Plant Physiol. (1989) 90, 1609-161 50032-0889/89/90/1 609/07/$01 .00/0

Received for publication July 12, 1988and in revised form April 14, 1989

Photoinhibition at Low Temperature inChilling-Sensitive and -Resistant Plants1

Suzan E. Hetherington*, Jie He, and Robert M. Smillie

Division of Horticulture, Sydney Laboratories, Commonwealth Scientific and Industrial Research Organisation,P. 0. Box 52, North Ryde, New South Wales 2113, Australia

ABSTRACT

Photoinhibition resulting from exposure at 70C to a moderatephoton flux density (300 micromoles per square meter per sec-ond, 400-700 nanometers) for 20 hours was measured in leavesof annual crops differing widely in chilling tolerance. The inci-dence of photoinhibition, determined as the decrease in the ratioof induced to total chlorophyll fluorescence emission at 693nanometers (F,/F,,,) measured at 77 Kelvin, was not confined tochilling-sensitive species. The extent of photoinhibition in leavesof all chilling-resistant plants tested (barley [Hordeum vulgareL.], broad bean [Vicia faba L.], pea [Pisum sativum L.], and wheat[Triticum aestivum L.]) was about half of that measured in chilling-sensitive plants (bean [Phaseolus vulgaris L.], cucumber (Cucu-mis sativus L.], lablab [Lablab purpureus L.], maize [Zea maysL.], pearl millet [Pennisetum typhoides (Burm. f.) Stapf & Hubbard], pigeon pea [Cajanus cajun (L.) Millsp.], sesame [Sesamumindicum L.], sorghum [Sorghum bicolor L. Moench], and tomato(Lycopersicon esculentum Mill.]). Rice (Oryza sativa L.) leaves ofthe indica type were more susceptible to photoinhibition at 70Cthan leaves of the japonica type. Photoinhibition was dependentboth on temperature and light, increasing nonlinearly with de-creasing temperature and linearly with increasing light intensity.In contrast to photoinhibition during chilling, large differences, upto 166-fold, were found in the relative susceptibility of the differ-ent species to chilling injury in the dark. It was concluded thatchilling temperatures increased the likelihood of photoinhibitionin leaves of both chilling-sensitive and -resistant plants. Further,while the photoinhibition during chilling generally occurred morerapidly in chilling-sensitive plants, this was not related directly tochilling sensitivity.

Many ofthe important annual crops cultivated in temperateclimates have been introduced from the tropics. In general,these plants are chilling-sensitive. They develop chilling injuryat temperatures below about 12°C, and occurrences ofchillingstress result in impaired plant growth performance (13). Inthe temperate zones recurrent periods of cold weather occur.

For crops such as cotton, maize, rice, and sorghum that are

subject to the agronomic practice of early spring planting thelikelihood of encountering chilling temperatures is increased.Similarly, for fresh produce crops such as beans, cucurbits,and tomatoes, early planting is often favored.The interaction of chilling temperatures and light on vege-

' Supported by a grant from the Australian Centre for IntemationalAgricultural Research.

tative tissue of tomato (15, 29) and cucumber (9) has beenidentified as a potentially more serious cause of injury thanchilling alone. When chilled in the light, leaves can becomephotoinhibited, a state characterized by depressed rates ofboth light-saturated and light-limited photosynthesis (12, 18)and reduced yields of Chl fluorescence measured either atroom temperature (28) or at 77 K (4, 16). Photoinhibition ismost likely to occur when the rate of photon capture signifi-cantly exceeds the throughput at exciton processing centersand energy dissipation by other means. At chilling tempera-tures photoinhibition can occur at light intensities below thatof full sunlight. At 5°C, for instance, light levels less than fullsunlight can within a few hours significantly decrease thequantum yield of photosynthesis in leaves of rice (5), maize(12), bean (18), and tomato (15). Up to a point, photoinhi-bition is completely reversible (1, 4), and this condition maybe thought of as an adaptive mechanism allowing someadjustment of photosynthetic processes to suit prevailing con-ditions of light, temperature and other environmental factors.However, prolonged exposure to light and low chilling tem-peratures can be debilitating and associated with severe andirreversible photoinhibition followed by photooxidative de-struction of Chl (1, 28) and ultimately cell death.

