The F685/F730 Chlorophyll Fluorescence Ratio as Indicator of Chilling Stress in Plants

(1)

The F685/F730 Chlorophyll Fluorescence Ratio as Indicator of Chilling Stress in Plants

GIOVANNI AGATI\ PIERO MAZZINGHI\ MICHELE L!PUCCI DI PAOLA2, FRANCO FUSI1, and

GIOVANNA CECCHI3

1 Istituto di Elettronica Quantistica - Consiglio Nazionale delle Ricerche, Via Panciatichi, 56/30, 50127 Firenze, Italy

2 Dipartimento di Biologia delle Piante Agrarie, Universira di Pisa, Viale delle Piagge, 23, 56124 Pisa, Italy

3Istituto di Ricerca sulle Onde Elettromagnetiche - Consiglio Nazionale delle Ricerche, Via Panciatichi, 64, 50127 Firenze, Italy

Received June 24, 1995 . Accepted Octobet 10, 1995

Summary

The response of chlorophyll fluorescence to chilling temperatures was evaluated by two different experiments. In the first, the F685/F730 and the FJFmchlorophyll fluorescence ratios were measured inPhaseo- Ius vulgarisL., cv. Mondragone plants under chilling stress at 4°C and moderate light (100 /lmol m-2s-l) up to 72 hours. FJFmdecreased linearly with chilling time indicating a photoinhibitory effect (no change was observed in the dark under the same conditions). F685/F730 underwent a rapid exponential decay followed by a linear slow decline. In a second experiment, the F685/F730 ratio, the total chlorophyll fluorescence, F685+F730, and the leaf temperature were monitored on a single leaf in a climate chamber as the temperature was decreased from 20 to 4°C. The experiment was run simultaneously on the chilling- sensitivePhaseolus vulgarisand on the chilling-tolerantPisum sativumL. (cv. Shuttle) plants. For both species two phases related to the leaf temperature can be distinguished: the first 4-hour period during which the leaf temperature decreased from 24 to 4°C, and a second period during which the leaf temperature slightly oscillated around 4°C. The behaviour of F685/F730 for the bean was quite different from that of the pea plant. During the first phase, it decreased markedly for the chilling-sensitive bean while a slight increase was observed for the chilling-resistant pea. In the following period, the F685/F730 values for the pea remained constant while those for the bean were found still to decrease. On the basis of our results, the use of the chlorophyll fluorescence ratio as indicator of plant chilling sensitivity can be envisaged.

Key words: Phaseolus vulgaris, Pisum sativum, chilling temperature, chlorophyll fluorescence, continuous monitoring, environmentaljactors, photoinhibition, stress detection in plants.

Abbreviations:F_o

=

initial fluorescence; Fm

=

maximum fluorescence; F_v

=

variable fluorescence (Fm-F_o;

ChI=chlorophyll; ChlFR= ratio between the red and near-ir bands of the chlorophyll fluorescence spectrum; F685 and F730

=

chlorophyll fluorescence intensity at 685 and 730 nm respectively; PPFD

=

^pho-

tosynthetic photon flux density; LEAF= Laser Excited Automatic Fluorometer.

Introduction

Temperature affects the photosynthetic capacity of plants by' a series of complex biochemical and biophysical processes (Oquist et al., 1983). Plants adapted to high-temperature environments undergo inhibition of photosynthesis when

exposed to low temperatures. The extent of inhibition is dependent on the species, age of leaves, chilling temperature regime and intensity of the environmental light (Oquist et al., 1987; Hetherington et al., 1989; Hodgson et al., 1989). A decrease in the photosynthetic rate was observed in many species of higher plants under chilling, no-freezing, temper-

(2)

atures in the dark. A more severe reduction occurs when chilling is induced under high light, since the susceptibility to photoinhibition is highly increased at low temperatures.

The mechanisms regulating the inhibition of photosynthesis under chilling are not completely understood. It seems definitely accepted that the primary target for chilling stress in the light is at the reaction center of PSII. On the other hand, storage at 0 °C in the dark appears to inhibit specifically the electron transport from water to PSII (Smillie and Nott, 1979).

