Chloroplast Small Heat Shock Protein Research Articles

AsHSP26.2, a creeping bentgrass chloroplast small heat shock protein positively regulates plant development.

The creeping bentgrass small heat shock protein AsHSP26.2 positively regulates plant growth and is a novel candidate for use in crop genetic engineering for enhanced biomass production and grain yield. Small heat shock proteins (sHSPs), a family of proteins with high level of diversity, significantly influence plant stress tolerance and plant development. We have cloned a creeping bentgrass chloroplast-localized sHSP gene, AsHSP26.2 responsive to IAA, GA and 6-BA stimulation. Transgenic creeping bentgrass overexpressing AsHSP26.2 exhibited significantly enhanced plant growth with increased stolon number and length as well as enlarged leaf blade width and leaf sheath diameters, but inhibited leaf trichomes initiation and development in the abaxial epidermis. These phenotypes are completely opposite to those displayed in the transgenic plants overexpressing AsHSP26.8, another chloroplast sHSP26 isoform that contains additional seven amino acids (AEGQGDG) between the consensus regions III and IV (Sun et al., Plant Cell Environ 44:1769-1787, 2021). Furthermore, AsHSP26.2 overexpression altered phytohormone biosynthesis and signaling transduction, resulting in elevated auxin and gibberellins (GA) accumulation. The results obtained provide novel insights implicating the sHSPs in plant growth and development regulation, and strongly suggest AsHSP26.2 to be a novel candidate for use in crop genetic engineering for enhanced plant biomass production and grain yield.

STAY-GREEN and light-harvesting complex II chlorophyll a/b binding protein are involved in albinism of a novel albino tea germplasm ‘Huabai 1’

‘Huabai 1’ is a new albino tea germplasm, with high amino acids in its albinism period, however, the albino mechanism remains unknown. To understand the albinism mechanism of ‘Huabai 1’, molecular-level studies were carried out to reveal the functions of CsLHCII and CsSGR in albinism. In this study, transmission electron microscopy (TEM) observations revealed that albino leaves of ‘Huabai 1’ are more damaged than that of ‘Baiye 1’. CsSGR overexpression in A. thaliana and N. benthamiana indicated that CsSGR is involved in the regulation of leaf albinism, and it may play an important role as a key regulator for the dismantling of photosynthetic chlorophyll - LHCP complexes. Moreover, CsLHCII overexpression promotes leaf regreening. The Yeast two-hybrid (Y2H) screening identified a chloroplast small heat shock protein (CssHSP, XM_028206028.1) which binds to CsSGR and CsLHCII respectively, forming CsSGR-CssHSP-CsLHCII protein complexes that may function in regulating albinism of ‘Huabai 1’.

Substrates of the chloroplast small heat shock proteins 22E/F point to thermolability as a regulative switch for heat acclimation in Chlamydomonas reinhardtii

Key messageWe have identified 39 proteins that interact directly or indirectly with high confidence with chloroplast HSP22E/F under heat stress thus revealing chloroplast processes affected by heat.Under conditions promoting protein unfolding, small heat shock proteins (sHsps) prevent the irreversible aggregation of unfolding proteins by integrating into forming aggregates. Aggregates containing sHsps facilitate the access of Hsp70 and ClpB/Hsp104 chaperones, which in ATP-dependent reactions disentangle individual proteins from the aggregates and assist in their refolding to the native state. Chlamydomonas reinhardtii encodes eight different sHsps (HSP22A to H). The goal of this work was to identify chloroplast-targeted sHsps in Chlamydomonas and to obtain a comprehensive list of the substrates with which they interact during heat stress in order to understand which chloroplast processes are disturbed under heat stress. We show that HSP22E and HSP22F are major chloroplast-targeted sHsps that have emerged from a recent gene duplication event resulting from the ongoing diversification of sHsps in the Volvocales. HSP22E/F strongly accumulate during heat stress and form high molecular mass complexes. Using differential immunoprecipitation, mass spectrometry and a stringent filtering algorithm we identified 39 proteins that with high-confidence interact directly or indirectly with HSP22E/F under heat stress. We propose that the apparent thermolability of several of these proteins might be a desired trait as part of a mechanism enabling Chlamydomonas chloroplasts to rapidly react to thermal stress.