Chilling-dependent photoinhibition has mainly been stud-ied in plants well known to be intolerant of chilling temper-atures, and this raises the question of whether or not adverseeffects of chilling and light on photosynthetic productivity areprimarily problems associated with chilling-sensitive plants.Photoinhibition ofphotosynthesis arising from the interactionof chilling and light has been shown to occur in leaves of thechilling-sensitive annual crops, bean (4, 18), cotton (18),cucumber (9, 28), maize (12), rice (5), and tomato (15, 29).Olive, an evergreen crop of warm temperate climates, is alsovulnerable to photoinhibition during chilling at 5°C (1). Incontrast, spinach, a chilling-resistant plant, was reported tobe insensitive to inhibition of photosynthetic processes inlight at 4°C (3). However, some recent studies have renderedthis distinction less clear. Two plants of temperate climates,Lemna minor and L. gibba were shown to be photoinhibitedat chilling temperatures (1 1, 16), although it is to be notedthat vegetative growth of these plants is largely restricted tothe warm months of the year and over-wintering is accom-plished by producing resting buds (turions) that sink to thebottom of ponds during winter. Photoinhibition during chill-ing was also demonstrated in two species of potato that aresomewhat more tolerant of chilling than the chilling-sensitiveplants mentioned above (26). Two relevant studies have been

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Plant Physiol. Vol. 90,1989

carried out on extremely chilling-resistant species. Leaves ofbarley exposed to high light intensity were photoinhibitedwhen irradiated at leaf temperatures below 1 2°C (19). In theother study, measurements of quantum yield at 25°C madeon shoots taken from exposed branches on the south side ofPinus sylvestris growing naturally in northern Sweden showedthat the quantum yield for photosynthesis fell during winterfrom a summer high of 0.057 to a winter low of 0.017 (10).Depletion ofChl in evergreen conifers in winter has also beenobserved, especially in trees exposed to high radiation levelsat high altitudes (27).The purposes of this study were to (a) ascertain if chilling-

resistant plants are also vulnerable to photoinhibition at chill-ing temperatures, (b) compare relative susceptibilities of dif-ferent species to chilling-induced photoinhibition, and (c)investigate if predisposition to photoinhibition at chillingtemperatures is closely related to susceptibility to chillinginjury in the dark. The approach adopted was to compareplant resistance to chilling in the dark with susceptibility tophotoinhibition at chilling temperatures. Comparisons weremade using leaves of 15 different tropical and temperateannual crop species encompassing a wide spectrum of chillingsensitivities. Some preliminary results were presented in asymposium paper (22).

MATERIALS AND METHODS

Plants and Growth Conditions

The chilling-sensitive plants used were bean Phaseolus vul-garis L. cv Redland Pioneer, dwarf bean (P. vulgaris cvWindsor Long Pod), cucumber (Cucumis sativus L. cv Palo-mar), lablab (Lablab purpureus L.), maize (Zea mays L. cvG390 Hybrid), pearl millet (Pennisetum typhoides (Burm. f.)Stapf & Hubbard), pigeon pea (Cajanus cajun (L.) Millsp.),indica rice (Oryza sativa L. cv Dee-geo-woo-gen and cv ErBai Ai), japonica rice (0. sativa cv Asahi), sesame (Sesamumindicum L.), sorghum (Sorghum bicolor L. Moench cv F64AHybrid), and tomato (Lycopersicon esculentum Mill. cvRouge de Marmande). The chilling-resistant plants used werebarley (Hordeum vulgare L. cv Weeah), broad bean (Viciafaba L. cv Coles Early), oat (Avena sativa L.), pea (Pisumsativum L. cv Greenfeast), and wheat (Triticum aestivum L.cv Gatcher). All plants other than rice were grown from seedduring summer under shade cloth in a glasshouse (tempera-ture range 18 to 28°C; maximum PFD 700 to 800 ,mol m-2s-'). The plants were grown in 20-cm diameter pots containinga mix of perlite, vermiculite, and peat (2:1:1, v:v:v) and werewatered by drip irrigation with half-strength modified Hoag-land solution. The rice was grown in soil in pots in anothersection of the glasshouse where the maximum PFD was 1000to 2000 ,umol m-2 s-'. Permanent flood was applied to riceseedlings at the three-leaf stage. For experiments, the penul-timate fully expanded leaves were harvested 5 to 6 h afterdawn. Plants used in the experiments were at the floweringor early fruiting stages.

