Expression of the chloroplast-localized small heat shock protein by oxidative stress in rice

Gene 245 (2000) 283–290 www.elsevier.com/locate/gene

Expression of the chloroplast-localized small heat shock protein by oxidative stress in rice Byung-Hyun Lee a, Sung-Hye Won a, Hyo-Shin Lee a, Mitsue Miyao b, Won-Il Chung c, In-Jung Kim c, Jinki Jo a, * a Department of Animal Science and Biotechnology, College of Agriculture, Kyungpook National University, Sankyukdong, Pukku, Taegu 702-701, South Korea b Department of Plant Physiology, National Institute of Agrobiological Resources (NIAR), Kannondai, Tsukuba 305-8602, Japan c Department of Biological Science, Korea Advanced Institute of Science and Technology, Taejon 305-701, South Korea Received 21 October 1999; received in revised form 20 December 1999; accepted 19 January 2000 Received by H. Uchimiya

Abstract A rice (Oryza sativa L. cv. Nakdong) cDNA clone, Oshsp26, encoding the chloroplast-localized small heat shock protein (smHSP) was isolated. Southern blot analysis of genomic DNA and the result of screening of a cDNA library indicated that the Oshsp26 gene is encoded by a single gene in the rice genome. The Oshsp26 gene was expressed following heat stress: the transcript level was highest when rice leaves were treated at high temperatures for 2 h at 42°C, and the transcripts became detectable after 20 min and reached a maximum level after 2 h. It was also found that the Oshsp26 gene was expressed following oxidative stress even in the absence of heat stress. Treatment of rice plants with methyl viologen (MV ) in the light and treatment with hydrogen peroxide (H O ), either in the light or in the dark, both caused a significant accumulation of the transcripts and the protein. Since 2 2 MV treatment in the light leads to the generation of H O inside the chloroplast, it is likely that H O by itself acts to induce the 2 2 2 2 expression of the Oshsp26 gene. These results suggest that the chloroplast smHSP plays an important role in protecting the chloroplast against damage caused by oxidative stress as well as by heat stress. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Active oxygen species; Molecular chaperone; Oryza sativa; Photosynthesis

1. Introduction All organisms respond to high temperature by inducing the synthesis of a group of proteins called heat shock proteins (HSPs) (Lindquist and Craig, 1988). The major HSPs synthesized in eukaryotes are classified into five conserved classes: HSP100, HSP90, HSP70, HSP60, and small HSPs (smHSPs) of 15–30 kDa (Lindquist and Craig, 1988). Among these, the most abundant in higher plants is a group of smHSPs that constitutes the HSP20 superfamily (Scho¨ffl and Key, 1982; Vierling, 1991). Plant smHSPs are all nuclear-encoded and classified into five multigene families, and localized to different

Abbreviations: ER, endoplasmic reticulum; HSP, heat shock protein; MV, methyl viologen; ORF, open reading frame; OsHSP, Oryza sativa heat shock protein; Oshsp, Oryza sativa heat shock protein gene; PS, photosystem; smHSP, small heat shock protein. * Corresponding author. Tel.: +82-53-950-5756; fax: +82-53-950-6750. E-mail address: [email protected] (J. Jo)

cellular compartments, including the cytosol, the chloroplast, the endoplasmic reticulum ( ER) and the mitochondrium ( Vierling, 1991). As in the case of mammalian and yeast smHSPs, plant smHSPs have a conserved carboxyl-terminal heat-shock domain of about 100 amino acid residues, which is homologous to a-crystallins of the vertebrate eye lens and contains two highly conserved subdomains, consensus I and II ( Vierling, 1991). The chloroplast smHSP is synthesized in the cytoplasm as a precursor and transported into the chloroplast. In addition to the carboxyl-terminal heat-shock domain, the chloroplast smHSP has another highly conserved domain, consensus III, which is predicted to form a methionine-rich amphipathic a-helix (Chen and Vierling, 1991). It has been shown that both plant and mammalian smHSPs as well as a-crystallins form 200–800 kDa multimeric complexes and that they exhibit molecular chaperone activity to prevent thermal aggregation of proteins and to facilitate refolding of denatured proteins ( Vierling, 1991). Recently, it has been recognized that the smHSPs