Heavy metal-induced oxidative stress on seed germination and seedling development: a critical review.

Heavy metal contamination in soils can influence plants and animals, often leading to toxicosis. Heavy metals can impact various biochemical processes in plants, including enzyme and antioxidant production, protein mobilization and photosynthesis. Hydrolyzing enzymes play a major role in seed germination. Enzymes such as acid phosphatases, proteases and α-amylases are known to facilitate both seed germination and seedling growth via mobilizing nutrients in the endosperm. In the presence of heavy metals, starch is immobilized and nutrient sources become limited. Moreover, a reduction in proteolytic enzyme activity and an increase in protein and amino acid content can be observed under heavy metal stress. Proline, is an amino acid which is essential for cellular metabolism. Numerous studies have shown an increase in proline content under oxidative stress in higher plants. Furthermore, heat shock protein production has also been observed under heavy metal stress. The chloroplast small heat shock proteins (Hsp) reduce photosynthesis damage, rather than repair or help to recover from heavy metal-induced damage. Heavy metals are destructive substances for photosynthesis. They are involved in destabilizing enzymes, oxidizing photosystem II (PS II) and disrupting the electron transport chain and mineral metabolism. Although the physiological effects of Cd have been investigated thoroughly, other metals such as As, Cr, Hg, Cu and Pb have received relatively little attention. Among agricultural plants, rice has been studied extensively; additional studies are needed to characterize toxicities of different heavy metals on other crops. This review summarizes the current state of our understanding of the effects of heavy metal stress on seed germination and seedling development and highlights informational gaps and areas for future research.

Agrobacterium rhizogenes rolB gene affects photosynthesis and chlorophyll content in transgenic tomato (Solanum lycopersicum L.) plants

Insertion of Agrobacterium rhizogenes rolB gene into plant genome affects plant development, hormone balance and defence. However, beside the current research, the overall transcriptional response and gene expression of rolB as a modulator in plant is unknown. Transformed rolB tomato plant (Solanum lycopersicum L.) cultivar Tondino has been used to investigate the differential expression profile. Tomato is a well-known model organism both at the genetic and molecular level, and one of the most important commercial food crops in the world.Through the construction and characterization of a cDNA subtracted library, we have investigated the differential gene expression between transgenic clones of rolB and control tomato and have evaluated genes specifically transcribed in transgenic rolB plants. Among the selected genes, five genes encoding for chlorophyll a/b binding protein, carbonic anhydrase, cytochrome b6/f complex Fe-S subunit, potassium efflux antiporter 3, and chloroplast small heat-shock protein, all involved in chloroplast function, were identified. Measurement of photosynthesis efficiency by the level of three different photosynthetic parameters (Fv/Fm, rETR, NPQ) showed rolB significant increase in non-photochemical quenching and a, b chlorophyll content. Our results point to highlight the role of rolB on plant fitness by improving photosynthesis.

Constitutive Expression of a Tomato Small Heat Shock Protein Gene LeHSP21 Improves Tolerance to High-Temperature Stress by Enhancing Antioxidation Capacity in Tobacco

It is well established that small heat shock proteins (sHSPs) play an important role in thermotolerance in various organisms due to their abundance and diversity. In the present study, a chloroplast small heat shock protein gene (LeHSP21) from tomato (Lycopersicon esculentum cv PKM-1) was constitutively expressed in tobacco (Nicotiana tabacum L. cv Wisconsin 38) plants via Agrobacterium-mediated transformation. When compared to wild-type control plants, transgenic tobacco plants constitutively expressing LeHSP21, driven by the cauliflower mosaic virus 35S promoter, exhibited improved tolerance to both high temperature and oxidative stress. Furthermore, when the seedlings were subjected to high temperature treatment, the activities of anti-oxidative enzymes and the content of proline were significantly higher in transgenic plants than those in the wild-type plants. Our results presented here demonstrate the feasibility of improving high temperature and oxidative stress tolerance in plants through the expression of LeHSP21 gene.

