doi:10.1093/aob/mcg020, available online at www.aob.oupjournals.org

Low Temperature Enhances Photosynthetic Down-regulation in French Bean(Phaseolus vulgaris L.) Plants

TSONKO TSONEV1, VIOLETA VELIKOVA1, KATYA GEORGIEVA1, PAUL F. HYDE 2 and

HAMLYN G. JONES 2 ,*

1Acad. M. Popov Institute of Plant Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, Bl. 21, 1113

So®a, Bulgaria and 2Division of Environmental and Applied Biology, Biological Sciences Institute, University of

Dundee, Dundee DD1 4HN, UK

Received: 3 July 2002 Returned for revision: 24 September 2002 Accepted: 21 October 2002 Published electronically: 12 December 2002

The mechanisms of photosynthetic adaptation to different combinations of temperature and irradiance duringgrowth, and especially the consequences of exposure to high light (2000 mmol m±2 s±1 PPFD) for 5 min, simulat-ing natural sun¯ecks, was studied in bean plants (Phaseolus vulgaris L.). A protocol using only short (3 min)dark pre-treatment was introduced to maximize the amount of replication possible in studies of chlorophyll ¯uor-escence. High light at low temperature (10 °C) signi®cantly down-regulated photosynthetic electron transportcapacity [as measured by the ef®ciency of photosystem II (PSII)], with the protective acclimation allowing thesimulated sun¯ecks to be used more effectively for photosynthesis by plants grown in low light. The greaterenergy dissipation by thermal processes (lower Fv¢/Fm¢ ratio) at low temperature was related to increased xantho-phyll de-epoxidation and to the fact that photosynthetic carbon ®xation was more limiting at low than at hightemperatures. A key objective was to investigate the role of photorespiration in acclimation to irradiance andtemperature by comparing the effect of normal (21 kPa) and low (1´5 kPa) O2 concentrations. Low [O2]decreased Fv/Fm and the ef®ciency of PSII (FPSII), related to greater PSII down-regulation in cold pre-treatedplants, but minimized further inhibition by the mild `sun¯eck' treatment used. Results support the hypothesisthat photorespiration provides a `safety-valve' for excess energy. ã 2003 Annals of Botany Company

Key words: Phaseolus vulgaris L., bean, low temperature, photosynthesis, down-regulation, high light, sun¯eck,chlorophyll ¯uorescence.

INTRODUCTION

In many habitats, crop and native plants are periodicallyexposed to low temperatures; this can signi®cantly reducetheir productivity (e.g. Buis et al., 1988; Fryer et al., 1995;Greaves, 1996; Haldiman, 1998). Among the many physio-logical mechanisms responsible for this is the potential fordamage to the photosynthetic apparatus caused by high light(Baker, 1994; Jones and Demmers-Derks, 1999). A com-bination of low temperature and light stress is thought tolead to photoinhibition and photo-oxidation since thenormal route for dissipation of absorbed radiant energy byphotosynthetic carbon metabolism is inhibited by lowtemperature (Baker, 1996). Although the reductions in theef®ciency of electron transport through photosystem II(PSII) that have been observed under such conditions havebeen termed photoinhibition, this term is now morecommonly reserved for actual long-term damage to thephotosynthetic system, including the D1 protein, or otherreductions in ef®ciency not reversible over an hour or so(e.g. Maxwell and Johnson, 2000). In many cases, thereduction in ef®ciency of electron transport through PSII ismore appropriately considered to be an acclimatory down-regulation involving reversible metabolic adjustment to thenew conditions, reducing the production of potentially

damaging reductants. This down-regulation can be readilyreversible as in the case of some DpH-mediated high energyquenching or state transitions (Horton et al., 1996), or it maydecay more slowly over periods from 30 min to severalhours.

The capacity of plants to tolerate low temperatures, and toadapt to them, varies among plant species and cultivars. Theability of French bean plants to tolerate short periods of highirradiance (simulating natural sun¯ecks) under conditionswhen utilization of the reducing power generated byphotosynthetic electron transport in carbohydrate produc-tion and growth is limited by low temperatures is investi-gated in this paper. Absorption of excess light can bedeleterious because it leads to increased production ofsinglet oxygen and reduced reactive oxygen species; thesereact with, and damage, proteins and other components ofphotosystems leading to true photoinhibitory damage.Critical conditions of low temperature and high light arecommonly experienced by arctic or alpine species in theirshort summer (Jones and Demmers-Derks, 1999) and bycrop species in temperate zones on cold spring mornings. Insuch situations, controlled dissipation of excess excitationenergy absorbed by the chlorophylls is essential to protectthe photosynthetic apparatus. French bean was chosen forthe present study because there is a substantial backgroundliterature on temperature/photoinhibition responses of thisspecies, which provides a good comparative basis for the