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have protective roles against a variety of stresses besides high temperatures. These include induction of the expression of 20–40 kDa HSPs by gamma-ray irradiation in tomatoes ( Ferullo et al., 1994), ER-localized smHSP-induction by cold storage in potato tubers (Berkel et al., 1994), cytosolic smHSP-induction by pathogenesis and ozone fumigation in parsley leaves ( Eckey-Kaltenbach et al., 1997), and the induction of mitochondrial and cytosolic smHSPs by oxidative stress in tomato suspension cultures (Banzet et al., 1998). Among a variety of stresses, oxidative stress appears to be crucial to plants, since most environmental stresses lead to generation of active oxygen species, which are highly toxic to cellular processes by adversely reacting with nucleic acids, lipid membranes and proteins (Davis and Goldberg, 1987). In plants, active oxygen species are inevitably generated during photosynthesis, and the major site of generation of these toxic species is the chloroplast under both unstressed and stressed conditions (Asada, 1994). Therefore, it is quite possible that the smHSPs, especially the chloroplast-localized smHSP, have protective functions against oxidative stress. In fact, it has recently been reported that the constitutive expression of the chloroplast smHSP could suppress photooxidative damage after heat treatment of tobacco plantlets (Miyao-Tokutomi et al., 1998). Here, we report the isolation and characterization of a cDNA clone for the chloroplast smHSP of rice. We also show that oxidative stress induces the synthesis of the chloroplast smHSP and propose that H O likely 2 2 acts as a signal for the gene expression.

2. Materials and methods 2.1. Plant growth and stress treatments Seeds of rice (Oryza sativa L. cv. Nakdong) were surface-sterilized in a sodium hypochlorite solution (available Cl: 5%) for 20 min, rinsed with tap water, and incubated in tap water at 25°C for 2 days. Germinated seeds were planted in soil and grown in a growth chamber on a 25°C day/22°C night cycle (day period being 16 h illumination at 350 mE m−2 s−1). For heat-stress treatments, rice leaves were incubated at designated temperatures in a shaking water bath under white light illumination at 200 mE m−2 s−1. The treated leaves were immediately frozen in liquid nitrogen and stored at −80°C until use. For oxidative-stress treatments, intact plants were misted with an aqueous solution containing either methyl viologen (MV, 1 mM ) or H O (10 mM ) and 2 2 0.01% Tween 20. Plants were then placed in growth chambers at 25°C and kept either in the dark or under illumination at 350 mE m−2 s−1. To obtain the root tissue, germinated seeds were grown on a nylon net floated on a liquid medium

containing half-strength MS (Murashige and Skoog, 1962).

inorganic

nutrients

2.2. Construction and screening of a rice cDNA library Poly(A)+ RNA (5 mg) was prepared from rice leaves which had been treated at 42°C for 2 h and used for the construction of a cDNA library. The library was constructed in l ZAPII (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. To obtain a hybridization probe, a DNA fragment encoding the C-terminal of the maize chloroplast smHSP was amplified by PCR on the basis of the nucleotide sequence published previously (Nieto-Sotelo et al., 1990). Primers used were 5∞-CAGATGCTGGACACGATGGA-3∞and 5∞-CACGCCGTTCTTGAGCTCG-3∞, and maize genomic DNA was used as a template. The resultant PCR product of 0.4 kb was ligated into a pGEM-T vector (Promega, Madison, WI ) and sequenced. The nucleotide sequence of the PCR product was completely identical to the published sequence (Nieto-Sotelo et al., 1990). The PCR product was labeled with [a-32P]dCTP by the Multiprime DNA labeling system (Amersham, UK ), and used as a probe for screening the cDNA library. Inserts from selected phage clones were subcloned into a phagemid vector (pBluescript SK−) using an in vivo excision system (Stratagene, La Jolla, CA). 2.3. DNA sequencing and sequence analysis Plasmids were isolated according to the standard protocol (Sambrook et al., 1989). The sequences of both strands of the isolated clones were determined by dideoxy chain termination methods (Sanger et al., 1977), using an ALFexpress Auto Cycle Sequencing Kit and an automated ALFexpress DNA sequencer (Pharmacia, Uppsala). Nucleotide and deduced amino acid sequences were analyzed using Genetyx software (SDC Software Development, Tokyo). 2.4. Northern blot analysis Total RNA was separated on a formaldehyde-denaturing agarose gel (Sambrook et al., 1989), blotted onto a nylon membrane (Hybond N, Amersham, UK ) and hybridized with either a 32P-labeled full-length Oshsp26 cDNA or a gene-specific probe (see below). To obtain a gene-specific probe for Oshsp26, a DNA fragment was amplified by PCR from the 3∞-noncoding region of the cDNA clone. Primers used were OsP-5 (5∞-CAGTAGACGAATGGCTG-3∞; see Fig. 1) and M13 universal primer. The PCR product of 0.2 kb was ligated into a pGEM-T vector (Promega, Madison, WI ), sequenced, and used as a gene-specific probe. Hybridization was carried out as described previously (Lee et al., 1998). The membrane was washed with 0.2×SSC and 0.1% SDS at 65°C for 1 h, exposed to an X-ray film or an