Overexpression of heat shock protein gene PfHSP21.4 in Arabidopsis thaliana enhances heat tolerance

Nuclear-encoded chloroplast small heat shock proteins (Cp-sHSPs) play important roles in plant stress tolerance due to their abundance and diversity. Their functions in Primula under heat treatment are poorly characterized. Here, expression analysis showed that the Primula Cp-sHSP gene, PfHSP21.4, was highly induced by heat stress in all vegetative and generative tissues in addition to constitutive expression in certain development stages. PfHSP21.4 was introduced into Arabidopsis, and its function was analysed in transgenic plants. Under heat stress, the PfHSP21.4 transgenic plants showed increased heat tolerance as shown by preservation of hypocotyl elongation, membrane integrity, chlorophyll content and photosystem II activity (Fv/Fm), increased seedling survival and increase in proline content. Alleviation of oxidative damage was associated with increased activity of superoxide dismutase and peroxidase. In addition, the induced expression of HSP101, HSP70, ascorbate peroxidase and Δ1-pyrroline-5-carboxylate synthase under heat stress was more pronounced in transgenic plants than in wild-type plants. These results support the positive role of PfHSP21.4 in response to heat stress in plants.

Molecular Characterization of Chenopodium album Chloroplast Small Heat Shock Protein and Its Expression in Response to Different Abiotic Stresses

Plants are sessile organisms that have to cope with different environmental stresses during their life cycle. Photosystem II is one of the most labile processes affected by abiotic stress. The chloroplast small heat shock proteins (Cp-sHSPs) are known to protect photosystem II and thylakoid membranes during heat stress. Previously, we reported several cis-regulatory elements in the promoter regions of Cp-sHSPs of Chenopodium album (Shakeel et al., Plant Physiol Biochem 49:898–908, 2011) and differential regulation of a novel Cp-sHSP family member, CaHSP26.13p, under heat and metal stress (Haq et al., Plant Cell Reports 31:1943–1957, 2012). To further explore the role of Cp-sHSPs in plant stress tolerance, we subjected C. album plants to salt, drought, or cold stress and characterized the accumulation of Cp-sHSP transcripts. Full-length Cp-sHSP transcripts were analyzed for the presence of characteristic domains of Cp-sHSPs and compared with previously known Cp-sHSP homologs and orthologs. Analysis showed that the CaHSP26.13p transcript is differentially regulated under heat, metal, cold, drought, or salt stress. Immunoblot analysis revealed the presence of the precursor proteins (∼26 kDa) synthesized from this transcript. The expression level varied depending on the conditions. Generally, transcript and protein levels were not correlated. This study demonstrates that a single C. album Cp-sHSP had multiple roles under a variety of environmental stresses.

A Dual Role for the Chloroplast Small Heat Shock Protein of Chenopodium album including Protection from Both Heat and Metal Stress

The chloroplast small heat shock proteins (Cp-sHSP) protect photosystem II and thylakoid membranes during heat and other types of stresses. The amount of Cp-sHSPs produced is correlated with plant thermotolerance. Cp-sHSPs of Chenopodium album have already been shown to be heat-regulated and have a novel promoter architecture containing some conserved regulatory motifs Shakeel et al. (Plant Physiol Biochem 49:898–908, 2011). To determine if similar or different Cp-sHSP isoforms protect plants against heat and metal stress, we isolated and characterized a novel Cp-sHSP isoform, CaHSP26.13p (GI # JX073659) from a Pakistani C. album ecotype and determined its expression under heat and metal stress by measuring transcript and protein levels. Presence of the same CaHSP26.13p transcript in heat- and metal-treated plants provided evidence of heat- and metal-regulated expression. Gene expression analysis indicated that Cp-sHSP transcript levels were maximum at 37 °C for 4 h (heat stress) and 20 mM cadmium (metal stress). Immunoblot analysis revealed that (1) maximum expression of precursor and processed protein was observed at 37 °C in the case of heat stress, (2) while only precursor protein of ~26 kDa was produced in the case of metal stress. The highest expression occurred when plants were treated with 15 mM cadmium. We speculate that post-translational regulation accounted for the lack of correlation between transcript and protein levels. This study demonstrates that differential regulation of the same C. album Cp-sHSP family member enables it to play a dual role in protecting the plant from heat and metal stress.