Measuring the fluorescence ratio F)F_m of the variable chlorophyll (Chi) fluorescence is a widely used method to monitor reversible or irreversible damage to PSII activity. In fact, this parameter equals the quantum yield for PSII photo- chemistry (Butler, 1978). F)F_m is remarkably constant among non-stressed plants, regardless of the species (F)F_m equal to about 0.8). On the other hand, it decreases drastically in plants kept few hours at chilling temperatures even under moderate light intensities (Hetherington et aI., 1989;

Somersalo and Krause, 1989).

The chilling induced reduction of F)F_{m ,} measured at room temperature (Neuner and Larcher, 1991; Sthapit et aI., 1995) or at 77 K (Hetherington et al., 1989; Huang et al., 1989), was suggested as a useful parameter to identify the plant sensitivity to low temperatures. Other Chi fluorescence parameters have been also indicated as plant chilling tolerance indexes. Hetherington et al. (1983) introduced the measurement of F" the maximal rate of the induced rise in Chi fluorescence, that markedly decreases in chilling-sensitive plants kept at 0

0c.

Relative chilling tolerance is defined as the time taken for Fe to decrease by 50% in leaves chilled at

o 0c.

More recently, Walker et al. (1990) evaluated the chilling tolerance in different tomato lines using the ratio of the initial to the peak fluorescence of the induction kinetics after nh at 2°C. Neuner and Larcher (1990) used the vitality index, Rfd = (fm-Q/fs, the photochemical quenching coefficient and the initial slope of the non-photochemical quenching coefficient as a monitoring method of screening soybean varieties for chilling susceptibility.

Recently, the chlorophyll fluorescence ratio (ChIFR), F685/F730, between the red and the near-infrared bands of the fluorescence spectrum has been proposed as stress indicator (Rinderle and Lichtenthaler, 1988). For example, ChlFR was observed to increase in plants under water stress or after mechanical injuries, with respect to controls (Lichtenthaler and Rinderle, 1988). Chilling stress in beans, on the contrary, was seen to induce a decrease of ChlFR (Lipucci di Paola et aI., 1992).

The use of the ChlFR in the detection of chilling stresses is attractive since it does not need predarkening of the leaf and therefore it would result more useful and advantageous with respect to Chi fluorescence induction measurements. In theory, it can also be used in remote sensing monitoring of vegetation (Valentini et aI., 1994). This work is an extension of our previous observation (Lipucci di Paola et aI., 1992) in order to accurately investigate the changes in the F685/F730 ratio induced by chilling temperatures under moderate light on a chilling sensitive plant. These data were compared with the fluorescence parameter F)Fm of the fluorescence induction kinetics under the same treatment. The potential use of

the ChlFR as screening parameter of plant chilling tolerance was also evaluated by measuring the F685/F730 ratio in the chilling-sensitive bean and the chilling-resistant pea species.

Materials and Methods

Plant material and growth conditions

Experiments were performed on 20-d old seedlings of bean(Pha- seolus vulgarisL., cv. Mondtagone) and pea (Pisum sativum L., cv.

Shuttle) at the stage of fully expanded primary leaves. Seeds were germinated in perlite and transplanted to perforated plastic pots (8 cm diameter) filled with expanded clay and placed in tanks (61 capacity, 24 plants per tank) with a continuously aerated nutrient solution. Seedlings were:frown in a climate chamber under continuous light (l00 !lmol m- s-I). The chamber temperature was set at 23±1°C and the relative humidity was 80%.

Chilling treatment

Chilling treatment was performed in the growth chamber at 4± 1°C and at a constant relative humidity of 80%. Experiments were done at low light intensity (lOO!lmolm-zs-') to minimise photoinhibitory effects. A separate batch of plants was used as control, maintained at 23 ± 1

0c,

relative humidity of 80%, in the same light conditions as stressed plants. Chilling stress was followed up to 3 days. All the experiments were conducted at atmospheric COz concentration.

Fluorescence measurements and data analysis

The Chi fluorescence ratio, F685/F730, was measured by the portable two-wavelengths fluorometer LEAF® (Laser Excited Auto- matic Fluorometer) (Loto, Florence, Italy) described previously (Li- pucci di Paola et aI., 1992). The instrument excites Chi fluorescence by a He-Ne laser and detects both the 685 and 730 nm fluorescence peaks by a couple of photomultiplier detectors preceded by suitable interference filters. An improved version of the LEAF instrument that uses a diode laser at 635 nm to excite fluorescence and a couple of photodiodes as detection system was also employed.

