Small heat shock proteins and stress tolerance in plants

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Small heat shock proteins and stress tolerance in plants Article in Biochimica et Biophysica Acta · September 2002 DOI: 10.1016/S0167-4781(02)00417-7 · Source: PubMed

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Review

Small heat shock proteins and stress tolerance in plants Weining Sun, Marc Van Montagu*, Nathalie Verbruggen1 Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Instituut voor Biotechnologie (VIB), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium Received 8 August 2001; received in revised form 2 May 2002; accepted 4 June 2002

Abstract Small heat shock proteins (sHsps) are produced ubiquitously in prokaryotic and eukaryotic cells upon heat. The special importance of sHsps in plants is suggested by unusual abundance and diversity. Six classes of sHsps have been identified in plants based on their intracellular localization and sequence relatedness. In addition to heat stress, plant sHsps are also produced under other stress conditions and at certain developmental stages. Induction of sHsp gene expression and protein accumulation upon environmental stresses point to the hypothesis that these proteins play an important role in stress tolerance. The function of sHsps as molecular chaperones is supported by in vitro and in vivo assays. This review summarizes recent knowledge about plant sHsp gene expression, protein structure and functions. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Chaperone; Embryogenesis; Transgenic plant

1. Introduction Upon heat stress, both prokaryotic and eukaryotic cells produce a group of proteins with a molecular mass of 15 to 42 kDa, designated small heat shock proteins (sHsps) [1]. Plants are characterized by unusually abundant and diverse sHsps that may reflect their need to quickly adapt to everchanging environmental conditions (such as temperature, light, and humidity). SHsps have been arranged in six classes based on DNA sequence similarity, immunological cross-reactivity, and intracellular localization [2,3]. sHsps are usually undetectable in vegetative tissues under normal growth conditions, but can be induced by environmental stresses and developmental stimuli. The correlation between the synthesis of sHsps and stress response led to the hypothesis that sHsps protect cells from the detrimental effects of stress. The mechanisms by which sHsps are involved in cell protection are not fully understood. Strong evidence support that sHsps function as molecular chaperones that bind to partially folded or denatured substrate *

Corresponding author. Tel.: +32-9-264-5170; fax: +32-9-264-5349. E-mail addresses: [email protected] (M. Van Montagu), [email protected] (N. Verbruggen). 1 Present address: Laboratoire de Physiologie et de Ge´ne´tique Mole´culaire des Plantes, Universite´ Libre de Bruxelles, CP 242, Boulevard du Triomphe, B-1050 Brussels, Belgium. Fax: + 32-2-650-2128.

proteins and thereby prevent irreversible aggregation or promote correct substrate folding [3 –7]. This review aims at summarizing the molecular and physiological data about plant sHsps.

2. Plant sHsp classes and their structure sHsps with a molecular mass of 15 to 42 kDa on denaturing polyacrylamide gel electrophoresis (PAGE) are the most dominant proteins produced in plants upon heat [2,8,9]. Scharf et al. [3] analyzed the complexity of the sHsps of Arabidopsis thaliana that surpasses by far that in any other organism investigated to date. The Arabidopsis genome contains 19 open reading frames, which code for sHsp-related proteins [3]. Plant sHsps are all encoded by nuclear genes and are divided into six classes. Three classes (classes CI, CII, and CIII) of sHsps are localized in the cytosol or in the nucleus [3,10] and the other three in the plastids (P) [10], the endoplasmic reticulum (ER) [11,12], and the mitochondria (M) [13 –15]. The organellar forms of sHsps appear to be unique to plants with the exception of the mitochondrial Hsp22 in Drosophila melanogaster. Classes CI and CII of cytosolic/nuclear sHsps are generally encoded by multigene families [10]. Within the same class, different members of sHsps share high sequence homology at the amino acid level. sHsps of the same class are also highly

0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 2 ) 0 0 4 1 7 - 7

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W. Sun et al. / Biochimica et Biophysica Acta 1577 (2002) 1–9

homologous among different plant species; however, sHsps from different classes share low sequence similarities [2,3, 10,16]. Nonetheless, all sHsps share a conserved 90-amino acid carboxyl-terminal domain called the a-crystallin domain (ACD) or heat shock domain, including the vertebrate a-crystallin proteins of the eye lens [3,10]. The ACD distinguishes sHsps from other small proteins induced by heat [17], can be subdivided into consensus I and consensus II domains separated by a hydrophilic domain of variable length [2,10], and consists of a h-sandwich of two antiparallel h-sheets [3]. The amino-terminal domains are quite divergent between classes. However, the unique consensus domains for each class of cytosolic/nuclear sHsps as well as the targeting peptides for the organelle-localized sHsps are found at the amino terminus [16]. A methionine-rich consensus III domain is present in the amino-terminal region of plastid-localized sHsps [10]. This methionine-rich domain has been suggested to form an amphipatic a-helix tertiary