The wheat chloroplastic small heat shock protein (sHSP26) is involved in seed maturation and germination and imparts tolerance to heat stress

The nuclear-encoded chloroplast small heat shock proteins (sHSPs) are present in all plant species from algae to angiosperms. Expression analysis shows that the wheat chloroplastic sHSP (HSP26) is highly inducible by heat stress in almost all the vegetative and generative tissues and is also expressed constitutively in certain developmental growth stages. We characterize wheat chloroplastic sHSP 26 through transgenic approach using Arabidopsis and report cloning of the promoter and its characterization. Transgenic Arabidopsis plants were substantially tolerant under continuous high temperature regimen than wild-type plants, as measured by photosystem II (PSII) activity, accumulation of more photosynthetic pigments, higher biomass and seed yield. Transgenic plants produced bold seeds under high temperature, having higher germination potential than the wild-type plants. Further, antisense Arabidopsis plants showed negligible tolerance even for non-lethal heat shock, impaired in basal thermo-tolerance, and accumulated less biomass and seed yield under normal growth conditions. Promoter analysis revealed the presence of several heat and other abiotic stress responsive cis-acting elements along with developmental stage and tissue-specific elements. Analysis of promoter through GUS reporter system in both transgenic rice and Arabidopsis further confirms the role of chloroplastic sHsp26 in heat and other abiotic stresses as well as during seed maturation and germination. Genome-wide expression analysis of overexpression Arabidopsis plants revealed that the transcriptome remained unchanged in the transgenic plants and the tolerance was due to the overexpression of chloroplastic heat shock protein (HSP) only.

Ecotypic variation in chloroplast small heat-shock proteins and related thermotolerance in Chenopodium album

Production of chloroplast-localized small heat-shock proteins (Cp-sHSP) is correlated with increased thermotolerance in plants. Ecotypic variation in function and expression of Cp-sHSPs was analyzed in two Chenopodium album ecotypes from cool vs. warm-temperate USA habitats [New York (NY) and Mississippi (MS) respectively]. P(et) was more heat tolerant in the MS than the NY ecotype, and MS ecotype derived proportionally greater protection of P(et) by Cp-sHSP during high temperatures. Four genes encoding Cp-sHSPs were isolated and characterized: CaHSP25.99n (NY-1) and CaHSP26.23n (NY-2) from NY ecotype, and CaHSP26.04m (MS-1) and CaHSP26.26m (MS-2) from MS ecotype. The genes were nearly identical in predicted amino-acid sequence and hydrophobicity. Gene expression analysis indicated that MS-1 and MS-2 transcripts were constitutively expressed at low levels at 25 °C, while no NY-1 and NY-2 transcripts were detected at this temperature. Maximum accumulation of NY-1 and NY-2 transcripts occurred at 33 °C and 40 °C for MS-1 and MS-2. Immunoblot analysis revealed that (1) protein expression was highest at 37 °C in both ecotypes, but was greater in MS than NY ecotype at 40 °C; and (2) import of Cp-sHSP into chloroplasts was more heat-labile in NY ecotype. The higher expression of one isoform in MS ecotype may contribute to its enhanced thermotolerance. Absence of correlation between protein and transcript levels, suggests the post-transcriptional regulation is occurring. Promoter analysis of these genes revealed significant variations in heat-shock elements (HSE), core motifs required for heat-shock-factor binding. We propose a correlation between unique promoter architecture, Cp-sHSP expression and thermotolerance in both ecotypes.

Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21

Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25° in relation to each other, suggesting a role for global dynamics in dodecamer function.

Duplication of the class I cytosolic small heat shock protein gene and potential functional divergence revealed by sequence variations flanking the α-crystallin domain in the genus Rhododendron (Ericaceae)

Positive selection in the -crystallin domain (ACD) of the chloroplast small heat shock protein (CPsHSP) gene was found in a previous study and was suggested to be related to the ecological adaptation of Rhododendron species in the subgenus Hymenanthes. Consequently, it was of interest to examine whether gene duplication and subsequent divergence have occurred in other sHSP genes, for example class I cytosolic sHSP genes (CT1sHSPs) in Rhododendron in Taiwan, where many endemic species have evolved as a result of habitat differentiation. A phylogeny of CT1sHSP amino acid sequences was built from Rhododendron, Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Vitis vinifera and other species for elucidation of the phylogenetic relationships among CT1sHSPs. Phylogenies of Rhododendron CT1sHSP nucleotide and amino acid sequences were generated for positive selection and functional divergence analysis, respectively. Positively selected sites and amino acid differences between types of Rhododendron CT1sHSPs were mapped onto the wheat sHSP16.9 protein structure. Average genetic distance (Dxy) and dN/dS ratios between types of Rhododendron CT1sHSP genes were analysed using sliding window analysis. Gene conversion was also assessed between types of Rhododendron CT1sHSPs. Two types of Rhododendron CT1sHSP were identified. A high level of genetic similarity and diversity within and flanking the ACD, respectively, between types of Rhododendron CT1sHSP were found. Main differences between the two types of Rhododendron CT1sHSPs were: (1) increased hydrophobicity by two positively selected amino acid sites and a seven-amino-acid insertion in the N-terminal arm; and (2) increased structural flexibility and solubility by a seven-amino-acid insertion in the N-terminal arm and one positively selected amino acid site in the C-terminal extension. Functional conservation of the ACD of Rhododendron CT1sHSP genes was inferred b

Dual role for tomato heat shock protein 21: protecting photosystem II from oxidative stress and promoting color changes during fruit maturation.

The tomato (Lycopersicon esculentum) chloroplast small heat shock protein (sHSP), HSP21, is induced by heat treatment in leaves, but also under normal growth conditions in developing fruits during the transition of chloroplasts to chromoplasts. We used transgenic tomato plants constitutively expressing HSP21 to study the role of the protein under stress conditions and during fruit maturation. Although we did not find any effect for the transgene on photosystem II (PSII) thermotolerance, our results show that the protein protects PSII from temperature-dependent oxidative stress. In addition, we found direct evidence of the protein's role in fruit reddening and the conversion of chloroplasts to chromoplasts. When plants were grown under normal growth temperature, transgenic fruits accumulated carotenoids earlier than controls. Furthermore, when detached mature green fruits were stored for 2 weeks at 2 degrees C and then transferred to room temperature, the natural accumulation of carotenoids was blocked. In a previous study, we showed that preheat treatment, which induces HSP21, allowed fruit color change at room temperature, after a cold treatment. Here, we show that mature green transgenic fruits constitutively expressing HSP21 do not require the heat treatment to maintain the ability to accumulate carotenoids after cold storage. This study demonstrates that a sHSP plays a role in plant development under normal growth conditions, in addition to its protective effect under stress conditions.

