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The Effect of Decreasing Temperature up to Chilling Values on the in vivo F685/F735 Chlorophyll Fluorescence Ratio in Phaseolus vulgaris and Pisum sativum: The Role of the Photosystem I Contribution to the 735 nm Fluorescence Band

Author(s): Giovanni Agati, Zoran G. Cerovic, Ismaël Moya Source: Photochemistry and Photobiology, 72(1):75-84. 2000.

Published By: American Society for Photobiology

DOI: 10.1562/0031-8655(2000)072<0075:TEODTU>2.0.CO;2 URL:

http://www.bioone.org/doi/full/10.1562/0031-

8655%282000%29072%3C0075%3ATEODTU%3E2.0.CO%3B2

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Photochemistry and Photobiology, 2000, 72(1): 75–84

The Effect of Decreasing Temperature up to Chilling Values on the

in vivo F685/F735 Chlorophyll Fluorescence Ratio in Phaseolus vulgaris and Pisum sativum: The Role of the Photosystem I Contribution to the 735 nm Fluorescence Band

Giovanni Agati*1, Zoran G. Cerovic2and Ismae¨l Moya2

1Istituto di Elettronica Quantistica—CNR, Sezione INFM di Firenze, Florence, Italy;

2Groupe Photosynthe`se et Te´le´de´te´ction, LURE/CNRS, Centre Universitaire Paris-Sud, Orsay cedex, France Received 1 February 2000; accepted 30 March 2000

ABSTRACT

The effect of leaf temperature (T), between 23 and 48C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 mE m22 s21) and low (30–35 mE m22 s21) light intensities in Phaseolus vulgaris and Pisum sativum. With decreasing temperature, an in- crease in the fluorescence yield at both 685 and 735 nm was observed. A marked change occurred at the longer emission band resulting in a decrease in the Chl fluores- cence ratio, F685/F735, with reducing T. Our fluorescence analysis suggests that this effect is due to a temperature- induced state 1–state 2 transition that decreases and in- creases photosystem II (PSII) and photosystem I (PSI) fluorescence, respectively. Time-resolved fluorescence life- time measurements support this interpretation. At a crit- ical temperature (about 68C) and low light intensity a sud- den decrease in fluorescence intensity was observed, with a larger effect at 685 than at 735 nm. This is probably linked to a modification of the thylakoid membranes, in- duced by chilling temperatures, which can alter the spill- over from PSII to PSI. The contribution of photosystem I to the long-wavelength Chl fluorescence band (735 nm) at room temperature was estimated by both time-resolved fluorescence lifetime and fluorescence yield measurements at 685 and 735 nm. We found that PSI contributes to the 735 nm fluorescence for about 40, 10 and 35% at the min- imal (F0), maximal (Fm) and steady-state (Fs) levels, re- spectively. Therefore, PSI must be taken into account in the analysis of Chl fluorescence parameters that include the 735 nm band and to interpret the changes in the Chl fluorescence ratio that can be induced by different agents.

INTRODUCTION

Analytical methods based on the measurement of chloro- phyll (Chl)† fluorescence are widely used to monitor the

¶Posted on the web on 7 April 2000.

*To whom correspondence should be addressed at: Istituto di Elet- tronica Quantistica—CNR, via Panciatichi 56/30, 50127 Firenze, Italy. Fax: 39-055-414612; e-mail: agati@ieq.fi.cnr.it

†Abbreviations: CFR, corrected fluorescence ratio; Chl, chlorophyll;

functionality of photosynthetic organisms (1). The most pop- ular techniques concern the detection of the fluorescence in- duction kinetics occurring upon exposure of dark-adapted photosynthetic systems to light (2,3). Various parameters of Chl fluorescence variations that are intimately correlated to changes in the activity of photosystem II (PSII), have been indicated as indexes of plant stress states. Useful information on the plant condition can also be obtained by the analysis of the Chl fluorescence spectrum under continuous light (4).

The Chl fluorescence ratio (FR) between the red (at about 685 nm) and the far-red (in the 730–740 nm range) emission bands was suggested as a new tool of plant stress detection (5). This method is particularly appealing since it could be used in remote sensing monitoring of vegetation. Remote detection of Chl fluorescence spectra was shown to be pos- sible by different fluorescence light detection and ranging systems (6,7). However, finding a correlation between changes in remote-sensed spectral fluorescence parameters and a particular plant stress condition is quite difficult. Be- cause of reabsorption at the shorter emission wavelengths, the Chl FR changes markedly with the leaf Chl content be- low about 250 mg m22, while it is quite insensitive to the pigment concentration for values higher than 300 mg m22 (8). A correct use of the Chl FR as plant stress indicator requires the complete knowledge of all factors that affect the Chl fluorescence spectrum. It was shown that environmental factors, such as light intensity and temperature, affect by themselves the Chl FR of healthy plants (9,10). Chl FR was observed to decrease in chilling stressed beans under light and was indicated as a potential index of plant chilling sen-

DAS, decay-associated spectra; F0, minimal fluorescence yield in the dark; Fm, maximal fluorescence yield; Fm9, maximal fluores- cence yield under light; Fs, fluorescence yield at the steady-state;

F685, red chlorophyll fluorescence intensity; F735, far-red chlo- rophyll fluorescence intensity; FR, fluorescence ratio between red and far-red chlorophyll emission; FWHM, full width at half max- imum; LHC, light-harvesting complex; NPQ, nonphotochemical quenching derived by the Stern–Volmer term; PPFD, photosyn- thetic photon flux density; PSI, photosystem I; PSII, photosystem II; qE, high-energy state chlorophyll fluorescence quenching; qI, photoinhibition chlorophyll fluorescence quenching; qN, nonpho- tochemical chlorophyll fluorescence quenching; qP, photochemi- cal chlorophyll fluorescence quenching; qT, state transition chlo- rophyll fluorescence quenching; T, leaf temperature.

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Figure 1. Experimental protocol for simultaneous fluorescence yield measurements at 685 and 735 nm. The arrow indicates the point at which red actinic light (650 nm) was switched on. The lower panel shows the T detected by a thermocouple on the adaxial leaf surface.

Inset shows the two spectral bands of integration of the fluorescence signal.

sitivity (11). The reliability of this method can be proved only after a complete understanding of the origin of the tem- perature-induced FR changes is achieved.

Until recently, room-temperature Chl emission at both 685 and 735 nm, in leaves, was considered coming from PSII with only a minor contribution from photosystem I (PSI) (1).

However, there are now evidences that the contribution of PSI is not negligible (12,13). At wavelengths longer than 700 nm the relative contribution of PSI to minimal fluores- cence yield in the dark (F0) was estimated to be between 30 and 50%, according to the species. Because of this, a change in the Chl FR is expected whenever a variation of PSII fluo- rescence occurs.