Determination of 'Dark' Chilling Tolerance

The time course of chilling-induced changes in Chl fluores-cence at O0C in darkness was followed by the decrease, meas-

ured at 0C, of F, the maximal rate of the induced rise in Chlfluorescence (7). Relative tolerance to this chilling stress wasdetermined by the time in h for a 50% decrease in Fr. Valuesgiven are the means of determinations made on 16 leaves foreach species. Standard errors of the mean are expressed aspercentages of the mean and ranged from ±5.2% to ± 10.5%.

Determination of Photoinhibition at Chilling Temperatures

Leaves were harvested and floated on water in subduedlight. Discs (1.3 cm diameter) or 2-cm lengths of leaf in thecase of monocotyledoneous species were cut and placed onwet filter paper supported on an aluminium plate (30 x 45 x0.3 cm). The leaf pieces were covered with thin polyethylenefilm. The plate was then placed in a controlled environmentcabinet and after temperature equilibration exposed to lightfrom metal halide lamps (Sylvania HID lamps, 1 500W/BT-56). Following the period of irradiation, photoinhibition ofthe leaf samples was measured by the decrease in the ratio ofthe induced to the maximum Chl fluorescence, Fv/Fmax, meas-ured at 77 K (4, 16). Two to four leaves were sampled fromeach species, and each experiment was repeated at least twice.Standard errors of the mean for Fv/Fma ranged between ±1.5% and ±9.9%.When photoinhibitory treatments were made at more than

one temperature, the leaf samples were placed on wet filterpaper on a thick aluminium plate (30 x 45 x 2 cm) heatedat one end and cooled at the other to produce a lineartemperature gradient along the plate. Otherwise, the leafsamples were treated as described above. Photoinhibition atdifferent light intensities was carried out by floating the leafsamples in Petri dishes that were sealed to light except for awindow in the dish lid through which the required PFD wasobtained by means of neutral density filters.

Chi Fluorescence Measurements

Changes in Fr during chilling at 0°C were measured usingan SF-10 fluorometer (Richard Brancker Research Ltd, Ot-tawa). Chl induction curves induced by red light (peak 680nm; PFD, 15 ,umol m-2 s-') were recorded on magnetic tapefor later replay via a fast A/D converter to an HP9826computer (Hewlett Packard, Sydney) programmed for calcu-lation of Fr.Induced Chl fluorescence of leaf samples frozen to 77 K

was measured using a bifurcated light-pipe system. Leaf sam-ples were attached to one end of a perspex rod, and after atleast 10 min in darkness the samples were immersed in liquidnitrogen. The rod was then attached to the bifurcated light-pipe. Actinic blue light (Corning 4-96 filter; 28 ,mol m-2 s-')was beamed via a shutter through one arm of the pipe andinduced Chl fluorescence emission from the leafsample meas-ured through the other arm by means of a photomultiplierprotected by a 693-nm interference filter and Corning 2-64red cut-off filter. Output from the photomultiplier was fed viaa fast A/D converter to the HP9826 computer, which wasprogrammed to record F0, F, and Fmax. Data collection wastriggered by opening the shutter, and, in order to have suffi-cient signal for direct coupling to the computer and to shortenthe time to reach Fmax, adequate actinic light intensity was

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CHILLING IN LIGHT

produced by using a blue wide-band filter rather than themore usual combination of blue filter plus narrow-band in-terference filter. This led to a small break through of actiniclight to the photomultiplier and control values for Fv/Fmaxreported here are 5 to 10% lower than values obtained usingthe two-filter combination (e.g. ref. 4).PFD (400-700 nm) was determined with a Quantum meter

Li- 185A (Li-Cor Inc. Lincoln, NE).

