RESUMO
Global warming poses a threat for crops, therefore, the identification of thermotolerance mechanisms is a priority. In plants, the core factors that regulate transcription under heat stress (HS) are well described and include several HS transcription factors (HSFs). Despite the relevance of alternative splicing in HS response and thermotolerance, the core regulators of HS-sensitive alternative splicing have not been identified. In tomato, alternative splicing of HSFA2 is important for acclimation to HS. Here, we show that several members of the serine/arginine-rich family of splicing factors (SRSFs) suppress HSFA2 intron splicing. Individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) combined with RNA-Seq revealed that RS2Z35 and RS2Z36, which make up a plant-specific clade of SR proteins, not only regulate HSFA2 but approximately 50% of RNAs that undergo HS-sensitive alternative splicing, with preferential binding to purine-rich RNA motifs. Single and double CRISPR rs2z mutant lines show a dysregulation of splicing and exhibit lower basal and acquired thermotolerance compared to wild type plants. Our results suggest that RS2Z35 and RS2Z36 have a central role in mitigation of the negative effects of HS on RNA splicing homeostasis, and their emergence might have contributed to the increased capacity of plants to acclimate to high temperatures.
RESUMO
Conditions that cause proteotoxicity like high temperature trigger the activation of unfolded protein response (UPR). The cytosolic (CPR) and endoplasmic reticulum (ER) UPR rely on heat stress transcription factor (HSF) and two members of the basic leucine zipper (bZIP) gene family, respectively. In tomato, HsfA1a is the master regulator of CPR. Here, we identified the core players of tomato ER-UPR including the two central transcriptional regulators, namely bZIP28 and bZIP60. Interestingly, the induction of ER-UPR genes and the activation of bZIP60 are altered in transgenic plants where HsfA1a is either overexpressed (A1aOE) or suppressed (A1CS), indicating an interplay between CPR and ER-UPR systems. Several ER-UPR genes are differentially expressed in the HsfA1a transgenic lines either exposed to heat stress or to the ER stress elicitor tunicamycin (TUN). The ectopic expression of HsfA1a is associated with higher tolerance against TUN. On the example of the ER-resident Hsp70 chaperone BIP3, we show that the presence of cis-elements required for HSF and bZIP regulation serves as a putative platform for the co-regulation of these genes by both CPR and ER-UPR mechanisms, in the case of BIP3 in a stimulatory manner under high temperatures. In addition, we show that the accumulation of HsfA1a results in higher levels of three ATG genes and a more sensitized induction of autophagy in response to ER stress which also supports the increased tolerance to ER stress of the A1aOE line. These findings provide a basis for the coordination of protein homeostasis in different cellular compartments under stress conditions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Solanum lycopersicum , Solanum lycopersicum/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Resposta a Proteínas não Dobradas , Retículo Endoplasmático/metabolismoRESUMO
Plants respond to higher temperatures by the action of heat stress (HS) transcription factors (Hsfs), which control the onset, early response, and long-term acclimation to HS. Members of the HsfA1 subfamily, such as tomato HsfA1a, are the central regulators of HS response, and their activity is fine-tuned by other Hsfs. We identify tomato HsfA7 as capacitor of HsfA1a during the early HS response. Upon a mild temperature increase, HsfA7 is induced in an HsfA1a-dependent manner. The subsequent interaction of the two Hsfs prevents the stabilization of HsfA1a resulting in a negative feedback mechanism. Under prolonged or severe HS, HsfA1a and HsfA7 complexes stimulate the induction of genes required for thermotolerance. Therefore, HsfA7 exhibits a co-repressor mode at mild HS by regulating HsfA1a abundance to moderate the upregulation of HS-responsive genes. HsfA7 undergoes a temperature-dependent transition toward a co-activator of HsfA1a to enhance the acquired thermotolerance capacity of tomato plants.