structure, with one face of the helix consisting of methionine and other hydrophobic residues and the opposite one entirely of strong hydrophilic residues. The methionine-rich amphipatic a-helix may be involved in substrate binding [18]. Plant sHsps have been considered not to be phosphorylated [2] until the recent observation that two forms of ZmHsp23.8-M (former name Hsp22) from maize can be phosphorylated [19]. These two forms of ZmHsp23.8-M result from the alternative splicing with a two-residue difference in the amino acid sequence and a 57-Da difference in molecular mass [18]. The phosphorylated forms represent 36% and 35% of the normal and alternatively spliced forms of Hsp22, respectively. Moreover, the amino acid sequence change creates a h-sheet in the alternatively spliced form. Whether this change is associated with an altered function of maize Hsp22 is under investigation. The plant sHsps, like those from other organisms, can form homo-oligomers with apparent molecular masses of

Fig. 1. (a) TaHsp16.9A-CI dimer. Monomers with two distinct conformations were observed in the oligomer and are shown in red and green. The N-terminal arm from the green monomer is shown in pale green—the corresponding residues from the red monomer were disordered. The swapped loop donated by the green monomer is indicated in cyan, and the corresponding loop from the red monomer in pink. The C-terminal extensions are shown in yellow and orange. (b) TaHsp16.9A-CI oligomer. Within the dodecamer, the dimers are arranged in two coaxial rings. Viewed down the center axis, the top ring consists of three dimers; two dimers are shown in blue and the third with the same color scheme as described above. The bottom ring has been lightened for clarity. The Cterminal extensions can be seen to tie the two rings together; the yellow extension interacts with another dimer within the same ring, and the orange with a dimer in the other ring. The a-helical N-terminal arms also connect subunits between the two rings. For further details, see Ref. [25]. Coordinates deposited in the PDB accession 1GME. Figures were made with Molscript [100]. This figure was prepared by Andy C. Hausrath and Elizabeth Vierling.


200 to 350 kDa [2,11,13,20– 22]. The common features of the ACD shared by the sHsps may be responsible for complex formation [2]. Analysis of the first crystal structure of a sHsp from Methanococcus jannaschii (Hsp16.5) revealed the formation of a hollow spherical complex composed of 24 subunits [23]. The carboxyl-terminal ACD is involved in contact with subunits and maintenance of the tertiary structure of the oligomeric complex [23]. Usually, plant sHsps form homo-oligomers with 12 subunits. Absence of hetero-oligomers with subunits from different sHsp classes in vivo and in vitro [21,24] suggests that determinants other than the ACD might be involved in oligomeric structure formation. The carboxyl-terminal deletion of PsHsp17.1-CII (former name Hsp17.7) and the amino-terminal Myc-tagged PsHsp18.1-CI and PsHsp17.1CII from Pisum sativum formed oligomers with altered sizes [24]. Thus, both the carboxyl-terminal extension of ACD and the amino-terminal region of sHsps may contain the information required for forming oligomeric structures. The first crystal structure of a plant sHsp has recently been published by van Montfort et al. [25] (Fig. 1A). In contrast to the spherical 24-mer structure of MjHsp16.5, the wheat TaHsp16.9A-CI (former name Hsp16.9) assembles into a dodecameric double disk (Fig. 1B). Each disk contains six ACDs organized in a trimer of dimers. Dimers are the building blocks for oligomer assembly and this feature seems to be conserved in plant sHsps [3,25]. Strand exchange, helix swapping, loose knots, and hinged extensions are involved in oligomer assembly [3,25]. Formation of large oligomeric structures and conformational changes are associated with the chaperone activity of sHsps. In vitro chaperone activity of the monomeric NtHsp18 of Nicotiana tabacum is lower than that of the oligomeric form as shown by less efficient suppression of thermal aggregation of the tested proteins [26]. The highly conserved ACD might be important for chaperone activity. In contrast, Escherichia coli that expresses only the amino-terminal part of the rice OsHsp16.9-CI gene fused to glutathione-S-transferase, is protected from heat shock [27]. In addition, mutations within the amino-terminal phenylalanine-rich region of the aB-crystallin abolished chaperone activity in vitro, without altering the oligomeric complex [28]. Therefore, the amino-terminal region is not only necessary for oligomerization but also for chaperone activity. Interaction of sHsps with their protein substrates seems to involve oligomer dissociation (which can be induced by heat) and reassembly with denatured proteins into larger complexes [3]. It is also unique for plant sHsps to form heat shock granules (HSGs), which are approximately 40 nm in diameter, during long-term heat stresses. Low et al. [7] have proposed that ongoing heat stress induces accumulation of unfolded proteins bound to sHsp oligomers in the cytoplasm that exceeds the refolding capacity of Hsp70/Hsp40. The complexes of denatured proteins –sHsp oligomers can be stored transiently in HSGs that disintegrate during the recovery period. HSGs are formed by aggregation of sHsps