RESULTS

Chilling Tolerances of Fifteen Crop Species

Fifteen annuals showing a wide range ofchilling sensitivitieswere chosen for the investigations reported in this paper.

Included were two cultivars of bean and also two of rice, onea japonica type and the other an indica type. The relativesusceptibilities of these plants to chilling injury at 0°C afterbeing grown together under the growth conditions describedin "Materials and Methods" are shown in Figure 1. Valuesfor relative chilling tolerance of leaves chilled at 0°C in dark-ness and under high relative humidity ranged from a fewhours for the more chilling-sensitive species to severalhundred hours for the more chilling-resistant ones. Pigeonpea, lablab, sesame, cucumber, bean, tomato, and maize were

the most intolerant; millet, sorghum, and rice were interme-diate in tolerance; and broad bean, wheat, oat, barley, andpea were extremely tolerant. The most tolerant plant, pea,showed a 166-fold greater chilling tolerance than the mostsusceptible plant, pigeon pea.

tissue to a moderate PFD (300 .tmol m-2 s-') at 7°C for 20 h,then freezing the tissue in liquid nitrogen and measuring theinduced Chl fluorescence. The results, shown in Figure 2,were obtained using leaves taken from the same plants as

those used for the determinations of chilling tolerance (Fig.1). Control values for leaf tissue kept in darkness at 7°C for20 h are also given in Figure 2. These values did not differsignificantly from values for Fv/Fmax determined on freshlyharvested leafmaterial (data not shown). In the light, however,evidence of photoinhibition can be seen from the decrease inFv/Fmax. There was a definite trend towards enhanced photo-inhibition at 7°C in the chilling-sensitive plants comparedwith the chilling-resistant plants. Nonetheless, it is importantto note that all of the species tested, whether chilling-sensitiveor not, were photoinhibited under the experimental condi-tions used. The two cultivars ofP. vulgaris showed almost thesame susceptibilities to photoinhibition. The rices too showedsimilar susceptibilities with the indica rice being the more

sensitive of the two. This trend in rice, that of higher suscep-tibility in the indica type, was confirmed in a comparison ofseveral indica and japonica varieties (22).The decrease in Fv/Fmax was linear in cucumber for at least

30 h and in barley for 45 h (data not shown).

Photoinhibition at Chilling Temperatures in Attached andDetached Leaves

The measurements shown in Figures 1 and 2 were madeon discs or pieces cut from leaves detached from the plants.

Photoinhibition at 70C of Fifteen Crop Species

The relative susceptibilities of the different crop species tophotoinhibition at 7°C were determined by exposing leaf

PIGEON PEALABLABSESAME

CUCUMBERBEAN (cv. 1)BEAN (cv. 2)

TOMATOMAIZE

PEARL MILLETSORGHUMRICE (jap)

RICE (Indica)WHEAT

OATBARLEY

PEA

0 100 200 300 400

CHIWNG TOLERANCE (h)

MAIZEPEARL MILLET

LABLABCUCUMBER

SESAMESORGHUM

PIGEON PEABEAN (cv. 1)

TOMATOBEAN (cv. 2)

TAlUAI

PEARICE (indica)

RICE Gap)BROAD BEAN

WHEATBARLEY

500

Figure 1. Chilling tolerances of 15 crop annuals. Relative chillingtolerances were determined as the time for a 50% decrease ininduced Chl fluorescence at 00C in the dark as described under"Materials and Methods." Chilling tolerance values for the first sevenspecies (pigeon pea to maize) are multiplied by 10. Species are listedin order of increasing chilling tolerance. Bean cv 1 is Windsor LongPod and cv 2 is Redlands Pioneer.

I I I I I I

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Fv/FmFigure 2. Photoinhibition in chilled leaves of 15 species. Leaf tissuewas kept at 70C under a PFD of 300 umol m-2 s-' for 20 h asdescribed under "Materials and Methods." Dark controls were keptat 70C in darkness for 20 h. Photoinhibition is indicated by the extentof decrease in F,/F,. at 77 K. Species are arranged in order ofdecreasing susceptibility to photoinhibition. Beans cv 1 and cv 2 areas given in Figure 1.