Assuntos
Fatores de Transcrição de Choque Térmico/genética , Solanum lycopersicum/genética , Transativadores/genética , Aclimatação , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Temperatura Alta , Solanum lycopersicum/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Termotolerância/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Wild relatives of crops thrive in habitats where environmental conditions can be restrictive for productivity and survival of cultivated species. The genetic basis of this variability, particularly for tolerance to high temperatures, is not well understood. We examined the capacity of wild and cultivated accessions to acclimate to rapid temperature elevations that cause heat stress (HS). We investigated genotypic variation in thermotolerance of seedlings of wild and cultivated accessions. The contribution of polymorphisms associated with thermotolerance variation was examined regarding alterations in function of the identified gene. We show that tomato germplasm underwent a progressive loss of acclimation to strong temperature elevations. Sensitivity is associated with intronic polymorphisms in the HS transcription factor HsfA2 which affect the splicing efficiency of its pre-mRNA. Intron splicing in wild species results in increased synthesis of isoform HsfA2-II, implicated in the early stress response, at the expense of HsfA2-I which is involved in establishing short-term acclimation and thermotolerance. We propose that the selection for modern HsfA2 haplotypes reduced the ability of cultivated tomatoes to rapidly acclimate to temperature elevations, but enhanced their short-term acclimation capacity. Hence, we provide evidence that alternative splicing has a central role in the definition of plant fitness plasticity to stressful conditions.
Assuntos
Processamento Alternativo/genética , Domesticação , Variação Genética , Precursores de RNA/genética , Solanum lycopersicum/genética , Solanum lycopersicum/fisiologia , Termotolerância/genética , Aclimatação , Alelos , Sequência de Bases , Estudo de Associação Genômica Ampla , Haplótipos/genética , Resposta ao Choque Térmico , Íntrons/genética , Polimorfismo Genético , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Transporte Proteico , Precursores de RNA/metabolismo , Plântula/fisiologia , TemperaturaRESUMO
KEY MESSAGE: Different genes coding for one ribosome biogenesis factor are differentially expressed and are likely under the control of distinct transcription factors, which contributes to the regulatory space for ribosome maturation. Maturation of ribosomes including rRNA processing and modification, rRNA folding and ribosome protein association requires the function of many ribosome biogenesis factors (RBFs). Recent studies document plant-specific variations of the generally conserved process of ribosome biogenesis. For instance, distinct rRNA maturation pathways and intermediates have been identified, the existence of plant specific RBFs has been proposed and several RBFs are encoded by multiple genes. The latter in combination with the discussed ribosome heterogeneity points to a possible function of the different proteins representing one RBF in diversification of ribosomal compositions. Such factor-based regulation would require a differential regulation of their expression, may be even controlled by different transcription factors. We analyzed the expression profiles of genes coding for putative RBFs and transcription factors. Most of the genes coding for RBFs are expressed in a comparable manner, while different genes coding for a single RBF are often differentially expressed. Based on a selected set of genes we document a function of the transcription factors AtMYC1, AtMYC2, AtbHLH105 and AtMYB26 on the regulation of different RBFs. Moreover, on the example of the RBFs LSG1 and BRX1, both encoded by two genes, we give a first hint on a differential transcription factor dependence of expression. Consistent with this observation, the phenotypic analysis of RBF mutants suggests a relation between LSG1-1 and BRX1-1 expression and the transcription factor MYC1. In summary, we propose that the multiple genes coding for one RBF are required to enlarge the regulatory space for ribosome biogenesis.
