Growth at moderately elevated temperature alters the physiological response of the photosynthetic apparatus to heat stress in pea (Pisum sativum L.) leaves

Authors


Pierre Haldimann. Fax: +41 31 631 4942; e-mail: pierre.haldimann@bluewin.ch

ABSTRACT

The impact of heat stress on the functioning of the photosynthetic apparatus was examined in pea (Pisum sativum L.) plants grown at control (25 °C; 25 °C-plants) or moderately elevated temperature (35 °C; 35 °C-plants). In both types of plants net photosynthesis (Pn) decreased with increasing leaf temperature (LT) and was more than 80% reduced at 45 °C as compared to 25 °C. In the 25 °C-plants, LTs higher than 40 °C could result in a complete suppression of Pn. Short-term acclimation to heat stress did not alter the temperature response of Pn. Chlorophyll a fluorescence measurements revealed that photosynthetic electron transport (PET) started to decrease when LT increased above 35 °C and that growth at 35 °C improved the thermal stability of the thylakoid membranes. In the 25 °C-plants, but not in the 35 °C-plants, the maximum quantum yield of the photosystem II primary photochemistry, as judged by measuring the Fv/Fm ratio, decreased significantly at LTs higher than 38 °C. A post-illumination heat-induced reduction of the plastoquinone pool was observed in the 25 °C-plants, but not in the 35 °C-plants. Inhibition of Pn by heat stress correlated with a reduction of the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Western-blot analysis of Rubisco activase showed that heat stress resulted in a redistribution of activase polypeptides from the soluble to the insoluble fraction of extracts. Heat-dependent inhibition of Pn and PET could be reduced by increasing the intercellular CO2 concentration, but much more effectively so in the 35 °C-plants than in the 25 °C-plants. The 35 °C-plants recovered more efficiently from heat-dependent inhibition of Pn than the 25 °C-plants. The results show that growth at moderately high temperature hardly diminished inhibition of Pn by heat stress that originated from a reversible heat-dependent reduction of the Rubisco activation state. However, by improving the thermal stability of the thylakoid membranes it allowed the photosynthetic apparatus to preserve its functional potential at high LTs, thus minimizing the after-effects of heat stress.

INTRODUCTION

High temperature (HT) negatively affects plant growth and survival and hence crop yield (Boyer 1982). According to a recent study each degree Centigrade increase in average growing season temperature may reduce crop yield by up to 17% (Lobell & Asner 2003). Although many factors may contribute to the negative correlation between crop yield and average temperature, direct HT effects on physiological processes probably play an important role. HT adversely affects various cell functions, but photosynthesis is well known to be particularly sensitive to heat stress (Berry & Björkman 1980; Quinn & Williams 1985). The problem of HT-dependent reduction of carbon assimilation is further exacerbated by the fact that under conditions with high solar irradiance leaf temperature can increase several degrees Centigrade above air temperature (Singsaas & Sharkey 1998; Leakey, Press & Scholes 2003). Only a few plant species have the capability to efficiently cool their leaves below air temperature (Radin et al. 1994; Wise et al. 2004). Therefore, studies aimed at better understanding how heat affects photosynthesis are, in the context of global warming, of increasing importance (Sharkey 2000).

As a result of changes related with the different solubility of CO2 and O2 and the kinetic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the rate of photorespiration (Pr) increases with increasing temperature, which reduces the net photosynthetic CO2 assimilation rate (Pn) (Monson et al. 1982; Jordan & Ogren 1984; Sage & Sharkey 1987). The reduction of Pn associated with an increase of Pr appears very quickly when a leaf is suddenly exposed to heat stress, but its magnitude is limited and Pn thereafter continuous to decrease over time due to the development of physiological perturbations (Schrader et al. 2004). Inhibition of photosynthesis by heat stress occurs both under photorespiratory and non-photorespiratory conditions (Kobza & Edwards 1987; Crafts-Brandner & Salvucci 2000), which provides additional evidence that reduced Pn at elevated leaf temperatures can only partially be explained by the greater rates of Pr at HT.

Heat stress can alter the organization of the thylakoid membranes (Schreiber & Berry 1977; Armond, Björkman & Staehlin 1980; Gounaris et al. 1984; Mohanty, Vani & Prakash 2002) and photosynthetic electron transport has been identified as being particularly sensitive to HT-dependent inhibition (Berry & Björkman 1980; Quinn & Williams 1985; Yordanov et al. 1986). Photosystem (PS) II is a notoriously thermolabile component of the thylakoid membranes (Berry & Björkman 1980; Havaux 1993; Havaux & Tardy 1996; Srivastava et al. 1997), whereas PSI is comparatively heat resistant (Havaux, Greppin & Strasser 1991; Havaux 1992, 1996). Heat not only damages the oxygen-evolving complex of PSII (Nash, Miyao & Murata 1985; Enami et al. 1994), but also impairs electron transfer within the PSII reaction centres (Bukhov, Sabat & Mohanty 1990; Pospíšil & Tyystjärvi 1999; Kouřil et al. 2004) and downstream of PSII (Sinsawat et al. 2004). Inactivation of PSII by HT is generally not rapidly reversible (Seemann, Berry & Downton 1984; Bilger, Schreiber & Lange 1987; Karim, Fracheboud & Stamp 1999; Sinsawat et al. 2004). However, thermal damage to PSII often appears only at rather HTs, especially when heat stress takes place in the presence of light (Bilger et al. 1987; Havaux et al. 1991; Havaux 1992; Law & Crafts-Brandner 1999; Crafts-Brandner & Law 2000), as it normally happens under natural conditions. Changes in lipid–protein interactions related with an increase of the fluidity of the thylakoid membranes at HT are thought to play a major role in heat-induced perturbations of the thylakoid membranes (Raison, Roberts & Berry 1982; Gounaris et al. 1984; Yordanov et al. 1986).

At moderately HT, when PSII activity is not impaired, inhibition of Pn may arise because of alterations in the permeability of the thylakoid membranes that leads to proton leakage (Pastenes & Horton 1996; Bukhov et al. 1999; Schrader et al. 2004). Moreover, it has been observed long ago that moderate heat stress results in a reduction of the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and that this reduction correlates with a reversible inhibition of Pn (Weis 1981a, b, 1982; Kobza & Edwards 1987). This phenomenon is not related with a thermal sensitivity of Rubisco per se, as the activity of the fully activated enzyme is robust against heat-dependent inhibition (Weis 1981a; Eckhardt & Portis 1997; Crafts-Brandner & Law 2000).