벼 엽록체 small HSP의 과발현에 의한 형질전환 식물체의 내열성 증가

엽록체 small HSP의 기능을 조사하기 위하여 벼로부터 분리한 엽록체 small HSP를 구성적으로 발현하는 형질전환 식물체를 제작하였다. 먼저 고온 스트레스 조건하에서의 형질전환 식물체의 내열성을 chlorophyll 형광으로 측정하여 분석하였다. Leaf disc를 고온 스트레스 조건에서 5분간 처리한 후, 광화학계 II의 불활성화를 나타내는 Fo 값과 증가치를 조사하였다. 형질전환 식물체는 고온 스트레스 하에서의 Fo 값의 증가가 현저하게 감소하였다. 또한 무균적으로 Petri dish에서 재배한 유식물체를 치사온도인 <TEX>$52^{\circ}C$</TEX>에서 45분간 처리한 후, <TEX>$20^{\circ}C$</TEX> 에서 계속적으로 배양하였을 때, wild-type 식물체는 전부 고사하였으나, 형질전환 식물체의 약 80%는 정상적으로 생존하였다. 또한 과발현된 Oshsp26 단백질의 축적량이 많을수록 내열성의 정도도 증가하였다. 이러한 결과는 엽록체 small HSP가 고온 스트레스 하에서 광합성기구를 보호함으로서 식물체의 내열성을 증가시키는데 있어서 중요한 기능을 담당하고 있음을 나타낸다. To investigate the function of chloroplast small heat shock protein (HSP), transgenic tobacco plants (Nicotiana tabacum L, cv. SR-1) that constitutively overexpress the rice chloroplast small HSP (Oshsp26) were generated. Effects of constitutive expression of the Oshsp26 on thermotolerance were investigated with the chlorophyll fluorescence. After 5-min incubation of leaf discs at high temperatures, an increase in the Fo level, indication of separation of LHCII from PSII, was mitigated by constitutive expression of the chloroplast small HSP When tobacco plantlets grown in Petri dishes were incubated at <TEX>$20^{\circ}C$/TEX> for 45 min and subsequently incubated at <TEX>$20^{\circ}C$/TEX> leaf color of wild-type plant became gradually white and all plantlets were finally died. Under the conditions in which all the wild-type plants died, more than 80% of the transformants remained green and survived. It was also found that the levels of Oshsp26 protein accumulated in transgenic plants were correlated with the degree of thermotolerance. These results suggest that the chloroplast small HSP plays an important role in protecting photosynthetic machinery, as a results, increases thermotolerance of whole plant during heat stress.

Variation in chloroplast small heat-shock protein function is a major determinant of variation in thermotolerance of photosynthetic electron transport among ecotypes of Chenopodium album.

Chloroplast small (low-molecular-weight) heat-shock proteins (csHsps) can protect photosynthetic electron transport (Pet), and quantitative variation in csHsps is correlated with thermotolerance of net photosynthesis and Photosystem II. However, the functional (i.e. protective) consequence of natural variation in csHsps is unknown. To investigate this, we used an in vitro assay to determine the contribution of csHsps to the tolerance of Pet to high temperatures in five ecotypes of Chenopodium album collected from habitats ranging from cool to warm, and we partitioned total Pet thermotolerance into basal and induced Pet components (without and with a pre-heat treatment, respectively, to induce csHsps). The ecotypes varied in total Pet thermotolerance and this was correlated with habitat temperature. Variation in total Pet thermotolerance was associated primarily with variation in induced Pet thermotolerance, and not with basal Pet thermotolerance. Variation in induced Pet was highly correlated with csHsp protection of Pet. Variation in csHsp function was associated with variation in csHsp content among ecotypes. These results are the first to demonstrate the direct functional consequences for natural variation in Hsps in plants, and show that functional variation is associated with evolutionary adaptation to specific habitats among ecotypes.

In vivo evidence from an Agrostis stolonifera selection genotype that chloroplast small heat-shock proteins can protect photosystem II during heat stress.

Previous in vitro experiments indicated that chloroplast small heat-shock proteins (sHsp) could associate with thylakoids and protect PSII during heat and other stresses, possibly by stabilizing the O2-evolving complex (OEC). However, in vivo evidence of sHsp protection of PSII is equivocal at present. Using previously characterized selection genotypes of Agrostis stolonifera Huds. that differ in thermotolerance and production of chloroplast sHsps, we show that both genotypes contain thylakoid-associating sHsps, but the heat-tolerant genotype, which produces an additional sHsp isoform not made by the sensitive genotype, produces a greater quantity of chloroplast and thylakoid sHsp. Following a pre-heat stress to induce sHsps, in vivo PSII function decreased less at high temperatures in the tolerant genotype. Differences in PSII thermotolerance in vivo were associated with increased thermotolerance of the OEC proteins and O2-evolving function of PSII, and not with other PSII proteins or functions examined. In vivo cross-linking experiments indicated that a greater amount of sHsp associated with PSII proteins during heat stress in the tolerant genotype. PSII was the most thermosensitive component of photosynthetic electron transport, and no differences between genotypes in the thermotolerance of other electron transport components were observed. These results indicate that in vivo chloroplast sHsps can protect O2 evolution and the OEC proteins of PSII during heat stress.