Assuntos
Arabidopsis/metabolismo , Ribossomos/metabolismo , Fatores de Transcrição/metabolismo , Regiões Promotoras Genéticas/genética , RNA Ribossômico/genética , Proteínas Ribossômicas/metabolismoRESUMO
Plants code for a multitude of heat stress transcription factors (Hsfs). Three of them act as central regulators of heat stress (HS) response in tomato (Solanum lycopersicum). HsfA1a regulates the initial response, and HsfA2 controls acquired thermotolerance. HsfB1 is a transcriptional repressor but can also act as co-activator of HsfA1a. Currently, the mode of action and the relevance of the dual function of HsfB1 remain elusive. We examined this in HsfB1 overexpression or suppression transgenic tomato lines. Proteome analysis revealed that HsfB1 overexpression stimulates the co-activator function of HsfB1 and consequently the accumulation of HS-related proteins under non-stress conditions. Plants with enhanced levels of HsfB1 show aberrant growth and development but enhanced thermotolerance. HsfB1 suppression has no significant effect prior to stress. Upon HS, HsfB1 suppression strongly enhances the induction of heat shock proteins due to the higher activity of other HS-induced Hsfs, resulting in increased thermotolerance compared with wild-type. Thereby, HsfB1 acts as co-activator of HsfA1a for several Hsps, but as a transcriptional repressor on other Hsfs, including HsfA1b and HsfA2. The dual function explains the activation of chaperones to enhance protection and regulate the balance between growth and stress response upon deviations from the homeostatic levels of HsfB1.
Assuntos
Resposta ao Choque Térmico/fisiologia , Proteínas de Plantas/fisiologia , Proteínas Repressoras/fisiologia , Solanum lycopersicum/metabolismo , Fatores de Transcrição/fisiologia , Eletroforese em Gel Bidimensional , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/fisiologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo RealRESUMO
HsfB1 is a central regulator of heat stress (HS) response and functions dually as a transcriptional co-activator of HsfA1a and a general repressor in tomato. HsfB1 is efficiently synthesized during the onset of HS and rapidly removed in the course of attenuation during the recovery phase. Initial results point to a complex regime modulating HsfB1 abundance involving the molecular chaperone Hsp90. However, the molecular determinants affecting HsfB1 stability needed to be established. We provide experimental evidence that DNA-bound HsfB1 is efficiently targeted for degradation when active as a transcriptional repressor. Manipulation of the DNA-binding affinity by mutating the HsfB1 DNA-binding domain directly influences the stability of the transcription factor. During HS, HsfB1 is stabilized, probably due to co-activator complex formation with HsfA1a. The process of HsfB1 degradation involves nuclear localized Hsp90. The molecular determinants of HsfB1 turnover identified in here are so far seemingly unique. A mutational switch of the R/KLFGV repressor motif's arginine and lysine implies that the abundance of other R/KLFGV type Hsfs, if not other transcription factors as well, might be modulated by a comparable mechanism. Thus, we propose a versatile mechanism for strict abundance control of the stress-induced transcription factor HsfB1 for the recovery phase, and this mechanism constitutes a form of transcription factor removal from promoters by degradation inside the nucleus.
Assuntos
DNA de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação/genética , Western Blotting , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Solanum lycopersicum/citologia , Solanum lycopersicum/genética , Proteínas de Plantas/genética , Ligação Proteica , Protoplastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genéticaRESUMO
Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 (Hsp70) and 90 (Hsp90) assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins with respect to the cytosolic chaperone-dependent regulation. Some preproteins such as pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins such as pSSU is more strongly dependent on Hsp70. The E3 ligase, C-terminus of Hsp70-interacting protein (Chip), appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable with the cytosolic unfolded protein response.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Solanum lycopersicum/metabolismo , Citosol/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
