A seed-specific transcription factor, HSFA9, anticipates UV-B light responses by mimicking the activation of the UV-B receptor in tobacco.

Sunflower heat shock factor A9 (HSFA9, hereafter A9) is a transcription factor involved in seed desiccation tolerance and longevity. A9 also links the regulation of seed maturation with that of seedling photomorphogenesis through visible light receptors. Analyses in transgenic Nicotiana tabacum (tobacco) indicated that A9 also affects responses mediated by NtUVR8, the receptor of ultraviolet light B (UV-B). We compared the effects of A9 and UV-B illumination on the nuclear localization of GFP-NtUVR8 in Nicotiana benthamiana leaves. We also used co-immunoprecipitation and limited proteolysis for analyzing the interaction between A9 and NtUVR8. We found that A9, by binding to NtUVR8, induced structural changes that resulted in enhancing the nuclear localization of NtUVR8 by hindering its nuclear export. The localization of UVR8 is crucial for receptor activation and function in Arabidopsis, where UV-B-activated nuclear UVR8 binds the E3 ubiquitin ligase COP1, leading to enhanced UV-B responses and photoprotection. A9 similarly activated NtUVR8 by enhancing COP1 binding without UV-B light. Seedlings and dark-germinated seeds that overexpress A9 showed primed UV-B light stress protection. Our results unveil a UV-B-independent activation mechanism and a role for UVR8 in plant seeds that might contribute to early stress protection, facilitating seedling establishment.

Heat Stress Factors Expressed during Seed Maturation Differentially Regulate Seed Longevity and Seedling Greening.

Heat Stress Factor A9 (A9), a seed-specific transcription factor contributing to seed longevity, also enhances phytochrome-dependent seedling greening. The RNA-seq analyses of imbibed-seed transcripts here reported indicated potential additional effects of A9 on cryptochrome-mediated blue-light responses. These analyses also suggested that in contrast to the A9 effects on longevity, which require coactivation by additional factors as A4a, A9 alone might suffice for the enhancement of photomorphogenesis at the seedling stage. We found that upon its seed-specific overexpression, A9 indeed enhanced the expected blue-light responses. Comparative loss-of-function analyses of longevity and greening, performed by similar expression of dominant-negative and inactive forms of A9, not only confirmed the additional greening effects of A9, but also were consistent with A9 not requiring A4a (or additional factors) for the greening effects. Our results strongly indicate that A9 would differentially regulate seed longevity and photomorphogenesis at the seedling stage, A9 alone sufficing for both the phytochrome- and cryptochrome-dependent greening enhancement effects.

SUMO-Dependent Synergism Involving Heat Shock Transcription Factors with Functions Linked to Seed Longevity and Desiccation Tolerance.

A transcriptional synergism between HaHSFA9 (A9) and HaHSFA4a (A4a) contributes to determining longevity and desiccation tolerance of sunflower (Helianthus annuus, L.) seeds. Potential lysine SUMOylation sites were identified in A9 and A4a and mutated to arginine. We show that A9 is SUMOylated in planta at K38. Although we did not directly detect SUMOylated A4a in planta, we provide indirect evidence from transient expression experiments indicating that A4a is SUMOylated at K172. Different combinations of wild type and SUMOylation site mutants of A9 and A4a were analyzed by transient expression in sunflower embryos and leaves. Although most of the precedents in literature link SUMOylation with repression, the A9 and A4a synergism was fully abolished when the mutant forms for both factors were combined. However, the combination of mutant forms of A9 and A4a did not affect the nuclear retention of A4a by A9; therefore, the analyzed mutations would affect the synergism after the mutual interaction and nuclear co-localization of A9 and A4a. Our results suggest a role for HSF SUMOylation during late, zygotic, embryogenesis. The SUMOylation of A9 (or A4a) would allow a crucial, synergic, transcriptional effect that occurs in maturing sunflower seeds.

Seed-specific transcription factor HSFA9 links late embryogenesis and early photomorphogenesis.