* For correspondence. Fax +44 (0)1382 344275, e-mail h.g.jones@dundee.ac.uk

ã 2003 Annals of Botany Company

Annals of Botany 91: 343±352, 2003

present extension to realistic sun¯eck treatments.Furthermore, it is an important crop whose growth intemperate regions is signi®cantly restricted by sensitivity tolow temperatures (e.g. Nishida and Murata, 1996). Thissensitivity is compounded by photoinhibition at high light(as shown here and elsewhere). Nevertheless, the sensitivitycan be modulated and there is good evidence that Phaseoluscan undergo substantial hardening or acclimatization to lowtemperatures (e.g. El-Saht, 1998). There is also geneticvariability in tolerance to low temperatures (Melo et al.,1997). The prime purpose of this paper, however, was toinvestigate the interacting effects of light environment andtemperature on photosynthesis.

Factors that enhance photoinhibition at low temperatureinclude reduced utilization of energy in photosyntheticcarbon metabolism; restricted rates of D1 protein turnover;decreased xanthophyll cycle activity; and inadequate ratesof removal of active oxygen species (Krause, 1994a). Theeffect of oxygen on the separate processes of photoinhibi-tion and photosynthetic down-regulation is poorly under-stood because of its dual role (Krause, 1994b). On the onehand it has protective effects through photorespiration andthe Mehler reaction (e.g. Osmond and Grace, 1995),although Ruuska et al. (2000) have suggested that theMehler reaction may have less effect than once thought. Onthe other hand, oxygen promotes photoinhibition mainly bytriggering D1 degradation and by increasing the probabilityof forming reactive oxygen species such as the superoxideanion radical (Brestic et al., 1995; Wiese et al., 1998).

Another important aspect of down-regulation is thedegree to which acclimatization to cold may enhanceprotective mechanisms. Acclimatization of the photosyn-thetic apparatus to low temperature and to high light is welldocumented but the mechanisms are still unclear. Theseinclude increased capacity of carbon assimilation at lowtemperature and increased activities of scavenger systems.OÈ quist et al. (1993a, b) reported that irrespective oftemperature (0±25 °C) or the state of cold-hardiness, thesusceptibility of photosynthesis in cereals to photoinhibitionis controlled by the redox state of QA, the primary PSIIacceptor. These authors suggested that the primary reasonfor photoinhibition at low temperature is an increase in theproportion of closed reaction centres due to slower rates ofCO2 assimilation at low temperature.

Many previous studies of the effects of high light at lowtemperatures have either used plants grown in growthchambers at unrealistically low irradiances (much lowerthan those in most natural environments), or have used`photoinhibitory' treatments with much higher irradiancesthan expected in natural environments (where irradiances ofapprox. 2000 mmol m±2 s±1 are rarely exceeded). Therefore,the main objectives of this study were to investigate thecapacity and control of photosynthetic down-regulation inbean plants grown at irradiances representative of naturalenvironments and in response to realistic simulatedsun¯ecks. Responses were compared in plants grown atnormal (22 °C) and low (10 °C) temperatures afteracclimation to different growth irradiances for 6 d. Aparticular aim was to determine the relative importance ofdifferent fates for the absorbed radiative energy, and

especially the potential role of oxygen and photorespirationin modulating the response of photosynthetic apparatus inplants pre-treated in cold conditions.

MATERIAL AND METHODS

Bean plants (Phaseolus vulgaris L. `Arcadia') were grownin 15 cm pots containing standard horticultural soil in aglasshouse at 22 °C. After 15 d, plants were divided intogroups and were kept for 6 d in a Sanyo (970/C/HQI;Gallenkamp, Loughborough, UK) growth chamber with 12 hdays under the following temperature (°C) and light (PPFD,mmol m±2 s±1) regimes: 10/100, 10/1000, 22/100 and22/1000. Relative humidity was maintained close to 60 %.The high irradiance treatment was more representative ofmany ®eld environments than the more usual growthchamber environments of 350 mmol m±2 s±1 or less (e.g.OÈ quist et al., 1992; OÈ quist and Huner, 1993). Chlorophyll¯uorescence and gas exchange were measured 2, 4 and 6 dafter moving plants into the chamber. Pigment contents andcatalase and peroxidase activities were determined at theend of the treatment period. The effect on photosynthesis ofa 5-min period of high irradiance (2000 mmol m±2 s±1),simulating a sun¯eck, was studied on the sixth day. Thisirradiance is close to the maximum likely to be experiencedby a leaf exposed to full sunlight (Jones, 1993). Twoindependent experiments for each growth temperature werecarried out giving four to eight replications for all variablesmeasured. Staggered plantings allowed further replicationson other dates in each experiment.