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with highly ordered and large cytosolic chaperone complexes [29]. HSG complexes contain mainly cytosolic sHsps from both classes CI and CII [29,30]. In addition to cytosolic sHsps, Hsp70 [29,30] and the heat stress transcription factor HSFA2 [31] are also present in the HSG complexes. The carboxyl-terminal domain of class CII sHsps and the formation of dodecamers are crucial for the heat-stress-induced auto-aggregation of these proteins and subsequent recruitment of class CI proteins into the HSG [24].

3. Plant sHsp production Under normal growth conditions, most sHsps cannot be detected in the vegetative tissues, but are rapidly produced in response to heat. Increasing the temperature to approximately 10 – 15 jC above the optimal growth temperature, which is usually in the sublethal range, induces the heat shock response. The extent of sHsp accumulation depends on the temperature and the duration of the stress period [32]. After the heat stress has been released, the sHsps are quite stable with half-lives of 30– 50 h [8,33], suggesting that sHsps may be important for the recovery as well [2]. Heat shock is not the only stimulus triggering sHsp gene expression and protein synthesis (Table 1). Some sHsps accumulate in response to osmotic stress. Expression of HaHsp17.6 (class CI) and HaHsp17.9 (class CII) genes is induced in water-stressed sunflowers and the mRNA levels correlate positively with the dehydration degree [34,35]. The corresponding sHsps are detected in stems and roots of stressed sunflowers. Mannitol and abscisic acid can induce the expression of HaHsp17.9-CII in in vitro-grown seedlings [36]. Cytosolic sHsps, which are homologous to HaHsp17.6-CI and HaHsp17.9-CII, accumulate constitutively in the resurrection plant Craterostigma plantagineum, but not in the desiccation-intolerant callus, unless treated with exogenous abscisic acid [37]. The levels of these C. plantagineum sHsps increased further in response to drought, suggesting that they are important for the drought tolerance and that induction of the genes is mediated by abscisic acid-dependent mechanisms. Genes coding for sHsps that are induced in response to osmotic stress also include cytosolic AtHsp17.7-CII and AtHsp17.6-CII in A. thaliana [former name At-HSP17.6A and At-HSP17.6-II; 38] and QsHsp17 (class CI) in young cork-oak seedlings [39]. In contrast to QsHsp17-CI, the two cytosolic class CII sHsps proteins of A. thaliana do not accumulate to detectable levels upon osmotic stress. A subset of sHsps, including two types of cytosolic classes CI and CII sHsps [38,39], a mitochondrial sHsp [40], and a chloroplast-localized sHsp [41], are induced by oxidative stress. Several sHsp-encoding genes are also induced by cold stress [42], heavy metal [43], ozone [44], UV radiation [39], and g-irradiation [40]. These observations suggest that sHsps may be associated with general abiotic stress responses.

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Table 1 Expression of sHsps in conditions other than heat stress Conditions

Plant species

sHsp gene/probes

Reference

Embryo development

Helianthus annuus

Arabidopsis thaliana

HaHsp17.6-CI HaHsp17.9-CII PsHsp18.1-CI and three related sHsps PsHsp17.7-CII and two related sHsps AtHsp17.4-CI, AtHsp17.6-CI AtHsp17.7-CII LpHsp17.7-CI homologue LpHsp17.3-CII homologue AtHsp17.4-CI, AtHsp17.6-CI AtHsp17.7-CII, AtHsp17.6-CII cDNA DF4-5 (class CI) HvHsp26.8-P, HvHsp26.9-P Hsp18.1 (class CI) MsHsp18.2-CI NtHsp18-CI cDNAs homologous to sHsp (class CI) ZmHsp17-CII (former name Hsp18) NtHsp18-CI LpHsp17.7-CI homologue LpHsp17.3-CII homologue LeHsp23.8-P (former name LeHsp21) TOM111 (class P) HaHsp17.6-CI HaHsp17.9-CII HaHsp17.6-CI homologues HaHsp17.9-CII homologues QsHsp17-CI AtHsp17.7-CII, AtHsp17.6-CII HaHsp17.6-CI HaHsp17.6-CI homologues HaHsp17.9-CII homologues QsHsp17-CI QsHsp17-CI Hsp22 (class M) Hsp17.9 (class CI) OsHsp26.6-P AtHsp17.7-CII CI19 (class ER) TOM66 (class CI) TOM111 (class P)/LeHsp23.8-P WAP20 (class ER) CsHsp17.5-CI Hsp18.1 (class CI) MsHsp18.2-CI AtHsp17.7-CII, AtHsp17.6-CIII