The aim of this work is to evaluate better the contribution of PSI to Chl fluorescence at physiological temperatures, and investigate its role during changes in the Chl FR induced by leaf temperature (T) variations and different actinic light lev- els. To do this, we performed both time-resolved and steady- state fluorescence measurements at 685 and 735 nm on bean and pea leaves attached to the plant. Modulated fluorescence signals at both red and far-red bands were continuously de- tected as the T was changed from room values to 48C under weak and moderate light intensities. Time-resolved fluores- cence measurements, acquired at 23 and 58C, permitted to separate the contribution of PSI, with lifetimes of the order of 100 ps or less, from that of PSII with longer lifetimes.

Results of this investigation gives useful information in or- der to explain the temperature effect on the Chl FR changes previously observed (9–11).

MATERIALS AND METHODS

Plant material. Bean (Phaseolus vulgaris L., cv Carioca) and pea (Pisum sativum L., cv Petit provenc¸al) plants were grown for 2 weeks in a growth chamber under a photosynthetic photon flux den- sity (PPFD) of 350mE m22s21with a 16 h photoperiod. The tem- perature was 20 and 168C during the day and night periods, respec- tively (80% relative humidity). Fluorescence measurements were performed on leaves attached to the plant.

Fluorescence yield measurements. Simultaneous measurement of the red and far-red Chl fluorescence from the upper leaf side was performed by the pulsed fluorometer, described in detail by Cerovic et al. (14), with the following modifications. The excitation beam was provided by a pulsed xenon lamp (1ms duration, repetition rate 53 Hz) filtered through a 340 nm interference filter (full width at half maximum [FWHM]510 nm, 03FIU008, Melles Griot, Cachan, France) and reflected to the leaf target by a UV-reflecting dichroic mirror (420 DCLP, Omega, Brattleboro, VT). The excitation lamp was attenuated by neutral density filters in order to avoid any actinic effect of the probe beam and still have a good fluorescence signal/

noise ratio. The pulse energy was measured to be 3 mJ m22. Part of the excitation beam was continuously monitored by a photodiode in order to correct the fluorescence signal for fluctuations in the exci- tation source. The collected fluorescence light was separated into the red and far-red components using a second dichroic mirror (DTGreen, Balzers, Zaventem, Belgium). Detection was performed by two identical photodiodes (S3590-01, Hamamatsu) through a red interference filter (FWHM522 nm, 682DF22, Omega) and a long- pass filter (RG9, Schott, Clichy, France) for the red and far-red com- ponent, respectively. Both photodiodes were protected by an UV- blocking filter (KV408, Schott). The spectral bands of integration of the two fluorescence signals are showed in the inset of Fig. 1. Con- sequently, the red band of Chl fluorescence was measured at 686 nm over a 15 nm bandwidth, while the far-red band was integrated over a 40 nm bandwidth at around 750 nm. For convenience and standardization to nomenclature in the literature, the two red and far-red measured fluorescence signals are indicated by F685 and F735, respectively.

Red light (peaked at 650 nm) from a 12 light emitting diode (HLMP-8150, Hewlett Packard, Les Ulis, France) array was used as actinic light with a PPFD ranging from 0 to 700mE m22s21. Satu- rating white light, 1 s pulses (3000mE m22s21), were provided by a fiber optic illuminator (KL1500 electronic, Schott).

The target leaf attached to the plant was kept in contact with a copper plate whose temperature was electronically controlled by a thermoelectric Peltier module. The T was monitored by a thermo- couple positioned on the adaxial leaf surface.

The experimental protocol for simultaneous fluorescence yield measurements at 685 and 735 nm is reported in Fig. 1. The leaf under investigation was dark adapted for at least 20 min, the F0and maximal fluorescence yield (Fm) levels were measured, and then the actinic light was switched on. At a steady-state fluorescence yield (Fs) the leaf was exposed to a saturating pulse in order to measure the maximum fluorescence yield under light (Fm9). After that, the T was changed from about 20 to 48C by steps of 4–58. Leaves were kept for a few minutes at the set temperature allowing for thermal equilibration before switching the Peltier system to a different tem- perature. The process was inverted with the same procedure to warm up the leaf to the initial temperature. The temperature change was then induced continuously from the higher to the lower value over a 4 min time range and back to the initial point. At each set tem- perature, Fm9 values were measured. The photochemical (qP) and nonphotochemical (qN) quenching parameters were calculated by qP 5(Fm9 2F)/(Fm9 2F0) and qN5(Fm2Fm9)/(Fm2F0) according to Schreiber et al. (15).

Lifetime fluorescence measurements. Time-resolved measure- ments of the Chl fluorescence decay on intact leaves at F0 were performed by the single photon counting system previously de- scribed (16). Excitation was provided by a pulsed diode laser at 635 nm (pulse duration,70 ps, repetition rate of 5 MHz). Detection was set alternatively on the short- or the long-wavelength fluorescence band by using a red (682 nm, FWHM530 nm) or a far-red (725 nm, FWHM535 nm) interference filter (Omega). Lifetime fluores- cence measurements at the steady-state level, Fs, were obtained by

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Photochemistry and Photobiology, 2000, 71(2) 77

Figure 2. DAS of four lifetime components derived by global de- convolution of Chl fluorescence decay. Open circles represent the total emission spectrum. Closed circles represent the mean Chl fluo- rescence lifetime spectrum.

Table 1. Component lifetime (ps) and fractional fluorescence intensity, Ri*, (%) for bean and pea leaves at 23 and 58C in steady-state conditions, Fs, measured at 685 and 735 nm. The uncertainties in the lifetimes and Riare610 and615%, respectively

Species T (8C) Band (nm)

Phaseolus vulgaris 23

685 735

5

685 735

Pisum sativum 23

685 735

5

685 735

tm 391 290 462 311 366 303 509 348

t1† t2

t3

t4

21 103 396 882

30 125 402 950

17 85 367 670

17 100 406 1130 R1

R2

R3 R4

5 13 71 11

6 36 52 6

6 9 66 19

7 38 47 8

2 14 68 16

,1 39 49 12

1 14 64 21

4 38 47 11

*The fractional fluorescence intensity Ri5Aiti/SiAitiis defined as the normalized integral of the exponential function Aiexp(2t/ti) of the ith lifetime component.

†Uncertainty ont1is estimated to be 10 ps according to the instrument sensitivity limit.

focussing the excitation beam to have a PPFD of about 100mE m22 s21 and after a preillumination period of about 1 h. Resolution of four different components, with lifetimes ranging between 20 ps and 1.1 ns, was obtained through deconvolution of the fluorescence de- cay curve by the Fluomarqt II software described in Camenen et al.

