. For each application of the method it is essential to establish a correlation, preferably based on a causal relation between the fluorescence parameters and the stress factor applied.
550
LUUK H. J. JANSSEN, HILUE E. WAMS, and PHIUP R. VAN HASSELT
Chlorophyll fluorescence can be used as a tool to study temperature effects on photosynthetic light reactions because thylakoid membrane organisation and functioning are influenced by temperature (Fork, 1979; Havaux and Lannoye, 1983; Murata, 1984). For tomato, a phase transition of thylakoid polar lipids was observed at 11°C, as determined by calorimetry, spin label and fluorescence techniques (Raison et al., 1986). Around the same temperature a maximum in delayed light emission was observed (Fork and Murata, 1990). This temperature is of physiological significance because it corresponds with the temperature limit below which chilling injury occurs in tomato (Lyons, 1973). Chlorophyll fluorescence induction of detached leaves has been used earlier as an assay for chilling injury of tomato (Smillie and Nott, 1979; Smillie et al., 1988). Chilling injury was accessed by the decrease with time of the initial fluorescence rise upon illumination, determined at o°c. The rate of decrease correlated with the chilling sensitivity of tomato species of different origin. More recently, Walker et al. (1990) observed that a similar parameter, the Fo / Fp ratio, increased during incubation at 2 0c. They also observed a positive correlation between this fluorescence parameter and chilling sensitivity of species differing in chilling sensitivity. An evaluation of techniques used to determine chilling injury in tomato (Kamps, 1987) showed that chlorophyll fluorescence was the most precise assay to quantify chilling injury. Electrolyte leakage and visual rating were less effective. Temperature effects on chlorophyll fluorescence induction at decreasing temperatures from 30° to 0 °C were previously studied with cucumber leaf discs anssen and van Hasselt, 1988). The fluorescence at the maximum of the induction kinetics (Fp) at non-saturating light conditions (10 ~mol· m- 2 ·s- l ) was determined. Two temperature breakpoints of Fp were observed. Photosynthesis was optimal between the two breakpoint temperatures. In a related study, it was shown that the breakpoint temperature of cucumber leaf discs was affected by growth conditions (van Hasselt et al., 1982). The aim of this study was to investigate if breakpoints of Fp , as observed in cucumber, would occur also in tomato, another chilling sensitive plant. In addition to chlorophyll fluorescence measurements, the temperature dependence of photosynthetic activity was determined at a low light intensity of 250 ~mol . m - 2. S - I. The light limitation of photosynthesis (von Caemmerer and Farquahar, 1981) makes it possible to compare temperature effects on the functioning of photosystems, as assessed by breakpoint temperatures, with the temperature effects on photosynthetic activity. Furthermore, effects of optimal and suboptimal growth conditions on the temperature of the breakpoints, photosynthesis and chlorophyll content were investigated to obtain more insight in the causal relations between these parameters and in the adaptation of the thylakoid membrane to growth conditions.
a
Seedlings were transplanted to 30 L tanks containing aerated nutrient solution. For explanation of the nutrient solution, see Smakman and Hofstra, 1982. Plants were grown in Conviron EF7H growth cabinets under two conditions: Firstly, 22/17°C day/night temperature and 300 Ilmol· m -2. S-I light intensity during a 16-hour day «optimal conditions» and secondly, 19/6°C day/night temperature and 100 Ilmol· m -2. S-I light intensity during a 8-hour day «suboptimal conditions». Ninety percent of the light intensity was provided by fluorescent lamps (Sylvania F72T12/CW/VHO) and 10 % by incandescent lamps (Sylvania, 60 Watt). It should be noted that the terms, optimal and suboptimal, are used as relative terms because maximal growth of tomato requires higher temperatures and light intensities. The suboptimal growth conditions resembled the most extreme conditions used to select tomato genotypes for cold tolerance by growth analyses (Smeets and Garretsen, 1986). All plants were in the same stage of development and had approximately 8 leaves at the time of measurement of chlorophyll fluorescence and photosynthesis. Optimally grown plants were 4 weeks old and suboptimally grown plants 8 weeks old. Leaf discs (0 7 mm) were punched from nearly expanded leaves (75 % of maximal leaf area). The second leaflet of the fifth leaf, counted from the top, was used for the experiments. Temperature control Temperature was controlled by a waterbath linked to a computer. Chlorophyll fluorescence and photosynthetic activity of leaf discs were measured simultaneously in two temperature controlled cuvets at decreasing temperatures from 30° to O°C, at intervals of 2°C (30°,28°, .. ,0 °C). The temperature was kept constant for 30 min during the measurements. The next temperature setting was reached by a decrease of 2 °C at a rate of 18°C· h -I. The leaf discs remained on the cuvet until the complete temperature range, from 30° to 0 °C, was completed. The time to perform the experiment, approximately 8 hours, can be relatively long because photosynthetic activity of leaf tissue samples was found to remain constant for several hours, provided that humidity is retained (SetHk and Sestak, 1971). Chlorophyll fluorescence Leaf discs were floated on tapwater during the chlorophyll fluorescence induction measurements (10 min). During the measurements 2 % oxygen was used because breakpoints were more pronounced in 2 % than in 21 % oxygen. The lower oxygen concentration had no effect on the temperatures where the breakpoints of Fp occurred (unpublished result). After dark adaptation for 20 min, chlorophyll fluorescence was induced by 10Ilmol·m- 2 ·s- 1 actinic light from a light emitting diode (Hewlett Packard HCMP0222) and measured by a photodiodelfilter combination (EG & G HUV 1l00BG/Schott RG-9). In the case of DCMU treatment, 50 IlM DCMU was added to the tapwater and discs were preincubated during 2 hours in the light (100 Ilmol· m -2. S-I). As a result, chlorophyll fluorescence quenching was completely inhibited at all temperatures. The actinic light intensity used to determine maximum fluorescence (Fm) of DCMUtreated leaf discs was also 10 Ilmol . m - 2 • S - I. Photosynthesis
Materials and Methods Plant material Seeds of tomato (Lycopersicon esculentum Mill genotype
Photosynthesis of 25 leaf discs (9.6cm 2) was measured at decreasing temperatures from 30° to O°C at intervals of 2°C (30°,28°, .. ,O°C). Leaf discs were floated on phosphate buffer (KH 2 P04 + K2 HP04 , 50 mM, pH = 5.0) in a temperature controlled cuvet in the light (250 Ilmol· m -2. S-I). Temperature de-
Temperature dependence of Chlorophyll fluorescence induction and Photosynthesis in Tomato
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Table 1: Mean breakpoint temperatures (oq, with standard deviations (±), of fluorescence, Fp. Values with same superscripts (a, b and c) do not significantly differ according to the studentized range test (0.05 level). Number of experiments as indicated. Chlorophyll fluorescence induction of tomato leaf discs was determined at decreasing temperatures from 30° to o°e. Plants were grown at optimal and suboptimal conditions as described in material and methods. growth condition
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breakpoint temperature LTB
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10
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HTB
optimal
15.5±3.5 n ~ 12
24.0±1.8C n <= 20
suboptimal
11.8±3.1' n-9
22.7±2.2C n = 11
b
temperature ( "C
Fig. 1: Chlorophyll fluorescence induction parameters, Fp and Fm , of leaf discs derived from optimally grown tomato plants. Chlorophyll fluorescence was induced by 10 !lmol· m - 2. S -1 actinic light and measured at decreasing temperatures from 30° to 0 0e. Chlorophyll fluorescence values were normalized to the highest value (loo%) found at O°e. Results of typical experiments are shown. (a): fluorescence at the maximum of the induction kinetics at non-saturating light conditions, Fp. (b): maximal chlorophyll fluorescence, Fm , of DCMU-treated leaf discs.
