Inhibition of photosynthesis by high temperature in oak (Quercus pubescens L.) leaves grown under natural conditions closely correlates with a reversible heat-dependent reduction of the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase
Institute of Plant Sciences, University of Bern, Altenbergrain 21, CH-3013 Bern, Switzerland
Pierre Haldimann. Fax: + 41 31 631 4942; e-mail: firstname.lastname@example.org
Inhibition of the net photosynthetic CO2 assimilation rate (Pn) by high temperature was examined in oak (Quercus pubescens L.) leaves grown under natural conditions. Combined measurements of gas exchange and chlorophyll (Chl) a fluorescence were employed to differentiate between inhibition originating from heat effects on components of the thylakoid membranes and that resulting from effects on photosynthetic carbon metabolism. Regardless of whether temperature was increased rapidly or gradually, Pn decreased with increasing leaf temperature and was more than 90% reduced at 45 °C as compared to 25 °C. Inhibition of Pn by heat stress did not result from reduced stomatal conductance (gs), as heat-induced reduction of gs was accompanied by an increase of the intercellular CO2 concentration (Ci). Chl a fluorescence measurements revealed that between 25 and 45 °C heat-dependent alterations of thylakoid-associated processes contributed only marginally, if at all, to the inhibition of Pn by heat stress, with photosystem II being remarkably well protected against thermal inactivation. The activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) decreased from about 90% at 25 °C to less than 30% at 45 °C. Heat stress did not affect Rubisco per se, since full activity could be restored by incubation with CO2 and Mg2+. Western-blot analysis of leaf extracts disclosed the presence of two Rubisco activase polypeptides, but heat stress did not alter the profile of the activase bands. Inhibition of Pn at high leaf temperature could be markedly reduced by artificially increasing Ci. A high Ci also stimulated photosynthetic electron transport and resulted in reduced non-photochemical fluorescence quenching. Recovery experiments showed that heat-dependent inhibition of Pn was largely, if not fully, reversible. The present results demonstrate that in Q. pubescens leaves the thylakoid membranes in general and photosynthetic electron transport in particular were well protected against heat-induced perturbations and that inhibition of Pn by high temperature closely correlated with a reversible heat-dependent reduction of the Rubisco activation state.
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Global warming, accompanied by an increased frequency of periods with exceptionally high temperatures, is one of the most important characteristics of the accelerated climatic changes. Climatic models predict that global temperature will continue to increase in the future. Hence, plants growing in temperate climates will more often be exposed to heat stress conditions. This will have both ecological and agricultural consequences. High temperatures adversely affect plant growth and survival in a number of ways, but the impact of heat stress on the photosynthetic apparatus is considered to be of particular significance because photosynthesis is often inhibited before other cell functions are impaired (Berry & Björkman 1980). In general, under conditions of high solar irradiance, leaf temperature can increase several degrees Centigrade above air temperature (Leakey, Press & Scholes 2003), which further enhances the problem of high-temperature-dependent reduction of carbon assimilation. Despite intensive research the physiological mechanisms involved in heat-dependent inhibition of photosynthesis still need to be clarified (Sharkey 2000).
Studies aimed at better understanding the physiological mechanisms involved in the inhibition of photosynthesis by heat stress, particularly those focusing on the role of a reduction of Rubisco activation, have concentrated on a few plant species including a variety of crop plants and were typically conducted with plants grown under a controlled environment. Accordingly, little information is thus far available concerning the effects of heat stress on the photosynthetic apparatus of leaves grown under natural conditions. This especially holds true for tree species. In the present study, we examined the response of the photosynthetic apparatus of oak (Quercus pubescens L.) leaves grown under natural conditions in a temperate climate to an elevation of leaf temperature. Besides Rubisco activation assays and immunoblot analysis of Rubisco activase, we employed combined measurements of gas exchange and chlorophyll a fluorescence to differentiate between inhibition of photosynthesis originating from heat effects on components of the thylakoid membranes and that resulting from effects on photosynthetic carbon metabolism.