PHOTOINHIBMON~I lDARK CONTROL

CHIWNG TOLERANCE* DARK, 0°CU* x1

U

UU

I

I

I

____j

II

--i

I

I

II

mI

I

I I I

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Plant Physiol. Vol. 90, 1989

It has already been demonstrated that the rate ofdevelopmentof dark chilling injury is the same in attached and detachedleaves provided both are chilled under the same experimentalconditions (25), but similar studies have not been reportedfor determinations of photoinhibition at chilling tempera-tures. In Table I it is shown that photoinhibition at 5°C is thesame in attached intact leaves of pigeon pea and in leaf discscut from leaves of similar age. For the whole plant only theexperimental attached leaf was maintained at 5°C while therest of the plant was kept at 20C. Both the experimentalattached leaf and leaf discs were chilled in a water-saturatedatmosphere. In attached leaves and leaf discs kept at 20C,values for Fv/Fmax remained high and photoinhibition wasslight. Data are also shown for an experiment with rice. Theextent of photoinhibition at 7°C in cut leaf pieces was com-parable to that in attached leaves. When kept in darkness at7C, neither leaf treatment showed a decrease in F,/Fma.

Comparison of Susceptibilities of Different Species toDark Chilling and to Photoinhibition at a ChillingTemperature

The data given in Figures 1 and 2 allow a direct comparisonto be made between the relative susceptibilities of the 15species to dark chilling injury on the one hand and to pho-toinhibition at a chilling temperature on the other hand.While chilling-sensitive plants were more susceptible thanresistant ones to both dark chilling and low-temperaturephotoinhibition, it is clear that the magnitudes of the differ-ences between chilling-sensitive and -resistant species for eachstress were very different. This is shown in Table II in whichis given the ratio of the chilling tolerance value (Fig. 1) to theextent of photoinhibition at 7C (Fig. 2) for each speciestested. While no functional meaning for this ratio is implied,the lack of any constancy in the ratio shows that a linearcorrelation does not exist between the two; in fact, for thefirst and last species listed in the table, these susceptibilitiesdiffer by more than two orders of magnitude ( 115-fold differ-ence for pea versus pigeon pea). From these results the con-clusion must be entertained that susceptibility of PSII to

Table I. Photoinhibition in Attached Leaves and Leaf PiecesPhotoinhibition, measured as FV/F,,, at 77 K, was compared in

attached leaves and leaf discs (1.3 cm dia.) of pigeon pea or cut leafpieces (2.0 cm in length) of rice (cv Er Bai Ai) as described in "Materialsand Methods." Attached leaves and leaf pieces were irradiated at aPFD of 275 (pigeon pea) or 300 (rice) MAmol m-2 s-' for 20 h at thetemperatures shown in the table. Plant temperature, except for theexperimental leaf, was 200C. n = 4 for pigeon pea and n = 6 for rice.Means ± SE are shown.

FV/F,,.Plant Experimental

Treatments Attached Leaf discs orleaves pieces

Pigeon pea 50C, light 0.24 ± 0.02 0.24 ± 0.02200C, light 0.68 ± 0.01 0.66 ± 0.02

Rice 70C, light 0.42 ± 0.01 0.43 ± 0.0170C, dark 0.74 ± 0.02 0.76 ± 0.01

200C, light 0.70 ± 0.02 0.70 ± 0.03200C, dark 0.79 ± 0.01 0.79 ± 0.01

Table II. Ratio of Susceptibility to Chilling in the Dark toSusceptibility to Photoinhibition at 7 °C

Ratios are values for chilling tolerance (Fig. 1) divided by values ofF,/F,. after photoinhibition at 70C (Fig. 2).