Rubisco activase is a stromal enzyme that plays an essential role in the process of Rubisco activation (Salvucci & Ogren 1996; Portis 2001; Spreitzer & Salvucci 2002; Portis 2003) and whose thermal sensitivity has been documented (Robinson & Portis 1989; Crafts-Brandner, van de Loo & Salvucci 1997; Salvucci et al. 2001). Inhibition of Pn at moderately HT may thus originate from an inhibition of Rubisco activation by Rubisco activase (Feller, Crafts-Brandner & Salvucci 1998; Law & Crafts-Brandner 1999; Crafts-Brandner & Salvucci 2000, 2002; Salvucci & Crafts-Brandner 2004a, b). Actually, the Rubisco activation state likely decreases under moderate heat stress because the activity of activase is not high enough by itself or because of physical impairment to compensate for the faster rate of Rubisco deactivation at HT (Crafts-Brandner & Salvucci 2000; Salvucci & Crafts-Brandner 2004a). That antisense plants lacking Rubisco activase are more heat-sensitive than their wild type counterparts supports this view (Sharkey et al. 2001).

Salvucci & Crafts-Brandner (2004b) reported that activase from plants native to warm regions is less sensitive to heat than activase from plants native to cold regions and that this correlates with the superior photosynthetic performance of the former plants at HT. However, the question whether a pre-acclimation at elevated temperatures improves the thermal stability of activase within a given plant species remains to be clarified. Moreover, the question whether inhibition of Pn by HT primarily originates from a reduction of the Rubisco activation state or an inhibition of electron transport is still a matter of debate (Salvucci & Crafts-Brandner 2004a, b; Schrader et al. 2004; Sinsawat et al. 2004; Wise et al. 2004). An increase in growth temperature often results in an upward shift of the optimum temperature for photosynthesis and electron transport, which enables the plants to perform well under the warmer conditions, provided that the shift does not correlate with a reduced photosynthetic capacity (Badger, Björkman & Armond 1982; Mawson & Cummins 1989; Yamasaki et al. 2002). Acclimation to elevated temperature also increases the critical temperature above which the photosynthetic apparatus gets irreversibly damaged by heat (Pearcy 1977; Süss & Yordanov 1986). The analysis of the response of the photosynthetic apparatus to heat stress in plants without or with a preacclimation at elevated temperature may thus help to get a better insight in the mechanisms involved in heat-dependent inhibition of Pn. In the present study, inhibition of Pn by heat stress was examined in pea (Pisum sativum L.) plants grown at control (25 °C) or moderately elevated temperature (35 °C). The impact of heat stress on the photosynthetic apparatus was assessed by measuring gas exchange and chlorophyll a fluorescence parameters and by complementing these biophysical measurements with the biochemical determination of the Rubisco activation state and immunoblot analysis of Rubisco activase.

MATERIALS AND METHODS

Plant material

Seeds of pea (Pisum sativum L., cv. Roi des Conserves) were germinated on moistened filter paper at 25 °C in darkness for 4 d. Seedlings were then planted in moist quartz sand in plastic trays and grown for 4–5 d in a growth chamber at 25 °C under a photosynthetic photon flux density (PPFD) of 150 µmol m−2 s−1 with a 14-h photoperiod. Thereafter, the plants were grown under the same light conditions in hydroponic culture as described elsewhere (Hildbrand, Fischer & Feller 1994). Control plants and plants acclimated to moderately elevated temperature were grown at a day/night temperature regime of 25/18 °C and 35/18 °C, respectively. The plants were grown up to 21 d at 25 °C and up to about 40 d at 35 °C. All the measurements were made on the most recently fully expanded leaves.

Gas exchange and chlorophyll a fluorescence measurements

Photosynthetic gas exchange measurements were performed on attached leaves using an open infrared gas analyser (IRGA) system (CIRAS-1; PP-Systems, Hitchin, UK). Measurements were made at a PPFD of 500 µmol m−2 s−1, a CO2 concentration of 350 µmol mol−1, and a relative humidity of 80%. Leaf temperature (LT) was adjusted to the desired level using the internal heating/cooling system of the analyser. When necessary, CO2 was injected in the circuit using the built-in injection system of the analyser. Measurements of chlorophyll (Chl) a fluorescence were performed simultaneously using a pulse amplitude modulated fluorometer (FMS-1; Hansatech, Kings Lynn, Norfolk, UK) as described elsewhere (Haldimann & Feller 2004). The following Chl a fluorescence parameters, defined and calculated as described in Maxwell & Johnson (2000), were monitored: Fv/Fm, the maximum quantum yield of the primary photochemistry of photosystem (PS) II; qP, a measure of the fraction of open PSII reaction centres (RCs) assuming that there is no connectivity between PSII units; Fv′/Fm′, the efficiency of excitation energy capture by open PSII RCs; ΦPSII, the quantum yield of PSII electron transport and NPQ, the non-photochemical fluorescence quenching.

When not otherwise specified, the samples were first dark-adapted at 25 °C for 30 min. This was done to determine the initial values of the minimum (F0) and maximum (Fm) fluorescence yield. The light unit of the gas analyser was then switched on and the samples illuminated at a PPFD of 500 µmol m−2 s−1 until steady-state photosynthesis was achieved. However, the samples were in any case illuminated for at least 30–45 min at 25 °C before subjecting them to a given treatment. In the experiment with a gradual increase of temperature, LT was first elevated from 25 °C to the next higher selected level at a rate of about 5 °C min−1 and then maintained constant for 30 min. Combined gas exchange and Chl a fluorescence measurements were performed at the end of this treatment, immediately before increasing LT to the next higher level. The same procedure was repeated for each of nine tested LTs in the range between 25 and 45 °C, with the same leaf being used throughout the whole experiment. In the experiments where temperature was increased rapidly rather than gradually, LT was directly increased to the desired level at a rate of about 5 °C min−1. LT was then maintained constant and records taken after 30 min when steady-state photosynthesis was achieved. Thereafter, LT was reduced to 25 °C and the values of the fluorescence parameters F0 and Fm determined after 30 min of dark-adaptation. A different leaf was used for each measurement at each of eight LTs selected in the range between 25 and 45 °C. In additional experiments of the same type, leaf tissue was collected immediately at the end of the 30 min heat-stress treatment and used for Rubisco activity measurements (see below). In the recovery experiments, samples that had been exposed to a given heat-stress treatment were subsequently allowed to recover at 25 °C, with gas exchange and Chl a fluorescence parameters being recorded over time between 0 and 30 min.