MATERIALS AND METHODS
All experiments were conducted with oak (Quercus pubescens L.) leaves collected during the morning hours from a tree located in the vicinity of the laboratory. Small twigs cut from the tree were immediately cut for a second time under water in order to avoid disturbances in the transpiration stream. The twigs, kept in a beaker containing water, were then transferred to the laboratory for measurements. Steady-state photosynthesis did not change over the day when measured at room temperature (25 °C) under the conditions described below. Fresh plant material was collected every day.
Gas exchange and chlorophyll a fluorescence measurements
Photosynthetic gas exchange measurements were performed using an open infrared gas analyser (IRGA) system (CIRAS-1; PP-Systems, Hitchin, UK). The net CO2 assimilation rate (Pn), the stomatal conductance (gs), the transpiration rate (Tr) and the intercellular CO2 concentration (Ci) were determined at a light intensity of 500 µmol photons m−2 s−1 (PAR), a constant CO2 concentration of 350 µmol mol−1 and a relative humidity of 80%. Leaf temperature was adjusted to the desired level using the internal heating/cooling system of the analyser. In the experiments in which the CO2 concentration was increased to a high level, CO2 was injected into the circuit using the built-in injection system of the gas analyser. Chlorophyll (Chl) a fluorescence measurements were performed simultaneously using a pulse amplitude modulated fluorometer (FMS-1; Hansatech, King's Lynn, Norfolk, UK). Combined measurements of gas exchange and Chl a fluorescence could only be performed following a modification of the light unit of the gas analyser that allowed the fibre optic of the fluorometer to slide in the unit and thereby come close to the adaxial surface of the sample enclosed in the leaf chamber. The following Chl a fluorescence expressions, defined and calculated as described in Maxwell & Johnson (2000), were monitored: Fv/Fm, the maximum quantum yield of the primary photochemistry of PSII; 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 PS II RCs; ΦPSII, the quantum yield of PSII electron transport and NPQ, the non-photochemical fluorescence quenching.
When not otherwise specified, each sample was first dark-adapted in the leaf chamber for 30 min at 25 °C. Thereafter, the weak measuring light beam of the fluorometer was switched on to determine the minimum fluorescence yield F0. Subsequently, the maximum fluorescence yield Fm was determined by applying a 1-s saturating light pulse via the fibre optic of the fluorometer. The light unit of the gas analyser was then switched on and the sample illuminated at a light intensity of 500 µmol photons m−2 s−1 until steady-state photosynthesis was achieved. However, in all cases the samples were illuminated for at least 30–45 min at a leaf temperature of 25 °C prior to start with the measurements. The initial values of gas exchange (Pn, gs,Tr,Ci) and Chl a fluorescence parameters (F0′, Fm′, Fs), as well as those of the fluorescence expressions derived from the latter (Fv′, qP, Fv′/Fm′, ΦPSII, NPQ) were first recorded immediately before starting with the heat stress treatment. In the experiment with gradually increased temperature, leaf temperature was first elevated from 25 °C to the next higher selected temperature at a rate of about 2.5 °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 prior to again increase the leaf temperature to the next higher selected level. The same procedure was repeated for each of nine tested leaf temperatures in the range between 25 and 45 °C, with the same leaf being used throughout the entire experiment. In the experiment in which temperature was increased rapidly rather than gradually, leaf temperature was directly increased to the desired level at a rate of about 2.5°C min−1. Leaf temperature was then maintained constant and records taken after 30 min when steady-state photosynthesis had been achieved. Thereafter, leaf temperature was reduced to 25 °C and the sample adapted to the dark for 30 min prior to again measuring the fluorescence parameters F0 and Fm. A different leaf was used for each measurement at each of five leaf temperatures selected in the range between 25 and 45°C. In additional experiments of the same type, immediately at the end of the 30 min heat stress treatment, the sample was harvested and used for Rubisco activity measurements as described below. For the recovery experiment, samples that had been rapidly exposed to 45 °C for 30 min were subsequently allowed to recover at 25 °C, with gas exchange and Chl a fluorescence parameters being recorded over time between 0 and 60 min. Parallel experiments of the same type were conducted for Rubisco activity measurements, with samples being collected after 0, 5, 15 and 30 min of recovery.