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pendent CO 2 gas exchange rate of the leaf discs determined with an infra-red gas analyzer (URAS 2T) in a closed system during 10 min, after aeration with ambient air for 20 min.
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Chlorophyll content Leaf pigment extracts were obtained by grinding 5 leaf discs in 4 mL cold acetone in the dark. The extracts were diluted with 4 mL acetone and 2 mL water. Cell fragments were removed by centrifugation at 1000 g for 10 min. Contents of chlorophyll a, band carotenoids x + c of the 80 % acetone supernatant were determined spectrophotometrically according to Lichtenthaler et aI., 1983.
Results
Chlorophyll fluorescence Fig. 1 a shows the temperature dependence of the first maximum in chlorophyll fluorescence induction, F p , of tomato leaf discs. The plants were grown at optimal conditions. Fp increased at decreasing temperatures, from 30° to around 24°C, followed by a slower increase or even a decrease from 24°C to around 15°C. Upon further lowering of the temperature to O°C, Fp increased steeply. As a consequence, two temperatures where a sudden change in temperature dependence of Fp occurred (breakpoints) were observed. A low temperature breakpoint (LTB) was found at about 15°C; a high temperature breakpoint (HTB) was found at about 24°C and two breakpoints of Fp were also found for leaf discs of suboptimally grown tomato plants. Maximal fluorescence (Fm) of DCMU-treated leaf discs of optimally grown tomato plants showed only one breakpoint, at approximately the same temperature where HTB of untreated leaf discs was observed (Fig. 1 b). Both the similarity in breakpoint temperatures and the similarity in the
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Fig. 2: Change of net carbon dioxide uptake rate of leaf discs from optimally grown tomato plants. Measurements were performed at decreasing temperatures from 30° to O°C. During the experiment, 25 leaf discs (9.6 cm2) were floated on phosphate buffer (50 mM, pH = 5.0) and aerated with ambient air. Result of a typical experiment is shown.
course of change of Fp and Fm with decreasing temperature suggest that HTB of untreated leaf discs and the breakpoint of DCMU-treated leaf discs have the same origin. Consequently, LTB of DCMU-treated leaf discs is absent. It should be noted that the rate of change at temperatures above HTB is greater for Fp than for Fm. Growth conditions of tomato plants affected the temperature of the breakpoints (Table 1). Compared with optimal growth conditions, suboptimal growth conditions caused a significant shift of LTB towards a 3.7 °C lower temperature. The decrease of 1.3 °C for HTB was not significant.
Photosynthesis Photosynthetic activity of leaf discs showed an optimum curve with temperature. An example of tomato plants grown under optimal conditions is shown in Fig. 2. Photosynthesis decreased at both low and high temperatures. The decrease of photosynthetic activity was stronger at low than at high temperatures. A maximal photosynthetic activity (Ama><) was observed at about 23°C.
552
Lum. H. J. JANSSEN, HILLIE E. WAMS, and PHILIP R. VAN HASSELT
Table 2: The highest carbon dioxide uptake rate (Amax) of tomato leaf discs at a light intensity of 2S0 Ilmol . m - 2 • S- I, determined in the temperature range from 30° to O°c. Mean values and standard deviations (±) are shown. Values with same superscripts (a and b) do not significantly differ according to the studentized range test (O.OS level). Number of experiments as indicated. Amax is expressed per leaf area as well as per chlorophyll content. In addition, the temperature range where photosynthesis exceeded 90 % of Amax is shown. Plants were grown at optimal and suboptimal conditions as described in material and methods. growth condition
Carbon dioxide uptake (Amax) Ilmol . m - 2 • S - 1 Ilmol· mg- I . h- I
4.4±0.Sb n=4 suboptimal 2.5±0.5a n=6 optimal
40.6±4.6b n=4 30.0±4.6a n = 6
A>90%A max temperature range 17°-28°C 13°-2SOC
Table 3: Total chlorophyll (Chla+b) and carotenoid (Carx+c) content of tomato leaves. Mean values and standard deviations (±) are shown. Values with same superscripts (a and b) do not significantly differ according to the studentized range test (O.OS level). Number of experiments as indicated. Plants were grown at optimal and suboptimal conditions as described in material and methods. growth condition optimal suboptimal
Chla+b
(mg· dm- 2) 3.9±0.l b n=4 3.0±0.2a n=4
Chla/b 3.9±OY
n=4 3.2±0.5a n=4
Carx+c
(mg· dm- 2) 0.69±0.06a n=4 0.49±0.01 a n=4
Chia+b Carx+c
S.7±OY n = 4 6.0±0.6a n = 4
Table 2 shows that A max was affected by the growth conditions of the tomato plants in two ways. Firstly, growth condition affected the absolute value of A max. Amax of suboptimally grown plants was lower than A max of optimally grown plants. On a leaf area base, the decrease of A max was 43 %. On a chlorophyll base, A max showed a smaller but still significant decrease of 26%. Secondly, the temperature where A max occurred differed. The shift in the temperature where A max was found is indicated by the temperature range where photosynthetic activity exceeded 90 % of A max; it was 13 ° - 25°C for suboptimal growth conditions and 17° - 28 °C for optimal growth conditions.
Chlorophyll content Optimally grown plants had a slightly higher chlorophyll a + b and carotenoid x + c content than suboptimally grown plants (Table 3). As mentioned above, this difference could not account for the difference in A max between optimally and suboptimally grown plants. The chlorophyll a/b ratio and the carotenoid x + c content tended to be higher for optimally grown plants but the differences were not significant. The chlorophyll/carotenoid ratio (a +b/x+c) was hardly affected by growth conditions.
