Photosynthesis Research 15:153-162 (1988) © Martinus Nijhoff Publishers, Dordrecht Printed in the Netherlands Regular Paper

Temperature-induced alterations of in vivo chlorophyll a fluorescence induction in cucumber as affected by D C M U L U U K H.J. J A N S S E N & P H I L I P R. V A N H A S S E L T Department of Plant Physiology, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands Received 18 June 1987; accepted in revised form 5 October 1987

Key words: chilling, chlorophyll a fluorescence, CO2 assimilation, Cucumis sativus L., her-

bicide, intact leaves Abstract. Induction of chlorophyll a fluorescence and photosynthesis as affected by tem-

perature were measured in cucumber leaf discs. Abrupt changes of the maximal variable fluorescence, Fv(p), and photosynthesis were observed around 9 ° and 21 °C when the temperature was decreased from 30 ° to 0 °C. The temperature-dependent maximal fluorescence of DCMU-treated leaf discs showed a single change around 21 °C. Temperature-induced chlorophyll a fluorescence alterations are discussed in relation to electron transport activity of the two photosystems and photosynthetic activity of the cucumber leaf discs. Abbreviations: DCMU - - 3-(3,4-dichlorophenyl)-l,l-dimethylurea, Fm - - maximal fluorescence, Fv(p) - - maximal variable fluorescence, qE - - energy-dependent fluorescence quenching, qQ - - Qa-dependent fluorescence quenching

Introduction

U p o n illumination o f d a r k - a d a p t e d leaves the chlorophyll a fluorescence intensity shows characteristic changes, k n o w n as the K a u t s k y effect. The changes during induction can provide i n f o r m a t i o n a b o u t the functioning o f the p h o t o s y n t h e t i c m e m b r a n e , due to the fact that chlorophyll a serves as an intrinsic fluorescent probe ( P a p a g e o r g i o u 1975). The fluorescence yield is influenced in a complex m a n n e r by events that are directly or indirectly related to photosynthesis (Krause a n d Weis 1984). Fluorescence intensity and p h o t o c h e m i s t r y alter in a c o m p l e m e n t a r y way. They are influenced by the oxido-reduction state o f the p r i m a r y electron acceptor o f p h o t o s y s t e m II, Qa (Duysens a n d Sweers 1963) a n d the trans thylakoid p r o t o n gradient. It was earlier observed that the maximal variable fluorescence, Fv(p), o f c u c u m b e r leaf discs, determined at different temperatures, shows changes between 0 ° and 3 0 ° C (van Hasselt, Woltjes and de J o n g 1982). Similar

154 alterations were observed in maize leaves (Havaux and Lannoye 1984). In the blue-green alga, Anacystis nidulans, these changes were attributed to phase transitions of the thylakoid membranes (Murata 1984). In higher plants, bulk phase transitions are unlikely to occur above 0 °C (Murata and Fork 1975). But in the thylakoid membranes of chilling sensitive plants gel phase domains may be formed above 0 °C, as reported for calorimetry, electron spin and fluorescence probe experiments (Raison and Orr 1986). In this study the effect of temperature-dependent chlorophyll fluorescence induction and photosynthesis was investigated in order to obtain insight in the origin of temperature-dependent changes of chlorophyll a fluorescence intensity. D C M U was applied to study temperature-dependent effects on chlorophyll fluorescence in the absence of electron transport.

Material and methods

Plant material Seeds of cucumber (Cucumis sativus L.), cv. groene scherpe, were germinated in the dark on moistened filter paper at 23 °C for 3 days. Seedlings were transplanted on 301. tanks with half strength Hoagland nutrient solution (Smakman and Hofstra 1982) under aeration. Cucumber plants were grown in a Conviron EF7H growth cabinet at 25 °/20 °C day/night temperature and at a photosynthetically active photon flux density of 260/rE • m -2 • s -~ during the 16-h day.

Preparation and handling of leaf discs Leaf discs (diameter 7 mm) were punched from nearly expanded (75% of maximal leaf area) leaves of 3 week old plants. The leaf discs were kept floating on 50 mM phosphate buffer, pH = 6.0, upper side up, during the measurements. Prior to the measurements, the leaf discs were held at room temperature for 30 minutes after punching. Two leaf discs were used for fluorescence measurements, photosynthesis and respiration were measured with 20 leaf discs, taken from the same leaf. Leaf discs were treated with D C M U by addition of 50#M D C M U from a 10mM stock solution in ethanol to the buffer and preincubated for 2h in the light (100 pE • m -2 • s-~). This resulted in a total loss of photosynthetic activity and no quenching of fluorescence occurred after a maximal fluorescence level was reached. The 0.5% ethanol in the buffer showed no effect on the CO2 uptake rate or the fluorescence induction response.