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instructions (Novagen, Madison, WI ). A DNA fragment encoding the mature protein portion of the OsHSP26 protein was amplified by PCR using an N-terminal primer, 5∞-CCCATATGCAGGAGAACAGGG-3∞ containing an NdeI site and a C-terminal primer, 5∞-TAGGATCCATTCGTCTACTGG-3∞ containing a BamHI site, and cDNA as a template. The amplified fragment was digested with NdeI and BamHI, and then subcloned into the expression vector pET28b (Novagen, Madison, WI ) to produce a fusion protein, His-tagged OsHSP26. After transformation into E. coli BL21(DE3) cells, induction of the fusion protein was directed by T7 polymerase, which was induced by 1 mM IPTG for 6 h. The recombinant 6×His–OsHSP26 protein was purified to homogeneity by repeated Ni2+ affinity chromatography, preparative SDS–PAGE, and electroelution. The purified recombinant 6×His– OsHSP26 protein was used to immunize New Zealand White rabbits according to standard methods (Sambrook et al., 1989). 2.7. Protein extraction and immunoblot analysis

Fig. 1. Nucleotide and deduced amino acid sequences of cDNA for the chloroplast smHSP of rice. Closed boxes indicate the translation initiation and termination sites. Horizontal arrows indicate the positions and orientations of primers, Os5N, Os3C and OsP-5, used for PCR amplification. The site of the putative polyadenylation signal is underlined. The nucleotide sequence data have been submitted to the DDBJ, EMBL and GenBank with the accession No. AB020973.

imaging plate and analyzed using an image analyzer (BAS2000, Fuji Film, Tokyo).

Tissues were ground into fine powder in liquid nitrogen with a mortar and pestle. The powder (200 mg) in a microfuge tube was supplemented with 50 mM Hepes, pH 7.5, 2 mM MgCl , 1 mM EDTA, 5% (w/v) polyvinyl2 pyrrolidone, 300 mM 2-mercaptoethanol, 2 mM PMSF and 10% glycerol to give a final volume of 1 ml. Samples were microcentrifuged at 16 000×g for 10 min and applied to 12% SDS–PAGE. After electrophoresis, gels were either stained with Coomassie brilliant blue R250 or transferred onto a nitrocellulose membrane (Protran, Schleicher & Schuell, Germany) for immunoblot analysis. Rabbit antiserum raised against the OsHSP26 fusion protein was used at a dilution of 1:2000. Bound antibodies were detected using a goat anti-rabbit alkaline phosphatase system (Promega, Madison, WI ).

2.5. Southern blot analysis

3. Results

Genomic DNA (5 mg) from rice leaves was digested with HindIII, SalI, or PstI, separated by electrophoresis on an 0.8% agarose gel and blotted onto a nylon membrane (Nytran-Plus, Schleicher & Schuell, Germany) by an alkaline transfer method. The membrane was hybridized with a 32P-labeled full-length Oshsp26 cDNA probe, washed with 0.2×SSC and 0.1% SDS at 60°C for 1 h and then autoradiographed.

3.1. Characterization of a cDNA clone for the chloroplast smHSP from rice

2.6. Expression of OsHSP26 protein in Escherichia coli and antibody production OsHSP26 protein was expressed in E. coli using a T7 expression system according to the manufacturer’s

A cDNA library of heat-treated rice leaves was screened using the PCR-amplified DNA fragment encoding the maize chloroplast smHSP as a probe. A total of 26 positive clones of various lengths were obtained after screening 3.5×104 recombinant phages. These clones show the same digestion patterns with restriction enzymes. Among them, five clones were sequenced and their sequences were found to be identical to each other, except the lengths of the 5∞-terminal region and of the poly (A)+ tail. The cDNA is 1000 bp long when its poly (A)+ tail is excluded (Fig. 1). A putative ATG