(17). Decay-associated spectra (DAS) were derived by the same pho- ton counting system using a monochromator (Jobin & Yvon HR 320, bandwidth 4 nm) in front of the photomultiplier to select the emission wavelength in the 670–760 nm range by 5 nm steps. A red longpass filter (RG665, 3 mm, Schott) was inserted in front of the monochromator to remove any diffused excitation light.

Individual analysis was performed for each decay, and then a global fit was applied to all the decays in the spectrum simulta- neously. The steady-state fluorescence spectrum was measured in the same spectral range by scanning the monochromator and inte- grating, over 1 s, the fluorescence emission at each wavelength by 1 nm steps. The spectra were repeated one time toward increasing wavelengths and one time toward decreasing wavelengths and summed. By this way fluorescence drifts during the time of mea- surement were minimized.

The T was controlled by keeping the lower side of the leaf in contact with a brass plate coupled to a Peltier system. For both bean and pea plants, measurements were recorded at the temperatures of 23 and 58C.

RESULTS AND DISCUSSION

PSI contribution to the far-red Chl fluorescence band:

time-resolved measurements

Time-resolved emission spectral analysis permits to separate various fluorescence components coming from the different photosynthetic compartments. It is therefore possible to es- timate the relative contribution of PSI to the two Chl fluo- rescence bands. Figure 2 shows the DAS of four lifetime components obtained by global deconvolution of the fluo- rescence decay curve for the bean plant, at 238C, in steady- state conditions. The DAS was quite similar to those previ- ously obtained on spinach leaves (18). The best fit for the fluorescence decay function was obtained with four com- ponents. The relative contribution to the fluorescence decay from each component is indicated by the fractional fluores- cence intensity Ri5Ai it/SiAI it defined as the normalized integral of the exponential function Aiexp(2t/ti) of the ith lifetime component. The spectrum of each component was derived by multiplying the Rispectrum by the total emission spectrum. As previously shown the components with life- time of the order of 100 ps or less, which peaked in the far- red, belong to PSI, while the ‘‘slow’’ components (0.4–1 ns) that peaked at 685 nm are attributed to PSII (18–24). The total emission spectrum and the spectrum of the mean Chl fluorescence lifetime are also reported in Fig. 2. Similar spectral curves were obtained for the pea plant. It is worth noting that the mean fluorescence lifetime at 735 nm is sig- nificantly lower than that at 685 nm (Table 1). This result, independent of the number of decay components assumed, agrees with that obtained from deconvolution showing a large contribution at longer wavelengths of the PSI fast com- ponents. The best fit parameters of Chl fluorescence decay at 685 and 735 nm for the bean and pea plants in steady- state conditions are given in Table 1.

The PSI contribution at 735 nm fluorescence at Fs and room temperature (238C) is calculated by (R11R )/2 SiRi and results to be 416 7% and 3966% for bean and pea, respectively.

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Table 2. Component lifetime (ps) and fractional fluorescence in- tensity, Ri*, (%) for bean and pea leaves at 238C and F0. The un- certainties in the lifetimes and Riare610 and615%, respectively Species

Band (nm)

Phaseolus vulgaris

682 725

Pisum sativum

682 725

tm 304 267 246 204

t1

t2

t3

t4

79 237 424 2306

65 175 381 3394 R1

R2 R3

R4

17 49 32 2

36 34 28 2

15 58 26 1

39 38 22 1

*The fractional fluorescence intensity Ri5Aiti/SiAitiis defined as the normalized integral of the exponential function Aiexp(2t/ti)

The best fit parameters of Chl fluorescence decay at F0 for the bean and pea plants are given in Table 2. In this case, a four-component deconvolution in the presence of a minor (1–2%) very long lifetime component cannot resolve the two PSI short components. Hence, only thet1decay component was used to calculate the PSI contribution to the long-wave- length fluorescence byR /1SiR .i It results to be 3665% and 3965% for bean and pea, respectively.

The results of the time-resolved measurements show that at the longer wavelength band of Chl fluorescence the con- tribution of PSI is in accordance with data reported previ- ously on various C3(including pea) and C4species by com- pletely different experimental approaches (12,13).

PSI contribution to the far-red Chl fluorescence band:

fluorescence yield measurements

It is widely accepted that the response of the two photosys- tems to the dark–light transition, that is the transition from open to closed reaction centers, is different. The fluorescence of PSII increases markedly while PSI fluorescence seems to have no variable parts (13,25–28). Assuming Butler and Ki- tajima’s model (27) to be valid also at room temperature, we can consider that the fluorescence at 685 nm is due to the PSII only, while fluorescence at 735 nm comes from both photosystems, that is:

F685[FPSII(685) F735[FPSII(735)1FPSI, where FPSI5FPSI(a)1FPSI(bwIII) and then PSI fluorescence is composed by an intrinsic part,a, due to direct absorption or energy transfer from the light-harvesting complex (LHC) and a fraction,bwIII, due to energy transfer from PSII. Con- sequently, making the hypothesis that PSI will remain almost constant during the rising transient of the fluorescence in- duction kinetics, we can derive the PSI contribution by plot- ting F735 vs F685 during the dark–light transition. The x–y plot results in a straight line that can be extrapolated back to the ordinate axis to give FPSI(a) (29). From this analysis, it results that PSI contributes to F735 for 436 6% and 40 64% at F0and 3664% and 3063% at Fs, for bean and

pea, respectively. At Fm the PSI contribution is reduced to 8–9%.

This second method used to estimate PSI relative fluores- cence at room temperatures could be questioned for the fol- lowing reasons: (1) changes in the PSI fluorescence during the fluorescence induction rise were postulated by Schreiber and Vidaver (30) as result of a rapid change in energy dis- tribution from PSII to PSI; (2) Malkin et al. (31) showed that possible differences in the kinetics at the two emission bands may be due to the fluorescence reabsorption effect (32). Because of attenuation of the excitation light, deeper leaf layers are expected to show slower fluorescence induc- tion kinetics than superficial layers. Furthermore, fluores- cence emitted inside the leaf will be reabsorbed at 685 nm by Chl itself. Thus, deeper layers with slower dark–light response will contribute more to F735 than to F685; (3) mea- surements on PSI–LHCI isolated complexes suggest a small contribution (10%) of PSI at 685 nm fluorescence (33); how- ever, in the whole leaf it will be markedly reduced by re- absorption; and (4) recently, Trissl (34) found a 13% in- crease in the fluorescence of PSI isolated systems from F0 to Fm, although direct extrapolation of this result to leaves is questionable.