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 collected immediately at the end of a given heat stress treatment or after a defined period of recovery following a 30-min treatment at 45 °C. The harvested leaf tissue was quickly cut in small pieces to facilitated extraction and extracted within about 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) polyvinylpyrrolidone, 1% (w/v) casein, 0.05% (v/v) Triton X-100, 1 m m phenyl methyl sulphonyl fluoride, and 20 µm leupeptine. An aliquot (30 µL) of the re-suspended extract was assayed at 30 °C either immediately, to determine initial Rubisco activity, or following a 10-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 activation state of Rubisco (Perchorowicz, Raynes & Jensen 1981) was calculated as the relative ratio of initial to total Rubisco activities. Western-blot analysis of Rubisco activase was performed essentially as described elsewhere (Feller et al. 1998). The primary antibody used for Rubisco activase detection was a polyclonal rabbit antibody raised against tobacco Rubisco activase and was kindly provided by Dr S. J. Crafts-Brandner and Dr M. E. Salvucci, ARS USDA, Phoenix, AZ, USA.
Photosynthetic gas exchange parameters as a function of leaf temperature
The net photosynthetic CO2 assimilation rate (Pn) decreased with increasing leaf temperature with the result that relative to the 25 °C-control it was more than 50 and 90% reduced at 40 and 45 °C, respectively (Fig. 1a). Increasing leaf temperature gradually as compared to rapidly did not basically alter the response of Pn to heat stress, showing that short-term acclimation did not help to diminish high-temperature-dependent inhibition of Pn. Above 30 °C, inhibition of Pn was accompanied by reduced stomatal conductance (gs) (Fig. 1b). However, even at the very high leaf temperature of 45 °C, the gs values were less than 20% (rapid temperature increase) and 50% (gradual temperature increase) reduced in comparison with those determined in the corresponding controls at 25 °C. The leaf transpiration rate (Tr) (Fig. 1c) and the intercellular CO2 concentration (Ci) (Fig. 1d) both increased with increasing leaf temperature, with the latter phenomenon becoming especially pronounced at temperatures higher than 35 °C. We would like to point out here that essentially the same results as described above and below were obtained in later experiments of the same type with oak tree seedlings (data not shown). This shows that using cut twigs rather than whole plants did not make any difference and that the results are not restricted to the tree used in the present study but can be generalized.
Chlorophyll a fluorescence parameters as a function of leaf temperature
Chlorophyll a fluorescence measurements revealed that the minimum Chl a fluorescence yield in the light-adapted state (F0′) was hardly affected by leaf temperature in the range between 25 and 35 °C (Fig. 2a). Above 35 °C, F0′ decreased with increasing leaf temperature with the result that at 45 °C it was about 10% reduced as compared with its initial value at 25 °C. The maximum fluorescence yield in the light-adapted state (Fm′) started to decrease already when leaf temperature increased above 30 °C and was more than 65% reduced at 45 °C as compared with 25 °C (Fig. 2b). As a result, the variable Chl a fluorescence yield (Fv′) decreased in parallel with Fm′ and was more than 80% reduced at 45 °C. The steady-state Chl a fluorescence yield in the light-adapted state (Fs) decreased almost linearly with increasing leaf temperature and was 30% reduced at 45 °C as compared with 25 °C (Fig. 2c). The changes of the Chl a fluorescence parameters in response to the elevation of leaf temperature were virtually the same when temperature was increased rapidly versus gradually, but at moderately high temperatures around 35 °C, Fm′ and hence also Fv′ were lower when temperature was increased gradually as compared to rapidly.