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Measurements of temperature-dependent fluorescence, photosynthesis and respiration Slow fluorescence induction (10 min), photosynthesis and respiration were measured at decreasing temperatures from 30 ° to 0 °C to determine the effect of temperature on cucumber leaf discs. The temperature was decreased by 2 °C, at a rate of 18 °C/h, between 30 ° and 0 °C; it was controlled by computer. After dark adaptation for 20 minutes chlorophyll fluorescence was induced by 10/~E • m -2 • s -1 light (655 ___ 20 nm) from a light emitting diode (Hewlett Packard HCMP0222) and measured with a photodiode/ amplifier combination (EG&G HUV 1100BG) equipped with a filter transmitting wavelengths > 690 nm (Schott RG-9). The slow fluorescence induction kinetics were registrated on a recorder. The first induced maximum, Fv(p), of cucumber leaf discs was taken as a temperature-dependent parameter. The maximal fluorescence after 5 seconds, Fm, was taken as a parameter of DCMU-treated leaf discs. The obtained values were related to the minimal (0%) and the maximal value (100%). Photosynthesis and respiration were measured by determining the CO2 concentration change in a closed system in the light (250 #E • m -2 • s-l), respective darkness for 10 minutes, after aeration in an open system for 20 minutes. An infrared gas analyzer (URAS 2T) was used to monitor the CO2 concentration.

Results

Fluorescence induction of control cucumber leaf discs The maximal variable fluorescence Fv(p) increased when temperature was decreased from 30 ° to around 18 °C. This increase was followed by a slower increase or occasionally a decrease of Fv(p) to around 11 °C. From 11 °C to 0 °C a further increase of the maximal variable fluorescence Fv(p) was evident. Two breakpoints in the temperature-dependent maximal variable fluorescence Fv(p) were present (Fig. 1). The initial fluorescence, Fo, was determined from fast registrations of the induction curve on a digital oscilloscope. Fo linearly increased with decreasing temperature (data not shown). The fluorescence was also measured at intervals of 1 instead of 2 °C to determine the position of the breakpoints more accurately. As a consequence, the duration of the experiment was increased from 10 to 21 h. Under these circumstances the increase in Fv(p) below 9 °C was followed by a decrease (Fig. 1 insert). The rate of decrease depended on duration of

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Fig. 1. Changesin maximalvariable fluorescence(Fv(p)) of cucumber leaf discs at decreasing temperature. Duration of experiment: 9.8 h. (insert: 21 h) Fig. 2. Breakpoints of maximal variable fluorescence(Fv(p) of cucumber leaf discs of 119 experiments. 83 (70%) low temperature breakpoints (B) were found (9.2 _+ 2.2 °C). 70 (59%) high temperature breakpoints (D) were found (20.8 + 4.4 °C).

cooling. Evidently, prolonged exposure at lower temperature caused a decrease of Fv(p). Breakpoint temperatures derived from the curve of the temperaturedependent maximal variable fluorescence Fv(p) of 119 experiments are given in an histogram (Fig. 2). Low temperature breakpoints were present around 9 °C (9.2 °C ___ 2.8 °C) in 70% of the experiments and high temperature breakpoints were present around 21 °C (20.8 °C ___ 4.4 °C) in 59% of the experiments. Occasionally, a breakpoint could not be determined since only insignificant changes in the slope of Fv(p) against temperature were measured. The positions of the two temperature breakpoints, present in a single measurement, were independent of each other. Fluorescence induction of DCMU-treated leaf discs The fluorescence of DCMU-treated cucumber leaf discs increased upon illumination, after dark adaptation, to a maximum, Fm. No fluorescence quenching occurred after the maximal value was reached. Fm increased with

