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BIOENERGETICS AND PHOTOSYNTHESIS

Heat Stress Induces an Aggregation of the Light-Harvesting Complex of Photosystem II in Spinach Plants¹

Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany (Y.T., X.W., Q.L., Z.Y., C.L.) and State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology (Z.C.), Chinese Academy of Sciences, Beijing 100101, People's Republic of China

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Whole spinach (Spinacia oleracea) plants were subjected to heat stress (25°C–50°C) in the dark for 30 min. At temperatures higher than 35°C, CO₂ assimilation rate decreased significantly. The maximal efficiency of photosystem II (PSII) photochemistry remained unchanged until 45°C and decreased only slightly at 50°C. Nonphotochemical quenching increased significantly either in the absence or presence of dithiothreitol. There was an appearance of the characteristic band at around 698 nm in 77 K fluorescence emission spectra of leaves. Native green gel of thylakoid membranes isolated immediately from heat-stressed leaves showed that many pigment-protein complexes remained aggregated in the stacking gel. The analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting demonstrated that the aggregates were composed of the main light-harvesting complex of PSII (LHCIIb). To characterize the aggregates, isolated PSII core complexes were incubated at 25°C to 50°C in the dark for 10 min. At temperatures over 35°C, many pigment-protein complexes remained aggregated in the stacking gel of native green gel, and immunoblotting analyses showed that the aggregates were composed of LHCIIb. In addition, isolated LHCII was also incubated at 25°C to 50°C in the dark for 10 min. LHCII remained aggregated in the stacking gel of native green gel at temperatures over 35°C. Massive aggregation of LHCII was clearly observed by using microscope images, which was accompanied by a significant increase in fluorescence quenching. There was a linear relationship between the formation of LHCII aggregates and nonphotochemical quenching in vivo. The results in this study suggest that LHCII aggregates may represent a protective mechanism to dissipate excess excitation energy in heat-stressed plants.

On the other hand, plants have evolved a series of mechanisms to protect photosynthetic apparatus against damage resulting from heat stress. When subjected to heat stress, a small heat shock protein is expressed and binds to the thylakoid membranes (Osteryoung and Vierling, 1994), and this binding protects PSII and, consequently, whole-chain electron transport (Heckathorn et al., 1998). Heat stress results in a state 1 to state 2 transition by which excitation energy transfer is in favor of PSI (Pastenes and Horton, 1996; Schrader et al., 2004). It has also been reported that the deepoxidation of violaxanthin (V) to zeaxanthin (Z) leads to increased tolerance of PSII and thylakoid membranes to heat stress (Havaux and Tardy, 1996; Havaux et al., 1996; Tardy and Havaux, 1997). In addition, isoprene synthesis in some plants increases tolerance of photosynthesis including PSII to heat stress (Singsaas et al., 1997; Sharkey et al., 2001). Furthermore, it has been reported that dephosphorylation of PSII in response to heat stress can be considered as a protective mechanism by which the repair process of PSII is facilitated (Rokka et al., 2000).

In this study, we report an aggregation of the light-harvesting complex of PSII (LHCII) that occurs both in isolated PSII core particles and LHCII and whole plants in response to heat stress. Our results suggest that the aggregation of LHCII seems to represent a protective mechanism to help dissipate excess excitation energy due to significant inhibition of CO₂ fixation under heat stress.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Effects of Heat Stress on Whole Plant

We first investigated the changes in CO₂ assimilation, PSII function, and 77 K chlorophyll fluorescence emission spectra of leaves, as well as native green gel of thylakoid membranes, after the whole spinach (Spinacia oleracea) plants were exposed to high temperatures in the dark for 30 min. Figure 1 shows the effects of heat stress on CO₂ assimilation rate. CO₂ assimilation rate decreased significantly with increasing temperatures over 35°C. For example, at 50°C, CO₂ assimilation rate decreased to near zero.

View larger version (9K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Effects of heat stress on CO2 assimilation rate of leaves of spinach plants. Seedlings grown at 25°C in the greenhouse were transferred to indicated temperatures in the chambers in the dark for 30 min, and then CO2 assimilation rate was determined. The values are mean ± SE of three independent experiments.

View larger version (11K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Effects of heat stress on Fo, Fm, and Fv/Fm of leaves of spinach plants. Seedlings grown at 25°C in the greenhouse were transferred to indicated temperatures in the chambers in the dark for 30 min, and then chlorophyll fluorescence parameters were determined. The values are mean ± SE of four independent experiments.