The agreement obtained between the above PSI relative fluorescence values and those from the time-resolved fluo- rescence measurements indicates, however, that the method used, even if approximate, is sufficiently valid. Further evi- dence can be derived by the behavior of the ratio between F685 and F735 compensated for the PSI contribution, named the corrected fluorescence ratio, CFR 5 F685/(F735 2 FPSI[a]), during the Kautsky kinetics. If PSI does not partic- ipate in the variable fluorescence, the CFR is expected to remain constant. In Fig. 3 the kinetics of CFR is reported along with that of the direct fluorescence ratio FR for bean and pea plants under different light intensity. It can be seen that correction of F735 for the constant fraction of PSI re- moves almost completely the variations observed in F685/

F735. Only small deviations (610%) from the expected con- stant CFR value are present in the initial transient.

Because of the significant contribution of PSI fluorescence to the far-red emission band at room temperature, a correct use of Chl fluorescence-based methods requires a precise definition of the spectral window of detection. Quantitative studies of variable Chl fluorescence, mainly due to PSII, can be largely affected when measured signals include the far- red emission if the PSI contribution is not taken into account.

Furthermore, interpretation of variation in the Chl FR must consider the relative contribution of both PSII and PSI.

The importance of the constant fraction of PSI fluores- cence atl .715 nm on the Chl FR was already addressed by Schreiber and Vidaver (30) estimating the effect on the fluorescence rise phase of different FPSI(a) correction values.

The analysis of the Chl FR was used to study the fluores- cence induction kinetics in leaves without, however, taking into account the effect of the PSI contribution (35–37).

Temperature-induced fluorescence changes:

fluorescence yield measurements

The temperature effect on the two Chl fluorescence bands was investigated under different actinic light intensities for

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Photochemistry and Photobiology, 2000, 71(2) 79

Figure 3. The Chl fluorescence ratio F685/F735, FR, (dashed lines) during the fluorescence induction kinetics. Arrows indicate the point at which actinic light was switched on. Solid lines show the Chl fluorescence ratio corrected for the PSI contribution at the F735 band, CFR5F685/(F7352FPSI[a]) (see text for definition). (a) Pea leaf under 200mE m22s21red actinic light. (b) Bean leaf under 35

mE m22s21red actinic light. Figure 4. Changes in fluorescence intensity and fluorescence ratio induced by variation of T for a bean leaf under 200mE m22s21. (a) T, (b) F685/F735 Chl fluorescence ratio (open triangles) and CFR (open squares) (see text for definition) and (c) fluorescence intensity at 685 and 735 nm. Fluorescence and fluorescence ratios are nor- malized to the initial value.

the bean and pea plants. The relative variation of the FR and fluorescence intensity corresponding to a change in the T at 200 mE m22 s21is reported in Figs. 4 and 5 for bean and pea, respectively. The CFR compensated for FPSI(a) is also reported. Fluorescence and fluorescence ratios were normal- ized to values at the initial temperature. The dynamic effect on fluorescence induced by temperature is different for the two species. In bean (Fig. 4), a monotonous increase of both 685 and 735 nm bands from 20 to 48C is observed, with a more pronounced response at F735 that makes the FR to decrease with decreasing T. This result was qualitatively in accordance with previous measurements using 635 nm laser excitation (10). The effect is reversible going back from 4 to 208C and similar for both the continuous and the step T changes. The spikes in the graphs are due to the saturating pulses for determination of quenching parameters. The var- iation with T of the CFR matches that of the FR but with a larger extent (Fig. 4b). Within the T range investigated changes in CFR and FR were 30 and 10%, respectively.

A more peculiar representation of T-induced fluorescence kinetics is observed in the pea plant (Fig. 5). Below about 158C, at each T reduction, a Kautsky-like fluorescence ki- netics is observed with a rapid increase in both F685 and F735 followed by a slower decline. The continuous reduc- tion of T from 20 to 48C causes a two-fold increase in the fluorescence intensity. It is worth noting that warming the leaf back to room temperature the fluorescence kinetics ex- hibits an opposite trend with a decrease to a minimum at first followed by a more gradual rising. The FR curve is parallel to that of the fluorescence intensity, as result of larg-

er changes in F685 with respect to F735. On the other hand, the CFR is completely correlated to the T changes (Fig. 5b).

Correction for the constant fluorescence contribution of PSI at 735 nm removes the initial T-induced rise in the FR and, again, amplifies the relative variation from about 10% for FR to 25% for CFR.

Under lower actinic light intensities (30–35mE m22s21), the T-induced fluorescence kinetics observed for both pea and bean were similar to that of pea at 200mE m22s21but with smaller changes in the fluorescence amplitude (Fig. 6).

The increase in fluorescence is delayed until T drops below 148C.

The effect of T on the CFR for bean and pea under dif- ferent light intensities is represented better in Fig. 7. Linear relationships for the CFR vs T curves are observed with slopes of the order of 0.018C21 and 0.0178C21for pea and bean, respectively. Under low light intensities curves are bi- phasic, changing the slopes at about 68C to 0.078C21 and 0.108C21for pea and bean, respectively (Fig. 7b).

The temperature dependence of the photochemical, qP, and nonphotochemical, qN, Chl fluorescence quenching, cal- culated at 685 nm, is reported in Fig. 8. In both bean and pea, at 200mE m22s21, decreasing T induces a reduction of qP to about 30% of the initial value. On the contrary, qN remains constant and high (about 0.9) in bean and increases at 48C in pea. At lower light intensity, a two-fold increase

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Figure 5. Changes in fluorescence intensity and fluorescence ratio induced by variation of T for a pea leaf under 200mE m22s21(a) T, (b) F685/F735 Chl fluorescence ratio (open triangles) and the CFR (open squares) (see text for definition) and (c) fluorescence intensity at 685 and 735 nm. Fluorescence and fluorescence ratios are normalized to the initial value.

Figure 7. Temperature profile of the Chl fluorescence ratio cor- rected for the PSI contribution at the F735 band, CFR5F685/(F735 2 FPSI[a]) (see text for definition). (a) Bean (diamonds) and pea (squares) at 200mE m22s21; data are fitted by a single regression line (dotted lines). (b) Bean at 35mE m22s21(triangles) and pea at 30 mE m22 s21 (circles); data are fitted by two regression curves above (dotted lines) and below (solid lines) 68C.

Figure 8. Photochemical, qP (circles), and nonphotochemical, qN (squares), quenching parameters as function of T for (a) bean at 200 mE m22s21, (b) bean at 35mE m22s21, (c) pea at 200mE m22s21 and (d) pea at 30mE m22s21red actinic light.

Figure 6. Temperature profile of the F685 Chl fluorescence band for pea at 200mE m22s21(squares), pea at 30mE m22s21(circles) and bean at 35mE m22s21(triangles).

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Photochemistry and Photobiology, 2000, 71(2) 81

in qNat 48C and constant values in qPare observed for both species.