ParametersFa'Fm andFJFmof the fluorescence induction kinetics were obtained using a pulse-amplitude modulations fluorometer (PAM-WI; H. Walz, Effeltrich, Germany) as reported previously (Schreiber, 1986).

At various time intervals of the chilling treatment, fluorescence was measured on each leaf by the LEAF device on both controls and stressed plants. The rapidity of the LEAF method allows for the col- lection of a large number of data on each leaf. Consequently, the F685/F730 value for each leaf was the average of about 30 measurements. The same leaves were then dark-adapted for 15 min and measured by the PAM instrument. Values of the different fluorescence parameters were the average on 6 leaves from 6 different plants.

All data were collected from the upper side of fully expanded primary leaves attached to the plant.

In a separate experiment, one plant of each species was continuously monitored while the chamber temperature was decreased from 23 to 4°C under 100 !lmolm- zs-1 and 80% of relative humidity.

The ChlFR and the total Chi fluorescence, F685+F730, were measured in automatic mode at regular 1 min time intervals from the beginning of the chilling treatment contemporaty on both bean and pea leaves by using two fiber optics LEAF fluorometers. The fiber optics tips of the two LEAF instruments were positioned at about 45° with respect to the leaf surface at a distance of about

(3)

F685/F730vst 0.10±0.01 1.27±0.23 -0.9±0.1 0.65±0.01 0.998

FJF_mvst -7.7±0.5 0.76±0.02 0.984

Table 1: Curve fitting parameters forF685/F730 and F)Fmversus time of chilling treatment inPhaseolus vulgaris.

2 mm in ordertoavoid shading of the leaf area under measurement.

At the same time the temperature of both leaves was also measured by two infrared thermometers (Land Cyclops Compac 3, Minolta) at a frequency of 1/60Hz.This experiment was aimedtoaccurately investigate the initial period during which the plant acclimates to 4 ·C starting from room temperature. The measurements was car- ried our up to 20 hours maintaining the chamber temperature at

4·C.

The variation ofF685/F730 and F)Fm versus the chilling time was analysed by fitting the mean values.

exponential fitting curve amplitude time con-

stant (h)

linear fitting curve slope intercept (xlO·J)

Results

Discussion

Comparison between F685/F730 and Fv/Fm

The effect of chilling temperatures on F685/F730 and Fj Fm under moderate light depends on the time of treatment.

For chilling times longer than about 6 hours the behaviour of the two chlorophyll fluorescence parameters is similar. In Phaseolus vulgaris,both factors decrease linearly. The decrease of FjFmwith chilling can be explained as due to a photoin-

Fluorescence and temperature continuous monitoring The results of the fluorescence continuous monitoring, as the environmental temperature was decreased and maintained at 4 ·C up to 20 hours, are shown in Figs. 2 a and 2 b for the bean and pea plants respectively. The ChlFR and the total F685+F730 ChI fluorescence are reported along with the leaf temperature as function of the chilling time. For both bean and pea, two phases related to the leaf temperature can be distinguished: 1) a first 4-hour period during which the leaf temperature exponentially decreased from 25 to 4·C, and 2) a second period during which the leaf temperature slightly oscillated around 4.c,The behaviour of ChI fluorescence is instead different for the two species. During the first phase, F685/F730 decreases markedly for the chilling-sensitive bean while a slight increase is observed for the chilling- resistant pea. In the following period, the ChlFR for the pea remained constant while those for the bean are found still to decrease. The total ChI fluorescence, F685+F730, increases in both pea and bean plants with decreasing leaf temperature to a maximum and then decreases to a plateau. However, in bean the fluorescence intensity increase is much smaller (50%vs. 130%) and shorter (2.5 vs. 5 h) than that observed in pea.

The relationship between ChlFR and the leaf temperature for the chilling-sensitive and chilling-tolerant species is better shown in Fig. 3 during the first phase.Itis evident that the slope of the decline in F685/F730 observed in bean increases when the leaf temperature becomes lower than 7·c' In pea, instead, F685/F730 is inversely correlated to the leaf temperature with a single regression line over the temperature range reported.

F685/F730 at time of the chilling. treatment longer than 6.5h is significant (0.025<P<0.05).

The ChI content, as measured previously (Lipucci et aI., 1992), was found to remain constant during the chilling treatment.

