Seed-specific expression patterns and regulation by ABI3 of an unusual late embryogenesis-abundant gene in sunflower.

We cloned the genomic sequences that correspond to a previously described group 1 late embryogenesis-abundant (Lea) cDNA from sunflower: Ha ds10. The Ha ds10 G1 gene had structural and gene-expression features that depart from those of other group 1 Lea genes. An intron was present at a conserved position but showed a much larger size (1024 bp). Transcription from the Ha ds10 G1 promoter was strictly seed-specific and it originated from at least two close initiation sites. The mRNAs accumulated from stages of embryogenesis that preceded seed desiccation. Ha ds10 G1 mRNA accumulation was moderately induced, by exogenous abscisic acid treatments, in immature seeds but not induced in seedlings. We observed unprecedented changes in Lea mRNA localization associated with seed desiccation: the homogeneous tissue distribution of Ha ds10 G1 mRNAs, which was characteristic of immature embryos, evolved later in embryogenesis to an asymmetric distribution within the cotyledons, with preferential mRNA accumulation in the cells of the palisade parenchyma and provascular bundles. We also showed that, in sunflower embryos, the Ha ds10 G1 promoter could be transiently activated by the Arabidopsis ABI3 transcription factor. We discuss the significance of these results regarding hypotheses of regulation and function of plant genes from the same family.

Dual regulation of a heat shock promoter during embryogenesis: stage-dependent role of heat shock elements.

Transgenic 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.