HSFA9 is a seed-specific transcription factor that in sunflower (Helianthus annuus) is involved in desiccation tolerance and longevity. Here we show that the constitutive overexpression of HSFA9 in tobacco (Nicotiana tabacum) seedlings attenuated hypocotyl growth under darkness and accelerated the initial photosynthetic development. Plants overexpressing HSFA9 increased accumulation of carotenoids, chlorophyllide, and chlorophyll, and displayed earlier unfolding of the cotyledons. HSFA9 enhanced phytochrome-dependent light responses, as shown by an intensified hypocotyl length reduction after treatments with continuous far-red or red light. This observation indicated the involvement of at least two phytochromes: PHYA and PHYB. Reduced hypocotyl length under darkness did not depend on phytochrome photo-activation; this was inferred from the lack of effect observed using far-red light pulses applied before the dark treatment. HSFA9 increased the expression of genes that activate photomorphogenesis, including PHYA, PHYB, and HY5. HSFA9 might directly upregulate PHYA and indirectly affect PHYB transcription, as suggested by transient expression assays. Converse effects on gene expression, greening, and cotyledon unfolding were observed using a dominant-negative form of HSFA9, which was overexpressed under a seed-specific promoter. This work uncovers a novel transcriptional link, through HSFA9, between seed maturation and early photomorphogenesis. In all, our data suggest that HSFA9 enhances photomorphogenesis via early transcriptional effects that start in seeds under darkness.

Heat shock transcription factors involved in seed desiccation tolerance and longevity retard vegetative senescence in transgenic tobacco.

MAIN CONCLUSION: Transcription factors normally expressed in sunflower seeds delayed vegetative senescence induced by severe stress in transgenic tobacco. This revealed a novel connection between seed heat shock factors, desiccation tolerance and vegetative longevity. HaHSFA9 and HaHSFA4a coactivate a genetic program that, in sunflower (Helianthus annuus L.), contributes to seed longevity and desiccation tolerance. We have shown that overexpression of HaHSFA9 in transgenic tobacco seedlings resulted in tolerance to drastic dehydration and oxidative stress. Overexpression of HaHSFA9 alone was linked to a remarkable protection of the photosynthetic apparatus. In addition, the combined overexpression of HaHSFA9 and HaHSFA4a enhanced all these stress-resistance phenotypes. Here, we find that HaHSFA9 confers protection against damage induced by different stress conditions that accelerate vegetative senescence during different stages of development. Seedlings and plants that overexpress HaHSFA9 survived lethal treatments of dark-induced senescence. HaHSFA9 overexpression induced resistance to effects of culture under darkness for several weeks. Only some homoiochlorophyllous resurrection plants are able to withstand this experimental severe stress condition. The combined overexpression of HaHSFA9 and HaHSFA4a did not result in further slowing of dark-induced seedling senescence. However, combined expression of the two transcription factors caused improved recovery of the photosynthetic organs of seedlings after lethal dark treatments. At later stages of vegetative development, HaHSFA9 delayed the appearance of senescence symptoms in leaves of plants grown under normal illumination. This delay was observed under either control or stress treatments. Thus, HaHSFA9 delayed both natural and stress-induced leaf senesce. These novel observations connect transcription factors involved in desiccation tolerance with leaf longevity.

Co-overexpression of two Heat Shock Factors results in enhanced seed longevity and in synergistic effects on seedling tolerance to severe dehydration and oxidative stress.

BACKGROUND: We have previously reported that the seed-specific overexpression of sunflower (Helianthus annuus L.) Heat Shock Factor A9 (HaHSFA9) enhanced seed longevity in transgenic tobacco (Nicotiana tabacum L.). In addition, the overexpression of HaHSFA9 in vegetative organs conferred tolerance to drastic levels of dehydration and oxidative stress. RESULTS: Here we found that the combined overexpression of sunflower Heat Shock Factor A4a (HaHSFA4a) and HaHSFA9 enhanced all the previously reported phenotypes described for the overexpression of HaHSFA9 alone. The improved phenotypes occurred in coincidence with only subtle changes in the accumulation of small Heat Shock Proteins (sHSP) that are encoded by genes activated by HaHSFA9. The single overexpression of HaHSFA4a in vegetative organs (which lack endogenous HSFA9 proteins) did not induce sHSP accumulation under control growth conditions; neither it conferred thermotolerance. The overexpression of HaHSFA4a alone also failed to induce tolerance to severe abiotic stress. Thus, a synergistic functional effect of both factors was evident in seedlings. CONCLUSIONS: Our study revealed that HaHSFA4a requires HaHSFA9 for in planta function. Our results strongly support the involvement of HaHSFA4a and HaHSFA9 in transcriptional co-activation of a genetic program of longevity and desiccation tolerance in sunflower seeds. These results would also have potential application for improving seed longevity and tolerance to severe stress in vegetative organs.