Plant Chilling TolerancePhotoinhibition at 7°C

Pigeon peaLablabSesameCucumberBeanaBeanbTomatoMaizePearl milletSorghumRicecRicedBroad beanWheatOatBarleyPea

10.514.714.915.511.914.121.149.2157141902174144877636311210

aWindsor Long Pod. b cv Redlands Pioneer. Ccv Asahi.d cv Dee-geo-woo-gen.

photoinhibition caused by chilling in light is not dependenton and perhaps is not even related to chilling sensitivity.

Temperature and Light Dependency of Photoinhibifton

As photoinhibition at 7C did not appear to be relateddirectly with chilling tolerance (Table II), the relationshipsbetween temperature, PFD, and photoinhibition were exam-ined in more detail. Figure 3 shows photoinhibition as afunction of temperature over the range of approximately 3 to21°C for two chilling-sensitive plants, cucumber and sesame,and two chilling-resistant ones, broad bean and pea. The PFDwas 275 jsmol m-2 s-' and the duration ofthe photoinhibitorytreatment was 20 h at all temperatures. The results confirmthat the chilling-resistant plants were susceptible to low-tem-perature photoinhibition though to a lesser extent than thechilling-sensitive plants and also showed that the photoinhi-bitory response to decreasing temperature was not linear butbecame increasingly severe as the temperature was decreased.

Figure 4 shows photoinhibition at 7°C in four species as afunction ofPFD. At low to moderate levels ofPFD, the extentof photoinhibition was linearly correlated with the PFD. Thefigure also shows that when the PFD rather than the temper-ature was varied, the two chilling-sensitive plants, cucumberand sorghum, were still more susceptible to photoinhibitionthan either oat or broad bean.

DISCUSSION

Dark Chilling Stress and Chilling in Light

The methods used in this study to compare respectivesusceptibilities to dark chilling stress and to photoinhibitioncaused by an interaction of light and chilling are based on

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CHILLING IN LIGHT

0.7

0.6 EA

0.5 -;, , . .

0L.4 -

CCM

0.3-

2 4 6 8 10 12 14 16 18 20 22

TEMPERATURE (°C)Figure 3. Photoinhibition as a function of the temperature in leafdiscs of cucumber, sesame, broad bean, and pea. Leaf discs wereirradiated with a PFD of 275 ,mol m-2 S-1 for 20 h at the temperatureindicated in the figure. Photoinhibition is indicated by the decrease ofF,/Fmae, at 77 K.

0.8

0.7 F

0.6 _

0.5 _

0.4 F

0.3 F

0.20 100 200 300

PFD (Amol m-2 s-1)Figure 4. Photoinhibition as a function of PFD for the chilling-sensi-tive plants cucumber and sorghum and chilling-resistant plants broadbean and oat. Leaf tissue at 70C was irradiated at PFD from 0 to 300gmol m-2 s-1 for 18 h. Values of r2 for linear correlations between Fv/Fmax and PFD were 0.98 for cucumber, 0.90 for sorghum, 0.99 forbroad bean, and 0.95 for oat.

studies previously published. Chilling leaves of various plantsat 0°C in the dark in a water-saturated atmosphere resultedin a progressive decrease in both the rate of rise, Fr, and thesize, F, of induced Chl fluorescence (20). The rate ofdecreasein Fr was related to the susceptibility of the species to chillinginjury. Thus the chilling-induced decline in Fr at 0°C hap-

pened rapidly in chilling-sensitive maize (7), bean, and cu-cumber (20) and, by comparison, much more slowly in chill-ing-resistant cabbage, kumquat, barley, wheat, and rye (20).Further, in cucumber leaves, the rapid decrease in F, observedat 0°C was not seen in leaves that were stored instead at ahigher, nonchilling temperature of 10°C (20). These experi-ments suggested that the rate of the decline in Fr duringchilling might be used as a measure of tolerances of leaves tochilling injury.Two kinds of studies were carried out in order to validate

