We show that seed-specific overexpression of the sunflower (Helianthus annuus) HaHSFA9 heat stress transcription factor (HSF) in tobacco (Nicotiana tabacum) enhances the accumulation of heat shock proteins (HSPs). Among these proteins were HSP101 and a subset of the small HSPs, including proteins that accumulate only during embryogenesis in the absence of thermal stress. Levels of late embryogenesis abundant proteins or seed oligosaccharides, however, were not affected. In the transgenic seeds, a high basal thermotolerance persisted during the early hours of imbibition. Transgenic seeds also showed significantly improved resistance to controlled deterioration in a stable and transgene-dependent manner. Furthermore, overexpression of HaHSFA9 did not have detrimental effects on plant growth or development, including seed morphology and total seed yield. Our results agree with previous work tentatively associating HSP gene expression with phenotypes important for seed longevity. These findings might have implications for improving seed longevity in economically important crops.

We report the cloning and functional characterization of the first heat-shock transcription factor that is specifically expressed during embryogenesis in the absence of environmental stress. In sunflower embryos this factor, HaHSFA9, trans-activated promoters with poor consensus heat-shock cis-elements, including that of the seed-specific Hahsp17.6G1 gene. Mutations that improved the heat-shock cis-element consensus at the Hahsp17.7G4 promoter impaired transient activation by HaHSFA9 in sunflower embryos. The same mutations did not affect heat-shock-induced gene expression of this promoter in transgenic tobacco plants but reduced the developmental activation by endogenous heat-shock transcription factors (HSFs) in seeds. Sunflower, and perhaps other plants such as tobacco, differs from the vertebrate animal systems in having at least one specialized HSF with expression and (or) activation patterns strictly restricted to embryos. Our results strongly indicate that HaHSFA9 is a transcription factor critically involved in the developmental activation of Hahsp17.6G1 and in that of similar target genes as Hahsp17.7G4.In eukaryotes, the heat-shock response and some developmental processes are under the control of a family of conserved DNA-binding proteins known as the heat-shock transcription factors (HSFs). 1 Although in some systems, as in Drosophila melanogaster, this regulation involves a single HSF (1), multigenic families of HSFs participate in vertebrate and in plant systems. These families have different sizes, which, together with particular gene expression and activation patterns for the HSFs, might have consequences in the degree of overlapping of regulatory functions mediated by these factors. The specific role of the different HSFs is mostly unknown, particularly for the plant HSFs, and for involvement in developmental processes, as the regulation of gene expression during embryogenesis (See for example, Ref. 2 and the reviews in Refs. 3 and 4).In vertebrate systems, three different HSFs (HSF1, HSF2, and HSF3) have ubiquitous expression patterns (for example, Refs. 5 and 6 and the review in Ref.3). A fourth HSF found in humans displays tissue-specific expression patterns, which suggested specialized functions but not related to embryogenesis (HSF4, Ref. 7). Plants contain the highest number of HSF genes in eukaryotes. This is inferred from in silico analyses from the fully sequenced Arabidopsis thaliana model and from functional analyses of different cloned HSFs in tomato, Arabidopsis, and other plants (reviewed in Refs. 4 and 8 and references therein). Plant HSFs share unique structural and phylogenetic relationships compared with the vertebrate HSFs (9). Fifteen of the 21 putative HSFs from A. thaliana thus contain an insertion of 21 amino acid residues in the oligomerization domain (characteristic of the plant class A HSFs), whereas class B HSFs have no such insertion. Gene expression studies for plant HSFs are very scarce, with only fragmentary data at the mRNA level and even scarcer reports for prote...

SummaryMost plant seeds tolerate desiccation, but vegetative tissues are intolerant to drastic dehydration, except in the case of resurrection plants. Therefore, changes in the regulation of genes normally expressed in seeds are thought to be responsible for the evolutionary origin of desiccation tolerance in resurrection plants. Here, we show that constitutive overexpression of the seed-specific HSFA9 transcription factor from sunflower is sufficient to confer tolerance to severe dehydration, outside of the developing seed context, to vegetative tissues of transgenic tobacco. Whole 3-week-old seedlings could survive severe dehydration. This was quantified as a water loss to 1.96 AE 0.05% of the initial water content, which corresponds to a water potential of » )40 MPa. Survival depended on the water potential, from 40% survival at » )20 MPa to 6.5% survival at » )40 MPa. Whole-seedling survival was limited by the dehydration sensitivity of the roots. Survival correlated with the ectopic expression of a genetic program involving seed-specific, small heat-shock proteins, but not late embryogenesis abundant proteins. The accumulation of sucrose or raffinose family oligosaccharides was not altered by HSFA9. The observed tolerance was achieved without a reduction of growth and development. Our results strongly support the previously suggested contribution of small heat-shock proteins to the desiccation tolerance of seeds. We provide a successful system for analyzing tolerance to severe dehydration in all vegetative organs of seedlings. We propose that HSFA9 is a potential genetic switch involved in the evolution of tolerance to vegetative desiccation.