80 70 60

0.22~

20 10

\J

^0.41

r--- t

---f---1

0.81 -D- Fv/Fm -t:JrooE---

0.72 --6- F685/F730 1.1

1.0

0.9 UJ(/J« 0.8 UJa:

() 0.7 UJ0 UJ> ^0.6 i=«

....J UJ 0.5 a:

0.4 0.3

0.2

0 30 40 50

TIME (h)

Fig. 1: Time course of the F685/F730 (.6.)and FJF_m (0) chlorophyll fluorescence ratios measured in Phaseolus vulgaris L., cv.

Mondragone during chilling at 4·C and 100~molm-2 S-I. Values are relative tocontrols and are the average±standard deviation of six different measurements.

F685/730 and F/Fm comparison

The F685/F730 and FjFm fluorescence parameters as function of the time of chilling treatment in bean are pre- sented in Fig. 1.Values are relative to controls. Itis evident that F685/F730 decreases quickly in the first few hours of the treatment and then more slowly.

A quite different behaviour is showed for FjFm that decreases linearly with. treatment. The decrement of FjFm⁼ (Fm-Fo)/Fm is mostly due to the decrease of Fm rather than to a variation ofFo (data not shown).

Curve fitting of the average values of the above experimental data results in the parameters reported in Table 1.

Values of FjFm are well fitted by a single linear curve with negative slope of -0.008. The best fit of F685/F730 consists of a rapid exponential decay followed by a slow linear decrement. In order to carefully evaluate the significance of the linear variation of F685/F730, we analysed the data by the one-way analysis of variance. It results that the change of

(4)

3 0 , - - - , - 0.46 1000

25!~

_{. 0}^a)oCD₀

o

0..

~ ²⁰ ⁰⁰ ^{. . 0}

::> \ 0

I- \

~ ¹⁵ ~ ⁰

~ \

:; \ 0

~ ~ ⁰

~ ¹⁰ ^\ 0

~ ~ ⁰

5 ~Cb

- TEMPERATURE -.- F685/F730

o F685+F730

0.44 0.42 0.40 0.38 ~

u.i?i 0.36 _u.~ 0.34 0.32 0.30

900

800 :i 700 ~_o u.f:2

600 Ji

~

500 400

300 15

10 TIME (h)

o 5

o+--~~~----,---~~_,_--~--,__~~---+0.28 20

3 0 , . . - - - , 1 . 0 8 600

:i 450 ~ 400

E

Ji

~

350 300 550

250 1.04 500

1.06

0.98 1.00 1.02 g,...

u.i?i

~u..

15 10

TIME (h) 5

b)

o-t---.,,--,--~~--,---~--,__~~o _ _-+0.94 20 25

;;:; 20~ a:::>

ffi~ 15 a.:;

wI-

~ 10 w...J

Fig.2:Variation of the chlorophyll fluorescence ratio, F685/F730 (e), total chlorophyll fluorescence, F685+F730 (0); and leaf temperature (solid line) during a continuous monitoring in a climate chamber ofPhaseolus vulgaris(a) and Pisum sativum (b) attached leaves. Light intensity was 100J.lmolm-²s-¹and relative humidity was 80%.

hibitory effect.Itis well known that chilling temperatures increase the plant sensitivity to photoinhibition so that also rel- atively low PPFD can inhibit the photosynthetic activity (Hetherington et aI., 1989; Ottander et aI., 1992; Somersalo and Krause, 1989; Powles et aI., 1983).

Photoinhibition consists of complex mechanisms aimed to protect the photosynthetic apparatus from excess light energy.

It can be seen as the result of concomitant damaging and repairing processes. Greer et al. (1986) already observed photoinhibition in Phaseolus vulgaris at 5·C and 140 Ilmol m-²s-1 and suggested that the inhibition was favoured by low temperatures by inactivation of the repair mechanism.

The dissipating mechanisms of photoinhibition are in competition with the radiative relaxation of chlorophyll.

Consequently, photoinhibition is accompanied by a reduction in the chlorophyll fluorescence. The ratio FjF_m was shown to be an useful indicator of photosynthesis inhibition under high-light intensity and physiological temperatures (Bjorkman, 1987), or at moderate light intensity and chilling

temperatures (Somersalo and Krause, 1989; Hetherington et aI., 1989). In our work, the presence of photoinhibition in beans at 4 ·C and 100 Ilmol m-2s-1 was than proved by the decrease of FjFm with the treatment. Moreover, in a test experiment performed in the dark we found that no change in FjFmwas induced by chilling alone (data not shown).