Chlorophyll ¯uorescence

Chlorophyll ¯uorescence was measured using a modu-lated system (FMS; Hansatech Ltd, Kings Lynn, UK). Initial(F0) and maximal (Fm) ¯uorescence yields were measuredon leaves that had been dark adapted for 30 min in thegrowth chamber. Other parameters of chlorophyll ¯uores-cence kinetics were measured in separate experiments usingthe environmentally controlled leaf chamber of a portablephotosynthesis system (Ciras-1; PP Systems, Hoddesdon,UK) at normal (21 kPa) or decreased (1´5 kPa) concentra-tions of O2, and at normal (22 °C) and low (10 °C)temperature using the following protocol: after insertion ofthe leaf in the chamber there was an initial 4-minillumination at the appropriate growth irradiance (100 or1000 mmol m±2 s±1), at the end of which CO2 assimilationand steady-state ¯uorescence (Fs) were determined and asaturating pulse (approx. 5000 mmol m±2 s±1) was given toobtain Fm¢ (quenched maximal ¯uorescence). The actiniclight was switched off and a 5-s pulse of weak far-red light(735 nm) was applied to give F0¢ (light-adapted initial¯uorescence) for calculation of FPSII (the quantum yield ofPSII). After 3 min dark adaptation and measurement of F0

followed by Fm, the sample was treated with high light(2000 mmol m±2 s±1) for 5 min, at the end of which CO2

assimilation, Fs, Fm¢ and F0¢ were measured as before. Aftera further 30 s in the dark, a saturating pulse was applied,followed by 5 s of weak far-red light to estimate Fm and F0.On the sixth day only, the dark recovery period was

344 Tsonev et al. Ð Low Temperature and Photosynthesis Down-regulation

extended by the use of saturating ¯ashes followed by far-redpulses after further periods in the dark of 0´5, 1, 2, 4, 8, 8and 11 min (giving a total of 35 min recovery). Gas mixtureswere obtained using Hastings Mass Flow controllers(Hampton, VA, USA) and stored in PVC beach balls untilrequired, with CO2 partial pressure maintained at 31 Paexcept where CO2-response curves were studied.

A 3-min initial dark adaptation was used to facilitatereplication; this gave generally similar Fv/Fm values to themore usual 20±30 min treatment (e.g. compare Fig. 1 andTable 1), although there was some evidence that it couldunderestimate Fv/Fm. In most cases, however, the use of a

3-min dark period led to only a 2 % reduction in Fv/Fm

compared with that obtained following a 30-min dark period(Fig. 3), although the underestimation did rise to about 9 %for the low temperature/high light treatment, presumablydue to slow relaxation.

CO2 exchange

Rates of CO2 assimilation and stomatal conductance weremeasured using the portable photosynthesis system (Ciras-1) 2 and 4 d after transfer of the plants to the growthchamber under irradiances (measured within the leafcuvette) corresponding to growth light at normal (21 kPa)and decreased (1´5 kPa) concentrations of O2, and at normal(22 °C) and low (10 °C) temperatures. Light- (seven pointswith 4 min adaptation) and CO2-response (11 points with3 min adaptation) curves were measured under the sameconditions on the sixth day. Respiration rates measured after35 min in the dark at the two O2 concentrations used werewithin 5 % of each other at both experimental temperaturesand did not differ signi®cantly.

Pigment extraction and analysis

Leaves were killed in liquid nitrogen, ground to a powderand stored at ±10 °C until required. Sub-samples (50 mg) offrozen shoot material were added to 500 ml 100 % methanolin a 1´5 ml Eppendorf vial and mixed for 1 min. A further500 ml 100 % methanol was added and the mixture mixedfor 1 min, after which it was spun down for 10 min at11 500 rpm. The supernatant was decanted and the resultantpellet resuspended in 500 ml 100 % methanol, mixed for1 min, and spun down for 10 min at 11 500 rpm. This wasrepeated once more, giving an extraction dilution of 25 mgf. wt shoot material per ml methanol. Samples were stored at±30 °C until analysis.