From the measurements of the fluorescence quenching pa- rameters, it is evident that the increase in fluorescence with decreasing T is due to a larger reduction of the primary quinone electron acceptor QA. This effect was attributed to a decrease of the thylakoid membrane fluidity induced by low temperatures, which inhibits the reoxidation of plasto- quinones (38). According to Huner et al. (39), the rising in the T-induced fluorescence kinetics derives from an increase in the PSII excitation pressure as a result of an imbalance between the energy supplied to the thylakoids and the energy used in carbon metabolism. This explains better the delay in fluorescence increase at lower light intensity when T is re- duced.

The temperature profile of qPand qNfor the pea plant at 200mE m22s21are quite similar to those reported by Bru¨g- gemann (40) at 370 mE m22 s21. On the contrary, Havaux (41) observed only a 5% reduction in the photochemical quenching of pea leaves at 48C and 120 mE m22 s21. This discrepancy is probably due to the different PPFD used since the low-temperature–induced reduction of QA is markedly enhanced by increasing the actinic light intensity. We con- firmed such an effect in bean between 15 and 200 mE m22 s21(data not shown).

The dependence of qN on T reported in the literature is quite variegated, changing with species and environmental conditions. The nonphotochemical quenching was seen to increase with decreasing T in both barley and maize leaves at high light intensities (42) or in pea at moderate light levels (40). But, below 8–108C and at 40mE m22s21, the nonpho- tochemical quenching decreased in spinach (43), tobacco (44) and tomato (45). We did not observe any change in qN with T when it was high at room temperature, as in bean at 200 mE m22 s21. A moderate increase in qNat low T was found for pea at 200mE m22s21, starting from a room tem- perature value of about 0.6. Large increases in qN at 48C were obtained when the initial qNwas quite low, in both pea and bean at low actinic light (Fig. 8b,d). The marked in- crease in qNexplains the sudden decrease in fluorescent in- tensity that occurs at a critical temperature of about 68C (Fig.

6).

A complete explanation of the qNvariation with T is pos- sible only by resolving the three main quenching compo- nents, namely the high-energy state fluorescence quenching, qE; the state transition quenching, qTand the photoinhibition quenching, qI. Bru¨ggemann and coworkers followed this ap- proach by time-resolution of qN dark relaxation (46,47).

However, it is not always possible to separate all the com- ponents (40), and the assumption about a negligible contri- bution of qTto qN(44) is questionable since at light inten- sities below 100 mE m22 s21 all the three qN components seem to be low and comparable (47,48). It was suggested that sudden changes in nonphotochemical components at a critical temperature between 5 and 108C are due to a de- crease in qE in correlation with a reduced zeaxanthin for- mation at low temperatures (43). This interpretation, how- ever, does not take into account the fact that the origin of the qEquenching relies on at least two different mechanisms:

one zeaxanthin-dependent and the other zeaxanthin-indepen- dent (48).

Our experimental data confirm previous evidences that the T effect on qNis more evident at low light intensities when the qE component is reduced and not saturated by a large transthylakoid pH gradient induced by a sufficiently high PPFD. There is, however, a contradiction between the in- crease in qNat 48C, in both pea and bean, at 30–35mE m22 s21and the decrease in qNbelow 88C observed by the Bru¨g- gemann’s group in various species at 40 mE m22 s21 (43–

45). A possible explanation for this can be found considering the different experimental protocols used. Here, the T change was operated after at least 15 min of actinic light irradiation at room temperature. During this period a signif- icant amount of zeaxanthin is probably produced (49) and a relative amount of qE is developed (50). Consequently, in our study the composition of qN before inducing the T de- crease was likely different from that in Bru¨ggemann’s ex- periments where the room temperature preillumination was not performed.

Useful information about the qNdependence on T can be derived by the analysis of the T-induced variation of the Chl fluorescence ratio. Because of the significant PSI contribu- tion to the F735 fluorescence band, any change in PSII fluo- rescence can produce a variation of FR. In fact, according to the model depicted above, for a change in F685, the pure PSII Chl fluorescence, there will be a corresponding pro- portional change in the PSII component of F735. This adds to the constant intrinsic PSI fluorescence, FPSI(a), leading to a variation in F685/F735. Such an effect could be respon- sible for the parallel increase in F685 and FR with T de- crease, observed in pea at 200 mE m22 s21 (Fig. 5), but it does not explain the antiparallel behavior of F685 and FR with T decrease in bean at the same actinic light intensity (Fig. 4). On the other hand, if T affected only PSII fluores- cence the CFR, where FPSI(a) is removed, would be expected to remain constant with temperature. Yet, relative reductions of CFR between about 20 and 45%, from 20 to 48C, has been found (Fig. 7). Therefore, the observed variation of the CFR with T indicates that the temperature effect on the Chl fluorescence concerns both photosystems and not only PSII.

The state transition quenching, qT(and spillover), among the various Chl fluorescence relaxation mechanisms, in- volves both photosystems by determining a redistribution of excitation energy in favor of PSI with respect to PSII. The main target of the other quenching processes is PSII only.

Therefore, we can suggest that, under our experimental con- ditions, state transitions contribute to the T-induced change in FR, and also CFR, by increasing F735 and decreasing F685. Because of the fast reversibility of the effect upon leaf warming to room temperature, photoinhibitory mechanisms can be ruled out.

Interestingly, for both species the rate of the T-induced change in CFR between 20 and 68C is the same under mod- erate and low PPFD (see slopes in Fig. 7). Consequently, within this temperature range the qEcomponent of qNseems not to be involved in the T effect on Chl fluorescence. Below 68C at low light intensity the marked increase in the CFR vs T slope (Fig. 7b) indicates that other relaxation mechanisms are now concerned.

The sudden drops in Chl fluorescence below 68C in bean at low PPFD and in pea occur at both emission bands but to a lower extent for F735. Chl fluorescence decreases were

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recently observed when the T was suddenly reduced by a 3 s contact with water at 58C at low light intensity (25mE m22 s21) (51). The effect of chilling on Chl fluorescence starting from the Fslevel is analogous to that observed during the continuous T change after the Peltier system was set at 48C, since an instantaneous fluorescence increase was followed by a slower decline. Destacking of the thylakoid membranes was observed to occur within a very short time after chilling of a chilling-sensitive plant (51). When the thylakoids are unstacked the protein complexes are homogeneously distrib- uted within the membrane and the excitation spillover from PSII to PSI is facilitated (26,52). This was found by Samson and Bruce (53) in spinach thylakoids destacked by Mg21 depletion, where an increase in the PSI absorption cross- section occurred along with a decrease in the PSII absorption cross-section. It is also known that membrane lipids undergo phase transition from fluid to gel state at low temperatures (54,55). This compacting of membrane lipids can make the Chl–protein complexes more close to each other and facili- tate spillover. According to such a mechanism induced by chilling T in a leaf, an increased contribution in PSI fluo- rescence and a corresponding decrease in PSII fluorescence is expected at the lower T. The above process can explain the increase in the CFR vs T slope below 68C (Fig. 7b) and contribute to the increase in qNmeasured at 48C (Fig. 8b,d) observed in pea and bean at low PPFD.