Protection of the photosynthetic apparatus from extreme dehydration and oxidative stress in seedlings of transgenic tobacco.

A genetic program that in sunflower seeds is activated by Heat Shock transcription Factor A9 (HaHSFA9) has been analyzed in transgenic tobacco seedlings. The ectopic overexpression of the HSFA9 program protected photosynthetic membranes, which resisted extreme dehydration and oxidative stress conditions. In contrast, heat acclimation of seedlings induced thermotolerance but not resistance to the harsh stress conditions employed. The HSFA9 program was found to include the expression of plastidial small Heat Shock Proteins that accumulate only at lower abundance in heat-stressed vegetative organs. Photosystem II (PSII) maximum quantum yield was higher for transgenic seedlings than for non-transgenic seedlings, after either stress treatment. Furthermore, protection of both PSII and Photosystem I (PSI) membrane protein complexes was observed in the transgenic seedlings, leading to their survival after the stress treatments. It was also shown that the plastidial D1 protein, a labile component of the PSII reaction center, and the PSI core protein PsaB were shielded from oxidative damage and degradation. We infer that natural expression of the HSFA9 program during embryogenesis may protect seed pro-plastids from developmental desiccation.

Repression by an auxin/indole acetic acid protein connects auxin signaling with heat shock factor-mediated seed 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.

Loss of function of the HSFA9 seed longevity program.

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 HaDREB2 transcription factor enhances basal thermotolerance and longevity of seeds through functional interaction with HaHSFA9.

BACKGROUND: Transcription 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. RESULTS: We 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. CONCLUSION: Our 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.

The ectopic overexpression of a seed-specific transcription factor, HaHSFA9, confers tolerance to severe dehydration in vegetative organs.

Most 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 +/- 0.05% of the initial water content, which corresponds to a water potential of approximately -40 MPa. Survival depended on the water potential, from 40% survival at approximately -20 MPa to 6.5% survival at approximately -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.

Distinct heat-shock element arrangements that mediate the heat shock, but not the late-embryogenesis induction of small heat-shock proteins, correlate with promoter activation in root-knot nematode feeding cells.

Genes coding small heat-shock proteins (sHSPs) show distinct behaviours with respect to environmental and developmental signals. Their transcriptional regulation depends on particular combinations of heat stress cis-elements (heat-shock elements; HSEs) but many aspects regarding their regulation remain unclear. Cyst and root-knot nematodes induce, in the roots of infected plants, the differentiation of special feeding cells with high metabolic activity (syncytia and giant cells, respectively), a process accompanied by extensive gene expression changes. The Hahsp17.7G4 (G4) promoter was active in giant cells and its HSE arrangements were crucial for this activation. In the present work, we provide further basis to associate giant cell expression with the heat-shock response of this gene class, by analysing additional promoters. The Hahsp17.6G1 (G1) promoter, not induced by heat shock, was silent in giant cells, while Hahsp18.6G2 (G2), which responds to heat shock, was specifically induced in giant cells. In addition, a mutated Hahsp17.7G4 promoter version (G4MutP) with a strong heat-shock induction was also induced in giant cells. The responses of the different promoters correlated with distinct HSE configurations, which might have implications on differential trans-activation. Furthermore, the shortest giant cell and heat-shock-inducible sHSP promoter version analysed in tobacco (-83pb Hahsp17.7G4) fully maintained its expression profile in Arabidopsis. Cyst nematodes did not induce the Hahsp17.7G4 promoter, revealing additional specificity in the nematode response. These findings, together with the fact that the class I sHSP products of endogenous genes accumulated specifically in tobacco giant cells, support the idea that these nematode-induced giant cells represent a transcriptional state very similar to that produced by heat shock regarding this class of genes. The high metabolic rate of giant cells may result in unfolded proteins requiring class I sHSPs as chaperones, which might, somehow, mimic heat-shock and/or other stress responses.

Improved resistance to controlled deterioration in transgenic seeds.

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.