Below 32 °C when leaf temperature was increased gradually or 35 °C when it was increased rapidly, high-temperature-dependent inhibition of Pn was not accompanied by a reduction of photosynthetic electron transport. Indeed, up to these temperatures the quantum yield of PSII electron transport (ΦPSII) was hardly affected by leaf temperature (Fig. 3a). Above 35 °C, ΦPSII decreased almost linearly with increasing leaf temperature. High-temperature-dependent inhibition of ΦPSII originated from both a reduction of the fraction of open PSII RCs (qP) (Fig. 3b) and a reduction of the efficiency of excitation energy capture by open PSII RCs (Fv′/Fm′) (Fig. 3c). High-temperature-dependent reduction of the Fv′/Fm′ ratio was associated with an increase of thermal dissipation of excess excitation energy, as reflected by an increase of non-photochemical fluorescence quenching (NPQ) (Fig. 3d). The qP values measured at 45 °C (Fig. 3b) indicate that the oak leaves had the capability to keep nearly 60% (gradual temperature increase) or 70% (rapid temperature increase) of the PSII RCs in an open configuration even when Pn was severely inhibited. At leaf temperatures higher than 30–35°C, the values of ΦPSII, Fv′/Fm′ and qP were somewhat lower when temperature was increased gradually as compared with rapidly (Fig. 3a–c), while the opposite was observed for the NPQ values (Fig. 3d).
The results described above show that the response of the photosynthetic apparatus to heat stress was essentially the same when temperature was increased rapidly versus gradually. Therefore, only the procedure with a rapid elevation of leaf temperature was used in further experiments. Figure 4 shows that the maximum quantum yield of the PSII primary photochemistry, as judged by measuring the Fv/Fm ratio, was only marginally affected by leaf temperature in the range between 25 and 45 °C. Actually, the Fv/Fm ratio was less than 6% reduced in the samples treated at 45 °C as compared with those treated at 25 °C. The heat-induced reduction of the Fv/Fm ratio originated from both a decrease of Fm and an increase of F0 (insert of Fig. 4).
Rubisco activation and western-blot analysis of Rubisco activase
The activity of fully carbamylated Rubisco (total Rubisco activity) was barely affected by leaf temperature in the range between 25 and 45 °C (Fig. 5a). On the contrary, the initial Rubisco activity clearly decreased with increasing leaf temperature. As a consequence, heat stress resulted in a significant reduction of the Rubisco activation state, which decreased from about 90% at 25 °C to less than 30% at 45 °C (Fig. 5b). Western-blot analysis of leaf extracts disclosed the presence of two Rubisco activase polypeptides in Q. pubescens(Fig. 6). In earlier studies conducted with other plant species, it was observed that heat stress at temperatures higher than 40 °C induces the formation of high-molecular-weight aggregates of activase and results in a redistribution of activase from the soluble to the insoluble fraction of extracts (Feller et al. 1998; Salvucci et al. 2001). No such effects were observed in the oak leaves, as a 60 min heat stress treatment at 40 or 45 °C did not significantly alter the profile of the Rubisco activase bands on the blot (Fig. 6). The same result was obtained when the gel was overloaded with protein (not shown). In the blot presented here, the two Rubisco activase bands appearing in the insoluble fraction of the extract were somewhat more pronounced in the sample treated at 45 °C than in the samples treated at 25 or 40 °C. However, most of the activase protein was always found in the soluble fraction of the extracts following incubation at 45 °C. Moreover, the phenomenon was not systematically observed. Therefore, the treatment at 45 °C did most likely not result in a significant redistribution of activase from the soluble to the insoluble fraction of extracts in oak leaves. That heat-induced redistribution of activase was so far predominantly observed for the smaller activase polypeptide (Rokka, Zhang & Aro 2001; Salvucci et al. 2001) supports this view. Likewise, the fact that in the soluble fraction of extracts the activase bands appeared more intense in the sample treated at 40 °C than in the two other samples was an artefact and must not be interpreted as an increase of the content of activase induced by heat stress.
Effects of an artificial increase of Ci
In a further experiment, we tested whether increasing the CO2 concentration would help the leaf to overcome heat-dependent inhibition of Pn. As expected, at a leaf temperature of 25 °C, an increase of Ci from about 280 to 880 µmol mol−1 resulted in a marked increase of Pn, but ΦPSII, Fv′/Fm′, qP and NPQ were virtually unaffected by the increase of Ci(Fig. 7), showing that photosynthetic electron transport activity was not altered. On the contrary, at a leaf temperature of 45 °C a comparable increase of Ci not only resulted in a large increase of Pn but also lead to a significant increase of ΦPSII, showing that photosynthetic electron transport activity was stimulated. Since the fraction of open PSII RCs (qP) increased only marginally in response to the elevation of Ci, the observed increase of ΦPSII originated for the most part from the high Ci-dependent increase of the light use efficiency of open PSII RCs (Fv′/Fm′) that was associated with a reduction of NPQ.