The effect of an elevation of LT in the dark on the Chl a fluorescence yield was measured on a leaf disc placed on moistened filter paper in a cuvette connected to a temperature-controlled water bath. The leaf disc was maintained in the dark at 24 °C for 30 min before starting with the treatment. LT was elevated at a rate of about 1 °C min−1. Chl a fluorescence was measured using a PAM-2000 fluorometer (Heinz Walz GbmH, Effeltrich, Germany), with only the weak modulated light beam being switched on and set at an extremely low intensity. As it has been shown that part of the heat-dependent increase of the fluorescence yield can originate from a non-photochemical reduction of the plastoquinone pool (Havaux 1992), records were taken 10 s after a 5-s illumination with far-red light.

Determination of light-dependent activation of Rubisco and western-blot analysis of Rubisco activase

Light-dependent activation of Rubisco was determined using a leaf disc of 1.5 cm in diameter. The sample was collected immediately at the end of a given heat stress treatment or following a 30 min period of recovery at 25 °C. Leaf tissue was extracted within less than 30 s using a glass homogenizer in 2 mL of CO2 free extraction buffer containing 100 m m Tricine, pH 8.0, 5 m m MgCl2, 0.1 m m ethylenediaminetetraacetic acid, 5 m m dithiothreitol, 1% (w/v) polyvinylpolypyrrolidone, 1% (w/v) casein, 0.05% (v/v) Triton X-100, 1 m m phenylmethylsulphonyl fluoride, and 20 µm leupeptine. An aliquot (30 µL) of the suspension was assayed at 30 °C either immediately, to determine initial Rubisco activity, or following a 5 min incubation period at 30 °C in an assay medium containing 10 m m NaHCO3 and Mg2+ but lacking ribulose-1,5-bisphosphate to determine the activity of fully carbamylated Rubisco (total Rubisco activity). Initial and total Rubisco assays were carried out following the method of Salvucci & Anderson (1987), with the exception that Triton X-100 and casein were not included in the assay medium. Assays were terminated after 30 s and incorporation of 14CO2 into acid-stable products was determined essentially as described by Salvucci & Anderson (1987). The Rubisco activation state (Perchorowicz, Raynes & Jensen 1981) was calculated as the ratio of initial to total Rubisco activities.

Leaf discs, 1.5 cm in diameter, were used for immunoblot analysis of Rubisco activase. The leaf discs, placed on moistened filter paper in Petri dishes floating on a temperature-controlled water bath, were first illuminated for 30 min at a PPFD of 500 µmol m−2 s−1 and a LT of 25 °C. The samples were then heat-stressed at different temperatures under the same irradiance for 30 or 60 min. This was achieved by transferring the samples to another water bath set at the appropriate temperature. Collected leaf tissue was immediately frozen in liquid nitrogen and subsequently stored at −80 °C until analysis. The light source was an Intralux 6000 lamp equipped with optical fibres (Volpi Manufacturing, Auburn, NY, USA). LT was measured with an infrared thermometer (Oakton TempTestr IR; Cole-Parmer International, Vernon Hills, IL, USA) and PPFD with a Li-Cor radiometer (Li-250; LiCor Inc., Lincoln, NE, USA). Extraction of leaf discs and western-blot analysis of Rubisco activase were performed essentially as described elsewhere (Feller et al. 1998). The soluble and insoluble fractions of extracts were separated by centrifugation. After washing the pellet with extraction buffer minus polyvinylpolypyrrolidone, the insoluble fraction was suspended in the same buffer in a volume equivalent to the starting extraction volume. This means that the soluble and insoluble lanes of the blots represent equivalent leaf areas. The primary antibody used for Rubisco activase detection was a polyclonal rabbit antibody raised against tobacco (Nicotiana tabaccum) Rubisco activase and was kindly provided by Dr S. J. Crafts-Brandner and Dr M. E. Salvucci, ARS USDA, Phoenix, AZ, USA.

Statistics

Statistical analyses were made using the GenStat software version 6.2 (Payne et al. 2000; Payne 2002).

RESULTS

Effect of growth temperature on photosynthetic activity

In comparison with the plants grown at 25 °C (25 °C-plants), the pea plants grown at 35 °C (35 °C-plants) displayed smaller leaves and were significantly retarded in growth. The younger leaves of the 35 °C-plants showed a healthy green appearance, but the older leaves died prematurely, with the leaf tissue progressively drying out and showing a white bleached appearance. This phenomenon was not observed in the 25 °C-plants. The most recently fully expanded leaves of the 35 °C-plants displayed at a LT of 25 °C a net photosynthetic CO2 assimilation rate (Pn) that was similar (PPFDs < 500 µmol m−2 s−1) or even slightly superior (PPFDs > 500 µmol m−2 s−1) to that determined in the same type of leaves in the 25 °C-plants (Fig. 1). Chl a fluorescence measurements revealed that the light response curves of ΦPSII, qP, Fv′/Fm′ and NPQ were nearly identical in both types of plants (Fig. 1; insert). However, at PPFDs greater than 500 µmol m−2 s−1, Fv′/Fm′ was somewhat higher in the 35 °C-plants than in the 25 °C-plants, while the opposite was observed for NPQ. Based on these measurements and unless specified otherwise, all the experiments with heat stress were conducted under a PPFD of 500 µmol m−2 s−1, i.e. an irradiance at which light was nearly saturating for Pn at 25 °C (Fig. 1). Illumination with higher PPFDs was avoided in order to minimize heat-induced photoinhibition.

Figure 1.

The light response of the net photosynthetic CO2 assimilation rate (Pn) determined in the most recently fully expanded leaves of pea plants grown at a day/night temperature regime of either 25/18 °C (•) or 35/18 °C (○). Each sample was illuminated for at least 30 min at a PPFD of 250 µmol m−2 s−1 and 25 °C before starting with the measurements. Measurements were made at a leaf temperature of 25 °C starting with the lowest PPFD. The inserts show the light response of: (a) the quantum yield of PSII electron transport (ΦPSII); (b) the fraction of open PSII reaction centres (qP); (c) the efficiency of excitation energy capture by open PSII reaction centres (Fv′/Fm′) and (d) the non-photochemical fluorescence quenching (NPQ). All records were taken at steady-state photosynthesis. Each data point represents the mean ± SE of three to five independent replicates.