Chlorophylls a and b, b-carotene, lutein, neoxanthin,violaxanthin, antheraxanthin and zeaxanthin were quanti-®ed by high pressure liquid chromatography using aprotocol based on that of Tsavalos et al. (1993). Thereversed-phase system consisted of a Waters C18 radialcompression module (4 mm; 8 3 100 mm) with a 200-mmC18 guard column using a solvent gradient of 10±100 %ethyl acetate in acetonitrile : water (9 : 1,v/v) over 25 min ata ¯ow rate of 1´0 ml min±1. Violaxanthin, antheraxanthin

F I G . 1. Variable to maximal chlorophyll ¯uorescence ratio (Fv/Fm) after6 d growth at low (10 °C) or high (22 °C) temperature, and either low(100 mmol m±2 s±1) or high (1000 mmol m±2 s±1) irradiance (during the12 h days). Measurements were made after 3-min dark adaptation andunder a normal (21 kPa) or decreased (1´5 kPa) concentration of O2 inthe air. Black and white columns: measurements made at the sametemperature as the growth temperature; hatched and stippled columns:measurements made at the contrasting temperature. Mean values 6 s.e.

of six replications from three independent experiments are presented.

TABLE 1. Variable to maximal chlorophyll ¯uorescence ratio (Fv/Fm), and initial (F0) and maximal (Fm) chlorophyll¯uorescence measured on the sixth day of treatment after 30-min dark adaptation at corresponding growth conditions

Tmeas. =Tgrowth (°C)

Imeas. = Igrowth

(mmol m±2 s±1)Fv/Fm

(relative units)F0

(relative units)Fm

(relative units)

10 100 0´815 6 0´0035b 56 6 1´2b 304 6 3´2c

10 1000 0´748 6 0´0094a 54 6 1´1b 219 6 9´6a

22 100 0´849 6 0´0016c 50 6 0´9b 330 6 7´2d

22 1000 0´837 6 0´0032bc 38 6 1´1a 241 6 7´0b

Mean values 6 s.e. of 12 replications from three independent experiments are presented. Values followed by the same letter do not differsigni®cantly at P = 0´05 according to the LSD test.

Tsonev et al. Ð Low Temperature and Photosynthesis Down-regulation 345

and zeaxanthin peaks were identi®ed and quanti®ed withstandard pigments supplied by VKI (Agern Alle 11, DK-2970 Hùrsholm, Denmark). All other pigments werecollected by fractionation, and identi®ed and quanti®edspectrophotometrically using reference spectra and extinc-tion coef®cients (Jeffrey et al., 1997). The de-epoxidationstate (deps) of the xanthophyll cycle pigments, violaxanthin(V), antheraxanthin (A) and zeaxanthin (Z) was calculatedas: deps = (A + Z)/(V + A + Z) (see Demmig-Adams andAdams, 1996).

Statistical analysis

Analysis of variance and other statistical tests wereconducted using Minitab 12.1 (Minitab Inc., StateCollege, PA, USA). Differences between treatmentswere assessed using ANOVA and least signi®cantdifferences (Snedecor and Cochrane, 1999).

RESULTS

Chlorophyll ¯uorescence

The photochemical ef®ciency of PSII, assessed as Fv/Fm

after a 30-min dark pre-treatment under normal growthconditions (Table 1) was close to the expected maximum forplants grown at low light. However, at least at low

temperature, there was evidence for photoinhibition ordown-regulation at high light, resulting mainly from adecrease in Fm with non-signi®cant changes in F0.

Figure 1 summarizes the effects of growth tempera-ture, measurement temperature, irradiance and oxygenconcentration on Fv/Fm measured using a 3-min darkperiod. Results were generally similar to those obtainedusing the standard 30-min pre-treatment (compare Fig. 1and Table 1; detailed results not shown) so allsubsequent data presented use the abbreviated 3-mindark protocol to allow maximal replication. As therewere few signi®cant trends over the 6 d (except forplants in the high temperature/high light treatment inwhich Fv/Fm increased from 0´76 on day 2 to 0´84 onday 6; P < 0´05), data for on the 3 d of measurementshave been averaged. The key observation was that Fv/Fm measured in 1´5 kPa O2 was consistently lower (by2±9 %) than that measured in normal oxygen (maineffect P < 0´01).