Discussion
Chlorophyll fluorescence Chlorophyll fluorescence induction is affected by many factors such as photosynthetic electron transport, the energy state of the thylakoid membrane, excitation energy transfer between photosystems and Calvin cycle activity (Krause and Weis, 1984). Explaining temperature effects on Fp is facilitated when some of these factors can be omitted. We assume that the thylakoid membrane is not energized when Fp is reached because the actinic light intensity was very low and Fp was reached within seconds after a dark period of 20 min (Sivak and Walker, 1983). As a consequence, Fp is only determined by photochemical fluorescence quenching, qQ (Duysens and Sweers, 1963; Krause and Weis, 1984), and effects of non photochemical fluorescence quenching, qN, can be neglected (Horton, 1985). In other words, the reduction state of the primary electron acceptor of photosystem II, QA, was controlling Fp. At Fp, maximal QA reduction reflects an equilibrium of PS II reducing activity and PS I oxidizing activity. PS I oxidizing activity is not yet affected by Calvin cycle activity; the onset of Calvin cycle activity coincides with the appearance of the second peak (M) in chlorophyll fluorescence induction (Sivak and Walker, 1983; Sivak et aI., 1985). QA reduction is controlled by two factors: the rate of photochemical electron transport, affecting the photochemical reduction of intersystem electron carrier pools, and the quantal distribution of excitation light within the photosynthetic apparatus, affecting the balance in activity of photosystem I and photosystem II. DCMU was added to analyze the role of photosynthetic electron transport in the breakpoints of Fp. In tomato leaf discs treated with DCMU, LTB was absent while HTB remained intact. Similar results were obtained with cucumber Ganssen and van Hasselt, 1988). Clearly, LTB was caused by a temperature induced alteration photosynthetic electron transport. Fp increased steeply at decreasing temperature below L TB, indicating an increased QA reduction at lower temperatures. The limited photosystem I QA oxidizing activity at lower temperatures could be caused by formation of gel phase domains in the lipid matrix of the thylakoid membrane, limiting electron transport by a decreased lateral diffusion of plastoquinone/ plastoquinol (Murata, 1984; Scoufflaire et al., 1985). Normally, lateral diffusion of plastoquinone/plastoquinol is not limiting electron transport, but the oxidation of plastoquinol at the cyt b/f complex itself (Haehnel, 1984; Barber, 1985). Low temperature induced local phase transitions of the thylakoid membrane (Chapman et al., 1983; Havaux and Lannoye, 1983; Martin, 1986; Fork and Murata, 1990) might explain the observed abrupt increase in Fp below LTB. It should be noted that LTB coincides with the temperature limit below which chilling injury occurs in tomato (Wolfe, 1978). This suggests that the shift of LTB from 16° to 12°C indicates a thylakoid membrane adaptation to suboptimal growth conditions which might be correlated with chillling tolerance. HTB was observed in both Fp of control leaf discs and in Fm of DCMU-treated leaf discs. Another factor than photosynthetic electron transport must be responsible for the
Temperature dependence of Chlorophyll fluorescence induction and Photosynthesis in Tomato
sudden change in temperature dependence of Fp at high temperature. HTB could be affected by an alteration in the quantal distribution of excitation light within the photosynthetic apparatus, decreasing the fraction of light received by photosystem II at higher temperatures. This state 1state 2 transition corrects for overexcitation of one of the photosystems and regulates the balance in activity of photosystem I and photosystem II (Barber, 1982). State transitions are influenced by excitation light quality (Malkin et ai., 1986) and temperature (Havaux, 1988). Temperature could overrule light quality effects so that the dark adapted state of the thylakoid membrane can influence photosynthesis in the light. Weis (1985) observed state changes of dark-adapted spinach leaves: at temperatures above 20°C, excitation light was distributed in favour of photosystem I (state 2). We suggest that HTB is caused by a similar temperature dependent change in the light distribution between the photosystems. This explanation is sustained by the observation that the rate of change at temperatures above HTB is greater for Fp than for Fm. An alteration in light distribution over the photosystems has a large effect on QA reduction because it affects both the QA oxidizing activity of photosystem I and the contrasting QA reducing activity of photosystem II. This additive effect on Fp was absent for Fm of DCMU-treated leaf discs. Breakpoint temperatures were affected by growth conditions. Compared with optimally grown tomato plants, suboptimally grown plants showed mainly a decrease in LTB, from 16° to 12°C. In contrast, cucumber showed a LTB at 7°C, which was hardly affected by growth conditions, but HTB was decreased from 21° to 16 °C (van Hasselt et aI., 1982). The fact that cucumber is more thermophilic than tomato could account for the different responses to suboptimal growth conditions. Photosynthesis
The optimum photosynthetic actiVIty of tomato was found at a rather low temperature, which was probably caused by the light limitation of 250 /lmol· m -2. S-1 (von Caemmerer and Farquahar, 1981). Light limitation of photosynthesis is essential for comparing temperature effects on the functioning of light reactions, as assessed by breakpoint temperatures, with temperature effects on photosynthesis; light saturation disguises temperature effects on photosystems and only shows temperature effects on enzymatic dark reactions of the Calvin cycle (von Caemmerer and Farquahar, 1981). At low temperatures, low photosynthetic activity coincided with high Fp values. Photosynthetic electron transport possibly was limited at low temperature, restricting NADPH and ATP supply. In contrast, Kee et al. (1986) observed that photosynthetic electron transport capacity of the photosystems in tomato at low temperatures exceeded that needed for photosynthesis, making a photosynthetic electron transport limitation of photosynthesis questionable. However, the significance of this in vitro measurements for the functioning of electron transport under in vivo conditions remains to be elucidated since mutual regulation mechanisms of light and dark reactions are present (Horton,
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1985; Foyer et aI., 1990). A second possible explanation for the observed decreased photosynthesis and increased Fp at low temperatures is an impairment of photosynthetic phosphorylation as a result of leakage or malfunctioning of the thylakoid membrane caused by phase transitions of local domains in the membrane (Peeler and Naylor, 1988; Fork and Murata, 1990). The decrease of photosynthesis at high, increasing temperatures has been ascribed to increased photorespiration (Sage and Sharkey, 1987), determined by ribulose-1,5-bisphosphate carboxylase/oxygenase temperature sensitivity. A higher rate of photorespiration requires more ATP which can be provided by an increased rate of photosynthetic cyclic electron transport (Bulte et ai., 1990). An altered light distribution in favour of photosystem I (state 2) at higher temperatures (Weis, 1985; Havaux, 1988) could stimulate ATP production and be functional in adaptation of the thylakoid membrane organisation. The temperature range for optimal photosynthesis decreased about 3 °C when plants were grown at suboptimal conditions. A similar decrease was observed for breakpoint temperatures of F p , indicating that photosynthesis and breakpoint temperature may be affected in the same way by alterations in the thylakoid membrane organisation. In addition, photosynthetic activity of suboptimally grown plants could be decreased by a low temperature induced loss of ribulosebisphosphate carboxylase/oxygenase activity (Sassenrath and Ort, 1990), which is not reflected in the chlorophyll fluorescence parameter Fp. Chlorophyll and carotenoid content
Differences in chlorophyll and carotenoid content could influence chlorophyll fluorescence results. However, breakpoint temperatures of Fp were determined by abrupt changes in the course of Fp with decreasing temperature from single discs. The absolute values of Fp , which were affected by chlorophyll a + b content, had no influence on the determination of breakpoint temperatures. Acknowledgements This investigation was supported by the Committee for Energy Consumption of the Dutch Ministry of Agriculture and Fisheries, and was carried out in cooperation with the Centre for Plant Breeding Research in Wageningen, the Netherlands. Our thanks are due to Prof. Dr. Ir. P. J. C. Kuiper for carefully reading the manuscript.