Net photosynthesis and chlorophyll a fluorescence parameters as a function of leaf temperature

Growth temperature hardly affected the temperature response curve of Pn(Fig. 2a). Pn started to decrease when LT reached about 30 °C and was more than 80% reduced at 45 °C as compared to 25 °C. Increasing LT gradually rather than rapidly did not diminish inhibition of Pn by HT. Reduction of Pn at elevated LTs did not arise because of stomatal limitations, as stomata closure, if occurring at all, was accompanied by an increase rather than a decrease of Ci (data not shown). At LTs higher than about 35 °C, inhibition of Pn was accompanied by a heat-dependent reduction of ΦPSII (Fig. 2b), which originated from both a decrease of qP (Fig. 2c) and a decrease of Fv′/Fm′ (Fig. 2d) that correlated with an increase of NPQ (data not shown). The HT-dependent reduction of ΦPSII was less pronounced than that of Pn, showing that electrons were flowing to alternative sinks. Between 25 and 35 °C an elevation of LT resulted in an increase rather than a decrease of ΦPSII (Fig. 2b). Neither growth temperature nor the mode of LT elevation appeared to have a significant effect on the temperature response curves of the different Chl a fluorescence parameters (Fig. 2b–d). However, at LTs higher than about 40 °C, the 25 °C-plants showed somewhat higher qP values when LT was increased rapidly as compared with gradually (Fig. 2c), whereas the opposite was observed for Fv′/Fm′ (Fig. 2d). Mainly the procedure with a rapid elevation of LT was used in the additional experiments presented below.

Figure 2.

Effect of leaf temperature on: (a) the net CO2 assimilation rate (Pn); (b) the quantum yield of PSII electron transport (ΦPSII); (c) the fraction of open PSII reaction centres (qP); and (d) the efficiency of excitation energy capture by open PSII reaction centres (Fv′/Fm′) in pea leaves grown at a day/night temperature regime of either 25/18 °C (•,▾) or 35/18 °C (○,▿). After photosynthesis had achieved steady state at 25 °C, leaf temperature was elevated to the indicated level either directly at a rate of about 5 °C min−1 using a different leaf for each of the eight selected temperatures (•,○) or gradually using the same leaf for each of the nine selected temperatures (▾,▿). In both cases records were taken at the end of a 30 min period at the indicated temperatures. Data are expressed in percent of the corresponding initial values determined at 25 °C. Each data point represents the mean ± SE of three to four independent replicates.

Thermal stability of thylakoid membrane components

In both types of plants the maximum quantum yield of the PSII primary photochemistry, as judged by measuring the Fv/Fm ratio (Kitajima & Butler 1975; Björkman & Demmig 1987), was hardly affected by LT in the range between 25 and 38 °C (Fig. 3). Actually, in the 35 °C-plants, even the treatment at the very high LT of 45 °C resulted in only a minor reduction of Fv/Fm. On the contrary, in the 25 °C-plants, Fv/Fm decreased abruptly when LT increased above 38 °C and was more than 40% reduced at 45 °C as compared to 25 °C. The reduction of Fv/Fm originated from both an increase of Fo and a decrease of Fm (Fig. 3; insert). In the 25 °C-plants, a treatment at temperatures higher than about 38 °C often resulted in a significant increase of the apparent Fo. This phenomenon was related with a heat-induced non-photochemical reduction of the plastoquinone (PQ) pool (see below, as well as Havaux 1996; Sazanov, Burrows & Nixon 1998; Bukhov, Samson & Carpentier 2000). In this case, the true F0, defined as the fluorescence yield when the primary quinone electron acceptor QA of PSII is fully oxidized, that is when all PSII reaction centres are in an open configuration, was determined after a 10 s period of darkness following a 5 s period of illumination with far-red (FR) light that preferentially excites PSI. The Fv/Fm ratio was always calculated using the ‘true Fo’.

Figure 3.

Effect of leaf temperature on the Fv/Fm ratio in pea leaves grown at a day/night temperature regime of either 25/18 °C (•) or 35/18 °C (○). The values of the minimum (F0) and the maximum (Fm) chlorophyll a fluorescence yield were first determined following dark-adaptation of the sample at 25 °C for 30 min. At this time, the Fv/Fm ratio ± SE was 0.800 ± 0.007 and 0.798 ± 0.010 in the plants grown at 25/18 °C and 35/18 °C, respectively. The sample was then illuminated until steady-state photosynthesis was achieved at 25 °C. Thereafter, leaf temperature was elevated to the indicated level at a rate of about 5 °C min−1 and then maintained constant for 30 min. Following this treatment the sample was again dark-adapted as described above prior to record the fluorescence parameters for a second time. The inserts show the effect of leaf temperature on F0 (a) and Fm (b). Each measurement was made using a different leaf. Data are expressed in percent of the corresponding initial values determined at 25 °C. Each data point represents the mean ± SE of three to four independent replicates.

In a further experiment, the changes of the Chl a fluorescence yield that occur during a light-to-dark transition were monitored in pea leaves that had been exposed in the light to a 30 min heat stress treatment at 42 °C (Fig. 4). At the end of the stress period, LT was set back to 25 °C and the actinic light source switched off. As expected, the fluorescence signal decreased instantaneously when the illumination with actinic light stopped, dropping to a level close to that corresponding to the initial F0 measured before starting with the heat stress treatment. However, in the 25 °C-plants, the signal thereafter started to increase again, reaching within a few minutes in darkness a level superior to that measured at the end of the heat stress treatment (Fig. 4a). The fluorescence signal decreased markedly when the sample was illuminated with FR light, but it started to increase again as soon as the illumination stopped. In the 35 °C-plants, the post-illumination changes of the fluorescence signal were minor and restricted to the first few minutes of darkness (Fig. 4b). In fact, the signal rapidly stabilized to a level corresponding to that of the initial F0. The small increase of the signal observed during the illumination with FR light was probably due to a weak stimulation of PSII.

Figure 4.

The changes of the chlorophyll a fluorescence yield monitored during a light-to-dark transition following a 30 min heat stress treatment at 42 °C in pea leaves grown at a day/night temperature regime of either 25/18 °C (a) or 35/18 °C (b). After the initial value of the minimum fluorescence yield F0 had been determined following a 30 min dark-adaptation at 25 °C, the sample was illuminated at a PPFD of 500 µmol m−2 s−1 and 25 °C until steady-state photosynthesis was achieved. Thereafter, leaf temperature was elevated to 42 °C for 30 min. At the end of this treatment, leaf temperature was rapidly set back to 25 °C and the actinic light switched off (large downward arrow), so that chlorophyll was then solely excited by the very weak modulated light beam of the fluorometer. The small upward and downward arrows indicate far-red light on and off, respectively. The dotted lines show the level of the fluorescence signal corresponding to the initial minimum fluorescence yield F0.

Monitoring the changes of the Chl a fluorescence yield that occur when LT is progressively elevated in the dark has for some time been employed as a tool to estimate the thermal stability of thylakoid membranes (i.e. Schreiber & Berry 1977; Bilger et al. 1987; Havaux 1992, 1993; Knight & Ackerly 2002). Using this technique for the pea plants showed that in the 25 °C-plants the fluorescence signal started to rise dramatically when LT reached about 40 °C, whereas in the 35 °C-plants a rapid increase of the signal was only observed at LTs higher than about 43 °C (Fig. 5), indicating that growth at moderately elevated temperature increases the thermal stability of the thylakoid membranes.