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decreasing temperatures from 30 ° to 0 °C (Fig. 3), but showed only one breakpoint around 18 °C. In contrast to control leaf discs, D C M U treated leaf discs showed no breakpoint around 9 °C. Figure 4 shows the breakpoints present in the temperature-dependent maximal fluorescence Fm of DCMU-treated leaf discs of 62 experiments. High temperature breakpoints were observed around 21 °C (20.6 °C + 2.1 °C) in 69% of the experiments, whereas no lower temperature breakpoints were found. Photosynthesis of leaf discs Net C O 2 uptake rate increased with decreasing temperature from 30 ° to around 22 °C; it remained constant to around 8 °C. The CO2 uptake rate decreased fast with decreasing temperatures below 8 °C. Changes in CO2 uptake followed a smooth curve with temperature, showing inflection points, and not abrupt changes as for Fv(p) and Fm (Fig. 5). Using the net CO2 uptake rate as a parameter, the temperature dependence of net photosynthesis was determined for 48 experiments (Fig. 6). A low temperature inflection point was present around 8 °C (8.2 °C + 2.22 °C) in 94% of the experiments and a high temperature inflection point was present 10C

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Fig. 3. Changesin maximalfluorescenceof DCMU-treated cucumber leaf discs at decreasing temperature. Duration of experiment: 9.0 h. Fig. 4. Breakpoints o[ maximal fluorescenceof DCMU-treated cucumber leaf discs of 62 experiments. 43 (69%) high temperature breakpoints (B) were found (20.6 _+ 2.1 °C).

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Fig. 5a. Net carbondioxide uptake rate o f cucumber lef discs at decreasing temperature and light intensity of250/~E • m -2 • s ~. 5b. Net carbondioxide production rate of cucumber leaf

discs at decreasing temperature in darkness. Duration of experiment; 9.8 h. Fig. 6. Inflection points of net carbondioxide uptake rate of cucumber leaf discs of 48 experiments. 45 (94%) low temperature inflectionpoints (8) were found (8.2 + 2.2°C). 31 (65%) high temperature inflectionpoints (m) were found (21.7 __+4.6 °C). around 22 °C (21.7 °C __+ 4.58 °C) in 65% of the experiments. The positions of both inflection points, present in a single measurement with 20 cucumber leaf discs, were independent of each other.

Respiration of control and D C M U treated leaf discs Respiration activity was measured as CO2 production in darkness for 10 minutes, after measuring the CO2 uptake rate in the light. Temperature decreased from 30 ° to 0 °C in steps of 2 °C. Respiration decreased exponentially with decreasing temperature (Fig. 5). No sudden changes in the temperature-dependent alterations in respiration were observed. D C M U did not alter the respiratory activity.

Discussion Fluorescence induction is influenced by many factors such as the reduction state of Qa (photochemical quenching (qQ) (Duysens and Sweers 1963)), the

159 energy state of the thylakoid membrane (energy-dependent quenching (qE) (Krause, Vernotte and Briantais 1982, Mathis 1986)), the rate of exitation energy transfer from photosystem II to photosystem I (State 1-State 2 transition (Barber 1982, Fork 1986)) and Calvin cyclus activity (Krause and Weis 1984). By excluding some of these factors by addition of DCMU, which inhibits electron transport beyond photosystem II, understanding of the mechanisms underlying the temperature dependent fluorescence alterations may be less tentative. The maximal variable fluorescence, Fv(p), reflects the fluorescence yield when the electron acceptor of photosystem II Qa is highly reduced and no thylakoid proton gradient is yet established (Sivak and Walker 1983). Fv(p) correlates with the capacity of photosystem II to drive electron transport in excess of the capacity of photosystem I. At high light intensity Fv(p) reflects maximal fluorescence caused by a complete reduction of Qa. At low light intensity, as applied in this study, Fv(p) may reflect a transient equilibrium between the electron flow from photosystem II to Qa and that from Qa to photosystem I (Mathis 1986). It cannot be excluded that the position of the breakpoints observed in Fv(p) may be influenced by the exciting light intensity, since at an higher light intensity photosystem II is more activated than photosystem I. This could influence the temperature-dependent redox state of Qa and as a consequence Fv(p). The onset of the Calvin cyclus activity upon illumination coincides with the appearance of the M peak in the fluorescence induction (Sivak and Walker 1983) of dark adapted leafs. Thus, Fv(p) should only be influenced by the balance in electron transport activity of the photosystems, the light energy distribution over the photosystems and not by Calvin cyclus activity. However, temperature-dependent changes in the level of Fv(p) and in Calvin cyclus activity can coincide when they are caused by an alteration in the organisation of membrane lipids. This interpretation is valid when the leaves are fully dark-adapted and photosynthesis is induced by the onset of the exciting light. The temperature-dependent change of Fv(p) was not altered when fluorescence induction was measured during 1 min instead of 10 min (data not shown). This indicates that effects of Calvin cyclus activity on Fv(p), after 20 min dark adaptation and at the low light intensity used in our experiments, are unlikely. The curve of Fv(p) against temperature showed two breakpoints (Figs. 1, 2). The low temperature breakpoint around 9 °C coincides with the temperature where thermophilic plants like cucumber are affected by chilling damage resulting in reduced growth, loss of membrane selective permeability (Harnischfeger and Jarry 1982) and photooxidation of leaf pigments (van Hasselt and Strikwerda 1976). Decreasing the temperature generally de-