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LUUK H. J. JANSSEN, HILUE E. W AMS, and PHIUP R. VAN HASSELT
VON CAEMMERER, S. and G. D. FARQUAHAR: Some relationships between biochemistry of photosynthesis and the gas exchange of leaves. Planta 153, 376-387 (1981). CHAPMAN, D. J., J. DE-FEUCE, and J. BARBER: Growth temperature effects on thylakoid membrane lipid and protein content of pea chloroplasts. Plant Physiol. 72, 225-228 (1983). DUYSENS, L. N. M. and H. E. SWEERS: Mechanisms of the two photochemical reactions in algae: a study by means of fluorescence. In: Jpn. Soc. Plant Physiol. Studies on microalgae and photosynthetic bacteria, 353-372. Univ. Tokyo Press, Tokyo (1963). FORK, D. C.: The influence of changes in the physical phase of thylakoid membrane lipids on photosynthetic activity. In: LYONS, J. M., G. GRAHAM, andJ. K. RAISON (eds.): Low Temperature Stress in Crop Plants, 215-229. Academic Press, New York (1979). FORK, D. C. and N. MURATA: The effect of light intensity on the assay of the low temperature limit of photosynthesis using msec delayed light emission. Photosyn. Res. 23, 319-323 (1990). FoYER, C., R. FURBANK, J. HARBINSON, and P. HORTON: The mechanisms contributing to photosynthetic control of electron transport by carbon assimilation in leaves. Photosyn. Res. 25, 83-100 (1990). HAEHNEL, W.: Photosynthetic electron transfer in higher plants. Annu. Rev. Plant Physiol. 35, 659-693 (1984). VAN HASSELT, P. R., J. WOLTJES, and F. DE JONG: The effect of temperature on chlorophyll fluorescence induction of cucumber lines differing in growth at suboptimal conditions. In: MARCELLE, R., H. CUJSTERS, and M. VAN POUCKE (eds.): Effects of Stress on Photosynthesis, 257-265. Martinus Nijhoff, Dr. W. Junk Publishers, The Hague (1982). HAvAUX, M.: Effects of temperature on the transition between state 1 and state 2 in intact maize leaves. Plant Physiol. 26, 245-251 (1988). HAVAUX, M. and R. LANNOYE: In vivo chlorophyll fluorescence and delayed light emission as rapid screening techniques for stress tolerance in crop plants. Z. Pflanzenziichtg. 95, 1-13 (1985). - - Temperature dependence of delayed chlorophyll fluorescence in intact leaves of higher plants: A rapid method for detecting the phase transition of the thylakoid membrane lipids. Photosyn. Res. 4, 257 -263 (1983). HORTON, P.: Interactions between electron transfer and carbon assimilation. In: BARBER, J. and N. R. BAKER (eds.): Photosynthetic Mechanisms and the Environment., 135-188. Elsevier, Amsterdam (1985). JANSSEN, L. H. J. and P. R. VAN HASSELT: Temperature-induced alterations of in vivo chlorophyll a fluorescence in cucumber as affected by DCMU. Photosyn. Res. 15, 153-162 (1988). KAMps, T., T. G. ISLEIB, R. C. HERNER, and K. C. SINK: Evaluation of techniques to measure chilling injury in tomato. Hort. Science 22(6}, 1309-1312 (1987). KEE, C. S., B. MARTIN, and D. R. aRT: The effects of chilling in the dark and in the light on photosynthesis of tomato: electron transfer reactions. Photosyn. Res. 8, 41- 51 (1986). KRAUSE, G. H. and E. WEIS: Chlorophyll fluorescence as a tool in Plant Physiol.: II. Interpretation of fluorescence signals. Photosyn. Res. 5, 139-157 (1984). LICHTENTHALER, H. K. and U. RrnoERLE: The role of chlorophyll fluorescence in the detection of stress conditions on plants. CRC Critical Rev. in Anal. Chern. 19, s29-s 89 (1988). LICHTENTHALER, H. K. and A. R. WELLBURN: Determination of total carotenoids and chlorophyll a and b of leaf extracts in different solvents. Biochem. Soc. Transitions 603,590-592 (1983). LYONS, J. M.: Chilling injury in plants. Annu. Rev. Plant Physiol. 24,445-466 (1973).