Pharbitis nil Craterostigma plantagineum

sHsp-1 (class CII) HaHsp17.6-CI homologues

[36] [46] [47] [47] [45] [38] [7] [7] [45] [38] [91] [92] [43] [43] [93] [94] [95] [93] [7] [7] [96] [96] [34] [34] [37] [37] [39] [38] [36] [37] [37] [39] [39] [40] [44] [41] [38] [97] [42] [42] [98] [83] [43] [43] W. Sun, unpublished [99] [37]

Pisum sativum Arabidopsis thaliana Lycopersicon esculentum Germination

Arabidopsis thaliana

Somatic embryogenesis

Pseudotsuga menziesii Hordeum vulgare Medicago sativa

Fruit maturation

Nicotiana tabacum Lilium Zea mays Nicotiana tabacum Lycopersicon esculentum

Osmotic stress

Helianthus annuus

Pollen development

Craterostigma plantagineum

Abscisic acid

Oxidative stress

Cold

Heavy metals

Photoperiod Normal temperature

Quercus suber Arabidopsis thaliana Helianthus annuus Craterostigma plantagineum Quercus suber Quercus suber Lycopersicon esculentum Petroselinum crispum Oryza sativa Arabidopsis thaliana Solanum tuberosum Lycopersicon esculentum Morus bombycis Castanea sativa Medicago sativa

Nomenclature of Arabidopsis thaliana sHsp genes and classification by intracellular localization of the corresponding proteins was adapted from Scharf et al. [3]: CI, CII, cytoplasm/nuclear class CI and CII; ER, endoplasmic reticulum; M, mitochondria; P, plastids/chloroplasts. Names of sHsp that are not (completely) sequenced have not been adapted, but the class is mentioned between parentheses.

In the absence of environmental stresses, synthesis of sHsps in plants is restricted to certain stages of development, such as embryogenesis, germination, pollen development, and fruit maturation (Table 1). Embryogenesis consists mainly of three stages. At the early stage, cell division and elongation are the major events. When seeds enter the mid-maturation stage, which is characterized by an abscisic acid peak, they become dormant and desiccate. At the late maturation stage, the reserve proteins and late

maturation-abundant proteins are synthesized. The mature seeds are resistant to dehydration. In Arabidopsis embryos, cytosolic AtHsp17.4-CI and AtHsp17.6-CI (former name AtHSP17.4 and HSP17.6), and AtHsp17.7-CII begin to accumulate when seeds enter mid-maturation stage and are abundant through the late maturation stage and in dry seeds [38,45]. In pea and sunflower, both cytosolic class I and class II sHsps appear in embryos during reserve synthesis and increase in abundance in mature seeds [36,46,47]. The