Interpretation of the change with chilling of the F685/

F730 ratio is complicated because of the scarce knowledge available on the physiological meaning of this fluorescence parameter. The main contribution to chlorophyll fluorescence in the red-far-red spectral region comes from PSII. The photosystem PSI seems to provide significant fluorescence, peaked at about 740 nm, only at low temperatures (Krause and Weis, 1991), so that the ratio between the shorter and the longer wavelength emission bands at 77 K is used to monitor changes in the energy distribution between PSI and PSII.

Several experimental evidences, however, suggest that at physiological temperatures the contribution of PSI to the 740 nm fluorescence is not negligible with respect to PSII fluores-

(5)

0.7 +----,---,---,----,---,----1

Fluorescence and temperature continuous monitoring The short-term effect of a decrease in temperature on the ChI fluorescence has been investigated simultaneously on the chilling-resistant pea and the chilling-sensitive bean. The decrement in F685/F730 observed in bean after 6.5 h (32%) is in accordance with that measured at the same time of chilling in the previous above experiment. It is evident from Fig. 3 that the effect of temperature on ChlFR is markedly different for the two species. In the chilling-sensitive bean, F685/F730 is directly correlated to the leaf temperature and there is a critical temperature, around 7·c' below which the slope of the regression line drastically increases. This is not the case for the chilling-tolerant pea in which a single inverse linear correlation between F685/F730 and leaf temperature is found. Also the behaviour of the total Chi fluorescence with leaf temperature is species dependent. In bean F685+F730 starts to decrease at about 7·c' while in pea it increases until the leaf temperature decreases to 4

.c.

The initial behaviour ofChlFR and F685+F730 in bean is analogue to that previously seen on Ficus benjaminiin the 14-25 ·C temperature range (Agati et aI., 1995). Explanation of these results is diffi- cult because a complete characterisation of the temperature profiles of the Chi fluorescence bands at the steady-state is lacking. In fact, most of the studies have been dedicated to the detection of the Chi fluorescence induction kinetics parameters at different temperatures. Itwas found that the steady-state ChI fluorescence in soybean increased with pro- gressive cooling from 20 to O·C (Neuner and Larcher, 1990).

This result was correlated to a large reduction of the photochemical quenching at lower temperatures while the non- photochemical quenching at the steady-state was only slightly affected by temperature. Analogue dependencies of the photochemical and non-photochemical fluorescence quenching on leaf temperature were reported by Koroleva et al. (1994) for a chilling tolerant species. Furthermore, decreasing leaf temperature induces a reduction of the thylakoid membrane fluidity which inhibits the reoxidation of plastoquinones Oquist, 1984; Demmig and Bjorkman, 1987; Somersalo and Krause, 1989).

The decrease of F685/F730 we observed in bean plants under chilling and moderate light treatment can then be explained as due to the inhibition ofPSII.

The variation in F685/F730 induced by chilling stress is opposite to that reported for all other stress conditions in which the Chi concentration is reduced (Rinderle and Lich- tenthaler, 1988; Lichtenthaler and Rinderle, 1988). In these cases, in fact, an increase in ChlFR is observed as due to a decrease in the fluorescence reabsorption at 690 nm (Agati et aI., 1993). In our experiment, the extent of the stress is too short to lead to a Chi degradation, as confirmed by pigment extractions (Lipucci di Paola et aI., 1992). Variations in the absorbance and scattering properties of the different Chi-protein complexes, going from room to chilling temperature, can not be ruled out. These effects could change the fluorescence reabsorption on the short-wavelength band (Agati et aI., 1993). However, in our experiment, we verified that the leaf transmittance spectra were the same before and after the chilling treatment.

30

10 15 20 25

LEAF TEMPERATURECC)

o 5

1.1 I--,~;---~======;l

Fig. 3: The chlorophyll fluorescence ratio, F685/F730 as function of the leaf temperature under moderate light intensity (l00Ilmol m-²s-I) inPhaseolus vulgaris(0) andPisum sativum(e).Solid lines are regression curves.

cence (Gently et aI., 1990; Pfiindel, 1995) and that variations in the ChlFR may reflect changes in the photosynthetic activity of the two photosystems (Agati et al., 1995).