MALKIN, S., A. TELFER, and J. BARBER: Quantitative analysis of state I-state 2 transitions in intact leaves using modulated fluorimetry: evidence for changes in the absorption cross-section of the two photosystems during state transitions. Biochim. Biophys. Acta 848, 48-57 (1986). MARTIN, B.: Arrhenius plots and the involvement of thermotropic phase transitions of the thylakoid membrane in chilling impairment of photosynthesis in thermophilic higher plants. Plant Cell Envir.9, 232-331 (1986). MURATA, N.: The lipid phase of photosynthetic membranes. In: SYBESMA, C. (ed.): Advances in Photosyn. Res., 131-138. Martinus Nijhof, Dr. W. Junk Publishers, The Hague (1984). OQUIST, G. and M. STRAND: Effects of frost hardening on photosynthetic quantum yield, chloroplast organisation and energy distribution between the two photosystems in Scots pine. Can. J. Bot. 64, 748-753 (1986). PEELER, T. C. and A. W. NAYLOR: A comparison of the effects of chilling on thylakoid electron transfer in pea (Pisum sativum L.) and cucumber (Cucumis sativus L.). Plant Physiol. 86, 147 -151 (1988). RAISON, J. K. and G. R. ORR: Phase transitions in thylakoid polar lipids of chilling sensitive plants. Plant Physiol. 80, 638-645 (1986). SAGE, R. F. and T. D. SHARKEY: The effect of temperature on the occurrence of O 2 and CO 2 insensitive photosynthesis in field grown plants. Plant Physiol. 84, 658-664 (1987). SASSENRATH, G. F. and D. R. aRT: The relationship between inhibition of photosynthesis at low temperature and the inhibition of photosynthesis after rewarming in chill-sensitive tomato. Plant Physiol. Biochem. 28, 457 - 465 (1990). SCOUFFLAIRE, c., R. LANNOYE, and J. BARBER: Influence of structural and physical properties of the thylakoid membrane on Qa oxidation. Photosyn. Res. 6,133-145 (1985). SETLlK, S. and Z. SESTAK: Use of leaf tissue samples in ventilated chambers for long term measurements of photosynthesis. In: SESTAK, Z., J. CATSKY, and P. G. JARVIS (eds.): Plant Photosynthetic Production: Manual of Methods, 316-342. Dr. W. Junk N.V., The Hague (1971). SIVAK, M. N., U. HEBER, and D. A. WALKER: Chlorophyll a fluorescence and light-scattering kinetics displayed by leaves during induction of photosynthesis. Planta 163, 419-423 (1985). SIVAK, M. N. and F. R. S. WALKER: Some effects of CO 2 concentration and decreased O 2 concentration on induction fluorescence in leaves. Proc. R. Soc. Lond. 217, 377-392 (1983). SMAKMAN, G. and J. J. HOFSTRA: Energy metabolism of plantago lanceolata as affected by change in root temperature. Physiol. Plant. 56,33 -37 (1982). SMEETS, L. and F. GARRETSEN: Growth analyses of tomato genotypes grown under low night temperatures and low light intensity. Euphytica 35,701-715 (1986). SMILLIE, R. M., S. E. HETHERINGTON, J. HE, and R. NOTT: Photoinhibition at chilling temperatures. Austr. J. Plant Physiol. 15, 207-222 (1988). SMILLIE, R. M. and R. NOTT: Assay of chilling injury in wild and domestic tomatoes based on photosystem activity of the chilled leaves. Plant Physiol. 63, 796-801 (1979). WALKER, M. A., D. M. SMITH, K. P. PAULS, and B. D. MCKERSIE: A chlorophyll fluorescence screening test to evaluate chilling tolerance in tomato. HortScience 25(3}, 334-338 (1990). WEIS, E.: Light and temperature induced changes in the distribution of excitation energy between photosystem I and photosystem II in spinach leaves. Biochim. Biophys. Acta 807, 118-126 (1985). WOLFE, J.: Chilling injury in plants: the role of membrane lipid fluidity. Plant Cell Envir. 1, 241-247 (1978).
T ernperature Dependence of Chlorophyll Fluorescence Induction and Photosynthesis in T ornato as Affected by T ernperature and Light Conditions During Growth LUUK H. J. JANSSEN, HILLIE
E. W AMS, and PHILIP R.
VAN HASSELT
Department of Plant Biology, University of Groningen, P.O. Box 14, 9750 AA Haren, the Netherlands Received August 12, 1991 . Accepted October 8,1991
Summary The temperature dependence of chlorophyll fluorescence induction and photosynthesis of tomato plants grown at different temperatures and light intensities was studied. Chlorophyll fluorescence induction and photosynthetic activity of leaf discs was determined between 0° and 30°C. Two breakpoints, around 16° and 24 °C, were observed when the maximum of chlorophyll fluorescence induction, Fp , was plotted against temperature. DCMU was used to study the role of photosynthetic electron transport on the breakpoints of Fp. Inhibition of photosynthetic electron transport by DCMU caused the disappearance of the breakpoint at 16°C. The temperature breakpoint at 24 °C remained intact. This result is used to discuss the mechanisms underlying the occurrence of the breakpoints. The low temperature breakpoint at 16°C was ascribed to electron transport limitation between photosystem I and photosystem II at temperatures below 16°C. The high temperature breakpoint was attributed to alterations in light distribution between photosystem I and photosystem II. An increased State II adaptation at temperatures above 24°C is believed to cause a decrease in Fp. Temperatures of both breakpoints were affected by growth conditions of the tomato plants. Compared with optimal growth conditions, suboptimal growth conditions caused a significant decrease in the low temperature breakpoint. Concomitantly, photosynthetic activity and leaf chlorophyll content were decreased. The possibility of using breakpoint temperatures to indicate an adaptation of the thylakoid membrane organisation and functioning to suboptimal growth conditions is discussed.
Key words: Tomato, Lycopersicon esculentum Mill genotype «IVT 1», chilling, cold acclimation, chlorophyll fluorescence induction, growth condition, herbicide, photosynthesis. Abbreviation list: A max = maximal photosynthetic activity; DCMU = 3-(3,4-dichlorophenyl)-I,I-dimethylurea; Fm = maximum fluorescence; Fo = constant fluorescence; Fp = maximum fluorescence induction; QA = primary electron acceptor of photosystem II; qQ = photochemical fluorescence quenching; qN = non photochemical fluorescence quenching.
Introduction Chlorophyll fluorescence measurements have been increasingly used to determine injury in plants due to stress as freezing, chilling, heat, drought and radiation (Havaux and Lannoye, 1985; Oquist and Strand, 1986; Lichtenthaler and Rinderle, 1988). The wide use of chlorophyll fluorescence © 1992 by Gustav Fischer Verlag, Stuttgart
measurements in stress research can be attributed to the advantages that the method provides: it is rapid, non-destructive and can be used on small samples of green plant material. For each application of the method it is essential to establish a correlation, preferably based on a causal relation between the fluorescence parameters and the stress factor applied.