Figure 5.

Effect of an elevation of leaf temperature in the dark on the chlorophyll a fluorescence yield in pea leaf discs obtained from plants grown at a day/night temperature regime of either 25/18 °C (•) or 35/18 °C (○). Each sample was first adapted to the dark at 24 °C for 30 min. Thereafter the weak modulated light beam of the fluorometer was switched on and leaf temperature gradually elevated at a rate of 1 °C min−1. Records were taken at each °C unit between 24 and 51 °C. The data were normalized to the initial value determined at 24 °C. Each data point represents the mean ± SE of three to four independent replicates.

Effects of an increase of Ci

Experiments examining the effects of an increase of Ci revealed that at a LT of 25 °C an elevation of Ci from about 250 to 1000 µmol mol−1 resulted in a significant increase of Pn(Fig. 6a), but hardly affected ΦPSII (Fig. 6b). On the contrary, at a LT of 42 °C, the high Ci-dependent enhancement of Pn (Fig. 6a) was accompanied by a significant increase of ΦPSII (Fig. 6b) that originated from both an increase of qP and an increase of Fv′/Fm′ (data not shown), showing that photosynthetic electron transport (PET) was stimulated. However, in the 25 °C-plants, photosynthesis remained strongly suppressed, with Pn reaching less than 25% of its control value at 25 °C and a low Ci. Accordingly, also ΦPSII showed at 42 °C and a high Ci a value that reached only about 36% of the control value. This is nevertheless 12% more when compared with the situation at a low Ci. In the 35 °C-plants, the Pn and ΦPSII values measured at 42 °C and a high Ci reached about 60% of the corresponding control values determined at 25 °C and a low Ci. In other words, at 42 °C, ΦPSII was more than two times greater at a high Ci than at a low Ci, demonstrating that in the 35 °C-plants PET was not the primary factor limiting Pn at high LTs.

Figure 6.

Effect of leaf temperature and intercellular CO2 concentration (Ci) on: (a) the net photosynthetic CO2 assimilation rate (Pn) and (b) the quantum yield of PSII electron transport (ΦPSII) in pea leaves grown at a day/night temperature regime of either 25/18 °C (grey bars) or 35/18 °C (black bars). Records were first taken when steady-state photosynthesis had been achieved at 25 °C and a low Ci (about 250 µmol mol−1). Ci was then increased to about 1000 µmol mol−1 and records taken after 5–10 min of equilibration. Thereafter, Ci was again decreased to the low level prior to increase the leaf temperature to 42 °C at a rate of about 5 °C min−1. During the heat stress treatment, Ci was first permanently maintained at the low level and records were taken after 30 min at 42 °C. Ci was then increased to the high level and records were again taken after 5–10 min of equilibration. The value of each column represents the mean of five independent replicates + SE. Values carrying different letters are significantly different at P < 0.01.

Effects of heat stress on Rubisco activation and Rubisco activase

Measurements of Rubisco activities revealed that heat stress did not affect the activity of fully activated Rubisco (data not shown). On the other hand, the initial Rubisco activity, measured immediately after the extraction of the samples, decreased with increasing LTs. As a result, the Rubisco activation state, namely the fraction of active Rubisco in a sample, decreased with increasing LTs (Fig. 7). The Rubisco activation state did not differ between the two types of plants, but at 42 °C it was somewhat higher in the 35 °C-plants than in the 25 °C-plants. Due to technical limitations related with the infrared gas analyser, it was not always possible to increase LT above 42–43 °C. This especially holds true for the 35 °C-plants. To keep up higher LTs it would have been necessary to increase air temperature above 50 °C, which was not feasible. Therefore, the impact of HT on Rubisco activase was determined using leaf discs that were subjected to different heat stress treatments on a water bath under a PPFD of 500 µmol m−2 s−1.

Figure 7.

Effect of leaf temperature on the Rubisco activation state in pea leaves grown at a day/night temperature regime of either 25/18 °C (•) or 35/18 °C (○). The Rubisco activation state was calculated as the ratio of initial to total Rubisco activities. After steady-state photosynthesis had been achieved at 25 °C, leaf temperature was elevated to the indicated level at a rate of about 5 °C min−1 and then maintained constant for 30 min prior to collect the sample for the measurements of the Rubisco activities. Each value represents the mean ± SE of three to four independent replicates. * indicates that the Rubisco activation state was significantly higher in the 35 °C-plants than in the 25 °C-plants at P < 0.05.

As observed in a previous study with pea plants (Salvucci et al. 2001), immunoblot analysis of Rubisco activase disclosed the presence of two activase polypeptides in the soluble fraction of leaf extracts from both types of plants, with the smaller polypeptide (about 41 kDa) appearing in much higher abundance than the larger polypeptide (about 44 kDa) (Fig. 8a). Whether the somewhat diffuse additional band appearing immediately below the 41 kDa band is a true extra activase polypeptide or simply the result of a proteolytic degradation is not clear. It has been reported that heat stress at temperatures higher than 40 °C induces the formation of high-molecular-weight aggregates of activase (Feller et al. 1998; Salvucci et al. 2001) and/or results in a redistribution of activase from the soluble to the insoluble fraction of extracts (Feller et al. 1998; Rokka, Zhang & Aro 2001; Salvucci et al. 2001). Heat-dependent formation of high-molecular-weight aggregates was not observed in the present study even in the samples that had been treated at 45 °C for 60 min (Fig. 8a & b). However, heat stress at 40 °C (mainly in the 25 °C-plants) or 45 °C (in both types of plants) indeed resulted in a redistribution of activase from the soluble (Fig. 8a) to the insoluble (Fig. 8b) fraction of extracts. As found in other studies (Rokka et al. 2001; Salvucci et al. 2001), the smaller activase polypeptide was more sensitive to a redistribution than the larger polypeptide, but in the 35 °C-plants significant amounts of the larger activase polypeptide were detected in the insoluble fraction of extracts obtained from the samples that had been treated at 45 °C. It appeared that in the 25 °C-plants the treatment at 45 °C resulted in a reduction of the content of Rubisco activase, as indicated by the disappearance of the 44 kDa polypeptide bands and the somewhat reduced intensities of the 41 kDa polypeptide bands.

Figure 8.

