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.

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BackgroundWe 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.ResultsHere 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.ConclusionsOur 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.