synthesis of sHsps along with seed maturation suggests that sHsps might be important for protection of cellular components against desiccation [45]. This hypothesis is supported further by the correlation of reduced sHsp levels with desiccation-intolerant phenotypes. In the desiccationintolerant abi3-6 mutant seeds, expression and accumulation of AtHsp17.4-CI, AtHsp17.6-CI, and AtHsp17.7-CII are abolished [38,45,48]. Such a reduction is not linked to a defect in embryogenesis because AtHsp17.4-CI can accumulate to wild-type levels in the desiccation-tolerant embryo-defective mutants lec2-1 and emb266, but not in fus3-3, lec1-2, and line 24 embryos, which are defective in embryogenesis and intolerant to desiccation [48]. sHsp genes are expressed under the control of the heat shock transcription factor (HSF) [49,50]. HSF acts through a highly conserved upstream response element, the heat shock-responsive element (HSE) [51], which consists of three contiguous and inverted repeats of a 5-bp sequence, defined as nGAAn [52,53]. In plants, the optimal HSE core consensus sequence was shown to be 5V-aGAAg-3V[54]. At least three units, resulting in 5V-nGAAnnTTCnnGAAn-3V, are efficient for the HSF binding [50]. HSF is present in a latent state under normal conditions, but upon heat stress, it is activated by trimerization, thus increasing the DNAbinding affinity and exposing the domains for transcription activity [49]. By using heterologous and homologous systems, both HSE-dependent and HSE-independent mechanisms have been shown to be involved in sHsp production during development. Synthetic HSEs could confer developmental regulation to a minimal cauliflower mosaic virus (CaMV) 35 S promoter in tobacco [55]. Promoter studies defined that the proximal HSE regions are necessary for the transcription activation at the late stage of embryogenesis, which coincides with seed desiccation [35]. This activation is probably mediated through binding of HSF, because mutations in the HSEs reduced the HSF-binding ability and, consequently, the promoter activation during development [56,57]. However, at the early embryogenesis stage, activation is not affected by mutations that abolished HSF binding, suggesting that other seed-specific trans-activator proteins are responsible for the regulation. HaHsp17.6 G1 is the only sHsp gene that does not respond to heat shock but to developmental regulation [57]. The HaHsp17.6 G1 promoter contains an imperfect HSE that is involved in developmental activation of HaHsp17.6 G1 [58], but HSE alone is insufficient to induce fully the HaHsp17.6 G1 promoter. Other cis-acting elements that are located upstream of HSE affect the promoter positively or negatively. HSF and other distinct trans-activating factors may cooperate in regulating HaHsp17.6 G1 during seed desiccation through the binding of HSE and these cis elements [58]. One of the transactivators defined to date turned out to be the transcription factor ABI3, which regulates various seed-specific genes [59,60]. The abi3 mutation affects the expression and accumulation of AtHsp17.4-CI, AtHsp17.6C-CI [45,48],

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and AtHsp17.7-CII [38] in Arabidopsis seeds. More direct evidence came from the investigation of the HaHsp17.7 G4 promoter activation in sunflower embryos that co-express Arabidopsis ABI3 and tomato LpHSFA1. The transient expression analysis showed that ABI3 and HSF activate synergistically the HaHsp17.7 G4 promoter in embryos [61]. ABI3-dependent activation requires the integrity of proximal and distal HSEs and the HSF activation domain. The carboxyl-terminal and amino-terminal domains of HSF and ABI3, respectively, are important for the co-activation. ABI3 by itself does not seem to have a major contribution, but only in concert with HSF through enhancement of the binding of HSF to HSE. ABI3 and HSF may interact with each other and ABI3 may act as a transcriptional coactivator by indirect DNA binding [61].

4. Function of plant sHsps As previously described, production and accumulation of sHsps in response to environmental stresses and developmental stimuli have been correlated with stress tolerance. The heat tolerance trait of Agrostis palustris was linked to the presence of additional Hsp25 polypeptides in heattolerant variants [62]. Positive relationships between sHsp levels and tolerance to heat [63,64] or desiccation [37] have been elucidated. The mechanisms of cell protection by sHsps are largely unknown. Some sHsps have been demonstrated to act as molecular chaperone in vitro [5,6] and in vivo [4,7]. Molecular chaperones are defined as ‘‘proteins that bind to and stabilize an otherwise unstable conformer of another protein and by controlled binding and release, facilitate its correct fate in vivo, be it folding, oligomeric assembly, transport to a particular subcellular compartment, or disposal by degradation’’ [65]. The ability to recognize and bind unfolded proteins, to suppress protein aggregation, and to influence the yield of protein folding are taken into account to define molecular chaperone activities [66]. Citrate synthase, mitochondrial malate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and firefly luciferase (LUC) are the most commonly used substrates for in vitro chaperone activity assay [22]. These proteins are denatured either by heat or by chemical reagents. Evaluation of the in vitro chaperone activity of sHsps is mainly achieved by comparing aggregation of these substrate proteins, inactivation of the enzyme activities, and refolding rate in the presence and absence of sHsps. LUC-producing Arabidopsis cells provide a nice cellular system for analysis of chaperone activity in vivo [4]. LUC is an ideal substrate for this activity assay because of the moderately inactivating temperature (39 –42 jC) that corresponds to that of sHsp induction in plants [4,41]. LUC refolding and, thus, reactivation depend on the chaperone activity. The high sensitivity of LUC activity measurements supports its application as chaperone substrate for in vivo studies. The in vivo