View larger version (8K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Effects of heat stress on the characterization of 77 K fluorescence emission spectra of leaves of spinach plants. Seedlings grown at 25°C in the greenhouse were transferred to indicated temperatures in the chambers in the dark for 30 min, and then 77 K fluorescence spectra were determined. A, The spectra were normalized at 680 nm. From the bottom to the top, the spectra were measured from the leaves of whole plants after exposure to 25°C, 35°C, 40°C, 45°C, or 50°C, respectively. The insert shows the 77 K fluorescence emission spectrum of the leaves of whole plants at 25°C. B, The fourth derivative of 77 K fluorescence emission spectra. C, The ratio F698/F680 derived from 77 K fluorescence emission spectra of leaves of spinach plants. The values are mean ± SE of five independent experiments.

View larger version (36K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Effects of heat stress on chlorophyll-protein complexes in thylakoid membranes separated by native green-gel (A). Thylakoid membranes were isolated from the leaves of whole plants after exposure to indicated temperatures in the chambers in the dark for 30 min. The five distinct bands contain: (1) LHCI and PSI core complexes; (2) PSI core complex; (3) trimers of LHCII; (4) PSII core complex; and (5) monomers of LHCII. FP, Free pigment. Effects of heat stress on the polypeptide composition of the chlorophyll aggregates that did not enter the stacking gel in native green-gel analyzed by SDS-PAGE (B) and the polypeptide composition of the chlorophyll aggregates that did not enter the stacking gel in native green gel analyzed by immunoblotting against CP43, CP47, D1, LHCIIb, and psaA (C). CP43, CP47, D1, and psaA were not detected in the aggregates.

To characterize the formation of the aggregates of LHCIIb induced by heat stress, we isolated PSII core particles and then incubated them at high temperatures in the dark for 10 min. Figure 5A shows the effects of heat stress on the native green gel of PSII core particles. According to 77 K fluorescence emission spectra of different gel bands, three distinct bands were separated: (1) the trimers of LHCII; (2) PSII core complex; and (3) the monomers of LHCII. At 25°C and 35°C, all the PSII core particles entered the gel completely. At 40°C, 45°C, and 50°C, however, some PSII core particles remained aggregated in the stacking gel. We also examined the composition of polypeptides of the aggregates. The aggregates in the stacking gel were thus denatured, and their polypeptides were analyzed by SDS-PAGE electrophoresis. Immunoblots against proteins from PSII indicate that D1, CP43, and CP47 proteins were not detectable in the stacking gel aggregates in both nonheated and heated PSII core particles, whereas the protein against the antibody of LHCIIb was detected. The content of LHCIIb increased with increasing temperatures over 35°C (Fig. 5B). These results indicate that heat stress induced an aggregation of LHCIIb when isolated PSII core particles were subjected to high temperature stress in the dark.

View larger version (63K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Effects of heat stress on chlorophyll-protein complexes in isolated PSII core particles separated by native green gel (A). Isolated PSII core particles were incubated at 25°C, 35°C, 40°C, 45°C, or 50°C in the dark for 10 min. The three distinct bands contain: (1) the trimers of LHCII; (2) PSII core complex; and (3) the monomers of LHCII. FP, Free pigment. Effects of heat stress on the polypeptide composition of the chlorophyll aggregates that did not enter the stacking gel in native green gel analyzed by immunoblotting against CP43, CP47, D1, and LHCIIb (B). CP43, CP47, and D1 were not detected in the aggregates. Effects of heat stress on the profiles of isolated LHCII separated by native green gel (C). Isolated LHCII was incubated at 25°C, 35°C, 40°C, 45°C, or 50°C in the dark for 10 min. The two distinct bands contain: (1) trimer; (2) monomers.

To further investigate the aggregation of LHCII induced by heat stress, we observed the formation of LHCII aggregates by using a fluorescence microscope. Figure 6 exhibits the changes in fluorescence images of isolated LHCII after incubation at high temperatures. At 25°C and 35°C, no aggregates of LHCII were observed. However, at 40°C, 45°C, and 50°C, massive aggregation of LHCII occurred and the size of the aggregates increased significantly with increasing temperatures.

View larger version (58K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Effects of heat stress on chlorophyll fluorescence images of isolated LHCII. Isolated LHCII was incubated at 25°C, 40°C, 45°C, or 50°C in the dark for 10 min, and then chlorophyll fluorescence images were determined. Bars in each plate = 5 µm.