550
LUUK H. J. JANSSEN, HILUE E. WAMS, and PHIUP R. VAN HASSELT
Chlorophyll fluorescence can be used as a tool to study temperature effects on photosynthetic light reactions because thylakoid membrane organisation and functioning are influenced by temperature (Fork, 1979; Havaux and Lannoye, 1983; Murata, 1984). For tomato, a phase transition of thylakoid polar lipids was observed at 11°C, as determined by calorimetry, spin label and fluorescence techniques (Raison et al., 1986). Around the same temperature a maximum in delayed light emission was observed (Fork and Murata, 1990). This temperature is of physiological significance because it corresponds with the temperature limit below which chilling injury occurs in tomato (Lyons, 1973). Chlorophyll fluorescence induction of detached leaves has been used earlier as an assay for chilling injury of tomato (Smillie and Nott, 1979; Smillie et al., 1988). Chilling injury was accessed by the decrease with time of the initial fluorescence rise upon illumination, determined at o°c. The rate of decrease correlated with the chilling sensitivity of tomato species of different origin. More recently, Walker et al. (1990) observed that a similar parameter, the Fo / Fp ratio, increased during incubation at 2 0c. They also observed a positive correlation between this fluorescence parameter and chilling sensitivity of species differing in chilling sensitivity. An evaluation of techniques used to determine chilling injury in tomato (Kamps, 1987) showed that chlorophyll fluorescence was the most precise assay to quantify chilling injury. Electrolyte leakage and visual rating were less effective. Temperature effects on chlorophyll fluorescence induction at decreasing temperatures from 30° to 0 °C were previously studied with cucumber leaf discs anssen and van Hasselt, 1988). The fluorescence at the maximum of the induction kinetics (Fp) at non-saturating light conditions (10 ~mol· m- 2 ·s- l ) was determined. Two temperature breakpoints of Fp were observed. Photosynthesis was optimal between the two breakpoint temperatures. In a related study, it was shown that the breakpoint temperature of cucumber leaf discs was affected by growth conditions (van Hasselt et al., 1982). The aim of this study was to investigate if breakpoints of Fp , as observed in cucumber, would occur also in tomato, another chilling sensitive plant. In addition to chlorophyll fluorescence measurements, the temperature dependence of photosynthetic activity was determined at a low light intensity of 250 ~mol . m - 2. S - I. The light limitation of photosynthesis (von Caemmerer and Farquahar, 1981) makes it possible to compare temperature effects on the functioning of photosystems, as assessed by breakpoint temperatures, with the temperature effects on photosynthetic activity. Furthermore, effects of optimal and suboptimal growth conditions on the temperature of the breakpoints, photosynthesis and chlorophyll content were investigated to obtain more insight in the causal relations between these parameters and in the adaptation of the thylakoid membrane to growth conditions.
a
Seedlings were transplanted to 30 L tanks containing aerated nutrient solution. For explanation of the nutrient solution, see Smakman and Hofstra, 1982. Plants were grown in Conviron EF7H growth cabinets under two conditions: Firstly, 22/17°C day/night temperature and 300 Ilmol· m -2. S-I light intensity during a 16-hour day «optimal conditions» and secondly, 19/6°C day/night temperature and 100 Ilmol· m -2. S-I light intensity during a 8-hour day «suboptimal conditions». Ninety percent of the light intensity was provided by fluorescent lamps (Sylvania F72T12/CW/VHO) and 10 % by incandescent lamps (Sylvania, 60 Watt). It should be noted that the terms, optimal and suboptimal, are used as relative terms because maximal growth of tomato requires higher temperatures and light intensities. The suboptimal growth conditions resembled the most extreme conditions used to select tomato genotypes for cold tolerance by growth analyses (Smeets and Garretsen, 1986). All plants were in the same stage of development and had approximately 8 leaves at the time of measurement of chlorophyll fluorescence and photosynthesis. Optimally grown plants were 4 weeks old and suboptimally grown plants 8 weeks old. Leaf discs (0 7 mm) were punched from nearly expanded leaves (75 % of maximal leaf area). The second leaflet of the fifth leaf, counted from the top, was used for the experiments. Temperature control Temperature was controlled by a waterbath linked to a computer. Chlorophyll fluorescence and photosynthetic activity of leaf discs were measured simultaneously in two temperature controlled cuvets at decreasing temperatures from 30° to O°C, at intervals of 2°C (30°,28°, .. ,0 °C). The temperature was kept constant for 30 min during the measurements. The next temperature setting was reached by a decrease of 2 °C at a rate of 18°C· h -I. The leaf discs remained on the cuvet until the complete temperature range, from 30° to 0 °C, was completed. The time to perform the experiment, approximately 8 hours, can be relatively long because photosynthetic activity of leaf tissue samples was found to remain constant for several hours, provided that humidity is retained (SetHk and Sestak, 1971). Chlorophyll fluorescence Leaf discs were floated on tapwater during the chlorophyll fluorescence induction measurements (10 min). During the measurements 2 % oxygen was used because breakpoints were more pronounced in 2 % than in 21 % oxygen. The lower oxygen concentration had no effect on the temperatures where the breakpoints of Fp occurred (unpublished result). After dark adaptation for 20 min, chlorophyll fluorescence was induced by 10Ilmol·m- 2 ·s- 1 actinic light from a light emitting diode (Hewlett Packard HCMP0222) and measured by a photodiodelfilter combination (EG & G HUV 1l00BG/Schott RG-9). In the case of DCMU treatment, 50 IlM DCMU was added to the tapwater and discs were preincubated during 2 hours in the light (100 Ilmol· m -2. S-I). As a result, chlorophyll fluorescence quenching was completely inhibited at all temperatures. The actinic light intensity used to determine maximum fluorescence (Fm) of DCMUtreated leaf discs was also 10 Ilmol . m - 2 • S - I. Photosynthesis
Materials and Methods Plant material Seeds of tomato (Lycopersicon esculentum Mill genotype
Photosynthesis of 25 leaf discs (9.6cm 2) was measured at decreasing temperatures from 30° to O°C at intervals of 2°C (30°,28°, .. ,O°C). Leaf discs were floated on phosphate buffer (KH 2 P04 + K2 HP04 , 50 mM, pH = 5.0) in a temperature controlled cuvet in the light (250 Ilmol· m -2. S-I). Temperature de-
Temperature dependence of Chlorophyll fluorescence induction and Photosynthesis in Tomato
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100
b
80
60
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Table 1: Mean breakpoint temperatures (oq, with standard deviations (±), of fluorescence, Fp. Values with same superscripts (a, b and c) do not significantly differ according to the studentized range test (0.05 level). Number of experiments as indicated. Chlorophyll fluorescence induction of tomato leaf discs was determined at decreasing temperatures from 30° to o°e. Plants were grown at optimal and suboptimal conditions as described in material and methods. growth condition
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breakpoint temperature LTB
20
0
10
20
30
HTB
optimal
15.5±3.5 n ~ 12
24.0±1.8C n <= 20
suboptimal
11.8±3.1' n-9
22.7±2.2C n = 11
b
temperature ( "C
Fig. 1: Chlorophyll fluorescence induction parameters, Fp and Fm , of leaf discs derived from optimally grown tomato plants. Chlorophyll fluorescence was induced by 10 !lmol· m - 2. S -1 actinic light and measured at decreasing temperatures from 30° to 0 0e. Chlorophyll fluorescence values were normalized to the highest value (loo%) found at O°e. Results of typical experiments are shown. (a): fluorescence at the maximum of the induction kinetics at non-saturating light conditions, Fp. (b): maximal chlorophyll fluorescence, Fm , of DCMU-treated leaf discs.