Reversibility of the inhibition of photosynthesis by heat stress
The reversibility of high-temperature-dependent inhibition of Pn was examined in leaves that had been exposed for 30 min to a heat stress treatment at 45 °C and in which Pn had decreased to less than 10% of its original value at 25 °C (Fig. 8). When leaf temperature was set back to 25 °C at the end of the treatment, Pn recovered to more than 50% of its original value within 10 min. After 45 min at 25 °C recovery attained more than 85%, but after 60 min it was still not complete. The insert of Fig. 8 shows that also the Chl a fluorescence parameters recovered from heat-induced changes. For instance, ΦPSII that had been nearly 60% reduced by the treatment at 45 °C progressively recovered to about 80% of its original value within the first 20 min of recovery at 25 °C. However, after this time, recovery was slower and ΦPSII remained reduced by more than 10% even after 60 min at 25 °C. Likewise, qP that had been 20% reduced by the heat stress treatment further decreased by about 10% within the first 5 min of recovery. Thereafter, qP started to increase again but remained about 10% reduced in comparison with its original value. On the contrary, the Fv′/Fm′ ratio almost fully recovered from heat-induced reduction within about 45 min. The pronounced NPQ that appeared at the end of the heat stress period markedly relaxed during the first 15–20 min of recovery and after 45 min at 25 °C it was at about its original level. The Rubisco activation state, which had decreased from about 90% to less than 30% at the end of the treatment at 45 °C, rapidly increased again when the heat stress was alleviated, with the result that nearly full recovery was achieved within approximately 15 min at 25 °C (Fig. 9).
Photosynthetic CO2 fixation before, during and after short-term heat stress
To determine how photosynthesis is responding to short-term leaf temperature fluctuations, we designed an experiment in which we measured Pn before, during and after a short-term period (10 min) during which leaf temperature was temporarily increased from 25 °C to a higher level selected in the range between 35 and 44 °C. In all the samples, the measurements started only after steady-state photosynthesis had been achieved at 25 °C following an illumination period of at least 30 min. Figure 10 shows that Pn decreased rapidly when leaf temperature was elevated to 35 °C or higher temperatures. In the treatment at 44 °C, Pn was about 40 and 70% reduced after 2 and 10 min at high temperature, respectively. Stomatal conductance was less than 20 and 30% reduced at the end of the treatments at 42 and 44 °C, respectively (data not shown). When leaf temperature was set back to 25 °C at the end of the short-term heat stress period, Pn recovered in all cases to its original value within less than 15 min. Actually, recovery was rather quick, since 5 min after the temperature had been reduced to 25 °C the Pn values of the samples treated at 40, 42 or 44 °C were all at about 80% of their original value, showing that the photosynthetic apparatus of the oak leaves had the capability to rapidly adjust to short-term leaf temperature fluctuations.
As expected, Pn decreased with increasing leaf temperature and was found to be more than 90% reduced at 45 °C as compared with 25 °C (Fig. 1a). Our finding that the response of Pn to heat stress was essentially the same when temperature was increased gradually versus rapidly is not consistent with the results obtained in similar experiments conducted with other plant species, in which, due to short-term acclimation processes, inhibition of Pn was less pronounced when temperature was increased gradually as compared with rapidly (Law & Crafts-Brandner 1999; Crafts-Brandner & Salvucci 2002). This discrepancy in the results obtained could originate from differences in the experimental protocol or may reflect a true differential response among plant species. However, it is equally likely that due to the exceptionally hot summer of 2003, during which our experiments were carried out, the oak leaves had become naturally acclimated to elevated temperatures, so that increasing temperature gradually or rapidly was not making a difference. Although gs decreased with increasing leaf temperature (Fig. 2b), inhibition of Pn by heat stress did not result from reduced leaf conductance, as high temperature-dependent reduction of gs was limited and accompanied by a significant increase of Ci (Fig. 3d), which rather reflects metabolic limitations. Other studies have reported that inhibition of Pn by heat stress does not arise because of stomatal limitations (Jiao & Grodzinski 1996; Law & Crafts-Brandner 1999; Crafts-Brandner & Law 2000).