The further inhibitory effect of 2000 mmol m±2 s±1 PPFDfor 5 min is shown in Fig. 2 where the ef®ciency of the openPSII reaction centres, Fv¢/Fm¢, is plotted as a percentage ofthe Fv¢/Fm¢ ratio before the high-light treatment. It is evidentthat the decline of Fv¢/Fm¢ was greater for plants grown atlow PPFD and that the inhibition was less at low O2

concentration than at normal O2.In all treatments the quantum yield of PSII (FPSII,

Table 2) was smaller when measured at low O2 concentra-tion, and was much greater at low light compared with highlight. However, after the simulated sun¯eck treatment, FPSII

was lower in the low-light-adapted than the high-light-adapted plants. The reduction in FPSII averaged 86 % forlow-light-adapted plants and only 44 % for the high-light-adapted plants.

In several cases an enhanced steady-state chlorophyll¯uorescence yield (Fs) was observed in plants grown in lowlight at the end of the 5-min high-light treatment, with theaverage increase being 27 6 3 % (P = 0´0056). There were,however, no differences in the responses at low and normalO2 concentrations (data not shown). There were also nodifferences in photochemical quenching (qP) betweenplants grown at low and high light, but qP was lower (by20 %) when measured at low O2 concentration (Table 2).The non-photochemical quenching (NPQ) was greatlydecreased by high light, especially at low temperatures,and was also decreased at low O2 concentration by anaverage of 32 % (Table 2).

Relaxation of Fv/Fm after the 5-min sun¯eck treatmentwas rapid and was more or less complete within 20±30 min(Fig. 3), indicating that any effect of the treatment on Fv/Fm

represented true down-regulation rather than photoinhibi-tory damage. The slightly higher ®nal values of Fv/Fm

compared with those prior to the photoinhibitory treatmentprobably result from the short dark period (3 min) used forthe ®rst (reference) Fm measurement. A characteristicdistinction in the relaxation between plants grown at lowand high temperature is the difference between treatments inthe ®rst case (P < 0´01) and the lack of differences in thesecond. The rate of recovery of PSII upon darkening wasclearly faster at high temperatures.

F I G . 2. Fv¢/Fm¢ at the end of a 5-min `sun¯eck' treatment (2000 mmolm±2 s±1 PPFD) as a percentage of the value before treatment for plantsgrown at low (10 °C) or high (22 °C) temperatures and under normal(21 kPa) or decreased (1´5 kPa) concentrations of O2 in the air. Blackand white columns: measurements made at the same temperature as thegrowth temperature; hatched and stippled columns: measurements madeat the contrasting temperature. Mean values 6 s.e. of six replications

from three independent experiments are presented.

346 Tsonev et al. Ð Low Temperature and Photosynthesis Down-regulation

Gas exchange

The changes in net photosynthetic rate before and afterthe 5-min high light treatment (Fig. 4) generally correlatewith the observed changes in FPSII, except for plants grownat 10 °C and treated at 22 °C whose photosynthetic rate waslow. This was related to the abnormally low stomatalconductances achieved in these particular treatments. Forexample, the 10/22 °C plants had a conductance of45 6 10 mmol m±2 s±1 compared with 460 6 50 mmol

m±2 s±1 in the plants grown and measured at 22 °C. The netphotosynthetic rate measured at 2000 mmol m±2 s±1 in plantsgrown at 22 °C and low light was lower than that in plantsgrown at the same temperature under high light.

Pigment content

For plants grown at both high and low temperatures for 6 dthe total Chl content was signi®cantly lower in plants grown

F I G . 3. Time course of recovery of Fv/Fm after 5-min `sun¯eck' treatment (2000 mmol m±2 s±1 PPFD) A, Growth temperature: 10 °C. B, Growthtemperature: 22 °C. Mean values 6 s.e. of six replications from three independent experiments are presented.

TABLE 2. Steady-state quantum yield of PSII under growth conditions (FPSII-steady), together with values of FPSII, qP andNPQ (FPSII*, qP* and NPQ*) at the end of the 5-min high-light treatment