Effect of leaf temperature on the form of Rubisco activase in the soluble (a) and insoluble (b) fractions of leaf extracts from pea plants grown at a day/night temperature regime of either 25/18 °C or 35/18 °C. The samples were illuminated for 30 min at a PPFD of 500 µmol m−2 s−1 and 25 °C prior to expose them to the indicated heat stress treatments under the same irradiance. Leaf tissue collected at the end of the treatment was rapidly frozen in liquid nitrogen and subsequently stored at −80 °C until homogenization and separation into soluble and insoluble fractions by centrifugation. Polypeptides in the two fractions were separated by SDS-PAGE and analysed for the presence of activase by western-blot analysis. Lanes were loaded with extract corresponding to equal amounts of leaf area. The positions of molecular weight standards are indicated at the left side of the figure.

Recovery from heat-induced inhibition of photosynthesis

Recovery from heat-dependent inhibition of Pn was analysed in an additional series of experiments in samples that had been exposed in the light to a 30 min heat stress treatment at either 40 or 42–43 °C. When the samples had been treated at 40 °C, Pn recovered within 30 min at 25 °C to nearly 70 and 90% of its original value in the 25 °C-plants and the 35 °C-plants, respectively (Fig. 9a). Differences between the two types of plants with regard to their capacity to recover from heat stress were also observed for ΦPSII (Fig. 9b) and Fv′/Fm′ (Fig. 9d), but hardly for qP (Fig. 9c). Recovery was generally rapid within the first 5 min at 25 °C, but much slower thereafter. Large differences appeared between the two types of plants with regard to their capacity to recover from a more severe heat stress treatment at 42–43 °C (Fig. 9a–d). Following such a treatment, the 35 °C-plants recovered almost like they did after the treatment at 40 °C, whereas the 25 °C-plants displayed virtually no recovery, with the fluorescence parameters even showing a further decrease.

Figure 9.

Recovery from heat-induced changes of: (a) the net photosynthetic CO2 assimilation rate (Pn); (b) the quantum yield of PSII electron transport (ΦPSII); (c) the fraction of open PSII reaction centres (qP); and (d) the efficiency of excitation energy capture by open PSII reaction centres (Fv′/Fm′) in pea leaves grown at a day/night temperature regime of either 25/18 °C (•,▾) or 35/18 °C (○,▿). After steady-state Pn had been achieved at 25 °C, leaf temperature was elevated to either 40 °C (•,○) or 42–43 °C (▾,▿) for 30 min. Thereafter leaf temperature was set back to 25 °C and recovery from heat-induced changes monitored over time. All data are expressed in percent of the corresponding initial values determined at 25 °C. Each data point represents the mean ± SE of three to four independent replicates.

In the recovery experiment described above (Fig. 9a), as well as in the experiment analysing the effects of an elevation of Ci (Fig. 6a), the treatment at 42–43 °C resulted in the 25 °C-plants in a complete inhibition of Pn. On the other hand, in other experiments, the same treatment resulted in only a partial inhibition of Pn (Fig. 1a). It appeared that mostly in the 25 °C-plants there were some differences among plant batches with regard to the extent of the inhibition of Pn by heat stress. This mainly hold true for temperatures higher than about 38 °C. In the range between 40 and 45 °C, the temperature response curve of Pn is rather steep (Fig. 1a) and heat can cause damage to PSII (Fig. 3) and alter thylakoid membrane properties (Figs 4 & 5). Therefore, it is expected that small differences in the tolerance of the photosynthetic apparatus to HT between the plants from different batches will significantly affect the extent of the inhibition of Pn observed at a given LT. It will obviously also affect the capacity of the leaves to recover from heat stress. However, after a heat stress treatment at temperatures higher than 40 °C, recovery was always much weaker in the 25 °C-plants than in the 35 °C-plants.

Figure 10a shows that in both types of plants the samples treated at 40 °C recovered almost completely from heat-dependent reduction of the Rubisco activation state within 30 min at 25 °C. In the 35 °C-plants, nearly the same observation was made for the samples that had been treated at 42–43 °C (Fig. 10b). Indeed, in this case, the Rubisco activation state increased from about 38% at the end of the stress period to more than 86% after 30 min of recovery, thus reaching a level close to that observed prior to the heat stress treatment (about 94%). In the 25 °C-plants, the samples treated at 42–43 °C recovered within the same time from 29% to about 45%, which means that the Rubisco activation state did not even reach half of its initial value (nearly 96%).

Figure 10.

The Rubisco activation state determined at steady-state photosynthesis in pea leaves grown at a day/night temperature regime of either 25/18 °C (grey bars) or 35/18 °C (black bars). The Rubisco activation state was not only measured before (control) and after a 30 min heat stress treatment (stress), but also following a 30 min period of recovery at 25 °C (recovery). During the stress phase leaf temperature was elevated to either 40 °C (a) or 42–43 °C (b). Illumination was kept constant at a PPFD of 500 µmol m−2 s−1. The Rubisco activation state was calculated as the ratio of initial to total Rubisco activities. The value of each column represents the mean + SE of three to four independent replicates. Values carrying different letters are significantly different at P < 0.01. * indicates that the Rubisco activation state was significantly higher in the 35 °C-plants than in the 25 °C-plants at P < 0.05.

DISCUSSION

The present study showed that in pea plants an elevation of growth temperature from 25 to 35 °C did not negatively affect the functioning of the photosynthetic apparatus of young mature leaves (Fig. 1), even though it resulted in a slow leaf development and lead to the premature death of the older leaves. This contrast with the situation in winter wheat (Triticum aestivum L.) where 35 °C was found to be a non-physiological temperature with leaf development being dramatically impeded and chlorophyll synthesis strongly suppressed (Yamasaki et al. 2002). A number of studies have shown that within the physiological range an increase of growth temperature results in upward shift of the temperature optimum for Pn and PET, but that the magnitude of the shift strongly depends on the plant species (Tieszen & Helgager 1968; Armond, Schreiber & Björkman 1978; Berry & Björkman 1980; Badger et al. 1982; Mawson & Cummins 1989; Yamasaki et al. 2002). In the pea plants, growth at 35/18 °C rather than 25/18 °C hardly altered the temperature response curves of Pn (Fig. 2a) and ΦPSII (Fig. 2b), showing that growth at moderately elevated temperature did not improve photosynthesis at high LTs.