Chl a fluorescence measurements are commonly used to assess heat-induced alterations of the thylakoid membranes and thermal damage to PSII (i.e. Schreiber & Berry 1977; Bilger et al. 1987; Havaux 1992, 1993; Feller et al. 1998). Our finding that F0′ showed a decrease rather than an increase in response to an elevation of leaf temperature (Fig. 2a) is consistent with the results obtained in a study with Arbustus unedo in which F0′ was found to increase only at temperatures higher than 47 °C (Bilger et al. 1987) and suggests that the thylakoid membranes of the oak leaves were not significantly damaged by the heat-stress treatments applied in our study. Such a conclusion must however, be taken with caution, as heat-dependent inhibition of photosynthetic carbon metabolism leading to the development of substantial NPQ may well have hidden the occurrence of a heat-dependent increase of F0′. On the other hand, our finding that the F0 values were only marginally affected by leaf temperature in the range between 25 and 45 °C (insert of Fig. 4) provides additional evidence that the thylakoid membranes of the oak leaves were rather resistant against irreversible heat-induced structural changes, which is in agreement with the observations made in other studies including a variety of tree species (Bilger et al. 1987; Faria et al. 1996; Dreyer et al. 2001).
Our finding that even the heat stress treatment at 45 °C resulted in a less than 6% reduction of the Fv/Fm ratio as compared to that determined in the 25 °C control (Fig. 4) shows that PSII was rather well protected against thermal damage and/or heat-dependent photo-inhibition. Hence, thermal inactivation of PSII contributed only marginally, if at all, to the observed inhibition of Pn at elevated leaf temperatures (Fig. 1a). Acclimation to high temperature has been demonstrated to significantly improve the thermal stability of thylakoid membranes and PSII (Süss & Yordanov 1986; Yordanov et al. 1986; Havaux 1993; Ghouil et al. 2003). Thus, besides a possible innate heat resistance, naturally acquired thermotolerance was probably an important factor that contributed to protect the thylakoid membranes of the oak leaves against thermal damage. Changes in fatty acid composition (Süss & Yordanov 1986; Murakami et al. 2000), accumulation of xanthophyll-cycle carotenoids (Havaux 1998), heat-shock proteins (Török et al. 2001; Heckathorn et al. 2002) and protective compatible solutes (Williams & Gounaris 1992) are among the factors that may have played a role in the phenomenon of in vivo-acquired thermotolerance. Light protection was certainly an additional important factor, as at low to moderate irradiance light significantly improves the resistance of PSII against thermal damage (Schreiber & Berry 1977; Weis 1982; Havaux et al. 1991).
Since thermal inactivation of PSII is known to be slowly reversible with substantial recovery often only being observed after several days (Seemann et al. 1984; Bilger et al. 1987; Karim et al. 1999), our finding that ΦPSII largely recovered from heat-induced reduction within less than an hour at 25 °C (insert of Fig. 8) provides further evidence that inhibition of photosynthetic electron transport was not the primary cause for the inhibition of Pn by high temperature in oak leaves. The result that Pn fully and rapidly recovered from short-term heat stress (Fig. 10) supports this view. On the other hand, reduced photosynthetic electron transport appears to be responsible for the fact that Pn did not fully recover from the inhibition induced by the 30 min heat stress treatment at 45 °C (Fig. 8). Indeed, after this treatment Pn and ΦPSII both remained more than 10% reduced even after 60 min of recovery at 25 °C (Fig. 8). The fraction of ΦPSII that did not recover from the heat stress was associated with reduced qp values (insert of Fig. 8). This result together with our finding that the treatment at 45 °C resulted in a limited reduction of the Fv/Fm ratio (Fig. 4) indicate that a small fraction of the PSII units got damaged during the heat stress treatment. However, it is worth mentioning that we did not observe any sustained reduction of the Fv/Fm ratio in experiments conducted under field conditions even when the temperature of the oak leaves had stabilized well above 40 °C for several hours, with leaf temperature however, not attaining 45 °C even on the warmest days of the exceptionally hot summer in 2003 when air temperature rose above 36 °C (Haldimann, Gallé & Feller, unpublished results).