What is the main target of photoinhibition is still under debate and it seems to depend markedly on the environmental conditions. Itwas observed that in vivoPSI is more resistant than PSII to strong light stress (Havaux and Eylet- ters, 1991). Under moderate high-light stress (420W/m²,Ih) of sugar maple leaves, the energy storage and the photosynthetic activity in PSI are augmented (Charland et aI., 1992), while PSII is specifically inhibited. At higher light intensities both PSII and PSI are inhibited, but the later at a lower extent.

Recently, it was observed that at low temperatures a prefer- ential inactivation of PSI occurs in both chilling-resistant (Havaux and Davaud, 1994) and chilling-sensitive plants (Te- rashima et al., 1994). The mechanism proposed for the PSI photoinhibition involves the inactivation of the electron ac- ceptor side without destruction of the P-700 complex (So- noike and Terashima, 1994). Consequently, being the PSI Chi fluorescence yield independent of the redox state of P-700 (Butler, 1978), no change in the ChI fluorescence at 730 nm can be ascribed to the potential chilling-induced photoinhibition of PSI.

In our experimental conditions, photoinhibition of PSII was proved to occur by the reduction of FJFm . Conse- quently, a decrease in the steady-state fluorescence emitted by PSII at both fluorescence maxima with chilling treatment is expected. As indicated above, the contribution of PSI to the longer-wavelength Chi fluorescence should remain constant under chilling. The larger quenching on the red fluorescence peak with respect to the far-red one under photoinhibition is confirmed by fluorescence measurements at 77 K (Ogren and

(6)

leading to an increase in the Chi fluotescence yield (Havaux and Gruszecki, 1993).

Understanding the change with temperature of F685/F730 is even more complicate and requires further investigations. It is however evident that different mechanisms operate on the two species during the chilling. In bean, at the initial decrease in ChlFR that is luckily due to a temperature effect only, a photoinhibitory effect adds below 7·C inducing a quenching of the total fluorescence and a more rapid decrease in F685/

F730. In pea, the temperature effect on F685/F730 is modest but opposite to that of bean. The reason for this difference probably relies on diverse mechanisms of energy dissipation, including PSII ~ PSI transfer, developed by the two species and on their dependence on leaf temperature (Holaday et aI., 1992; Koroleva et aI., 1994; Adams et aI., 1995).

F685/F730 as screening parameter for chilling tolerance Large attention have been devoted to the development of screening methods to identifY the plant sensitivity to low temperatures (Hetherington et aI., 1983; Walker et aI., 1990;

Neuner and Larcher, 1990; Hetherington et aI., 1989; Sthapit et al., 1995). At present, all the procedures used are based on the measurement of Chi fluorescence induction parameters and, therefore, their practical application is limited by the requirement of predarkening of the leaf.

Our data on plants with different chilling susceptibility in- dicate a new, rapid method for the screening of the plant chilling tolerance.

We observed (Figs. 2 and 3) that the temperature effect on F685/F730 is species dependent, being opposite in the chilling-resistant pea with respect to the chilling-sensitive bean.

Although more experimental work is needed to understand the physiological meaning of the change of F685/F730 with temperature in relation to the plant chilling sensitivity, the usefulness of this parameter can be envisaged. We demon- strated that the F685/F730 ratio between the two Chi fluorescence peaks can be used to monitor chilling stresses under light. The measurement of F685/F730 by two wavelengths fluorometers, as the LEAF instrument, presents the advan- tage, with respect to fluorescence kinetics measurements, of no needs for predarkening of the plant. A large number of leaves can then be sampled in a short time improving the sta- tistics of data. Moreover, this method can result useful, in principle, in remote sensing of vegetation. The reliability of the method must be checked on a large number of different species and the suitable parameter, such as the slope of the F685/F730 vs. leaf temperature curve, to be used as index of chilling sensitivity must be found.

References

ADAMS, W. W. lll, A. HOEHN, and B. DEMMIG-ADAMS: Chilling temperatures and the xanthophyll cycle. A comparison of warm- grown and overwintering spinach. Aust.J.Plant Physiol. 22,75- 85 (1995).

AGATI, G., F. FUSI, P. MAZZINGHI, and M. LIPUCCI DI PAOLA: A simple approach to the evaluation of the reabsorption of chlorophyll fluorescence spectra in intact leaves.J. Photochem. Photo- bioI. B: BioI. 17, 163-171 (1993).