Temperature-induced fluorescence changes: time- resolved fluorescence analysis

The results of time-resolved measurements at 23 and 58C under 100 mE m22s21, reported in Table 1, confirm that a reduction of T induces a redistribution of excitation energy in favor of PSI. It can be seen that from room- to low tem- peratures there is no significant change in the mean fluores- cence lifetime, tm, at 735 nm and in the PSI related short lifetime components. On the other hand, the relative fluo- rescence yield of PSI (R11 R2) shows a small increase at 58C. This is characteristic of static changes involving energy redistribution by a state 1 to state 2 transition (and/or spill- over), which augments the PSI antenna size without affect- ing the mean lifetime (56). At 685 nm,tmincreases from 23 to 58C mainly because of an increase in the lifetime of the slowest component, which is known to be a PSII component.

The increase in both R4 and t4 with lowering T indicates more reduced QA, in accordance with the lower qP values measured at low temperatures (Fig. 8a,c). The larger change int4with T decrease, found in pea with respect to bean, is correlated to a larger change in reduction state of QAoccur- ring in the former (compare qPvs T slopes in Fig. 8a,c). The presence of spillover from PSII to PSI is difficult to be as- sessed by the lifetime measurements. Spillover would result in a quenching of PSII fluorescence and a decrease in the PSII lifetimes, but this decrease is largely compensated by an increase in lifetime (and yield) due to larger reduction of QA.

Effect of detection wavelength on Chl fluorescence parameters

As noted previously (13), when fluorescence is detected above 700 nm, the presence of a non-negligible contribution

of PSI to Chl fluorescence can affect the calculation of pa- rameters in which fluorescence values enter as absolute quantities. Accordingly, it is expected that at 735 nm the variable fluorescence relative to the maximum fluorescence, Fv/Fm 5(Fm 2F0)/Fm, and the quantum yield of PSII non- cyclic electron transport given by (Fm 2Fs)/Fm (57) be un- derestimated with respect to 685 nm. Indeed, we found that Fv/Fm at 685 nm (0.869 6 0.010 as average and standard deviation on eight determinations) was 10% larger than at 735 nm (0.79260.012) in agreement with Genty et al. (12).

The same relative difference was observed in (Fm 2 Fs)/Fm (0.7846 0.048 and 0.7126 0.037 at 685 and 735 nm, re- spectively). Particular care must be taken when the nonpho- tochemical quenching is derived by the Stern–Volmer term, NPQ 5 Fm/Fm9 2 1. For example, in bean and pea under 200 mE m22 s21, with high nonphotochemical quenching, that is relatively low values of Fm9, we noticed that NPQ calculated at 685 nm was 65% higher than the value calcu- lated at 735 nm. On the other hand, photochemical and non- photochemical quenching calculated according to Schreiber et al. (15), that is as ratio of differential fluorescence signals, show the same values at both Chl fluorescence bands be- cause a cancellation of the constant PSI fluorescence at 735 nm occurs.

It is interesting to investigate whether the changes in qP and qNinduced by a reduction of T depend on the fluores- cence detection wavelength. By comparing the quenching parameters determined at 685 and 735 nm at room- and chilling temperatures, it is seen that qPvariations are similar at the two fluorescence bands. For qN, on the contrary, the increase observed at low T in pea and bean, at low PPFD, is affected by the detection wavelength since it is more marked, even by 35%, at 685 nm than at 735 nm. This result provides further evidence that lowering T to chilling values changes the fluorescence of both PSII and PSI by a different extent.

CONCLUSIONS

We have confirmed, by different experimental approaches, that the contribution of PSI to Chl fluorescence in the far- red band at physiological temperatures is not negligible with respect to the PSII contribution.

Because of the presence of a PSI contribution to the lon- ger wavelength Chl emission, attention must be paid in the calculation of some fluorescence parameters, such as NPQ and Fv/Fm, when detection over 700 nm is performed. We also showed that the relative changes in the nonphotochem- ical quenching induced by particular environmental factors can be markedly different if detected on the 685 or the 735 nm fluorescence band.

The effect of a reduction of T on the steady-state Chl fluorescence under moderate light intensity, is the same as adding more light to the photosynthetic apparatus, i.e. a larg- er reduction of QA and a relative increase in fluorescence yield. Even for small changes from room- to chilling tem- peratures a decrease in the F685/F735 fluorescence ratio is induced. Our fluorescence analysis suggests that this effect is due to a temperature-induced state 1–state 2 transition that decreases and increases PSII and PSI fluorescence, respec-

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Photochemistry and Photobiology, 2000, 71(2) 83

tively. Time-resolved fluorescence lifetime measurements support this interpretation.

At a critical temperature and low light intensity a sudden decrease in fluorescence yield was observed with a larger effect at 685 nm than at 735 nm. This is probably linked to a modification of the thylakoid membranes, induced by chill- ing temperatures that can alter the spillover from PSII to PSI.

Our study shows that, under appropriate experimental conditions, detection of the Chl fluorescence spectral shape can provide information on the photosynthetic activity of both PSII and PSI photosystems. The Chl FR can, therefore, potentially detect any plant stress that modifies the deacti- vation mechanisms of PSII, as observed under photoinhibi- tion and chilling injury (10,11). However, the use of the Chl FR as screening parameter of plant chilling tolerance pre- viously suggested (11) must be reconsidered. The present study shows that short-term changes in the Chl fluorescence spectrum induced by low T are not directly correlated to the chilling-sensitivity of the species. The effect of T on the Chl fluorescence yield and ratio is due to mechanisms of energy dissipation in the photosynthetic apparatus that depends crit- ically on the experimental conditions—such as light inten- sity, preillumination period and rate of T change—at which the investigation is performed. Accordingly, the differences observed formerly in the FR temperature profile between pea and bean (11) were probably due to the diverse environment with respect to this experiment. More marked differences in the Chl FR between chilling-sensitive and chilling-resistant species could likely be found under long-term chilling con- ditions.

Acknowledgements The present work was supported by an EC TMR grant for the access to LURE (Laboratoire pour l’Utilisation du Rayonnement E´ lectromagne´tique)/CNRS, ORSAY, France, pro- jects BF 032-96, BF 023-97 and BF 022-98. We thank Dr. Claudia Biagi, Dr. David Gaveau and Dr. Gwendal Latouche for help with fluorescence measurements. Z.G.C. and I.M. would like to acknowl- edge the support of the CNRS through the GDR 1536 ‘‘FLUO- VEG’’.

REFERENCES

1. Krause, G. H. and E. Weis (1991) Chlorophyll fluorescence and photosynthesis: the basics. Annu. Rev. Plant Physiol. Plant Mol.

