Arabidopsis class I small heat shock proteins: Regulation and functional analysis during seed development

The goal of this dissertation was to analyze the regulation and function of cytoplasmic class I small heat shock proteins (sHSPs) during seed development in Arabidopsis thaliana. Results show that two class I sHSPs accumulate in late seed maturation, persist in the dry seed and decline rapidly during germination. HSP17.4 accounts for 90% of total class I sHSP in the dry seed. The temporal pattern of sHSP accumulation during seed development suggests that HSP17.4 may help establish seed properties that are acquired during late seed maturation, such as dormancy or desiccation tolerance. Several mutants with reduced seed dormancy were determined to accumulate wild type levels of HSP17.4, however, all desiccation intolerant seeds analyzed had decreased levels of HSP17.4. Thus, HSP17.4 reduction correlates with desiccation intolerance. In total, these data suggest that HSP17.4 is not sufficient for seed dormancy and that it may be necessary for desiccation tolerance. The localization and regulation of HSP17.4 were examined in developing Arabidopsis seeds by transforming plants with hsp17.4 promoter fused to the β-glucuronidase (GUS) gene. HSP17.4::GUS expression was detected in the cotyledons early in seed development and eventually throughout the embryo. Arabidopsis embryos showed a much different pattern of HSP17.4::GUS expression in response to heat indicating distinct mechanisms regulate sHSP transcription during heat shock and during development. To analyze seed specific transcriptional activator regulation of HSP17.4 transcription, HSP17.4::GUS transgenic plants were crossed to seed transcriptional activator mutants. Results showed aberrant localization of HSP17.4::GUS in fus3-3 and lec1-2 seeds and negligible levels in abi3-6. These results strongly implicate AB13 in the transcriptional regulation of HSP17.4. To analyze more specifically HSP17.4 function, transgenic antisense technology was used to suppress hsp17.4 expression to 30--50% of wild type. These lines exhibited a reduced dormancy phenotype as assayed by reduced sensitivity to germination on ABA and by the ability of fresh seed to germinate. These data provide insight into the localization, regulation and function of HSP17.4 during seed maturation. The seed-specific transcriptional activator ABI3 is implicated in controlling hsp17.4 expression during development. Overall, these results demonstrate the importance of HSP17.4 during seed maturation, and establish a role for sHSPs in dormancy