AGATI, G., P. MAZZINGHI, F. FUSI, and I. AMBROSINI: The F685/

F730 chlorophyll fluorescence ratio as a tool in plant physiology:

response to physiological and environmental factors. J. Plant.

Physiol. 145,228-238 (1995).

BJORKMAN,0.: Low-temperature chlorophyll fluorescence in leaves and its relationship to photon yield of photosynthesis in photoinhibition. In: KYLE, D. J.,

c.

B. OSMOND, and C.J. ARNTZEN (eds.): Photoinhibition, pp. 123-144. Elsevier Science Publishers B.Y., Amsterdam (1987).

BUTLER, W. L.: Energy distribution in the photochemical apparatus of photosynthesis. Ann. Rev. Plant Physiol. 29, 345-378 (1978).

CHARLAND, M.,K.VEERANJANEYULU, and R. M. LEBLANC: Simulta- neous determination ofin vivoactivities of PSI and PSII following high-light stress. In: MURATA, N. (ed.): Research in Photosynthe- sis, pp. 459-462. Kluwer Academic Publishers, Dordrecht (1992).

DEMMIG, B. and O. BJORKMAN: Comparison of the effect of excessive light on chlorophyll fluorescence (77K) and photon yield ofO₂evo- lution in leaves of higher plants. Planta 171, 171-184 (1987).

GENTLY, B.,J. WONDERS, and N. R. BAKER: Non-photochemical quenching of F⁰ in leaves is emission wavelength dependent: con- sequences for quenching analysis and its interpretation. Photo- synth. Res. 26, 133-139 (1990).

GREER, D. H.,J.A. BERRY, and O. BJORKMAN: Photoinhibition of photosynthesis in intact bean leaves: role of the light and temperature, and requirement for chloroplast-protein synthesis during recovery. Planta 168,253-260 (1986).

HAvAux, M. andA. DAVAUD: Photoinhibition of photosynthesis in chilled potato leaves is not correlated with a loss of Photosystem- II activity. Photosynth. Res. 40,75-92 (1994).

HAvAux, M. and M. EYLETTERS: Is the in vivoPhotosystem I function resistant to photoinhibition?An answer from photoacoustic and far-red absorbance measurements in intact leaves. Z.Natur- forsch. 46c,1038-1044 (1991).

HAvAux, M. andW.1.GRUSZECKI: Heat- and light-induced chlorophyll afluorescence changes in potato leaves containing high or low levels of the carotenoid zeaxanthin: indications of a regula- toty effect of zeaxanthin on thylakoid membrane fluidity. Photo- chern. Photobiol. 58,607-614 (1993).

HETHERINGTON, S. E.,J. HE, and R. M. SMILLIE: Photoinhibition at low temperature in chilling-sensitive and -resistant plants.

Plant Physiol.90,1609-1615 (1989).

HETHERINGTON, S. E., R. M. SMILLIE,A.K. HARDACRE, and H.A.

EAGLES: Using chlorophyll fluorescence in vivo to measure the chilling tolerances of different populations of maize. Aust. J.

Plant Physiol. 10,247-256 (1983).

HODGSON, R. A.J. andJ. K. RAISON: Inhibition of photosynthesis by chilling in moderate light: a comparison of plants sensitive and insensitive to chilling. Planta 178, 545-552 (1989).

HOLADAY, A. S.,W. MARTINDALE, R. ALRED,A. L. BROOKS, and R.

C.LEEGOOD: Changes in activities of enzymes of carbon metabo- lism in leaves during exposure of plants to low temperature. Plant Physiol. 98, 1105-1114 (1992).

HUANG,L.-K.,C.B. OSMOND, andI.TERASHIMA: Chilling injury in mature leaves of rice. II. Varietal differences in the response to in- teractions berween low temperature and light measured by chlorophyll fluorescence at 77 K and the quantum yield of photosynthesis. Aust.J.Plant Physiol. 16, 339-352 (1989).

KOROLEVA, O.Y.,W. BRUGGEMANN, and G. H. KRAUSE: Photoinhi- bition, xanthophyll cycle and in vivo chlorophyll fluorescence quenching of chilling-tolerant Oxyria digyna and chilling-sensi- tiveZea mays. Physiol. Plant 92, 577- 584 (1994).