Tgrowth

(°C)Igrowth

(mmol m±2 s±1) O2 (kPa)FPSII-steady

(relative units)FPSII*

(relative units)qP*

(relative units) NPQ*

10 100 21 0´63 6 0´01 0´07 6 0´01 0´17 6 0´02 2´15 6 0´3510 100 1´5 0´41 6 0´08 0´06 6 0´00 0´15 6 0´01 1´50 6 0´5910 1000 21 0´15 6 0´02 0´14 6 0´02 0´23 6 0´08 0´61 6 0´4110 1000 1´5 0´12 6 0´01 0´10 6 0´00 0´19 6 0´01 0´24 6 0´1122 100 21 0´78 6 0´01 0´13 6 0´01 0´30 6 0´09 2´48 6 0´4822 100 1´5 0´75 6 0´01 0´10 6 0´01 0´25 6 0´04 2´40 6 0´1622 1000 21 0´43 6 0´05 0´18 6 0´02 0´55 6 0´08 2´14 6 0´1222 1000 1´5 0´26 6 0´02 0´12 6 0´03 0´37 6 0´23 1´40 6 0´13

All values ( 6 s.e.) were averaged over the 3 d of measurement at O2 concentrations of either 21 or 1´5 kPa.

Tsonev et al. Ð Low Temperature and Photosynthesis Down-regulation 347

at high light than those grown at low light (Table 3). Theratio of chlorophyll a/b did not change signi®cantly as aresult of low-temperature or high-light treatment (data notshown). Concentrations of total carotenoids and totalxanthophyll were greater for plants grown at low tempera-ture. There were much larger increases in antheraxanthinand zeaxanthin in plants in the high light treatmentsresulting in a clearly enhanced de-epoxidation state, withdeps reaching 0´78 at high light and low temperature.

Figure 5 shows the relationship between some character-istics of chlorophyll ¯uorescence and the deps state of thexanthophyll cycle for the different treatments. A closeinverse correlation (R2 = 0´98) existed between the quantumyield of PSII and deps, as well as between the ef®ciency ofopen PSII units (Fv¢/Fm¢) and deps. There was also a verystrong correlation (R2 = 0´99) between F0¢/Fm¢ and the de-epoxidation state of the xanthophyll cycle.

DISCUSSION

A combination of gas-exchange, chlorophyll ¯uorescenceand biochemical analysis was used to examine changes inphotosynthetic responses of French bean plants grown atlow or high light to a simulated sun¯eck treatment, and theinteractions of these responses with temperature and oxygenconcentration. The experiment was designed to mimic theeffect of sun¯ecks on such plants in their natural environ-ments, and to determine whether temperature or growthirradiance affect the photosynthetic responses to suchsun¯ecks. Another aim was to determine whether anyphotosynthetic responses observed were a consequence ofphotoinhibitory damage to the system or whether theyrepresent protective down-regulation, and if this depends ongrowth temperature. The rapid recovery of most photosyn-thetic variables, including Fv/Fm, on darkening indicates

F I G . 4. Net photosynthetic rate at growth light and at the end of 5-min `sun¯eck' treatment (2000 mmol m±2 s±1 PPFD) measured under a normal(21 kPa) or decreased (1´5 kPa) concentration of O2. Mean values 6 s.e. of six replications from three independent experiments are presented.

348 Tsonev et al. Ð Low Temperature and Photosynthesis Down-regulation

that the observed responses to the sun¯ecks largely re¯ectedpositive regulatory responses which regulate the utilizationand dissipation of incident radiant energy, rather thandamage to PSII.

A chronic decrease in the ef®ciency of photosyntheticelectron transport through PSII (as indicated by Fv/Fm) wasfound in bean plants grown at high light and low tempera-ture (Fig. 1), with this effect being largely attributable to thegreater reduction in Fm than F0 (Table 1). This can beinterpreted as an increase in the rate constant for thermaldissipation (Vavilin et al., 1995; Saccardy et al., 1998) and/or inactivation of reaction centres of PSII (Le Gouallec et al.,1991). The general reduction in both Fm and F0 in the high-light conditions indicates changes in antenna size whichminimize absorbance of incident light (Verhoeven et al.,1996; Neidhardt et al., 1998). Following the simulatedsun¯eck treatment a further large decrease in quantumef®ciency of PSII was observed, especially at low tempera-ture. Note that any effect of this sun¯eck treatment had beentotally reversed within 20±30 min (Fig. 3).

Several factors could have enhanced down-regulation atlow temperature. The main factor is likely to be the greatlyreduced capacity for utilization of incident radiant energy inphotosynthetic carbon metabolism (Fig. 4), indicated by thefact that, following a ten-fold increase in incident light, netassimilation only doubled at low temperature comparedwith a nearly nine-fold increase at 22 °C (Fig. 4). Leavescould, therefore, use nearly all the extra energy at hightemperature but not at low temperature. Other possiblefactors include changes in respiratory metabolism (Mawson,1993), decreased activity of the xanthophyll cycle, insuf®-cient scavenging of active oxygen (Krause, 1994a) and areduced rate of enzymatic PSII repair processes (Aro et al.,1990). The decrease in the quantum yield (FPSII) observedafter the sun¯eck treatment at low temperature indicates thata decreased fraction of excitation energy absorbed by PSIIantennae is utilized in PSII photochemistry, while thereduced ef®ciency of open PSII units (Fv¢/Fm¢) is evidencefor enhanced energy dissipation in light-harvesting antennae(Schreiber et al., 1994; Demmig-Adams and Adams, 1996).