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pendent CO 2 gas exchange rate of the leaf discs determined with an infra-red gas analyzer (URAS 2T) in a closed system during 10 min, after aeration with ambient air for 20 min.
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Chlorophyll content Leaf pigment extracts were obtained by grinding 5 leaf discs in 4 mL cold acetone in the dark. The extracts were diluted with 4 mL acetone and 2 mL water. Cell fragments were removed by centrifugation at 1000 g for 10 min. Contents of chlorophyll a, band carotenoids x + c of the 80 % acetone supernatant were determined spectrophotometrically according to Lichtenthaler et aI., 1983.
Results
Chlorophyll fluorescence Fig. 1 a shows the temperature dependence of the first maximum in chlorophyll fluorescence induction, F p , of tomato leaf discs. The plants were grown at optimal conditions. Fp increased at decreasing temperatures, from 30° to around 24°C, followed by a slower increase or even a decrease from 24°C to around 15°C. Upon further lowering of the temperature to O°C, Fp increased steeply. As a consequence, two temperatures where a sudden change in temperature dependence of Fp occurred (breakpoints) were observed. A low temperature breakpoint (LTB) was found at about 15°C; a high temperature breakpoint (HTB) was found at about 24°C and two breakpoints of Fp were also found for leaf discs of suboptimally grown tomato plants. Maximal fluorescence (Fm) of DCMU-treated leaf discs of optimally grown tomato plants showed only one breakpoint, at approximately the same temperature where HTB of untreated leaf discs was observed (Fig. 1 b). Both the similarity in breakpoint temperatures and the similarity in the
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10
20
30
tefTl)erature ( "C
Fig. 2: Change of net carbon dioxide uptake rate of leaf discs from optimally grown tomato plants. Measurements were performed at decreasing temperatures from 30° to O°C. During the experiment, 25 leaf discs (9.6 cm2) were floated on phosphate buffer (50 mM, pH = 5.0) and aerated with ambient air. Result of a typical experiment is shown.
course of change of Fp and Fm with decreasing temperature suggest that HTB of untreated leaf discs and the breakpoint of DCMU-treated leaf discs have the same origin. Consequently, LTB of DCMU-treated leaf discs is absent. It should be noted that the rate of change at temperatures above HTB is greater for Fp than for Fm. Growth conditions of tomato plants affected the temperature of the breakpoints (Table 1). Compared with optimal growth conditions, suboptimal growth conditions caused a significant shift of LTB towards a 3.7 °C lower temperature. The decrease of 1.3 °C for HTB was not significant.
Photosynthesis Photosynthetic activity of leaf discs showed an optimum curve with temperature. An example of tomato plants grown under optimal conditions is shown in Fig. 2. Photosynthesis decreased at both low and high temperatures. The decrease of photosynthetic activity was stronger at low than at high temperatures. A maximal photosynthetic activity (Ama><) was observed at about 23°C.
552
Lum. H. J. JANSSEN, HILLIE E. WAMS, and PHILIP R. VAN HASSELT
Table 2: The highest carbon dioxide uptake rate (Amax) of tomato leaf discs at a light intensity of 2S0 Ilmol . m - 2 • S- I, determined in the temperature range from 30° to O°c. Mean values and standard deviations (±) are shown. Values with same superscripts (a and b) do not significantly differ according to the studentized range test (O.OS level). Number of experiments as indicated. Amax is expressed per leaf area as well as per chlorophyll content. In addition, the temperature range where photosynthesis exceeded 90 % of Amax is shown. Plants were grown at optimal and suboptimal conditions as described in material and methods. growth condition
Carbon dioxide uptake (Amax) Ilmol . m - 2 • S - 1 Ilmol· mg- I . h- I
4.4±0.Sb n=4 suboptimal 2.5±0.5a n=6 optimal
40.6±4.6b n=4 30.0±4.6a n = 6
A>90%A max temperature range 17°-28°C 13°-2SOC
Table 3: Total chlorophyll (Chla+b) and carotenoid (Carx+c) content of tomato leaves. Mean values and standard deviations (±) are shown. Values with same superscripts (a and b) do not significantly differ according to the studentized range test (O.OS level). Number of experiments as indicated. Plants were grown at optimal and suboptimal conditions as described in material and methods. growth condition optimal suboptimal
Chla+b
(mg· dm- 2) 3.9±0.l b n=4 3.0±0.2a n=4
Chla/b 3.9±OY
n=4 3.2±0.5a n=4
Carx+c
(mg· dm- 2) 0.69±0.06a n=4 0.49±0.01 a n=4
Chia+b Carx+c
S.7±OY n = 4 6.0±0.6a n = 4
Table 2 shows that A max was affected by the growth conditions of the tomato plants in two ways. Firstly, growth condition affected the absolute value of A max. Amax of suboptimally grown plants was lower than A max of optimally grown plants. On a leaf area base, the decrease of A max was 43 %. On a chlorophyll base, A max showed a smaller but still significant decrease of 26%. Secondly, the temperature where A max occurred differed. The shift in the temperature where A max was found is indicated by the temperature range where photosynthetic activity exceeded 90 % of A max; it was 13 ° - 25°C for suboptimal growth conditions and 17° - 28 °C for optimal growth conditions.
Chlorophyll content Optimally grown plants had a slightly higher chlorophyll a + b and carotenoid x + c content than suboptimally grown plants (Table 3). As mentioned above, this difference could not account for the difference in A max between optimally and suboptimally grown plants. The chlorophyll a/b ratio and the carotenoid x + c content tended to be higher for optimally grown plants but the differences were not significant. The chlorophyll/carotenoid ratio (a +b/x+c) was hardly affected by growth conditions.