The above results clearly demonstrate that heat stress induced an aggregation of LHCII. It has been shown that the aggregation of isolated LHCII leads to quenching of chlorophyll fluorescence yield (for review, see Horton et al., 2005). We thus investigated the changes in chlorophyll fluorescence quenching of isolated LHCII after it was incubated at high temperatures in the dark for 10 min. Figure 7 shows the effects of heat stress on fluorescence quenching of isolated LHCII. There was no significant change in fluorescence quenching at 30°C and 35°C. However, fluorescence quenching increased significantly with increasing temperatures over 35°C. It has been demonstrated that antimycin A inhibits the formation of aggregation of isolated LHCII, resulting in a decrease in fluorescence quenching (Ruban et al., 1992). Thus, addition of antimycin A is supposed to decrease fluorescence quenching of isolated LHCII during heat stress. Indeed, we observed that addition of antimycin A resulted in a decrease in chlorophyll fluorescence quenching of isolated LHCII during heat stress (Fig. 7).

View larger version (11K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Effects of heat stress on fluorescence quenching of isolated LHCII in the absence or presence of 50 µM antimycin A. Isolated LHCII was incubated at 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C in the dark for 10 min, and then chlorophyll fluorescence was determined.

View larger version (7K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Effects of heat stress on NPQ of spinach leaves in the absence or presence of DTT (A), the ratio (A + Z)/(V + A + Z) of spinach leaves in the absence or presence of DTT (B), the ratio F698/F680 of spinach leaves in the absence or presence of antimycin A (C), NPQ of spinach leaves in the absence or presence of antimycin A (D), and the relationship between NPQ and F698/F680 of spinach leaves in the absence or presence of antimycin A (E). DTT treatment was performed by immersing the cut end of a petiole in a 3-mM DTT solution for 2 h at 25°C at a light intensity of 50 µmol m–2 s–1, and then DTT-treated and non-DTT-treated leaves were exposed to indicated temperatures in the dark for 30 min. To eliminate the involvement of the deepoxidation of V during measurement of NPQ, the detached leaves were first accumulated with DTT, and then the leaves were accumulated with antimycin A by immersing the cut end of a petiole in a 5 mM antimycin A solution for 2 h at 25°C at a light intensity of 50 µmol m–2 s–1. Thereafter, the leaves treated with both antimycin A and DTT were exposed to indicated temperatures in the dark for 30 min. NPQ was determined as described in "Materials and Methods." The values are mean ± SE of four independent experiments.

We further investigated the relationship between the formation of the aggregates of LHCII and the heat-induced NPQ in vivo. It has been shown that antimycin A inhibits the formation of LHCII aggregation in vitro and decreases the F₆₉₈/F₆₈₀ ratio (Ruban et al., 1992). Thus, we used antimycin A to inhibit the formation of the LHCII aggregates during heat stress and to examine whether the inhibition of formation of the LHCII aggregates decreased the formation of NPQ. To eliminate the involvement of the deepoxidation of V during measurement of NPQ, the detached leaves were allowed to accumulate antimycin A after the leaves were accumulated with DTT. The formation of the LHCII aggregates was quantitated by the ratio of F₆₉₈/F₆₈₀ from 77 K fluorescence emission spectra of leaves (Ruban and Horton, 1992; Ruban et al., 1994). Antimycin A indeed decreased the ratio of F₆₉₈/F₆₈₀ during exposure to high temperatures, suggesting that antimycin A partially inhibited the formation of the LHCII aggregates (Fig. 8C). At the same time, antimycin A also significantly inhibited an increase in NPQ (Fig. 8D). There was a clear linear relationship between the ratio of F₆₉₈/F₆₈₀ and NPQ (Fig. 8E). These results suggest that heat-induced NPQ in the presence of DTT was associated with the formation of the LHCII aggregates.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
In this study, the results showed that heat stress induced the appearance of a new band peaking at about 698 nm based on the 77 K fluorescence emission spectra (Fig. 3). Moreover, the native green gel of thylakoid membranes isolated immediately from heat-stressed leaves of whole spinach plants demonstrated that heat stress induced the aggregation of some chlorophyll-protein complexes. The analyses of SDS-PAGE and western blotting further showed that this aggregation was composed of the main light-harvesting complex of LHCII (Fig. 4). The experiments on isolated PSII core complex and LHCII further showed that heat stress indeed induced an aggregation of LHCII (Fig. 5). Thus, both in vivo and in vitro studies clearly demonstrated that heat stress induced an aggregation of LHCII.