Gain of function approaches that have been published by our laboratory determined that HSFA9 (Heat Shock Factor A9) activates a genetic program contributing to seed longevity and to desiccation tolerance in plant embryos. We now evaluate the role(s) of HSFA9 by loss of function using different modified forms of HaHSFA9 (sunflower HSFA9), which were specifically overexpressed in seeds of transgenic tobacco. We used two inactive forms (M1, M2) with deletion or mutation of the transcription activation domain of HaHSFA9, and a third form (M3) with HaHSFA9 converted to a potent active repressor by fusion of the SRDX motif. The three forms showed similar protein accumulation in transgenic seeds; however, only HaHSFA9-SRDX showed a highly significant reduction of seed longevity, as determined by controlled deterioration tests, a rapid seed ageing procedure. HaHSFA9-SRDX impaired the genetic program controlled by the tobacco HSFA9, with a drastic reduction in the accumulation of seed heat shock proteins (HSPs) including seed-specific small HSP (sHSP) belonging to cytosolic (CI, CII) classes. Despite such effects, the HaHSFA9-SRDX seeds could survive developmental desiccation during embryogenesis and their subsequent germination was not reduced. We infer that the HSFA9 genetic program contributes only partially to seed-desiccation tolerance and longevity.

The plant hormone auxin regulates growth and development by modulating the stability of auxin/indole acetic acid (Aux/IAA) proteins, which in turn repress auxin response factors (ARFs) transcriptional regulators. In transient assays performed in immature sunflower embryos, we observed that the Aux/IAA protein HaIAA27 represses transcriptional activation by HaHSFA9, a heat shock transcription factor (HSF). We also found that HaIAA27 is stabilized in immature sunflower embryos, where we could show bimolecular fluorescence complementation interaction between native forms of HaIAA27 and HaHSFA9. An auxin-resistant form of HaIAA27 was overexpressed in transgenic tobacco seeds, leading to effects consistent with down-regulation of the ortholog HSFA9 gene, effects not seen with the native HaIAA27 form. Repression of HSFs by HaIAA27 is thus likely alleviated by auxin in maturing seeds. We show that HSFs such as HaHSFA9 are targets of Aux/IAA protein repression. Because HaHSFA9 controls a genetic program involved in seed longevity and embryonic desiccation tolerance, our findings would suggest a mechanism by which these processes can be auxin regulated. Aux/IAA-mediated repression involves transcription factors distinct from ARFs. This finding widens interpretation of auxin responses. H aHSFA9 and the ortholog factors (HSFA9) are specialized heat shock transcription factors (HSFs) that are expressed only in seeds and perform functions during embryogenesis at normal growth temperature. The heat stress response in plants involves multiple HSFs, but HSFA9 does not have a role in the vegetative response to high temperature (1, 2). In Arabidopsis, transcription of the HSFA9 gene is activated by ABA-insensitive 3 (ABI3), a key regulator controlling late-seed development (2). Target genes of HSFA9 encode different heat shock proteins (HSP) (1-5). Gain of function (3, 4) and loss of function (5) approaches determined that in sunflower (Helianthus annuus L.) and tobacco (Nicotiana tabacum L.), HSFA9 activate transcription of specific small heat-shock protein (shsp) genes. Our previous studies (3-5) indicated that HSFA9 factors are involved in the control of a genetic program that regulates seed longevity and embryonic desiccation tolerance. This program includes genes that encode different HSP but not late embryogenesis abundant (LEA) proteins (3-5). To search for additional transcription factors (TFs) involved in the regulation of this process, we used a yeast two-hybrid system to identify embryo TFs that interact with HaHSFA9. Surprisingly, we found that the auxin/ indole acetic acid (Aux/IAA) protein HaIAA27 interacts with HaHSFA9.Aux/IAA are unstable proteins that are further destabilized in response to the major naturally occurring auxin, indole-3-acetic acid (IAA) (6). Aux/IAA proteins act as nuclear-localized transcriptional repressors of auxin response factors (ARFs) (7). In the current model of Aux/IAA function, auxin alleviates repression of ARFs by inducing Aux/IAA degradation in the 26S proteasome (i.e., refs. 8-10 an...