In agreement with earlier findings (i.e. Berry & Björkman 1980; Badger et al. 1982; Süss & Yordanov 1986; Yamasaki et al. 2002; Sinsawat et al. 2004), measurements of the Fv/Fm ratio revealed that growth at moderately elevated temperature efficiently protected PSII against heat-dependent inactivation (Fig. 3). Short-term acclimation to HT has been shown to significantly improve the thermal stability of PSII (Havaux 1993; Law & Crafts-Brandner 1999) and to reduce inhibition of Pn by heat stress (Law & Crafts-Brandner 1999; Crafts-Brandner & Salvucci 2002). Therefore, we expected that mostly in the 25 °C-plants increasing LT gradually rather than rapidly would help to diminish heat-dependent inhibition of Pn and PET. That the mode of LT elevation hardly affected the temperature response curves of Pn (Fig. 2a) and PET (Fig. 2b), suggests that there might be differences among plant species with regard to their capacity to develop thermal tolerance during short-term acclimation to HT. Alternatively, the time of acclimation to HT in the treatment with a gradual elevation of LT might have been too short to allow the pea plants to improve the thermal stability of their photosynthetic apparatus. However, the finding that a short-term pre-treatment at moderately HT significantly improved the thermotolerance of PSII in Solanum tuberosum leaves (Havaux 1993) indicates that thermal protection can be rapidly built-up. In any case, thermal damage to PSII can only marginally, if at all, explain the inhibition of Pn by heat stress in the 35 °C-plants, whereas it contributed to reduce Pn in the 25 °C-plants, although probably only at temperatures higher than about 38 °C (Fig. 3). Our finding that the 35 °C-plants, unlike the 25 °C-plants, had the capability to efficiently recover from heat-dependent inhibition of Pn (Fig. 9) supports this conclusion, as thermal damage to PSII has been shown to be only slowly reversible, if at all (Seemann et al. 1984; Bilger et al. 1987; Karim et al. 1999; Sinsawat et al. 2004).

In the 25 °C-plants, heat stress induced a post-illumination non-photochemical reduction of the PQ pool (Fig. 4a), which is consistent with the results obtained in other studies (Havaux et al. 1991; Havaux 1992, 1996; Sazanov et al. 1998; Yamane et al. 2000; Bukhov et al. 2000). Non-photochemical reduction of the PQ pool probably reflects a heat-induced activation of cyclic electron transport around PSI, whose function might be to balance proton leakage originating from a heat-dependent alteration of the permeability of the thylakoid membranes (Bukhov et al. 1999; Schrader et al. 2004). Donation of electrons from stromal reductants to the intersystem electron transport chain may perhaps also partially compensate for the reduced supply of electrons associated with a thermal inactivation of PSII. Heat-induced cyclic electron transport may thus be essential for the maintenance of an adequate ATP/ADP ratio and for the activation of NPQ at high LTs. However, as this probably occurs at the expense of stromal reductants (Schrader et al. 2004), the depletion of reductants might well have been an important factor involved in the inhibition of Pn by heat stress in the 25 °C-plants (see Pastenes & Horton 1996; Schrader et al. 2004). That non-photochemical reduction of the PQ pool was not observed in the 35 °C-plants (Fig. 4b) suggests that proton leakage did probably not occur in these plants and that inhibition of Pn at high LTs did not arise because of the depletion of stromal reductants. It appears therefore that growth at moderately elevated temperature increased the thermal stability of the thylakoid membranes. Our finding that growth at 35 °C, when compared with 25 °C, resulted in an upward shift of the critical temperature above which heat stress in the dark resulted in a rapid rise of the Chl a fluorescence yield (Fig. 5) supports this view. Other studies have shown that acclimation to elevated temperatures significantly improves the thermal stability of the thylakoid membranes and PSII (i.e. Süss & Yordanov 1986; Yordanov et al. 1986; Havaux 1993; Ghouil et al. 2003; Sinsawat et al. 2004). In this respect, changes in fatty acid composition (Süss & Yordanov 1986; Murakami et al. 2000), as well as the accumulation of heat-shock proteins (Heckathorn et al. 2002; Török et al. 2001) and of protective compatible solutes (Williams & Gounaris 1992; Park et al. 2003) may play an important role. The accumulation of zeaxanthin, a carotenoid, is also considered as an important factor (Havaux et al. 1996; Havaux 1998), but accumulation of this pigment was not observed in Zea mays leaves grown at 41 °C (Sinsawat et al. 2004).

Our observation that in the 35 °C-plants an increase of Ci resulted in a significant increase of Pn at high LT (Fig. 6a) and that the stimulation of carbon assimilation was accompanied by a large increase of ΦPSII (Fig. 6b) provides perhaps the best evidence that in these plants the primary factor limiting photosynthesis at high LTs was neither reduced PET nor the occurrence of thylakoid membrane leakiness (see also Haldimann & Feller 2004). Crafts-Brandner & Law (2000) earlier demonstrated that the stimulation of Pn by an increase of Ci at high LT occurs both under photorespiratory and non-photorespiratory conditions. In the 25 °C-plants, an increase of Ci at a LT of 42 °C stimulated Pn (Fig. 6a) and ΦPSII (Fig. 6b) to a much lesser extent than in the 35 °C-plants, demonstrating that a high Ci does not help to overcome inhibition of Pn that originates from reduced PET (Fig. 3) and/or from a heat-dependent alteration of the permeability of the thylakoid membranes (Fig. 4a).

From the results discussed above, it appears that in the 35 °C-plants the observed reduction of ΦPSII with increasing LTs (Fig. 2b) mainly originated from a down-regulation of PSII activity that developed in response to a reversible heat-dependent inhibition of CO2 assimilation in the Calvin cycle (Figs 2a & 9a). At moderately high LTs, this conclusion does probably also hold true for the 25 °C-plants. On the other hand, the heat-dependent reduction of PET observed in the 25 °C-plants at higher LTs (Fig. 2b) most probably originated from both a reversible down-regulation of PSII activity and an irreversible inactivation of PSII (Fig. 3), which was possibly accompanied by a permanent impairment of other components of the thylakoid membranes (Sinsawat et al. 2004). The relative contribution of each of the two factors to the reduction of PET can perhaps best be estimated in recovery experiments, which disclose the after-effects of heat stress (Fig. 9a–d). Down-regulation of PSII activity appeared as a reversible reduction of the light use efficiency of open PSII reaction centres (Figs 2d & 9d), which was associated with an increase of NPQ (data not shown) that reflects thermal dissipation of excess excitation energy. The stimulation of thermal energy dissipation allowed the photosynthetic apparatus to keep a large fraction of the PSII reaction centres in an open configuration (Figs 2c & 9c) even when Pn was strongly suppressed by heat. This probably contributed to protect the photosynthetic apparatus against heat-dependent photoinhibition. As found in other studies (Leakey et al. 2003; Haldimann & Feller 2004), we observed that HT-dependent inhibition was more pronounced for Pn (Figs 2a & 9a) than for ΦPSII (Figs 2b & 9b). This shows that electrons were flowing to alternative sinks, which represents another mechanism by which the photosynthetic apparatus can protect itself against heat-induced photoinhibition (see Osmond et al. 1997; Wingler et al. 2000; Ort & Baker 2002).