KRAUSE, G. H. and E. WEIS: Chlorophyll fluorescence and photosynthesis: the basics. Ann. Rev. Plant Physiol. Plant. Mol. BioI.

42,313-349 (1991).

LICHTENTHALER, H. K. and U. RtNDERLE: The role of chlorophyll fluorescence in the detection of stress conditions in plants. CRC Crit. Rev. Anal. Chern. 19 (Suppl.I),S29-S85 (1988).

(7)

LIpUCCI DI PAOLA, M., P. MAZZINGHI, A. PARDOSSI, and P. VER- NIERl: Vegetation monitoring of chilling stress by chlorophyll fluorescence ratio. EARSeL Adv. Rem. Sens. 2, 2-6 (1992).

NEUNER, G. and W LARCHER: Determination of differences in chilling susceptibility of two soybean varieties by means ofin vivo chlorophyll fluorescence measurements.

J.

Agron. Crop Sci. 164, 73-80 (1990).

- - The effect of light, during and subsequent to chilling, on the photosynthetic activity of two soybean cultivars, measured byin vivo chlorophyll fluorescence. Photosyntherica 25, 257-266 (1991).

OGREN, E. and G. OQUIST: Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. III. Chlorophyll fluorescence at 77 K. Physiol.

Plant. 62, 193-200 (1984).

OQUIST, G.: Effects of low temperature on photosynthesis. Plant Cell Environ. 6, 281-300 (1983).

OTTANDER,C,T. HUNDAL, B. ANDERSSON, N. P. A. HUNER, and G. OQUIST: On the susceptibility of photosynthesis to photoinhibition at low temperatures. In: MURATA, N. (ed.): Research in Photosynthesis, pp. 455 -458. KIuwer Academic Publishers, Dordrecht (1992).

PFUNDEL, E.: PSI fluorescence at room temperature: possible effects on quenching coefficients. In: MATHIS, P. (ed.): Proceedings of the 10th International Congress on Photosynthesis. KIuwer Aca- demic Publishers, Dordrecht (in press).

POWLES, S. B.,

J.

A. BERRY, and O. BJoRKMAN: Interaction between light and chilling temperature on the inhibition of the photosynthesis in chilling-sensitive plants. Plant Cell Environ. 6, 117-123 (1983).

RINDERLE,U.and H.K. LICHTENTHALER: The chlorophyll fluorescence ratio F690-F735 as a possible stress indicator. In: LICH- TENTHALER, H. K. (ed.): Applications of Chlorophyll Fluores- cence, pp. 189-196. KIuwer Academic Publishers, Dordrecht (1988).

SCHREIBER, U.: Detection of rapid induction kinetics with a new type of high-frequency modulated chlorophyll fluorometer. Pho- tosynth. Res. 9,261-272 (1986).

SONOIKE, K. and1.TERASHIMA: Mechanism of photosystem-I photoinhibition in leaves ofCucumis sativusL..Planta 194, 287-293 (1994).

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).

SOMERSALO, S. and G. H. KRAUSE: Photoinhibition at chilling temperature. Planta 177, 409-416 (1989).

STHAPIT, B. R.,

J.

R. WITCOMBE, and

J.

M. WILSON: Methods of se- lection for chilling tolerance in Nepalese rice by chlorophyll fluorescence analysis. Crop Sci. 35,90-94 (1995).

TERASHIMA,1.,S. FUNAYAMA, andK. SONOIKE: The site of photoinhibition in leaves of Cucumis sativusL. at low temperatures is photosystemI, not photosystem II. Planta 193,300-306 (1994).

VALENTINI, R., G. CECCHI, P. MAZZINGHI, G. SCARASCIA MUG- NOZZA, G. AGATI, M. BAZZANI,P. DE ANGELlS, F.FUSI, G. MAT- TEUCCI, andV.RAIMONDI: Remote sensing of Chlorophyllafluo- rescence of vegetation canopies: 2. physiological significance of fluorescence signal in response to environmental stresses. Remote Sens. Environ. 47, 29-35 (1994).

WALKER, M. A., D. M. SMITH,K.P.PAULS, and B. D. MCKERSIE: A chlorophyll fluorescence screening test to evaluate chilling tolerance in tomato. Hort. Sci. 25, 334-339 (1990).