Discussion
Chlorophyll fluorescence Chlorophyll fluorescence induction is affected by many factors such as photosynthetic electron transport, the energy state of the thylakoid membrane, excitation energy transfer between photosystems and Calvin cycle activity (Krause and Weis, 1984). Explaining temperature effects on Fp is facilitated when some of these factors can be omitted. We assume that the thylakoid membrane is not energized when Fp is reached because the actinic light intensity was very low and Fp was reached within seconds after a dark period of 20 min (Sivak and Walker, 1983). As a consequence, Fp is only determined by photochemical fluorescence quenching, qQ (Duysens and Sweers, 1963; Krause and Weis, 1984), and effects of non photochemical fluorescence quenching, qN, can be neglected (Horton, 1985). In other words, the reduction state of the primary electron acceptor of photosystem II, QA, was controlling Fp. At Fp, maximal QA reduction reflects an equilibrium of PS II reducing activity and PS I oxidizing activity. PS I oxidizing activity is not yet affected by Calvin cycle activity; the onset of Calvin cycle activity coincides with the appearance of the second peak (M) in chlorophyll fluorescence induction (Sivak and Walker, 1983; Sivak et aI., 1985). QA reduction is controlled by two factors: the rate of photochemical electron transport, affecting the photochemical reduction of intersystem electron carrier pools, and the quantal distribution of excitation light within the photosynthetic apparatus, affecting the balance in activity of photosystem I and photosystem II. DCMU was added to analyze the role of photosynthetic electron transport in the breakpoints of Fp. In tomato leaf discs treated with DCMU, LTB was absent while HTB remained intact. Similar results were obtained with cucumber Ganssen and van Hasselt, 1988). Clearly, LTB was caused by a temperature induced alteration photosynthetic electron transport. Fp increased steeply at decreasing temperature below L TB, indicating an increased QA reduction at lower temperatures. The limited photosystem I QA oxidizing activity at lower temperatures could be caused by formation of gel phase domains in the lipid matrix of the thylakoid membrane, limiting electron transport by a decreased lateral diffusion of plastoquinone/ plastoquinol (Murata, 1984; Scoufflaire et al., 1985). Normally, lateral diffusion of plastoquinone/plastoquinol is not limiting electron transport, but the oxidation of plastoquinol at the cyt b/f complex itself (Haehnel, 1984; Barber, 1985). Low temperature induced local phase transitions of the thylakoid membrane (Chapman et al., 1983; Havaux and Lannoye, 1983; Martin, 1986; Fork and Murata, 1990) might explain the observed abrupt increase in Fp below LTB. It should be noted that LTB coincides with the temperature limit below which chilling injury occurs in tomato (Wolfe, 1978). This suggests that the shift of LTB from 16° to 12°C indicates a thylakoid membrane adaptation to suboptimal growth conditions which might be correlated with chillling tolerance. HTB was observed in both Fp of control leaf discs and in Fm of DCMU-treated leaf discs. Another factor than photosynthetic electron transport must be responsible for the
Temperature dependence of Chlorophyll fluorescence induction and Photosynthesis in Tomato
sudden change in temperature dependence of Fp at high temperature. HTB could be affected by an alteration in the quantal distribution of excitation light within the photosynthetic apparatus, decreasing the fraction of light received by photosystem II at higher temperatures. This state 1state 2 transition corrects for overexcitation of one of the photosystems and regulates the balance in activity of photosystem I and photosystem II (Barber, 1982). State transitions are influenced by excitation light quality (Malkin et ai., 1986) and temperature (Havaux, 1988). Temperature could overrule light quality effects so that the dark adapted state of the thylakoid membrane can influence photosynthesis in the light. Weis (1985) observed state changes of dark-adapted spinach leaves: at temperatures above 20°C, excitation light was distributed in favour of photosystem I (state 2). We suggest that HTB is caused by a similar temperature dependent change in the light distribution between the photosystems. This explanation is sustained by the observation that the rate of change at temperatures above HTB is greater for Fp than for Fm. An alteration in light distribution over the photosystems has a large effect on QA reduction because it affects both the QA oxidizing activity of photosystem I and the contrasting QA reducing activity of photosystem II. This additive effect on Fp was absent for Fm of DCMU-treated leaf discs. Breakpoint temperatures were affected by growth conditions. Compared with optimally grown tomato plants, suboptimally grown plants showed mainly a decrease in LTB, from 16° to 12°C. In contrast, cucumber showed a LTB at 7°C, which was hardly affected by growth conditions, but HTB was decreased from 21° to 16 °C (van Hasselt et aI., 1982). The fact that cucumber is more thermophilic than tomato could account for the different responses to suboptimal growth conditions. Photosynthesis
The optimum photosynthetic actiVIty of tomato was found at a rather low temperature, which was probably caused by the light limitation of 250 /lmol· m -2. S-1 (von Caemmerer and Farquahar, 1981). Light limitation of photosynthesis is essential for comparing temperature effects on the functioning of light reactions, as assessed by breakpoint temperatures, with temperature effects on photosynthesis; light saturation disguises temperature effects on photosystems and only shows temperature effects on enzymatic dark reactions of the Calvin cycle (von Caemmerer and Farquahar, 1981). At low temperatures, low photosynthetic activity coincided with high Fp values. Photosynthetic electron transport possibly was limited at low temperature, restricting NADPH and ATP supply. In contrast, Kee et al. (1986) observed that photosynthetic electron transport capacity of the photosystems in tomato at low temperatures exceeded that needed for photosynthesis, making a photosynthetic electron transport limitation of photosynthesis questionable. However, the significance of this in vitro measurements for the functioning of electron transport under in vivo conditions remains to be elucidated since mutual regulation mechanisms of light and dark reactions are present (Horton,
553
1985; Foyer et aI., 1990). A second possible explanation for the observed decreased photosynthesis and increased Fp at low temperatures is an impairment of photosynthetic phosphorylation as a result of leakage or malfunctioning of the thylakoid membrane caused by phase transitions of local domains in the membrane (Peeler and Naylor, 1988; Fork and Murata, 1990). The decrease of photosynthesis at high, increasing temperatures has been ascribed to increased photorespiration (Sage and Sharkey, 1987), determined by ribulose-1,5-bisphosphate carboxylase/oxygenase temperature sensitivity. A higher rate of photorespiration requires more ATP which can be provided by an increased rate of photosynthetic cyclic electron transport (Bulte et ai., 1990). An altered light distribution in favour of photosystem I (state 2) at higher temperatures (Weis, 1985; Havaux, 1988) could stimulate ATP production and be functional in adaptation of the thylakoid membrane organisation. The temperature range for optimal photosynthesis decreased about 3 °C when plants were grown at suboptimal conditions. A similar decrease was observed for breakpoint temperatures of F p , indicating that photosynthesis and breakpoint temperature may be affected in the same way by alterations in the thylakoid membrane organisation. In addition, photosynthetic activity of suboptimally grown plants could be decreased by a low temperature induced loss of ribulosebisphosphate carboxylase/oxygenase activity (Sassenrath and Ort, 1990), which is not reflected in the chlorophyll fluorescence parameter Fp. Chlorophyll and carotenoid content
Differences in chlorophyll and carotenoid content could influence chlorophyll fluorescence results. However, breakpoint temperatures of Fp were determined by abrupt changes in the course of Fp with decreasing temperature from single discs. The absolute values of Fp , which were affected by chlorophyll a + b content, had no influence on the determination of breakpoint temperatures. Acknowledgements This investigation was supported by the Committee for Energy Consumption of the Dutch Ministry of Agriculture and Fisheries, and was carried out in cooperation with the Centre for Plant Breeding Research in Wageningen, the Netherlands. Our thanks are due to Prof. Dr. Ir. P. J. C. Kuiper for carefully reading the manuscript.
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