Substitution of conserved methionines by leucines in chloroplast small heat shock protein results in loss of redox-response but retained chaperone-like activity.

During evolution of land plants, a specific motif occurred in the N-terminal domain of the chloroplast-localized small heat shock protein, Hsp21: a sequence with highly conserved methionines, which is predicted to form an amphipathic alpha-helix with the methionines situated along one side. The functional role of these conserved methionines is not understood. We have found previously that treatment, which causes methionine sulfoxidation in Hsp21, also leads to structural changes and loss of chaperone-like activity. Here, mutants of Arabidopsis thaliana Hsp21 protein were created by site-directed mutagenesis, whereby conserved methionines were substituted by oxidation-resistant leucines. Mutants lacking the only cysteine in Hsp21 were also created. Protein analyses by nondenaturing electrophoresis, size exclusion chromatography, and circular dichroism proved that sulfoxidation of the four highly conserved methionines (M49, M52, M55, and M59) is responsible for the oxidation-induced conformational changes in the Hsp21 oligomer. In contrast, the chaperone-like activity was not ultimately dependent on the methionines, because it was retained after methionine-to-leucine substitution. The functional role of the conserved methionines in Hsp21 may be to offer a possibility for redox control of chaperone-like activity and oligomeric structure dynamics.

Does the chloroplast small heat shock protein protect photosystem II during heat stress in vitro?

It has been suggested that the function of the chloroplast-localized small heat shock protein (sHsp) is to protect photosystem II (PSII) from heat inactivation. This paper reports that addition of purified sHsp protein to isolated thylakoid membranes gave no protection of PSII and questions that there is any direct effect of the sHsp on PSII. The opinion is forwarded that the primary role for the chloroplast-localized sHsp may not even be protection of PSII.

Photosynthetic Thermotolerance is Quantitatively and Positively Correlated with Production of Specific Heat-shock Proteins Among Nine Genotypes of Lycopersicon (Tomato)

We recently showed that the chloroplast small heat-shock protein (herein referred to as chlp Hsp24) protects photosystem 2 (PS2) during heat stress, and phenotypic variation in production of chlp Hsp24 is positively related to PS2 thermotolerance. However, the importance of chlp Hsp24 or other Hsps to other aspects of photosynthesis and overall photosynthetic thermotolerance is unknown. To begin investigating this and the importance of genetic variation in Hsp production to photosynthetic thermotolerance, the production of several prominent Hsps and photosynthetic thermotolerance were quantified in nine genotypes of Lycopersicon, and then the relationships between thermotolerance of net photosynthetic rate (P N) and production of each Hsp were examined. The nine genotypes exhibited wide variation in P N thermotolerance and production of each of the Hsps examined (chlp Hsp70, Hsp60, and Hsp24, and cytosol Hsp70). No statistically significant relationship was observed between production of chlp Hsp70 and P N thermotolerance, and only a weak positive relationship between cytosolic Hsp70 and P N was detected. However, significant positive relationships were observed between production of chlp Hsp24 and Hsp60 and P N thermotolerance. Hence natural variation in production of chlp Hsp24 and Hsp60 is important in determining variation in photosynthetic thermotolerance. This is perhaps the first evidence that chlp Hsp60 is involved in photosynthetic thermotolerance, and these in vivo results are consistent with previous in vitro results showing that chlp Hsp24 protects PS2 during heat stress.