Heat shock proteins (Hsps) are molecular chaperones primarily involved in maintenance of protein homeostasis. Their function has been best characterized in heat stress (HS) response during which Hsps are transcriptionally controlled by HS transcription factors (Hsfs). The role of Hsfs and Hsps in HS response in tomato was initially examined by transcriptome analysis using the massive analysis of cDNA ends (MACE) method. Approximately 9.6% of all genes expressed in leaves are enhanced in response to HS, including a subset of Hsfs and Hsps. The underlying Hsp-Hsf networks with potential functions in stress responses or developmental processes were further explored by meta-analysis of existing microarray datasets. We identified clusters with differential transcript profiles with respect to abiotic stresses, plant organs and developmental stages. The composition of two clusters points towards two major chaperone networks. One cluster consisted of constitutively expressed plastidial chaperones and other genes involved in chloroplast protein homeostasis. The second cluster represents genes strongly induced by heat, drought and salinity stress, including HsfA2 and many stress-inducible chaperones, but also potential targets of HsfA2 not related to protein homeostasis. This observation attributes a central regulatory role to HsfA2 in controlling different aspects of abiotic stress response and tolerance in tomato.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Fatores de Transcrição/genética , Secas , Perfilação da Expressão Gênica , Fatores de Transcrição de Choque Térmico , Temperatura Alta , Solanum lycopersicum/fisiologia , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Secretion of proteins is a central strategy of bacteria to influence and respond to their environment. Until now, there has been very few discoveries regarding the cyanobacterial secrotome or the secretion machineries involved. For a mutant of the outer membrane channel TolC-homologue HgdD of Anabaena sp. PCC 7120, a filamentous and heterocyst-forming cyanobacterium, an altered secretome profile was reported. To define the role of HgdD in protein secretion, we have developed a method to isolate extracellular proteins of Anabaena sp. PCC 7120 wild type and an hgdD loss-of-function mutant. We identified 51 proteins of which the majority is predicted to have an extracellular secretion signal, while few seem to be localized in the periplasmic space. Eight proteins were exclusively identified in the secretome of wild-type cells, which coincides with the distribution of type I secretion signal. We selected three candidates and generated hemagglutinin-tagged fusion proteins which could be exclusively detected in the extracellular protein fraction. However, these proteins are not secreted in the hgdD-mutant background, where they are rapidly degraded. This confirms a direct function of HgdD in protein secretion and points to the existence of a quality control mechanism at least for proteins secreted in an HgdD-dependent pathway.
Assuntos
Anabaena/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Anabaena/genética , Proteínas da Membrana Bacteriana Externa/genética , Sistemas de Secreção Bacterianos/genética , Transporte Biológico/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Periplasma/metabolismo , Transporte ProteicoRESUMO
Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the heat stress protein 70 and 90 (Hsp70 or Hsp90) families assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins in respect to the cytosolic chaperone dependent regulation. Some preproteins like pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins like pSSU is more strongly dependent on Hsp70. The E3 ligase Chip appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable to the cytosolic unfolded protein response.
RESUMO
tRNA-NTs (tRNA nucleotidyltransferases) are required for the maturation or repair of tRNAs by ensuring that they have an intact cytidine-cytidine-adenosine sequence at their 3'-termini. Therefore this enzymatic activity is found in all cellular compartments, namely the nucleus, cytoplasm, plastids and mitochondria, in which tRNA synthesis or translation occurs. A single gene codes for tRNA-NT in plants, suggesting a complex targeting mechanism. Consistent with this, distinct signals have been proposed for plastidic, mitochondrial and nuclear targeting. Our previous research has shown that in addition to N-terminal targeting information, the mature domain of the protein itself modifies targeting to mitochondria and plastids. This suggests the existence of an as yet unknown determinate for the distribution of dual-targeted proteins between these two organelles. In the present study, we explore the enzymatic and physicochemical properties of tRNA-NT variants to correlate the properties of the enzyme with the intracellular distribution of the protein. We show that alteration of tRNA-NT stability influences its intracellular distribution due to variations in organelle import capacities. Hence the fate of the protein is determined not only by the transit peptide sequence, but also by the physicochemical properties of the mature protein.
Assuntos
Organelas/enzimologia , Organelas/metabolismo , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Dicroísmo Circular , Biologia ComputacionalRESUMO
Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.
Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plantas/metabolismo , Anabaena/metabolismo , Cianobactérias/ultraestrutura , Microscopia Eletrônica , Chaperonas Moleculares/metabolismo , Periplasma/metabolismo , Transporte Proteico/fisiologia , Tilacoides/metabolismoRESUMO
Heat stress transcription factors (Hsfs) regulate gene expression in response to environmental stress. The Hsf network in plants is controlled at the transcriptional level by cooperation of distinct Hsf members and by interaction with chaperones. We found two general mechanisms of Hsf regulation by chaperones while analyzing the three major Hsfs, A1, A2, and B1, in tomato (Solanum lycopersicum). First, Hsp70 and Hsp90 regulate Hsf function by direct interactions. Hsp70 represses the activity of HsfA1, including its DNA binding, and the coactivator function of HsfB1 in the complex with HsfA2, while the DNA binding activity of HsfB1 is stimulated by Hsp90. Second, Hsp90 affects the abundance of HsfA2 and HsfB1 by modulating hsfA2 transcript degradation involved in regulation of the timing of HsfA2 synthesis. By contrast, HsfB1 binding to Hsp90 and to DNA are prerequisites for targeting this Hsf for proteasomal degradation, which also depends on a sequence element in its carboxyl-terminal domain. Thus, HsfB1 represents an Hsp90 client protein that, by interacting with the chaperone, is targeted for, rather than protected from, degradation. Based on these findings, we propose a versatile regulatory regime involving Hsp90, Hsp70, and the three Hsfs in the control of heat stress response.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/genética , Fatores de Transcrição/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Solanum lycopersicum/metabolismo , Proteínas de Plantas/genética , Mapeamento de Interação de Proteínas , Protoplastos/metabolismo , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
In plants, a family of more than 20 heat stress transcription factors (Hsf) controls the expression of heat stress (hs) genes. There is increasing evidence for the functional diversification between individual members of the Hsf family fulfilling distinct roles in response to various environmental stress conditions and developmental signals. In response to hs, accumulation of both heat stress proteins (Hsp) and Hsfs is induced. In tomato, the physical interaction between the constitutively expressed HsfA1 and the hs-inducible HsfA2 results in synergistic transcriptional activation (superactivation) of hs gene expression. Here, we show that the interaction is strikingly specific and not observed with other class A Hsfs. Hetero-oligomerization of the two-component Hsfs is preferred to homo-oligomerization, and each Hsf in the HsfA1/HsfA2 hetero-oligomeric complex has its characteristic contribution to its function as superactivator. Distinct regions of the oligomerization domain are responsible for specific homo- and hetero-oligomeric interactions leading to the formation of hexameric complexes. The results are summarized in a model of assembly and function of HsfA1/A2 superactivator complexes in hs gene regulation.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Complexos Multiproteicos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Solanum lycopersicum/genética , Estresse Fisiológico/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Cromatografia em Gel , Reagentes de Ligações Cruzadas/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Modelos Biológicos , Dados de Sequência Molecular , Proteínas de Plantas/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Fatores de Transcrição/genéticaRESUMO
HsfA2 is a heat stress (hs)-induced Hsf in peruvian tomato (Lycopersicon peruvianum) and the cultivated form Lycopersicon esculentum. Due to the high activator potential and the continued accumulation during repeated cycles of heat stress and recovery, HsfA2 becomes a dominant Hsf in thermotolerant cells. The formation of heterooligomeric complexes with HsfA1 leads to nuclear retention and enhanced transcriptional activity of HsfA2. This effect seems to represent one part of potential molecular mechanisms involved in its activity control. As shown in this paper, the activity of HsfA2 is also controlled by a network of nucleocytoplasmic small Hsps influencing its solubility, intracellular localization and activator function. By yeast two-hybrid interaction and transient coexpression studies in tobacco (Nicotiana plumbaginifolia) mesophyll protoplasts, we found that tomato (Lycopersicon esculentum) Hsp17.4-CII acts as corepressor of HsfA2. Given appropriate conditions, both proteins together formed large cytosolic aggregates which could be solubilized in presence of class CI sHsps. However, independent of the formation of aggregates or of the nucleocytoplasmic distribution of HsfA2, its transcriptional activity was specifically repressed by interaction of Hsp17.4-CII with the C-terminal activator domain. Although not identical in all aspects, the situation with the highly expressed, heat stress-inducible Arabidopsis HsfA2 was found to be principally similar. In corresponding reporter assays its activity was repressed in presence of AtHsp17.7-CII but not of AtHsp17.6-CII or LpHsp17.4-CII.