It should be pointed out that in addition to an appearance of a new band at 698 nm in the 77 K fluorescence emission spectra in heat-stressed leaves, we observed that heat stress also induced an increase in two bands at around 685 and 694 nm (Fig. 3B). Similar results were observed in the 77 K fluorescence emission spectra of LHCII crystals that show several distinct fluorescence bands at around 687 and 694 nm (Pascal et al., 2005). An increase in these two bands in heat-stressed leaves was not due to the aggregation of CP43 and CP47 of PSII, because no CP43 and CP47 were detected in the aggregates using immunoblotting (Fig. 4). It has been suggested that these bands may be associated with the presence of different emitting species arising from changes in chlorophyll configuration (Pascal et al., 2005).

Although great progress has been made in the characterization of the LHCII aggregates in vitro (for review, see Horton et al., 2005), very little is known about their occurrence in vivo. According to the fluorescence emission band with a peak at around 700 nm, several studies have suggested that LHCII aggregation occurs in vivo. For example, a reversible quenching at around 700 nm was observed upon formation of high energy quenching in thylakoid membranes isolated from photoinhibited spinach leaves (Ruban et al., 1991) and in intact whole leaves of the tropical plants Guzmaina monostachis (Ruban et al., 1993; Ruban and Horton, 1995). The formation of a new band at 699 nm in 77 K fluorescence emission spectra was also observed under strong CO₂ deficit (iffel and Vácha, 1998). The results in this study clearly show that LHCII aggregation occurred in vivo during heat stress. To the best of our knowledge, until now, there is no report showing that heat stress leads to an aggregation of LHCII in vivo.

It has been suggested that an aggregation of isolated LHCII results in a significant increase in nonphotochemical dissipation of excitation energy (Horton et al., 1996). In this study, our results also suggest that the aggregation of isolated LHCII induced by heat stress resulted in a significant increase in nonphotochemical dissipation of excitation energy (Fig. 7). Furthermore, we investigated whether the LHCII aggregates induced by heat stress lead to a significant increase in nonphotochemical dissipation of excitation energy in vivo by examining the changes in NPQ after the leaves were exposed to heat stress in the dark. Our results show that in the absence of DTT, heat stress resulted in a significant increase in NPQ of the leaves that was accompanied by a significant increase in the ratio (Z + A)/(V + A + Z), suggesting that this increased NPQ was associated with increased deepoxidation of V. Comparison of NPQ values between non-DTT and DTT-treated leaves demonstrates that about one-half of this increased NPQ was contributed by the deepoxidation of V (Fig. 8, A and B). However, when the deepoxidation of V was inhibited by DTT, NPQ still existed and increased significantly with increasing temperatures over 35°C (Fig. 8, A and B), indicating that heat stress induced a kind of NPQ that was independent of the deepoxidation of V. Our results clearly demonstrate that this xanthophyll cycle-independent NPQ was linearly related to the formation of the LHCII aggregates (Fig. 8, C–E).

Our results show that compared to a substantial decrease in CO₂ assimilation rate during heat stress, F_v/F_m showed only a slight decrease at 50°C (Figs. 1 and 2), which means that the energy input by light exceeds the demand of CO₂ fixation metabolism. Thus, the heat-stressed plants are potentially exposed to excess excitation energy, which will inevitably result in damage to photosynthetic apparatus if excess excitation energy cannot be dissipated safely. The xanthophyll cycle has been shown to be a photoprotective dissipation process and plays an important role in dissipating excess excitation energy in the antennae complexes of PSII as heat (Demmig-Adams and Adams, 1992; Horton et al., 1996; Gilmore, 1997; Niyogi et al., 1998). Our results show that heat stress induced an enhancement of the deepoxidation of V, and this xanthophyll cycle-related thermal dissipation in heat-stressed plants contributed about one-half to the total NPQ. On the other hand, thermal dissipation from the LHCII aggregates in heat-stressed plants contributed a significant part to the total NPQ (Fig. 8). Obviously, the LHCII aggregates will help to dissipate excess excitation energy in heat-stressed plants. In addition, our results show that an aggregation of LHCII started to occur at temperatures over 35°C, while F_v/F_m started to decrease slightly only at 50°C (Figs. 2–4), suggesting that an aggregation of LHCII occurred prior to heat damage to PSII. Therefore, our results suggest that the LHCII aggregates induced by heat stress seem to represent a protective mechanism by which excess excitation energy in heat-stressed plants resulting from significant inhibition of CO₂ fixation can be dissipated.