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initiation codon was found at nucleotide position 107. The sequence around this triplet fits with the consensus sequence for the translation initiation site in plant genes, i.e. AACAATGGC (Lu¨tcke et al., 1987). If translation starts at this ATG triplet, the cDNA has only one uninterrupted coding region of 720 bp which encodes a polypeptide of 239 amino acid residues with a predicted molecular mass of 26 639 (Fig. 1). Hereafter, this cDNA is designated as Oshsp26 cDNA, and its translation product as the OsHSP26 protein. The OsHSP26 protein has a structure characteristic of the chloroplast smHSP (Fig. 2). In addition to the consensus regions I and II, a region highly homologous to the consensus region III of the chloroplast smHSP ( Vierling, 1991) is present at position 87–110 of the OsHSP26 protein. Suzuki et al. (1998), on the basis of protein sequencing, have shown that the N-termini of the mature forms of the chloroplast smHSP of Arabidopsis (AtHSP21) and pea ( HSP21) are Gln45 and Gln50, respectively. As judged from the sequence homology shown in Fig. 2, the N-terminus of the mature form of OsHSP26 protein is Gln47. The amino acid sequences around the putative N-terminus (AAQE) are highly conserved in the chloroplast smHSPs of monocotyledonous plants (Fig. 2).

Taken together, we conclude that the OsHSP26 protein is the chloroplast smHSP. 3.2. Analysis of the chloroplast smHSP gene in rice genome To determine the number of the Oshsp26 gene in the genome of rice, we performed Southern hybridization using as a probe a full-length cDNA of Oshsp26 (Fig. 3). One or two distinct hybridizing bands were detected upon digestion with three restriction enzymes, HindIII, SalI, or PstI, of which cleavage sites were not present in the cDNA sequence. These results could be an indication of the presence of a small gene family for Oshsp26. However, the intensity of one of the two bands detected upon digestion with HindIII and PstI was much lower than the other. In addition, all of the 26 positive clones screened from the cDNA library showed the same digestion pattern with restriction enzymes. Thus, it is more likely that Oshsp26 is encoded by a single gene. The appearance of two distinct bands might result from the presence of HindIII and PstI sites in some intron(s) in the Oshsp26 gene. The presence of one gene for the chloroplast smHSP has previously been reported in Arabidopsis (Osteryoung et al., 1993), while pea and

Fig. 2. Alignment of the deduced amino acid sequences of the chloroplast smHSPs from seven plant species. The maize sequence is taken from Nieto-Sotelo et al. (1990), barley from Kruse et al. (1993), wheat from Nguyen et al. (1993), Arabidopsis from Chen and Vierling (1991), pea from Vierling et al. (1988), and tobacco from Lee et al. (1998). An arrowhead indicates the putative processing site and boxes indicate the consensus regions I, II and III ( Vierling, 1991). Asterisks represent identical amino acid residues and dashes indicate gaps introduced to optimize alignment.

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Fig. 3. Southern blot analysis of genomic DNA of rice plants. Genomic DNA (5 mg) was digested with HindIII (H ), SalI (S), or PstI (P) and separated on an 0.8% agarose gel. The full-length Oshsp26 cDNA was used as a hybridization probe.

soybean ( Vierling et al., 1988), barley ( Kruse et al., 1993), tobacco (Lee et al., 1998), and wheat (Nguyen et al., 1993) contain two genes. 3.3. Expression of the chloroplast smHSP gene under heat stress Expression of the chloroplast smHSP gene under heat-stress conditions was investigated by Northern blot analysis. As shown in Fig. 4A, transcripts were not detected at the control temperature of 25°C, but were accumulated at temperatures of 39°C and higher. The amount of transcripts accumulated was at a maximum at 42°C and decreased significantly at 45°C. Fig. 4B shows changes in the level of transcripts during heat treatment at 42°C and the subsequent recovery treatment at 25°C. The transcripts were barely detected within the first 20 min, increased to reach a maximum level after 2 h, and then decreased during the next 1 h of the heat treatment. After a temperature reduction to 25°C, the amounts of transcripts declined rapidly, to reach a barely detectable level after 3 h. Similar kinetics of accumulation of transcripts, i.e. the relatively slow appearance of transcripts after the onset of heat stress and the stable accumulation of transcripts for a long time period, have been reported for other plant smHSPs, namely the cytosolic smHSP of the soybean (Scho¨ffl