Our finding that high LTs resulted in a reduction of the Rubisco activation state (Fig. 7), whereas the activity of fully activated Rubisco was hardly affected by heat (data not shown) is consistent with the results obtained in other studies (i.e. Law & Crafts-Brandner 1999; Crafts-Brandner & Law 2000; Crafts-Brandner & Salvucci 2000, 2002; Haldimann & Feller 2004). That the response of the Rubisco activation state to an elevation of LT was similar in the 25 °C-plants and the 35 °C-plants (Fig. 7) is consistent with our finding that the temperature response curve of Pn was pretty much the same in both types of plants (Fig. 2a) and shows that growth at moderately elevated temperature hardly contributed to diminish heat-dependent Rubisco inactivation. The results discussed above provide evidence that in the 35 °C-plants heat-dependent reduction of the Rubisco activation state did not arise because of an inhibition of PET or the occurrence of thylakoid membrane leakiness. It follows that in the 35 °C-plants Rubisco inactivation was probably a primary cause for the inhibition of Pn at high LTs (Fig. 2a). Our finding that in these plants the Rubisco activation state (Fig. 10) and Pn (Fig. 9a) nearly fully recovered from heat-dependent reduction within 30 min at 25 °C supports this view and is consistent with the results obtained in a study with Quercus pubescens leaves grown under natural conditions (Haldimann & Feller 2004). In the 25 °C-plants, irreversible inhibition of PET associated with thermal damage to PSII (Figs 3 & 6b) and perhaps also damage to other components of the photosynthetic electron transport chain (Sinsawat et al. 2004) appeared to be an important factor involved in the heat–dependent reduction of the Rubisco activation state that occurred at LTs higher than about 40 °C (Figs 7 & 10b). Indeed, following a 30 min heat stress treatment at 42–43 °C, the Rubsico activation state remained in these plants reduced by more than 50% relative to its initial value, even after a 30 min period of recovery at 25 °C (Fig. 10b). On the other hand, the finding that the Rubisco activation state fully recovered from the reduction induced by the heat stress treatment at 40 °C (Fig. 10a), suggests that PET was probably not a limiting factor for Rubisco activation at moderately high LTs. However, we cannot exclude that the reduction of the Rubisco activation state observed in 25 °C-plants at moderately HT did not at least partly originate from physiological changes related with the occurrence of reversible heat-dependent thylakoid membrane leakiness. On the other hand, the fact that the reduction of the Rubisco activation state induced by moderate heat stress usually correlates with increased levels of RuBP and decreased levels of 3-phosphoglycerate (Kobza & Edwards 1987; Law & Crafts-Brandner 1999; Crafts-Brandner & Law 2000; Sharkey et al. 2001; Crafts-Brandner & Salvucci 2002) would not support this latter view.

Heat-dependent reduction of the Rubisco activation state that originates neither from limited PET nor from the occurrence of thylakoid membrane leakiness might not only be associated with an increased rate of Rubisco inactivation at HT, but also with a reduced activation of Rubisco by Rubisco activase (Crafts-Brandner & Salvucci 2000; Salvucci & Crafts-Brandner 2004a, b). Evidence has been provided that heat stress can result in the formation of high-molecular-weight aggregates of Rubisco activase (Feller et al. 1998; Salvucci et al. 2001) and/or lead to a redistribution of activase from the soluble to the insoluble fraction of extracts (Feller et al. 1998; Rokka et al. 2001; Salvucci et al. 2001), suggesting that thermal denaturation of activase may play a role in HT-dependent reduction of Rubisco activation. In the present study, formation of high-molecular-weight aggregates appeared neither in the 35 °C-plants nor in the 25 °C-plants, even following a 60 min treatment at the very high LT of 45 °C (Fig. 8a & b). On the other hand, heat stress indeed resulted in a redistribution of activase polypeptides from the soluble to the insoluble fraction of extracts (Fig. 8a & b). The role of a redistribution of activase that may arise because of a reversible association of activase polypeptides with the thylakoid membranes (Rokka et al. 2001) needs to be further clarified, as in Q. pubescens leaves grown under natural conditions heat-dependent reduction of Rubisco activation was not associated with a redistribution of activase polypeptides (Haldimann & Feller 2004). It is possible that Rubisco activase from thermophylous Q. pubescens is more thermotolerant than activase from pea, as suggested by the differential thermal stability of activase observed between plants from contrasting thermal environments (Salvucci & Crafts-Brandner 2004b). However, it is equally likely that activase from Q. pubescens is actually not more thermotolerant than activase from pea, but that in Q. pubescens the enzyme was better protected against thermal alterations due to the development under natural conditions of a more efficient protective system, including for instance the accumulation of heat-shock proteins and other favourable changes in the cellular environment within the chloroplasts.

To conclude, in this paper we have shown that in pea plants growth at moderately elevated temperature hardly contributed to diminish inhibition of Pn by heat stress that was associated with a reversible heat-dependent reduction of the Rubisco activation state. However, by improving the thermal stability of heat-sensitive thylakoid membrane components growth at moderately elevated temperature significantly minimized the after-effects of heat stress. It has been shown that not only growth at moderately elevated temperatures, but also a pre-acclimation of the plants at elevated temperatures considerably improves the thermal stability of the thylakoid membranes and photosynthetic electron transport (i.e. Süss & Yordanov 1986; Havaux 1993). Therefore, our results suggest that under natural conditions, where the plants typically become progressively acclimated to elevated temperatures, reversible heat-dependent reduction of the Rubisco activation state is probably in many plant species a primary factor involved in the inhibition of photosynthesis by heat stress, but definite proof will have to be provided by experiments conducted in the field.

ACKNOWLEDGMENTS

The authors thank Dr S. J. Crafts-Brandner and Dr M. E. Salvucci (Western Cotton Research Laboratory, U.S. Department of Agriculture, Phoenix, AZ, USA) for the generous gift of the Rubisco activase antibodies, as well as the following members of the Institute of Plant Sciences in Bern: L. Zimmermann for the valuable help in statistical analyses, Dr S. Hörtensteiner for useful advice concerning the biochemical work and R. Hölzer for expert technical assistance in western-blot analyses. This work was part of the project ‘Thermoak’ within the ‘NCCR Climate’, a co-operative research program supported by the Swiss National Science Foundation. P.H. had a Swiss Federal Research Fellowship.

Ancillary