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...

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...

A small heat shock protein (sHSP) gene from sunflower, Ha hsp17.6 G1, showed expression patterns that differ from what is known for members of this gene family. The mRNAs of this gene accumulated in seeds during late desiccation stages of zygotic embryogenesis but not in response to heat shock in vegetative tissues. The failure to respond to heat shock was independent of the developmental stage after germination and shock temperature. Nuclear run-on analyses demonstrated that transcription from the Ha hsp17.6 G1 promoter is not induced by heat shock. This agrees with the presence, in this promoter, of sequences with little similarity to heat shock elements. Our results show an evolutionary divergence, in the regulation of plant sHSP genes, which has originated stress-responsive genes and nonresponsive members within this gene family. We discuss implications for mechanisms controlling the developmental regulation of sHSP genes in plants.One of the characteristics of the plant heat shock response is the synthesis of a large number of different, but evolutionarily related, polypeptides of 17-30 kDa (the sHSPs).1 In contrast, animals express only one to four sHSPs upon heat shock. The diversification in plants of heat-inducible sHSP genes could be a consequence of sesility; because plants cannot move away from heat, they would have evolved a battery of specialized "stress genes," the sHSPs. These are expressed in response to heat in all subcellular compartments and could allow plants to cope better with the stress conditions on site (for review, see Ref. 1). In animal and plant systems, heat shock genes encoding proteins of higher molecular weight, for example the HSP70s, have been shown to contain heat-inducible and noninducible members (2, 3). In the case of plant sHSPs the evidence for the existence of genes that are not induced by heat shock is weak and indirect, as it is based on the detection in seeds of sHSP isoforms that are different from the heat shockinduced polypeptides (4,5).In addition to being part of the heat shock response, some plant sHSP genes have been shown to be expressed at normal growth temperatures during zygotic embryogenesis (4 -7). Developmental regulation studies of plant sHSP genes are scarce. So far, only two plant sHSP promoters and 5Ј-flanking sequences have been reported to confer regulation to chimeric genes in maturing seeds: those from soybean Gm hsp17. 3B (8) and sunflower Ha hsp17.7 G4 (9). Initial studies have pointed to common control elements between the heat shock response and activation during embryogenesis. For example, the functional implication of HSEs in both processes is supported by results of deletion analysis (8, 9). Other observations point to the involvement in embryos of distinct control elements, for example, the effect of abi3 mutations on sHSP accumulation in Arabidopsis seeds (10). It is also clear that not all plant sHSP genes are developmentally regulated during embryogenesis, at least for the most systematically analyzed sunflower (9) and Arabidopsis ge...