160 creases photosynthesis and, as a consequence, N A D P H consumption. The increase of the maximal variable fluorescence Fv(p) can be explained by an increase of the percentage-reduced Qa, due to a decrease of its reoxidation rate. Electron transport from Qa to N A D P ÷ can be inhibited by low temperature-induced formation of gel phase domains in the lipid matrix of the thylakoid membrane (Scoufflaire, Lannoye and Barber 1985) affecting lateral plastoquinone diffusion. Calvin cyclus activity can also be inhibited by a decrease in N A D P H and ATP production. In tomato chloroplasts the chemical reactions involved in carbondioxide fixation were more inhibited by low temperature than the electron transport capacity of the photosystems (Kee, Martin and Oft 1986). In cucumber leaf discs, however, a decrease of the Fv(p) level was observed at prolonged exposure at temperatures below 9°C (Fig. 1 insert) indicating electron transport inhibition. This phenomenon has been used for screening leaves of plants for low temperature and chilling sensitivity and is believed to be derived from inhibition of the water splitting system (Smillie and Nott 1979). Such a decrease in electron donation from the water splitting system to photosystem II reaction centers will result in a decrease of the percentage-reduced Qa and may offer an explanation for the observed decrease of Fv(p). In the prescence of D C M U , Fv(p) still increased with decreasing temperature but a low temperature breakpoint was absent. The increase may be caused by gradual structural effects of decreasing temperature on the thylakoid membranes. As electron transport is completely inhibited by D C M U (Ridley and Horton 1984), it can be concluded that electron transport is a prerequisite for the occurrence of the low temperature breakpoint. The absence of a low temperature breakpoint in 30% of the experiments with control leaf discs may be explained by the complementary effects of low temperature (increase of Fv(p)) and prolonged exposure to this temperature (decrease of Fv(p)) on Fv(p). A high temperature breakpoint was observed around 21 °C in the temperature-dependent course of both Fv(p) and Fm in the absence and in the presence of D C M U . The fact that the high temperature breakpoint remained in the presence of D C M U can be explained as follows. D C M U inhibits electron transport from Qb to plasquinone and results in a maximal reduction of Qa upon illumination (Pallett and Dodge 1980). Electron transport beyond photosystem II is inhibited, and as a result of the lack of electron donation and the loss of electrons to N A D P +, photosystem I becomes oxidized (Ridley and Horton 1984). Thus, linear and cyclic electron transport will be inhibited by D C M U as indicated by the absence of pHinduced fluorescence quenching after reaching a maximal fluorescence level.

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A possible explanation for the observed changes in the maximal fluorescence around 21 °C can be a temperature-dependent change in the structural organization of the light harvesting complexes. It was observed (Weis 1985) that state changes of dark-adapted spinach leaves corresponded mainly with state 1 below 20 °C and state 2 above 20 °C. Similarly, the dark-adaptated state of the cucumber leaf discs may be mainly state 2 at temperatures above the high temperature breakpoint and state 1 below this temperature breakpoint. State changes enable a regulation of linear and cyclic electron transport (Barber 1982). At temperatures above the high temperature breakpoint cyclic electron transport may be stimulated by a state 2 membrane dark adaptation and NADPH production and photosynthesis could be reduced. The absence of a high temperature breakpoint in 40% of the experiments with control leaf discs (31% for DCMU treated) may be caused by an incomplete state 2 membrane dark adaptation. The role of the temperature-dependent state transitions is currently under investigation.

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