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Fig. 4. Expression of transcripts of the chloroplast smHSP gene under heat-stress conditions in rice. (A) Temperature dependence. Rice leaves were treated for 2 h at designated temperatures. (B) Time course during heat treatment and the subsequent recovery treatment. Rice leaves were treated at 42°C for 3 h, and then further incubated at 25°C. At designated times, samples were withdrawn for analysis. (C ) Expression in various organs. Hydroponically grown rice plants were treated for 2 h at designated temperatures. L, S, and R indicate leaves, stems and roots, respectively. Total RNA (5 mg) was separated by electrophoresis and probed with the gene-specific probe.

and Key, 1982) and the mitochondrial smHSP of the pea (Lenne et al., 1995). The chloroplast smHSP gene was expressed not only in leaves but also in stems and roots in response to heat stress at 42°C for 2 h (Fig. 4C ), and not in these regions in the absence of heat stress. The levels of transcripts on the RNA basis, however, were high in leaves, moderate in roots and low in stems. 3.4. Oxidative-stress-induced expression of the chloroplast smHSP gene To investigate the response of the Oshsp26 gene to oxidative stress, rice plants were subjected to two different treatments, i.e. with MV (1 mM ) or H O 2 2 (10 mM ). It is known that illumination of leaves in the presence of MV leads to the generation of superoxide anions and H O around photosystem I (PS I ) in the 2 2 chloroplast (Asada, 1994; Kleczkowski, 1993). As shown in Fig. 5A, when plants were treated with MV in the light, a significant amount of transcripts of the chloroplast smHSP was accumulated. Since the transcripts were not detected at all after MV treatment in

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form in leaves. The 21 kDa band of the OsHSP26 protein was also detected in leaves which had been subjected to oxidative-stress treatments, i.e. leaves treated with MV in the light and those treated with H O in both the light and dark. As in the case of the 2 2 transcript level, the levels of OsHSP26 protein accumulated by two different oxidative-stress treatments were almost the same.

4. Discussion

Fig. 5. Expression of the chloroplast smHSP gene in rice leaves under oxidative-stress conditions. (A) Northern blot. Rice plants were misted with a solution containing either MV or H O and then placed under 2 2 illumination at 350 mE m−2 s−1 (L) or in darkness (D) for 2 or 5 h. (−) represents control plants which were treated in the same way but in the absence of MV and H O . Total RNA (20 mg) was prepared 2 2 from leaves of treated plants, separated by electrophoresis and probed with the gene-specific probe. (B) Immunoblot. Rice plants were treated with MV or H O as above, but for 8 h. For comparison, rice leaves 2 2 were exposed to heat stress (HS) at 42°C for 3 h (H ). After treatments, total proteins (40 mg) were extracted from leaves and subjected to immunoblotting. C denotes rice leaves subjected to the control treatments. E. coli denotes the lysate of E. coli cells that expressed the recombinant OsHSP26 protein (10 mg protein).

the dark, the effects of the treatment in the light are ascribable to the generation of active oxygen species but not to MV itself. In the case of the H O treatments, 2 2 the transcripts were accumulated in leaves both in the light and the dark, though the transcript level was slightly higher in the light. The levels of transcripts accumulated by MV treatment in the light and H O 2 2 treatment in the light were almost comparable. The accumulation of the OsHSP26 protein in response to oxidative stresses was analyzed by immunoblotting using anti-OsHSP26 protein antibody (Fig. 5B). The antibody detected a single band of 21 kDa in cell lysates of E. coli that expressed the OsHSP26 fusion protein. The molecular mass of the fusion protein is nearly identical to the calculated value of the mature protein portion of OsHSP26. Also in heat-treated leaves, a single band of 21 kDa was detected. These observations indicate that the antibody used was highly specific to the chloroplast smHSP and that the OsHSP26 protein expressed on heat stress was processed to the mature