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system also makes it possible to evaluate a specific molecular chaperone in the chaperone network. Several classes of molecular chaperones exist in eukaryotes, including Hsp100/Clp proteins, Hsp90, Hsp70, Hsp60/chaperonins, calnexin, nucleoplasmin, sHsps and co-chaperones Hsp40/ DnaJ, GrpE, and Cpn60, which are accessory proteins mediating activity of specific chaperones [17]. Members from different families often cooperate as chaperone machines, for example the Hsp70 and Hsp40 [67 – 71]. In contrast to the Hsp60 and Hsp70 [65], sHsp chaperone activities are ATP independent [72]. The current model for the function of sHsps is that sHsps can bind selectively nonnative proteins, prevent their aggregation, and maintain them in a state competent for ATPdependent refolding by other chaperones [2,5,41,73,74]. Experiments performed in vitro with sHsps from diverse organisms have demonstrated that sHsps are particularly effective in preventing thermal aggregation of other proteins by an ATP-independent mechanism [5,72]. sHsps can undergo temperature-dependent conformational changes, resulting in either dissociation of the oligomeric complex [75] or alteration of the surface hydrophobicity [5,76] and increase in the affinity for substrate binding. For PsHsp18.1CI, the exposed hydrophobic sites at the conserved consensus II region [2] can bind heat-denatured substrates [5]. Suppression of conformational changes resulted in lower substrate-binding and chaperone activities [26]. Analysis of the crystal structure of TaHsp16.9A-CI revealed four hydrophobic sites that would be exposed when the oligomer disassembles into dimers, three of which were identified as putative substrate-binding sites [25]. The TaHsp16.9A-CI dodecamer also undergoes a reversible temperature-dependent dissociation into smaller components, and similar to PsHsp18.1-CI, it can form high molecular weight complexes with a model substrate [25]. The observation that the oligomeric structures of sHsp complexes formed under nonstress conditions become dissociated at high temperatures suggests that new spherical structures may be formed by reassociation of sHsp dimers with unfolded substrate proteins [25,75], thus avoiding protein aggregation. Conformational changes do not depend on high temperature only. The oligomeric conformation of the Arabidopsis AtHsp25.3-P (former name HSP21) changes when exposed to heat and oxidative stresses in vitro [80]. This change is caused by sulfoxidation of the methionines in the conserved methionine-rich amphipatic helix [18]. Similar conformational changes occur in vivo when AtHsp25.3-P-overproducing plants are challenged by heat [77]. The role of sulfoxidation is unknown. Methionines act probably as primitive antioxidant system [78] and sulfoxidation will modulate the binding between methionine-rich helix and ligands [18]. In vitro, sHsps prevent weakly or not the inactivation of model enzymes, such as citrate synthase, mitochondrial malate dehydrogenase, and LUC. sHsps can bind nonnative

proteins on their surface and maintain them in a foldingcompetent state [5,73]. PsHsp18.1-CI seems to be more effective than Hsp70 in hampering proteins from thermal aggregation, but PsHsp18.1-CI alone cannot refold the heatdenatured LUC, unless rabbit reticulocyte lysate or wheat germ extract is present in the refolding mix [5]. AtHsp17.6CCI can prevent inactivation of LUC in vivo, suggesting that other chaperone systems are involved, such as Hsp70, which is constitutively expressed in Arabidopsis cells [4]. Recently, a minimal chaperone system, composed of PsHSP18.1-CI and either the eukaryotic Hsp cognate (HSC)/Hsp70 plus DnaJ homologs Hdj1 and Ydj1 or the prokaryotic DnaK plus DnaJ and GrpE, could refold heat-denatured LUC [41]. Data collected so far favor the model in which sHsps bind to nonnative proteins and create a reservoir of refoldable proteins. The substrates are subsequently passed to Hsp70 and co-chaperones for ATP-dependent folding [41]. The hypothesis is supported by in vivo data as shown by Forreiter et al. [4]. LUC produced in Arabidopsis cells remains more active and recovers more quickly from heat stress when protoplasts are co-producing Hsp70 and AtHsp17.6-CI. This is true for sHsps from prokaryotes [74,76] and eukaryotes different from plants [73]. In support to this model, HSGs contain Hsp70 in addition to cytosolic sHsps [7,29,30]. One of the possible mechanisms for sHsps to protect other proteins from denaturation is through the assembling of hollow spheres. This mechanism implies that certain proteins or RNAs critical for cell survival under stress may be trapped into or on the surface of the sphere [79]. By the formation of HSGs, sHsps co-localize with Hsp70, making the cooperation between two types of chaperones more efficient. The substrate specificity is quite low for cytosolic sHsps. The diverse amino-terminal structures of sHsps may well be suited for binding a wide range of nonnative proteins [7,79]. Mitochondria- and chloroplast-localized sHsps have been shown to protect electron transport chain in mitochondria [63] and Photosystem II electron transport during heat stress [6], respectively. A chloroplastic 22-Ku Hsp from Chenopodium album, which is localized in the thylakoid lumen, interacts specifically with the thermolabile oxygenevolving complex of Photosystem II [80]. This chloroplastic Hsp can protect the oxygen-evolving complex from heat stress-induced damage, but fails to reactivate the heatdenatured Photosystem II. In addition to the chaperone function, sHsps may have other roles. DcHsp17.7-CI has been suggested to be involved in translational control upon heat shock [64].