An important aspect of the present experiments wastreatment with 1´5 kPa O2 to inhibit photorespiration (PR)but not normal respiratory metabolism or the Mehlerreaction (Brestic et al., 1995; Wiese et al., 1998), underthe hypothesis that the loss of photorespiration as a sink forelectrons might lead to photoinhibitory damage or enhanceddown-regulation (see Ort and Baker, 2002). It is uncertain,however, whether low oxygen actually decreases electron¯ow, or whether all energy is diverted on a one-to-one basisinto photosynthetic metabolism (Sharkey and Vassey, 1989;Osmond and Grace, 1995). In all treatments, the ratiosFv/Fm and Fv¢/Fm¢ measured under 1´5 kPa O2 were smallerthan those measured at normal O2 concentration (Fig. 1),suggesting that photorespiration does indeed provide asigni®cant `safety-valve' for excess energy that cannot bereplaced by compensating increased CO2 ®xation (Ort andBaker, 2002). Interestingly, however, the further decrease inFv/Fm after 5 min high-light treatment (Fig. 2) was greaterat 21 kPa O2, with NPQ being smaller. Nevertheless, theabsolute value of Fv/Fm remained smaller at low O2

concentrations. According to Goh et al. (1999) the ¯uores-cence-derived rates of electron transport do not necessarilycorrespond with the rate of CO2 ®xation, and this appears tobe the case here. Other forms of electron transport through

F I G . 5. Relationship between the chlorophyll ¯uorescence variablesF¢v/F¢m, F¢0/F¢m, FPSII and qP, and a measure of the de-epoxidation stateof the xanthophyll cycle components (A+Z)/(V+A+Z). Mean values 6

s.e. are presented.

TABLE 3. Quantities of chlorophyll [in mol g±1 f. wt (3104)], b-carotene and xanthophylls (lutein, violaxanthin,antheraxanthin, zeaxanthin, neoxanthin) per total Chl and the de-epoxidation state [(A + Z)/(A + V + Z)]

Tgrowth

(°C) Igrowth Chlorophyll b-Carotene Lutein Violaxanthin Antheraxanthin Zeaxanthin Neoxanthin(A + Z)/

(A + V + Z)Total

xanthophyllsTotal

carotenoids

10 100 30´6b 0´053b 0´103b 0´017a 0´006b 0´009a 0´028b 0´464b 0´163bc 0´216ab

10 1000 23´5a 0´036a 0´097b 0´015a 0´017c 0´035c 0´025ab 0´777c 0´188c 0´224b

22 100 31´4b 0´048b 0´098b 0´012a 0´001a 0´004a 0´023ab 0´294a 0´138ab 0´186a

22 1000 25´7a 0´054b 0´076a 0´017a 0´007b 0´013b 0´020a 0´534b 0´132a 0´186a

s.e.d. 1´70 0´008 0´008 0´003 0´002 0´003 0´003 0´051 0´012 0´018

Standard errors of difference within columns (s.e.d.) were calculated from ANOVAs (four to ®ve replicates). Within a column, different superscriptsindicate values that are signi®cantly different (LSD at P < 0´05).

Tsonev et al. Ð Low Temperature and Photosynthesis Down-regulation 349

PSII, such as the Mehler±ascorbate±peroxidase cycle,which depend on O2 as the electron acceptor (Schreiberet al., 1995; Asada et al., 1998; Ort and Baker, 2002) couldalso contribute to the O2-sensitivity of the Fv/Fm ratio. Thisis supported by data for single guard cells (Goh et al., 1999).It is notable that no signi®cant differences were observed inO2 sensitivity of Fv/Fm at low (10 °C) or normal (22 °C)temperature when measured at normal CO2 concentration.Although the effect of reduced CO2 concentrations was notinvestigated in the present study, Savitch et al. (2000) usedcombinations of such treatments with reduced O2 concen-trations to show that both photorespiration and the Mehlerreaction can mitigate the sensitivity to photoinhibition inwheat plants grown in high light. A possible decrease in thechlororespiratory oxidation of the plastoquinone pool as aresult of low oxygen should also be considered (BuÈchel andGarab, 1996).