We have isolated a full-length cDNA for the Oshsp26 gene that encodes the chloroplast smHSP of rice ( Fig. 1). Oshsp26 showed the expression pattern in leaves that is characteristic of genes of plant smHSPs induced by heat stress, namely relatively slow induction after the onset of heat stress and stable accumulation of transcripts for a long time period during heat stress ( Fig. 4). In addition, its deduced amino acid sequence shares features characteristic of the chloroplast smHSP ( Fig. 2) and its 26 kDa translation product was processed to the mature form of 21 kDa in leaves (Fig. 5B). Oshsp26 was also expressed following heat stress in the stem and the root, and significant levels of transcripts were accumulated (Fig. 4C ). The expression in a nonphotosynthetic tissue suggests that the protective function of the chloroplast smHSP is not limited to photosynthesis. Our results thus confirm a previous suggestion by Kruse et al. (1993) and Vierling et al. (1986) that the chloroplast smHSP protects both the photosynthetic and non-photosynthetic tissues against damage caused by heat stress. In this study, we demonstrated that the chloroplast smHSP was synthesized and accumulated in leaves during oxidative stress ( Fig. 5). This is the first report showing its expression by oxidative stress at the transcript as well as protein levels. We employed two different oxidative-stress treatments, namely MV treatment in the light and H O treatment in the light and 2 2 dark, and both treatments induced the expression of the chloroplast smHSP. It is well known that MV inhibits the PS I reaction by intercepting electron transfer from ferredoxin to NADP+, and then reduces O to the 2 superoxide anion, O− ( Kleczkowski, 1993). Superoxide 2 anions thus generated are readily dismutated to H O 2 2 by the action of superoxide dismutases inside the chloroplast (Asada, 1994). Thus, the MV treatment corresponded to the generation of H O inside the chloroplast. 2 2 The H O treatment, on the other hand, exposed all the 2 2 cellular compartments to H O . Therefore, it is likely 2 2 that H O itself is responsible for the expression of the 2 2 Oshsp26 gene in both cases. A previous study reported that treatments with MV and H O both induced the 2 2 synthesis of the mitochondrial and cytosolic smHSPs but not the chloroplast smHSP (Banzet et al., 1998). This observation might have resulted from plant materi-

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als used. In the previous study, tomato suspension culture cells at the beginning of the exponential phase of growth were used, and it is likely that chloroplasts of these cells were not fully developed so that the chloroplast smHSP, if any, could not be detected. On the other hand, it was also reported that treatment with H O was more effective in inducing the synthesis of 2 2 smHSPs than treatment with MV (Banzet et al., 1998). This observation might have resulted from conditions of illumination during treatment with MV in the previous study, since the amount of active oxygen species generated in the presence of MV depends on the illumination intensity. Since H O is able to diffuse freely 2 2 through membranes (Levin et al., 1994), H O generated 2 2 inside the chloroplast by the MV treatment likely diffuses to the cytoplasm and/or the nucleus. In contrast, O− is 2 highly reactive and cannot diffuse over long distances. Taken together, we propose that H O is a mediator of 2 2 oxidative stress for the gene expression. During the course of photosynthesis, various active oxygen species are generated inside the chloroplast (Asada, 1994). Photosystem II (PS II ) is most sensitive to active oxygen species, and a protein of the PS II reaction center (D1 protein) is susceptible to attack by these reactive species under illumination, leading to photoinhibition of photosynthesis (Miyao, 1994). It has previously been demonstrated that induction of the chloroplast smHSP by heat stress renders resistance to photoinhibition (Stapel et al., 1993) and that the chloroplast smHSP can be accumulated under illumination with strong light even under non-heat-stress conditions (Downs et al., 1998). In addition, the protective function of the chloroplast smHSP against photooxidative stress has been proposed using transgenic tobacco plants that constitutively express the protein (Miyao-Tokutomi et al., 1998). On the other hand, the protection of PS II electron transport by the chloroplast smHSP during heat stress has been demonstrated by an in vitro experiment (Heckathorn et al., 1998), though its mechanism remains unsolved. Therefore, it is possible that the chloroplast smHSP plays an important role in protecting the photosynthetic machinery, especially PS II, against damage caused by photooxidative stresses as well as by heat stress. Plants have several acquired mechanisms, such as the xanthophyll cycle, the active oxygen scavenging system in the vicinity of PS I, and photorespiration (Asada, 1994), to protect the photosynthetic machinery during photooxidative stress. In addition to these protective mechanisms, the chloroplast smHSP may be involved in protecting the photosynthetic machinery under severe photooxidative-stress conditions. Acknowledgements This work was supported by grant of Post-Doc. Program of Kyungpook National University (1998),

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and in part by the Ministry of Agriculture and Forestry of Korea.

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