It is not clear how heat stress induced an aggregation of LHCII. Studies with isolated LHCII and thylakoids have shown that LHCII aggregation was stimulated by low intrathylakoid pH and/or by Z (Horton et al., 1996). Because heat treatment in this study was performed in the dark, it is not possible that there was a low intrathylakoid pH. In addition, we observed that no Z was induced during heat stress in the dark. Therefore, it is not likely that the LHCII aggregation in heat-stressed plants was associated with low intrathylakoid pH and Z. Here, we tentatively propose that heat stress may induce conformational changes in LHCII itself that lead to a specific association between hydrophobic domains of different LHCII, thus resulting in the aggregation of LHCII.

In terms of reversibility of the LHCII aggregates in heat-stressed plants, we observed that heat-induced increase in the ratio F₆₉₈/F₆₈₀ recovered nearly to the level at 25°C after these heat-stressed plants were returned to 25°C, suggesting that the LHCII aggregates induced by heat stress were largely reversible. Accordingly, the decreased CO₂ assimilation rate also almost recovered to the level at 25°C. Similarly, a reversible quenching at around 700 nm was observed after a subsequent dark recovery for high light-treated spinach leaves (Ruban et al., 1991). Thus, the reversibility of the LHCII aggregates further suggests that an aggregation of LHCII can be considered to be an adaptation mechanism in response to heat stress, where CO₂ assimilation is inhibited and excitation energy is excess.

In conclusion, both in vivo and in vitro experiments in this study showed that heat stress induced an aggregation of LHCII. Moreover, there was a linear relationship between the formation of LHCII aggregates and NPQ in vivo and in vitro. In addition, heat stress substantially inhibited CO₂ assimilation rate but only slightly decreased F_v/F_m. The results in this study suggest that an aggregation of LHCII may represent a protective mechanism to dissipate excess excitation energy in heat-stressed plants.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Materials and Growth Conditions

Spinach (Spinacia oleracea) plants were grown in the soil in a greenhouse at 25°C ± 1°C with photosynthetic photon flux density of 300 µmol m^–2 s^–1, a relative humidity of 75% to 80%, and a photoperiod of 14/10 h light/dark. For the convenience of heat treatment for whole plants, some seedlings were grown in plastic pots (30 cm in diameter, 25 cm tall) filled with soil. All the seedlings were fertilized with enough NPK nutrients and watered with enough water so that they were grown well. The seedlings after growth for 2 months were subjected to various experiments. All the measurements on physiological and biochemical parameters were carried out on the youngest fully expanded leaves.

Light-Saturated CO₂ Assimilation Rate

CO₂ assimilation rate was made on a fully expanded attached leaf of spinach seedlings using an open system (Ciras-1, PP Systems). After exposure to high temperatures in the dark for 30 min, the whole plants were returned to 25°C, and gas exchange was then analyzed. The light-saturating CO₂ assimilation rate was made at a CO₂ concentration of 360 µL L^–1, 25°C with a relative humidity 80% and saturating light (800 µmol m^–2 s^–1). The measurements lasted approximately 10 min, during which no significant recovery was observed on these parameters.

Pigment Analyses

Leaf samples were taken and immediately frozen in liquid nitrogen. Leaf samples were extracted in ice-cold 100% acetone, and the pigment extracts were filtered through a 0.45-µm membrane filter. Pigments were separated and quantified by HPLC, following the protocol described in a previous article (Lu et al., 2001).

Isolation of Thylakoid Membranes, PSII Core Particles, and LHCII

Leaves were homogenized in a medium containing 0.4 M Suc, 50 mM Tricine, pH 7.6, and the homogenate was centrifuged at 500g for 2 min to remove large debris. The supernatant was centrifuged at 3,000g for 10 min. The pellet was washed twice with the medium (50 mM Tricine, 10 mM NaCl, 5 mM MgCl₂, pH 7.6) at 10,000g for 10 min. The resulting washed pellet was thylakoid membranes and was resuspended in a minimal volume of the same medium with 10% glycerol frozen in liquid and stored at –80°C for further use. All procedures were carried at 0°C to 4°C. PSII core particles and LHCII were isolated according to Ghanotakis et al. (1984) and Simidjiev et al. (1997), respectively. The isolated complexes were stored in 0.5-mL Eppendorf tubes under –80°C.