SummaryTransgenic tobacco expression was analysed of chimeric genes with point mutations in the heat shock element (HSE) arrays of a small heat shock protein (sHSP) gene from sunflower: Ha hsp17.7 G4. The promoter was developmentally regulated during zygotic embryogenesis and responded to heat stress in vegetative tissues. Mutations in the HSE affected nucleotides crucial for human heat shock transcription factor 1 (HSF1) binding. They abolished the heat shock response of Ha hsp17.7 G4 and produced expression changes that demonstrated dual regulation of this promoter during embryogenesis. Thus, whereas activation of the chimeric genes during early maturation stages did not require intact HSE, expression at later desiccation stages was reduced by mutations in both the proximal (-57 to -89) and distal (-99 to -121) HSE. In contrast, two point mutations in the proximal HSE that did not severely affect gene expression during zygotic embryogenesis, eliminated the heat shock response of the same chimeric gene in vegetative organs. Therefore, by site-directed mutagenesis, it was possible to separate the heat shock response of Ha hsp17.7 G4 from its developmental regulation. The results indicate the co-existence, in a single promoter, of HSF-dependent and -independent regulation mechanisms that would control sHSP gene expression at different stages during plant embryogenesis.

Hahsp17.6G1 is the promoter of a small heat stress protein (sHSP) from sunflower (Helianthus annuus) that is activated during zygotic embryogenesis, but which does not respond to heat stress. We report here the cloning of a transcription factor (TF), sunflower drought-responsive element binding factor 2 (HaDREB2), by one-hybrid interaction with functional cis-elements in Hahsp17.6G1. We have analyzed the functional interaction between HaDREB2 and a second transcription factor, sunflower heat stress factor A9 (HaHSFA9), which was previously assigned to the regulation of Hahsp17.6G1. HaDREB2 and HaHSFA9 synergistically trans-activate the Hahsp17.6G1 promoter in bombarded sunflower embryos. This synergistic interaction is heat stress factor (HSF) specific and requires the binding of both factors to the promoter. The C-terminal region of HaHSFA9 is sufficient for the HSF specificity. Our results represent an example of a functional interaction between members of the Apetala 2 (HaDREB2) and HSF (HaHSFA9) families of transcription factors. We suggest new roles in zygotic embryogenesis for specific members of the AP2 transcription factor family.The functional assignment of individual transcription factors (TFs) from multimember families is one of the major goals in postgenomic analyses of plants. We focus our attention on factors that are involved in the developmental induction of small heat stress protein (sHSP) genes during zygotic embryogenesis. In plants, as in other eukaryotes, HSPs of different molecular masses, including the sHSPs (17-30 kD), are expressed not only during the heat shock response but also during development in the absence of exogenous stresses (for review, see Sun et al., 2002).

BackgroundTranscription factor HaDREB2 was identified in sunflower (Helianthus annuus L.) as a drought-responsive element-binding factor 2 (DREB2) with unique properties. HaDREB2 and the sunflower Heat Shock Factor A9 (HaHSFA9) co-activated the Hahsp17.6G1 promoter in sunflower embryos. Both factors could be involved in transcriptional co-activation of additional small heat stress protein (sHSP) promoters, and thus contribute to the HaHSFA9-mediated enhancement of longevity and basal thermotolerance of seeds.ResultsWe found that overexpression of HaDREB2 in seeds did not enhance longevity. This was deduced from assays of basal thermotolerance and controlled seed-deterioration, which were performed with transgenic tobacco. Furthermore, the constitutive overexpression of HaDREB2 did not increase thermotolerance in seedlings or result in the accumulation of HSPs at normal growth temperatures. In contrast, when HaDREB2 and HaHSFA9 were conjointly overexpressed in seeds, we observed positive effects on seed longevity, beyond those observed with overexpression of HaHSFA9 alone. Such additional effects are accompanied by a subtle enhancement of the accumulation of subsets of sHSPs belonging to the CI and CII cytosolic classes.ConclusionOur results reveal the functional interdependency of HaDREB2 and HaHSFA9 in seeds. HaDREB2 differs from other previously characterized DREB2 factors in plants in terms of its unique functional interaction with the seed-specific HaHSFA9 factor. No functional interaction between HaDREB2 and HaHSFA9 was observed when both factors were conjointly overexpressed in vegetative tissues. We therefore suggest that additional, seed-specific factors, or protein modifications, could be required for the functional interaction between HaDREB2 and HaHSFA9.