the use of this experimental approach, namely, (a) chilling-induced changes measured by decreases in F, were comparedwith independent methods of assessing chilling injury, and(b) evidence for a relationship between the extent of thedecrease in Fr and plant chilling tolerance was sought bydetermining chilling-induced change in F, for a range ofclosely related plant species whose relative chilling tolerancescould be inferred either from the climate of their naturalhabitats or from their agricultural performances in differentclimates. Thus, the extent of the decrease in Fr of leaves ofchilled maize seedlings was shown to be correlated with twoindices of chilling damage, the degree of postchilling inhibi-tion of seedling growth and ratings for visual damage to thefoliage (6). Several studies have related changes in Fr at 0°Cto expected differences in chilling tolerance. In a study ofwildpotato species native to the Andean region of South America,a clear gradient was found of increasing cold tolerance asindicated by the rate of Fr decrease in chilled leaves and thealtitude at which the plants grew naturally (23). A highlandrace of maize also showed slower rates of Fr decrease duringchilling compared with races from warmer climates (7). In acomparison of commercial maizes, the line Corn Belt Dentwhich is cultivated in warm regions showed a faster declinein Fr with chilling than Northern Flint, a line which iscommonly included in hybrids used in cooler maize growingareas (7).As to the mechanism responsible for the decrease in Fr

during chilling, this can probably be attributed to inhibitionof Hill reaction activity. Induced Chl fluorescence, which attemperatures above freezing originates from PSII only, in-creases in yield as QA, the acceptor for PSII, becomes reduced.Thus any specific inhibition of PSII photoreductive activityshould result in a decrease in Fr. Consistent with this is theobservation that when intact tomato leaves were chilled fordifferent times and the chloroplasts then isolated, there was adecrease in photoreductive activity of the chloroplast mem-branes that was linearly related to the time for which theleaves were chilled (24).The use of Fv/Fmax decrease at 77 K as a measure of

photoinhibition of PSII is based on studies from a number oflaboratories, notably those of Bjorkman (2, 4) and Oquist(16). By carrying out the measurement at 77 K, damageoccurring only at the reaction centers of PSII or PSI isdetected; specificity for impairment to PSII is obtained by thechoice of 693 nm as the wavelength for the fluorescencemeasurements. The other commonly used indicator of pho-toinhibition is a decrease in the quantum yield for photosyn-thesis. For a range ofspecies whose leaves were photoinhibitedto different extents, the two measurements, that is decrease

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in quantum yield and decrease in Fv/Fmax at 77 K, were highlycorrelated (2).The results shown in Figure 2 cast doubt on the supposition

that photoinhibition of PSII at low temperatures is attribut-able to chilling stress. Cellular injury resulting from chillingin the absence of light is generally confined to chilling-sensi-tive plants at temperatures below about 1 2°C (13). Yet, leavesof chilling-resistant plants began to develop photoinhibitionat a moderate PFD of275 ,umol m-2 s-' at temperatures below1 8°C (Fig. 3). Further, while chilling-sensitive plants generallyshowed greater susceptibility (Fig. 2), photoinhibition of cu-cumber and sesame was not confined to the chilling-sensitivetemperature range and was evident both above and below12°C (Fig. 3). The lack ofa linear relationship between chillingstress in the dark and in the light for the species examined(Table II) again indicates that photoinhibition at low temper-ature is not specifically associated with or caused by thedevelopment of chilling injury. This point is further exempli-fied by a comparison of the extent of photoinhibition forselected individual species. Photoinhibition at 7°C was com-parable in tomato, bean, oat, and pea, yet the last two plantsare extremely chilling-resistant compared with tomato andbean based on either the determined values for dark chillingtolerance (Fig. 1) or their widely acknowledged differences insusceptibility to chilling injury.