5. Modulation of plant sHsp production by genetic modification Heat shock response is a conserved reaction of cells against elevated temperatures by transient reprogramming of the cellular activities to cease the normal protein synthesis and to synthesize a set of Hsps [50]. Upon heat shock,


some sHsps can reach up to 1% of the total leaf or root cell proteins [8,9]. In the absence of stress, sHsps are produced strictly at certain developmental stages. Accumulating data suggest that sHsp production is correlated with stress tolerance, assuming that altered Hsp levels may affect stress tolerance. However, there are few gain-of-function reports about enhanced stress tolerance through genetically modified production of plant sHsps (Table 2). Given that Hsps production is regulated through HSFs, transgenic Arabidopsis plants were engineered that expressed constitutively AtHSF1 or AtHSF3 [81,82]. Constitutive expression of the chimeric gene AtHSF1-h-glucuronidase (GUS), AtHSF3 or AtHSF3-GUS, but not AtHSF1 or AtHSF4, caused derepression of heat shock response and accumulation of a set of Hsps under normal growth conditions; as a consequence, thermotolerance was conferred to transgenic plants [81,82]. Because a set of Hsps, including sHsps and Hsp70, were constitutively produced in these transgenic plants, it was impossible to determine the contribution of sHsps to the observed increased thermotolerance. In heterologous systems, the bacterial thermotolerance in E. coli is increased by the produced recombinant rice OsHsp16.9-CI-GUS fusion protein [25]. Overexpression of the chestnut CsHsp17.5 gene enhanced bacterial viability under heat and cold stress [83]. The protective effects of both sHsps are associated with increased stability of soluble proteins. The expression of the carrot DcHsp17.7 (class CI) gene was genetically modified [64] and a significantly higher thermotolerance was obtained in DcHsp17.7-CI overexpressing plants. Moreover, plants overexpressing DcHsp17.7-CI in antisense orientation were less tolerant to heat shock. The protein levels of DcHsp17.7-CI and its homolog DcHsp17.9-CI were lower than in wild-type and overproducing plants. These experimental data support that DcHsp17.7-CI may play an important role in thermotolerance. However, enhancement of thermotolerance was not preceded by the synthesis of large quantities of DcHsp17.7CI mRNA or proteins. The decreased thermotolerance seemed to be a consequence of overall reduction in Hsp synthesis, especially of sHsps in the DcHsp17.7-CI antisense transgenic plants. These observations suggest that

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sHsps have probably other functions than in stress tolerance. Transgenic Arabidopsis plants overexpressing AtHsp17.7-CII accumulate high levels of AtHsp17.7-CII mRNA and protein under normal growth conditions [38]. The AtHsp17.7-CII-overexpressing plants showed increased tolerance to drought and salt stress, but not to heat stress. Endogenous AtHsp17.7-CII proteins naturally accumulated in seeds along with the acquisition of dehydration resistance [38]. Probably, AtHsp17.7-CII functions as a molecular chaperone upon osmotic stress in overproducing plants and during seed maturation, when proteins are prone to be denatured upon dehydration and rehydration, as supported by in vitro assays [38]. In contrast, Alamillo et al. [37] reported that overproduction of seed-stored sunflower sHsps in tobacco failed to confer water stress tolerance. Heat and osmotic stresses are always accompanied with oxidative stress. The adverse effects of various environmental stresses are mediated, at least in part, by an enhanced generation of the reactive oxygen species (ROS) (O2 S, H2O2, and SOH) [84]. O2 S dismutates to O2 and H2O2 in the presence of superoxide dismutase. In turn, H2O2 and O2 S can react to generate the hydroxyl radical SOH, the most potent oxidant, which attacks most macromolecules, provoking membrane damage and lesions in DNA, affecting protein synthesis and stability, and leading to metabolic dysfunction and cell death. The role of sHsps in oxidative stress tolerance is highlighted by observations that production and accumulation of plant sHsps increase in response to H2O2 treatment in vitro (Table 1). Chloroplasts are the main sites for generating ROS under both stress and unstressed conditions [85]. Conceivably, the chloroplast-localized sHsps might be involved in oxidative stress tolerance. The unique methionine-rich amphipatic a-helix structure of chloroplastic sHsps and the ability of undergoing conformational changes upon oxidant treatments allow oxidative stress to be counteracted [77,86]. In strong support, overproduction of AtHsp25.3-P in Arabidopsis conferred tolerance to combined heat and high light, which induce oxidative stress [77]. The oxidative stress-inducible expression of cytosolic [38,39] and mitochondrial sHsps [40] suggests that these sHsps may also contribute to oxidative stress tolerance. In mammalian cells, sHsps can decrease the intracellular ROS levels via the glutathione-dependent path-