Biol. 42, 313–349.

2. Govindjee (1995) Sixty-three years since Kautsky: chlorophyll a fluorescence. Aust. J. Plant Physiol. 22, 131–160.

3. Renger, G. and U. Schreiber (1986) Practical applications of fluorometric methods to algae and higher plant research. In Light Emission by Plants and Bacteria (Edited by Govindjee, J.

Amesz and D. C. Fork), pp. 587–619. Academic Press, Orlando.

4. Rinderle, U. and H. K. Lichtenthaler (1988) The chlorophyll fluorescence ratio F690–F735 as a possible stress indicator. In Applications of Chlorophyll Fluorescence (Edited by H. K.

Lichtenthaler), pp. 189–196. Kluwer Academic, Dordrecht.

5. Lichtenthaler, H. K. and U. Rinderle (1988) The role of chlo- rophyll fluorescence in the detection of stress conditions in plants. CRC Crit. Rev. Anal. Chem. 19, 29–85.

6. Cerovic, Z. G., G. Samson, F. Morales, N. Tremblay and I.

Moya (1999) Ultraviolet-induced fluorescence for plant moni- toring: present state and prospects. Agronomie 19, 543–578.

7. Lu¨ deker, W., K. P. Gu¨nther and H.-G. Dahn (1995) Comparison of different detection set-ups for laser-induced fluorescence monitoring of vegetation. EARSeL Adv. Remote Sens. 3, 32–41.

8. Gitelson, A. A., C. Buschmann and H. K. Lichtenthaler (1998) Leaf chlorophyll fluorescence corrected for re-absorption by

means of absorption and reflectance measurements. J. Plant Physiol. 152, 283–296.

9. Agati, G., P. Mazzinghi, F. Fusi and I. Ambrosini (1995) The F685/730 chlorophyll fluorescence ratio as a tool in plant phys- iology: response to physiological and environmental factors. J.

Plant Physiol. 145, 228–238.

10. Agati, G. (1998) Response of the in vivo chlorophyll fluores- cence spectrum to environmental factors and laser excitation wavelength. Pure Appl. Opt. 7, 797–807.

11. Agati, G., P. Mazzinghi, M. Lipucci di Paola, F. Fusi and G.

Cecchi (1996) The F685/F730 chlorophyll fluorescence ratio as indicator of chilling stress in plants. J. Plant Physiol. 148, 384–

390.

12. Genty, B., J. Wonders and N. R. Baker (1990) Non-photochem- ical quenching of Fo in leaves is emission wavelength depen- dent: consequences for quenching analysis and its interpretation.

Photosynth. Res. 26, 133–139.

13. Pfu¨ndel, E. (1998) Estimating the contribution of photosystem I to total leaf chlorophyll fluorescence. Photosynth. Res. 56, 185–195.

14. Cerovic, Z. G., M. Bergher, Y. Goulas, S. Tosti and I. Moya (1993) Simultaneous measurement of changes in red and blue fluorescence in illuminated isolated chloroplasts and leaf pieces:

the contribution of NADPH to the blue fluorescence signal. Pho- tosynth. Res. 36, 193–204.

15. Schreiber, U., U. Schliwa and W. Bilger (1986) Continuous re- cording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorom- eter. Photosynth. Res. 10, 51–62.

16. Briantais, J.-M., J. Dacosta, Y. Goulas, J.-M. Ducruet and I.

Moya (1996) Heat-stress induces in leaves an increase of the minimum level of chlorophyll fluorescence, Fo: a time resolved analysis. Photosynth. Res. 48, 189–196.

17. Camenen, L., Y. Goulas, G. Guyot, Z. G. Cerovic, G. Schmuck and I. Moya (1996) Estimation of the chlorophyll fluorescence lifetime of plant canopies: validation of a deconvolution method based on the use of a 3-D canopy mockup. Remote Sens. En- viron. 58, 157–168.

18. Schmuck, G. and I. Moya (1994) Time-resolved chlorophyll fluorescence spectra of intact leaves. Remote Sens. Environ. 47, 72–76.

19. Hodges, M. and I. Moya (1986) Time-resolved chlorophyll fluo- rescence studies of photosynthetic membranes: resolution and characterisation of four kinetic components. Biochim. Biophys.

Acta 849, 193–202.

20. Hodges, M. and I. Moya (1988) Time resolved chlorophyll fluo- rescence studies of pigment protein complexes from photosyn- thetic membranes. Biochem. Biophys. Acta 935, 41–52.

21. Holzwarth, A. R., J. Wendler and W. Haehnel (1985) Time- resolved picosecond fluorescence spectra of the antenna chlo- rophylls in Chlorella vulgaris—resolution of photosystem I fluorescence. Biochim. Biophys. Acta 807, 155–167.

22. Holzwarth, A. R. (1987) Picosecond fluorescence spectroscopy and energy transfer in photosynthetic antenna pigments. In The Light Reactions (Edited by J. Barber), pp. 95–157. Elsevier, Amsterdam.

23. Moya, I., J.-M. Briantais and R. Garcia, (1981) Comparison between lifetime spectra of chloroplasts and subchloroplasts par- ticles at21968C and 208C. In Photosynthesis I. Photophysical Processes—Membrane Energization (Edited by G. Akoyunog- lou), pp. 163–172. Balaban, Philadelphia.

24. Turconi, S., N. Weber, G. Schweitzer, H. Strotmann and A. R.

Holzwarth (1994) Energy transfer and charge separation kinetics in photosystem I. 2. Picosecond fluorescence study of various PSI particles and light-harvesting complex isolated from higher plants. Biochim. Biophys. Acta 1187, 324–334.

25. Barber, J., S. Malkin and A. Telfer (1989) The origin of chlo- rophyll fluorescence in vivo and its quenching by the photosys- tem II reaction centre. Phil. Trans. R. Soc. London B 323, 225–

448.

26. Briantais, J.-M., C. Vernotte, G. H. Krause and E. Weis, (1986) Chlorophyll a fluorescence of higher plants: chloroplasts and leaves. In Light Emission by Plants and Bacteria (Edited by

(11)

Govindjee, J. Amesz and D. C. Fork), pp. 539–583. Academic Press, Orlando.

27. Butler, W. L. (1978) Energy distribution in the photochemical apparatus of photosynthesis. Annu. Rev. Plant Physiol. 29 345–

378.

28. Dau, H. (1994) Molecular mechanisms and quantitative models of variable photosystem II fluorescence. Photochem Photobiol.

60, 1–23.

29. Kitajima, M. and W. L. Butler (1975) Excitation spectra for photosystem I and photosystem II in chloroplasts and the spec- tral characteristics of the distribution of quanta between the two photosystems. Biochim. Biophys. Acta 408, 297–305.