In some treatments the rates of net CO2 assimilationmeasured at 1´5 kPa O2 were lower than expected, and didnot differ signi®cantly from those measured at 21 kPa O2.These observations are evidence for inhibition of photo-synthesis at low oxygen concentrations. Similar effects ofsub-optimal concentrations of O2 were noted by Marocoet al. (1997) in other C4 plants. These authors showed thatinhibition of photosynthesis below the optimum wasassociated with increased reduction of the quinone A pool(QA), decreased ef®ciency of open reaction centres anddecreased activity of PSII. Reduction of FPSII, over-reduction of QA and decreased CO2 assimilation havebeen reported by Dietz et al. (1985) at low O2 concentra-tions in the C3 plants Spinacea oleracea, Asarum euro-paeum and Helianthus annuus. In experiments with beanplants under drought conditions, Brestic et al. (1995) alsoshowed that decreasing the oxygen content of the air from21 to 2 kPa, to inhibit photorespiration, decreased the linearelectron transport rate. A de®ciency in ATP caused by over-reduction of electron carriers limiting cyclic electron ¯owwas proposed as a possible reason for the inhibition ofphotosynthesis in the absence of O2 in spinach (Ziem-Hanckand Heber, 1980) and Chroomonas sp. (Suzuki and Ikawa,1993). However, the mechanisms of inhibition of photo-synthetic activity at low O2 partial pressures are not yetclear.

The increase in the ratio of total carotenoids to chloro-phyll in response to growth at low temperature was morepronounced at high than at low PPFD. This may re¯ect anadaptation mechanism permitting plants to reduce lightabsorption in the antenna on the one hand, and to increasethe capacity for photoprotection on the other (Haldiman,1998). Carotenoids act as scavengers of triplet chlorophylland singlet oxygen, and the xanthophyll cycle pigmentszeaxanthin and antheraxanthin mediate the harmless dissi-pation of excess excitation energy as heat (Demmig-Adamsand Adams, 1996). The increased contents of zeaxanthinand antheraxanthin in plants grown under 1000 mmol m±2 s±1

PPFD and the consequently enhanced deps were closelycorrelated with FPSII and also with Fv¢/Fm¢ and F0¢/Fm¢(Fig. 5). These results support the hypothesis that xantho-phyll cycle activity makes an important contribution toenergy quenching in French bean exposed to short periods

of very high light (Adams and Demmig-Adams, 1995;Verhoeven et al., 1997). The increased concentration ofb-carotene could contribute to the ¯uidization of thechloroplast membrane at low temperature (Strzalka, 1994).

The present data showing a decreased quantum ef®ciencyof PSII and a decreased rate of CO2 assimilation under highlight at low temperature are evidence for signi®cant down-regulation in the photosynthetic apparatus in plants grown inlow light. There were also indications of greater oxidativestress in low-temperature/high-light conditions, with plantsshowing an increased H2O2 concentration and slightdecreases in the activities of catalase and peroxidase (datanot shown). The close correlation between the thermaldissipation in the antenna (1 ± Fv¢/Fm¢) and the de-epoxidation state of the xanthophyll cycle supports thehypothesis that xanthophyll cycle activity makes an import-ant contribution to quenching of excess energy.Nevertheless, quenching in the reaction centres might alsobe signi®cant (Bukhov et al., 2001; Peterson and Havir,2001). Relaxation of this type of quenching tends to beslower than zeaxanthin-dependent antenna quenching and isindicated by the slow phase relaxation shown for the lowtemperature/high light treatment in Fig. 3. The signi®cantcontribution of Fm to the decline in Fv/Fm also supports thissuggestion.

There was no evidence that the simulated sun¯eck lasting5 min led to any irreversible damage to PSII, even for plantsgrown at low temperature and low light. A low O2

concentration enhanced PSII down-regulation in cold pre-treated plants, as expressed by a reduction in Fv/Fm andFPSII, but minimized further inhibition by the short sun¯ecktreatment used. These results support the hypothesis thatphotorespiration provides a `safety-valve' for excess energythat cannot be replaced by compensating increased CO2

®xation.

ACKNOWLEDGEMENTS

This research was supported by NATO Scienti®c Divisiongrant LST.CLG 974973. We are grateful to Dr RichardParsons for help with generating the gas mixtures and toWendy M. James for technical support.

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