Table 2 Genetic modification of sHsps expression sHsp gene

Expression in organism

Stress tolerance

Reference

GST-OsHsp16.9(CI) CsHsp17.5-CI DcHsp1.7.-CI Antisense-DcHsp17.7-CI AtHsp2-5.3-P AtHsp17.7-CII Seed-stored HaHsps AtHSF1 AtHSF1-GUS AtHSF3

Escherichia coli Escherichia coli Daucus carota Daucus carota Arabidopsis thaliana Arabidopsis thaliana Helianthus annuus Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana

Increased thermotolerance Increased thermotolerance Increased thermotolerance Decreased thermotolerance Increased tolerance to heat under high light condition Increased osmotolerance, no effect in thermotolerance No effect in water stress tolerance No effect in thermotolerance Increased thermotolerance Increased thermotolerance

[27] [83] [84] [84] [77] [38] [37] [81] [81] [82]

8


way [87]. In addition, Hsp27, which is the major sHsp in mammalian cells exposed to oxidative stress, also protects against apoptosis by interfering with cytochrome c release from mitochondria. Recently, the regulation of the integrity of the F-actin network by Hsp27 has been suggested to be involved in the retention of cytochrome c in mitochondria [88]. Hamilton and Heckathorn [89] demonstrated that in plants, electron transport of mitochondrial complex I was protected by sHsps upon salt stress, with an effect similar to that of the antioxidants ascorbate, gluthathione, a-tocopherol, as well as the superoxide dismutase and catalase enzymes. This result provided evidence that mitochondrial sHsps may protect cellular proteins through a mechanism involving ROS scavenging. It is not clear whether the expression of sHsp genes during oxidative stress is induced by H2O2, which may function as a signaling molecule [84,89,90], or by the damaged proteins. Probably, both mechanisms are involved [40].

6. Perspectives sHsps are regarded as stress proteins with a potential to protect cells from stress damage. Initially detected upon heat, sHsp production has now been observed upon many abiotic stresses, at specific developmental stages in reproductive organs and in seeds, and in leaves of unstressed resurrection plants. Sometimes, modified sHsp levels have been associated with better stress tolerance. High levels of sHsps in Arabidopsis, as a result of overproducing HSFs, increased the basal level of thermotolerance, although they failed to increase the acquired thermotolerance [81,82]. This observation suggests that components other than sHsps and/ or physiological changes that occur in plants, which have been subjected to sublethal heat shock, are also important for thermotolerance. sHsps may coordinate with other components to cope with the stress challenge. Until now, the proteins in the oxygen-evolving complex of Photosystem II may be the only putative in vivo substrates identified for chloroplastic sHsps [61]. No in vivo substrates for sHsps localized in the cytosol, nucleus, endoplasmic reticulum, and mitochondria have been identified. Certainly, the identification of real substrates will help us to understand the role of plant sHsps in stress tolerance and development and to unravel their impressive multiplicity. Knowing sHsp substrates may also help understand whether the same sHsp plays the same role upon different conditions as well as to recognize differences between plant species. Furthermore, it is unclear whether identical conformational changes for oligomer dissociation and oligomer rearrangement with protein substrates differ for the same sHsp that is produced upon different types of stress. Little is known about the reason why plants contain unusually abundant sHsps, what may be the distinct function of different sHsps, and whether they cooperate in stress conditions. We are just at the beginning of under-

standing the functions of sHsps. Many open questions remain to be answered and a better understanding of sHsps might provide a powerful tool to unravel mechanisms used by plants to cope with stress as well as to modify their stress tolerance.

Acknowledgements We thank Elizabeth Vierling and Andy C. Hausrath for the generous gift of Fig. 1A,B, Klaus-Dieter Scharf for critical reading of the manuscript, and Martine De Cock for help in preparing it.

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