Consistent with the separation ofphotoinhibition at chillingtemperatures with chilling stress in the absence of light areobservations that the sites of primary damage to the photo-synthetic electron pathway are different depending on whetherthe exposure to chilling is made in darkness or in light. Bothstresses, dark chilling and chilling in light, cause decreases ininduced Chl fluorescence (in dark [20], in light [28]) andinhibition ofPSII activity (in dark [24], in light [8]). However,the response of induced Chl fluorescence at 77 K to these twostress conditions is quite different. In maize, the induced Chlfluorescence of leaves was drastically reduced by an exposureto 0°C in the dark for 24 h, but if the leaves were then frozento 77 K and induced Chl fluorescence again measured, therewas no decrease in Fv/Fma below that of control, unchilledtissue, thus indicating that no damage in fact had occurred tothe reaction center of PSII (21). In contrast, Fv/Fmax rapidlydeclined in leaves chilled in light (Fig. 2). This suggests chillingin light damages the reaction center of PSII (and also of PSI),while dark chilling inhibits the supply of electrons to thereaction center. 'Dark' chilling injury then, which presumablyoccurs in the light as well as in the dark, is mainly confinedto chilling-sensitive plants exposed to temperatures belowabout 12°C. In the light, however, additional stress damagemay take place, specifically to the photosystem reaction cen-ters, in both chilling-sensitive and -resistant plants. Thegreater susceptibility of the former group of plants to chillingin light (Figs. 2 and 3) may be due to a greater sensitivity tolight rather than to their obviously greater susceptibility tochilling stress, especially as differences between the two groupsof plants were maintained over a range of light intensities(Fig. 4). Further studies are required to establish the physio-logical basis for this difference and also to explain why sus-ceptibility to photoinhibition increases with decreasingtemperature.

Implications for Cultivation in the Field

A major finding of this study was that a wide range ofannual crop plants originating from both warm and temperateclimates are subject to photoinhibition at low temperatures.As photoinhibition has the potential to decrease photosyn-thetic productivity, this result appears to have some importantimplications for agriculture. At high risk are plants introducedto temperate climates from the tropics, examples being to-mato, various cucurbits, beans, maize, and rice. These plantsare generally more susceptible to photoinhibition at chillingtemperatures than plants from cooler climes (Fig. 2) and inthe field following a decrease in temperature, photoinhibitorydamage may be exacerbated by chilling-induced dehydrationof the leaves. Water stress, like low temperatures, rendersphotosynthetic tissues more susceptible to photoinhibition(17), and chilling-sensitive plants are particularly susceptibleto water loss when first exposed to chilling temperatures (14).This complication was avoided in the present experiments bycarrying out photoinhibitory treatments at close to watersaturation, but it may be an important factor in some circum-stances in the field, for instance with the onset of cool, drywinds.

Studies ofplants in controlled environments have indicatedthe potential role of the interaction of light and chilling inlimiting photosynthetic productivity in the field. Based onmeasurements of photoinhibition which developed in leavesof tomato plants kept under moderate light and low temper-atures approximating those prevailing during a Mediterraneanwinter, Yakir et al. (30) concluded that photoinhibition is animportant factor limiting the productivity of tomatoes andother chilling-sensitive plants in winter. Bongi et al. (1) studiedthe decrease in quantum yield in controlled low temperatureenvironments of leaves of 3-month-old olive plants. Theyconcluded that while the leaves of this evergreen crop areapparently less susceptible to photoinhibition during chillingstress than short-lived leaves ofchilling-sensitive annual crops,photoinhibition nonetheless is potentially a major influenceon photosynthetic productivity of olives cultivated in a Med-iterranean or similar climate. While published accounts ofactual photoinhibitory damage in crops appear to be lacking,we have recently recorded photoinhibition during winter inleaves of several citrus species growing near Gosford, NewSouth Wales (330 25' S) and also in rice growing at Yanco,New South Wales (34° 33' S) in midsummer following thepassage of a cold front (SE Hetherington, RM Smillie, J He,and M Lipucci di Paola, unpublished results).Of particular interest is the finding that major chilling

resistant crops such as wheat and barley are susceptible tophotoinhibition if the temperature is low enough (Fig. 2).Even extremely frost-tolerant species can apparently be simi-larly affected as evidenced by the development of photoinhi-bition coinciding with the onset of winter in needles of Pinussylvestris (10). Further studies are needed to assess whetheror not photoinhibition induced by low temperatures caninfluence yields oftemperate crops cultivated in cool climates.

ACKNOWLEDGMENT

The technical assistance of Ms. R. Nott is gratefully acknowledged.

1614 HETHERINGTON ET AL.

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CHILLING IN LIGHT

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