Despite the evidence presented above that heat-induced inactivation of photosynthetic electron transport can only marginally, if at all, explain the inhibition of Pn at elevated temperatures (Fig. 1a), one may still argue that under heat stress conditions electron transport might have been reversibly inhibited and could hence have been a primary limiting factor for photosynthesis. That this was not the case is demonstrated by our finding that at 45°C ΦPSII markedly increased when Pn was stimulated by increasing Ci (Fig. 7). Crafts-Brandner & Law (2000) observed earlier that increasing Ci completely reversed heat-dependent inhibition of Pn in cotton (Gossypium hirsutum L.) and moreover demonstrated that the phenomenon occurs both under photorespiratory and non-photorespiratory conditions.
High-temperature-dependent reversible thylakoid membrane leakiness that may alter the ATP and NAD(P)H levels is another physiological perturbation that could have played a role in the reduction of Pn at high leaf temperatures (see Pastenes & Horton 1996; Bukhov et al. 1999; Schrader et al. 2004). Such subtle changes in the permeability of the membranes would not have been detected by measuring F0 or Fm and may have been difficult to recognize from changes of F0′. Proton leakage is expected to reduce high-energy state fluorescence quenching (qE), which is directly related with the build-up of a proton gradient (ΔpH) across the membrane. Thus, if significant leakage had occurred in the oak leaves, then the Chl a fluoresecence yield at steady-state (Fs) would likely have increased in response to an elevation of leaf temperature, as observed when the formation of a ΔpH is inhibited chemically by using uncoupling agents. Therefore, our finding that Fs progressively decreased with increasing leaf temperature (Fig. 2c) suggests that the thylakoid membranes were most probably also protected against heat-dependent leakiness. On the other hand, heat stress has been reported to induce a cyclic electron transport around PSI that can balance the loss of protons, so that a sizeable ΔpH can be maintained despite membrane leakage (Bukhov et al. 1999). Consequently, proton leakage might still have occurred in the oak leaves. Heat-dependent activation of cyclic electron transport has been shown to correlate with a significant increase of the apparent F0 following a light-to-dark transition (Sazanov, Burrows & Nixon 1998). Therefore, since we did not observe such a phenomenon in the oak leaves, we can reasonably assume that the permeability of the thylakoid membranes was indeed not significantly altered by heat stress.
The results discussed above indicate that the high-temperature-dependent reduction of ΦPSII (Fig. 3a) mainly originated from a reversible down-regulation of PSII activity that developed in response to an inhibition of photosynthetic carbon metabolism by heat stress. Down-regulation was associated with the development of significant NPQ (Fig. 3d) leading to a reduction of the light use efficiency of open PSII RCs (Fig. 3c). NPQ significantly relaxed under heat stress conditions when photosynthetic electron transport increased in response to a stimulation of CO2 assimilation by an increase of Ci (Fig. 7) or when CO2 fixation recovered from heat-dependent inhibition (insert of Fig. 8), showing that thermal dissipation of absorbed excitation energy was modulated as a function of carbon assimilation activity. The capacity of the oak leaves to safely dissipate excess excitation energy certainly largely contributed to protect PSII from heat-dependent photo-inhibition by helping the photosynthetic apparatus to keep a large fraction of the PSII RCs in an open configuration (Fig. 3b) even when Pn was dramatically reduced by heat stress. Our finding that the high-temperature-dependent decrease of ΦPSII (Fig. 3a and insert of Fig. 8) was less pronounced than that of Pn (Fig. 1a and Fig. 8) is in good agreement with the results obtained by Leakey et al. (2003) and indicates that electrons were flowing to alternative sinks, among which photorespiration was probably the most important. Electron flow to alternative sinks certainly also contributed to protect PSII against heat-dependent photo-inhibition (see Osmond et al. 1997; Wingler et al. 2000; Ort & Baker 2002).