30. Schreiber, U. and W. Vidaver (1976) The I-D fluorescence tran- sient. An indicator of rapid energy distribution changes in pho- tosynthesis. Biochim. Biophys. Acta 440, 205–214.

31. Malkin, S., P. A. Armond, H. A. Mooney and D. C. Fork (1981) Photosystem II photosynthetic unit sizes from fluorescence in- duction in leaves. Plant Physiol. 67, 570–579.

32. Virgin, H. I. (1954) The distortion of fluorescence spectra in leaves by light scattering and its reduction by infiltration. Phy- siol. Plant. 7, 560–570.

33. Croce, R., G. Zucchelli, F. M. Garlaschi, R. Bassi and R. C.

Jennings (1996) Excited state equilibriation in the photosystem I–light-harvesting I complex: P700 is almost isoenergetic with is antenna. Biochemistry 35, 8572–8579.

34. Trissl, H. W. (1997) Determination of the quenching efficiency of the oxidized primary donor of photosystem I, P7001: impli- cations for the trapping mechanism. Photosynth. Res. 54, 237–

240.

35. Bradbury, M. and N. R. Baker (1981) Analysis of the slow phases of the in vivo chlorophyll fluorescence induction curve.

Changes in the redox state of photosystem II electron acceptors and fluorescence emission from photosystems I and II. Biochim.

Biophys. Acta 635, 542–551.

36. Bradbury, M. and N. R. Baker (1983) Analysis of the induction of chlorophyll fluorescence in leaves and isolated thylakoids:

contributions of photochemical and non-photochemical quench- ing. Proc. R. Soc. London B 220, 251–264.

37. Buschmann, C. and H. Schrey (1981) Fluorescence induction kinetics of green and etiolated leaves by recording the complete in vivo emission spectra. Photosynth. Res. 1, 233–241.

38. Havaux, M. and W. I. Gruszecki (1993) Heat- and light-induced chlorophyll-a fluorescence changes in potato leaves containing high or low levels of the carotenoid zeaxanthin—indications of a regulatory effect of zeaxanthin on thylakoid membrane fluid- ity. Photochem. Photobiol. 58, 607–614.

39. Huner, N. P. A., D. P. Maxwell, G. R. Gray, L. V. Savitch, M.

Krol, A. G. Ivanov and S. Falk (1996) Sensing environmental temperature change through imbalances between energy supply and energy consumption: redox state of photosystem II. Physiol.

Plant. 98, 358–364.

40. Bru¨ ggemann, W. (1992) Low-temperature limitations of pho- tosynthesis in 3 tropical Vigna species: a chlorophyll fluores- cence study. Photosynth. Res. 34, 301–310.

41. Havaux, M. (1987) Effects of chilling on the redox state of the primary electron acceptor QAof photosystem II in chilling-sen- sitive and resistant plant species. Plant Physiol. Biochem. 25, 735–743.

42. Labate, C. A., M. D. Adcock and R. C. Leegood (1990) Effects

of temperature on the regulation of photosynthetic carbon assim- ilation in leaves of maize and barley. Planta 181, 547–554.

43. Bru¨ ggemann, W. and O. Y. Koroleva (1995) Chilling sensitivity of violaxanthin deepoxidation inhibits the development of en- ergy-dependent chlorophyll fluorescence quenching in vivo.

Plant Physiol. Biochem. 33, 251–259.

44. Bru¨ ggemann, W. and F. P. Wolter (1995) Decrease of energy- dependent quenching, but no major changes of photosynthesis parameters in Arabidopsis thaliana with genetically engineered phosphatidylglycerol composition. Plant Sci. 108, 13–21.

45. Bru¨ ggemann, W. and P. Linger (1994) Long-term chilling of young tomato plants under low light. IV. Differential responses of chlorophyll fluorescence quenching coefficients in Lycoper- sicon species of different chilling sensitivity. Plant Cell Physiol.

35, 585–591.

46. Hodges, M., G. Cornic and J.-M. Briantais (1989) Chlorophyll fluorescence from spinach leaves: resolution of non-photochem- ical quenching. Biochim. Biophys. Acta 974, 289–293.

47. Quick, W. P. and M. Stitt (1989) An examination of factors contributing to non-photochemical quenching of chlorophyll fluorescence in barley leaves. Biochim. Biophys. Acta 977, 287–

296.

48. Jahns, P. and G. H. Krause (1994) Xanthophyll cycle and en- ergy-dependent fluorescence quenching in leaves from pea plants grown under intermittent light. Planta 192, 176–182.

49. Demmig-Adams, B., K. Winter, A. Kru¨ger and F.-C. Czygan (1989) Zeaxanthin synthesis, energy dissipation, and photopro- tection of photosystem II at chilling temperatures. Plant Physiol.

90, 894–898.

50. Koroleva, O. Y., W. Bru¨ ggemann and G. H. Krause (1994) Pho- toinhibition, xanthophyll cycle and in vivo chlorophyll fluores- cence quenching of chilling-tolerant Oxyria digyna and chilling- sensitive Zea mays. Physiol. Plant. 92, 577–584.

51. Yun, J.-G., T. Hayashi, S. Yazawa, Y. Yasuda and T. Katoh (1997) Degradation of photosynthetic activity of Saintpaulia leaf by sudden temperature drop. Plant Sci. 127, 25–38.

52. Murata, N. and K. Satoh, (1986) Absorption and fluorescence emission by intact cells, chloroplasts, and chlorophyll-protein complexes. In Light Emission by Plants and Bacteria (Edited by Govindjee, J. Amesz and D. C. Fork), pp. 137–159. Aca- demic Press, Orlando.

53. Samson, G. and D. Bruce (1995) Complementary changes in absorption cross-sections of photosystems I and II due to phos- phorylation and Mg21-depletion in spinach thylakoids. Biochim.

Biophys. Acta 1232, 21–26.

54. Fork, D. C. (1979) The influence of changes in the physical phase of thylakoid membrane lipids on photosynthetic activity.

In Temperature Stress in Crop Plants (Edited by J. M. Lyons, D. Graham and J. K. Raison), pp. 215–231. Academic Press, New York.

55. Murata, N. and J. Yamaya (1984) Temperature-dependent phase behavior of phosphatidylglycerols from chilling-sensitive and chilling-resistant plants. Plant Physiol. 74, 1016–1024.

56. Hodges, M., J.-M. Briantais and I. Moya (1987) The effect of thylakoid membrane reorganisation on chlorophyll fluorescence lifetime components: a comparison between state transitions, protein phosphorylation and the absence of Mg21. Biochim. Bio- phys. Acta 893, 480–489.

57. Genty, B., J.-M. Briantais and N. R. Baker (1989) The relation- ship between the quantum yield of photosynthetic electron trans- port and quenching of chlorophyll fluorescence. Biochim. Bio- phys. Acta 990, 87–92.

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