Chlorophyll Fluorescence Measurements

Chlorophyll fluorescence quenching analyses of leaves were measured with a PAM-2000 chlorophyll fluorescence system (Heinz Walz). After a dark adaptation period of 10 min F_o was determined by a weak red light. F_m of a dark-adapted leaf was measured during a subsequent saturating pulse of white light (8,000 µmol m^–2 s^–1 for 0.8 s). The leaf was then continuously illuminated with actinic light at an intensity of 800 µmol m^–2 s^–1 for about 5 min. The steady-state fluorescence was thereafter recorded, and a second saturating pulse of white light (8,000 µmol m^–2 s^–1 for 0.8 s) was imposed to determine the F_m level in the light-adapted state (F_m'). F_v/F_m and NPQ were calculated as (F_m – F_o)/F_m and (F_m/F_m') – 1, respectively. Fluorescence nomenclature was according to van Kooten and Snel (1990).

The 77 K chlorophyll fluorescence spectra of leaves were measured with a spectrofluorometer (Hitachi F-4500). All spectra were recorded at 77 K with the spectral bandwidth of emission monochromator of 5 nm. The excitation wavelength was 436 nm. The spectra distributions of the emission monochromator and detector (photomultiplier) were corrected by the program designed for model F-4500 fluorescence spectrophotometer by Hitachi.

Chlorophyll fluorescence quenching of isolated LHCII was monitored using a Walz PAM-101 fluorometer as described by Ruban et al. (1994), and fluorescence quenching was quantified as the difference in fluorescence yield divided by the amplitude of F_m, (F – F')/F', where F is the level of fluorescence for a sample diluted into 0.03% DM at 25°C, and F' is the actual level of fluorescence recorded at different higher temperatures.

Native Green-Gel Electrophoresis

The native green-gel electrophoresis of thylakoid membranes, PSII core particles, and LHCII were determined according to the procedure of Tanaka et al. (1987) using a tube gel (diameter 1 cm) with 8% resolving gel and 4% stacking gel. Solubilization medium contains 2 mM Tris-maleate, pH 7.0, 0.5% SDS, and 10% glycerol. Solubilization medium was added to yield a ratio of SDS:chlorophyll of 10:1 (w/w). The electrode medium contains 25 mM Tris, pH 8.3, 192 mM Gly, and 0.1% lithium dodecyl sulfate. After samples and solubilization medium were mixed gently, the electrophoresis was carried out immediately.

Observation of LHCII Aggregates

Aggregation of LHCII induced by heat stress was observed by using a fluorescence microscopy (Olympus BX-61). The aggregates were imaged using an excitation range of 530 to 550 nm and an emission range of 590 to 750 nm. The fluorescence images were taken at 100-fold magnification using a digital camera. The samples of LHCII were diluted in 10 mM Tricine medium, pH 7.8, containing 0.03% DM, and chlorophyll concentration was 2 mg mL^–1.

SDS-PAGE and Immunological Analyses

Samples were solubilized in the presence of 6 M urea and separated by SDS-PAGE (Laemmli, 1970) using 15% (w/v) acrylamide gels with 6 M urea. For immunoblotting, polypeptides were electrophoretically transferred to polyvinylidene difluoride membranes (Millipore), and proteins were detected with antibodies raised against CP43, CP47, D1, LHCIIb, and psaA.

Determinations of Protein and Chlorophyll

Chlorophyll content was measured in 80% (v/v) acetone according to Porra et al. (1989). Protein content was determined by the dye-binding assay according to Bradford (1976).

	ACKNOWLEDGMENTS

 
We thank our colleagues at the Institute of Botany, Lixin Zhang and Chunhong Yang, for discussion during carrying out this study. We also thank the two anonymous reviewers and the editor for critical comments on the manuscript.

Received October 2, 2006; accepted November 26, 2006; published December 1, 2006.

	FOOTNOTES

 
¹ This work was supported by the Chinese Academy of Sciences (Frontier Project of the Knowledge Innovation Engineering no. KJCX2–SW–w29) and the Program of 100 Distinguished Young Scientists (to C.L.).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Congming Lu (lucm{at}ibcas.ac.cn).

www.plantphysiol.org/cgi/doi/10.1104/pp.106.090712

^* Corresponding author; e-mail lucm{at}ibcas.ac.cn; fax 86–10–62595516.

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