Our findings that inhibition of Pn by heat stress (Fig. 1a) closely correlated with a reduction of the Rubisco activation state (Fig. 5b) and that the activity of fully activated Rubisco was not significantly altered by heat stress (Fig. 5a) are in good agreement with the results obtained in other studies conducted with different plant species (Feller et al. 1998; Law & Crafts-Brandner 1999; Crafts-Brandner & Law 2000; Crafts-Brandner & Salvucci 2000, 2002). It has been proposed that under moderate heat stress conditions Rubisco activation is reduced because the activity of activase is not high enough by itself or because of physical impairment to compensate for the faster de-activation of Rubisco at high temperatures (Crafts-Brandner & Salvucci 2000; Salvucci & Crafts-Brandner 2004a). Our results suggest that when the components of the thylakoid membranes are protected against heat-induced alterations, this reasoning is not only valid for moderate but also for severe heat stress conditions. The exposure of leaves or chloroplasts to temperatures higher than 40 °C has been shown to induce the formation of high-molecular-weight aggregates of activase and to result in 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). These effects suggest that thermal denaturation of activase plays a role in the reduction of Rubisco activation at high leaf temperatures. Western-blot analysis of Rubisco activase revealed that in Q. pubescens elevated leaf temperatures neither induced the formation of high-molecular-mass complexes nor resulted in a major redistribution of activase from the soluble to the insoluble fraction of the extracts (Fig. 6), indicating that activase was resistant or protected against heat-induced denaturation. Our finding that Rubisco activation fully and rather rapidly recovered from high-temperature-dependent inhibition upon alleviation of the heat stress (Fig. 9) supports this view. A direct role of Rubisco activase in the reduction of Rubisco activation at high leaf temperatures can however, not be ruled out, as heat stress might have induced subtle conformational changes of activase that cannot be detected by immunoblot analysis but that negatively affect Rubisco activation by disturbing subunit associations between Rubisco and activase (see Crafts-Brandner & Salvucci 2000; Salvucci & Crafts-Brandner 2004a). Likewise, heat stress might have reduced Rubisco activase activity by altering the quaternary structure of activase from the more active associated state to the less active dissociated state (Crafts-Brandner et al. 1997). The mechanisms involved in heat-dependent reduction of Rubisco activation need to be further clarified, as also subtle conformational changes of the Rubisco enzyme may impede the interactions between Rubisco and activase. In any case, it appears that in the oak leaves reversible heat-dependent reduction of Rubisco activation played a major role in the inhibition of Pn at elevated leaf temperatures. However, as earlier observed in cotton leaves by Crafts-Brandner & Law (2000), we found that Rubisco activation (Fig. 9) recovered more rapidly than Pn (Fig. 8) from heat-dependent inhibition, which indicates that other factors were also playing a role. Finally, by providing evidence that inhibition of Rubisco activation rather than electron transport is the primary functional limitation of photosynthesis at high temperature in oak leaves, our study contributes to the ongoing debate on how heat stress affects photosynthesis (see Salvucci & Crafts-Brandner 2004a, b; Schrader et al. 2004; Wise et al. 2004).
To conclude, in this paper we have shown that in Q. pubescens leaves grown under natural conditions in a temperate climate the thylakoid membranes in general and photosynthetic electron transport in particular are rather well protected against heat-induced perturbations and that inhibition of carbon assimilation at elevated leaf temperatures closely correlates with a reversible high-temperature-dependent reduction of the Rubisco activation state. The capability of Q. pubescens to preserve the functional potential of its photosynthetic apparatus under heat stress conditions is probably an important factor for the remarkable capacity of this tree species to grow and survive in hot and dry habitats.
The authors thank Dr S. J. Crafts-Brandner and Dr M. E. Salvucci (Western Cotton Research Laboratory, US Department of Agriculture, Phoenix, AZ, USA) for the generous gift of the Rubisco activase antibodies, Stephan Hörtensteiner for useful advice concerning the biochemical work and Regina Hölzer for expert technical assistance in western-blot analysis. 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.