Functional interaction between two transcription factors involved in the developmental regulation of a small heat stress protein gene promoter.

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.

Induction of the Hahsp17.7G4 promoter by root-knot nematodes: involvement of heat-shock elements in promoter activity in giant cells.

Root-knot nematodes feed from specialized giant cells induced in the plants that they parasitize. We found that the promoter of the Hahsp17.7G4 gene, which encodes a small heat-shock protein involved in embryogenesis and stress responses, directed GUS expression in tobacco galls induced by the root-knot nematode Meloidogyne incognita. In roots containing a GUS reporter fusion to the Hahsp17.7G4 promoter, 10% of the galls stained for GUS expression 1 to 3 days after infection and the fraction stained increased to 60 to 80% 17 to 20 days after infection. A DNA fragment from -83 to +163, which contains heat-shock element (HSE) core sequences, is sufficient to support a promoter activity largely restricted to giant cells within the galls. Two-point mutations in HSE cores, previously reported to abolish the heat-shock response and to strongly reduce the embryogenesis response of the same promoter, did not reduce expression in giant cells. This suggests a distinct regulation of the promoter by nematodes. However, additional point mutations located at positions crucial for binding of heat-shock transcription factors (HSFs) caused a severe decrease in the nematode response. These results demonstrate that HSEs are involved in the promoter activation in giant cells and suggest that HSFs may mediate this response.

A seed-specific heat-shock transcription factor involved in developmental regulation during embryogenesis in sunflower.

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.

Selective activation of the developmentally regulated Ha hsp17.6 G1 promoter by heat stress transcription factors.

Using two well-characterized heat stress transcription factors (Hsfs) from tomato (Lycopersicon peruvianum; LpHsfA1 and LpHsfA2), we analyzed the transcriptional activation of the Ha hsp17.6 G1 promoter in sunflower (Helianthus annuus) embryos. In this system, we observed transient promoter activation only with LpHsfA2. In contrast, both factors were able to activate mutant versions of the promoter with improved consensus Hsf-binding sites. Exclusive activation by LpHsfA2 was also observed in yeast (Saccharomyces cerevisiae) without other Hsfs and with a minimal Cyc1 promoter fused to the Ha hsp17.6 G1 heat stress cis-element. Furthermore, the same promoter mutations reproduced the loss of activation selectivity, as observed in sunflower embryos. The results of in vitro binding experiments rule out differential DNA binding of the two factors as the explanation for the observed differential activation capacity. We conclude that the specific sequence of this heat stress cis-element is crucial for Hsf promoter selectivity, and that this selectivity could involve preferential transcriptional activation following DNA binding. In sunflower embryos, we also observed synergistic transcriptional activation by co-expression of LpHsfA1 and LpHsfA2. Mutational analyses of the Ha hsp17.6 G1 promoter, combined with in vitro binding assays, suggest that mixed oligomers of the two factors may be involved in promoter activation. We discuss the relevance of our observations for mechanisms of developmental regulation of plant heat stress protein genes.

Reversible heat-induced inactivation of chimeric beta-glucuronidase in transgenic plants.

We compared the expression patterns in transgenic tobacco (Nicotiana tabacum) of two chimeric genes: a translational fusion to beta-glucuronidase (GUS) and a transcriptional fusion, both with the same promoter and 5'-flanking sequences of Ha hsp17.7 G4, a small heat shock protein (sHSP) gene from sunflower (Helianthus annuus). We found that immediately after heat shock, the induced expression from the two fusions in seedlings was similar, considering chimeric mRNA or GUS protein accumulation. Surprisingly, we discovered that the chimeric GUS protein encoded by the translational fusion was mostly inactive in such conditions. We also found that this inactivation was fully reversible. Thus, after returning to control temperature, the GUS activity was fully recovered without substantial changes in GUS protein accumulation. In contrast, we did not find differences in the in vitro heat inactivation of the respective GUS proteins. Insolubilization of the chimeric GUS protein correlated with its inactivation, as indicated by immunoprecipitation analyses. The inclusion in another chimeric gene of the 21 amino-terminal amino acids from a different sHSP lead to a comparable reversible inactivation. That effect not only illustrates unexpected post-translational problems, but may also point to sequences involved in interactions specific to sHSPs and in vivo heat stress conditions.