Role of an ABI3 homologue in dormancy maintenance of yellow-cedar seeds and in the activation of storage protein and Em gene promoters

ABI3/VP1 proteins are members of a large group of transcription factors that act as intermediaries in regulating abscisic acid (ABA)-responsive genes during seed development, including those involved in reserve deposition, acquisition of desiccation tolerance and dormancy induction. CnABI3, an ABI3/VP1 gene homologue was recently cloned from yellow cedar, a conifer species that produces seeds that are deeply dormant at maturity. Here, we investigated whether the conifer ABI3/VP1 gene homologue shares characteristics with its angiosperm counterparts. CnABI3 was synthesized exclusively in seeds, with no detectable protein in leaves and roots. Stable expression of the CnABI3 gene in two transgenic tobacco lines previously transformed with chimeric constructs (vicilin and napin 5 regions linked to a beta-glucuronidase (GUS) gene-coding region) showed that the ectopic expression of the CnABI3 protein strongly activated both the vicilin and napin storage protein gene promoters in leaves and other vegetative tissues. GUS activities were up to more than 1000-fold of those in control plants. ABA had a synergistic effect, further enhancing GUS activity levels. When expressed transiently in yellow-cedar embryos, CnABI3 activated the expression of a chimeric Em-GUS gene in the presence of ABA. The role of CnABI3 in dormancy maintenance of yellow-cedar seeds was examined by monitoring the expression of the CnABI3 gene at the mRNA and protein levels before, during and after dormancy termination. CnABI3 protein was present in the megagametophyte and embryo of dormant mature and warm stratified seed, but declined during subsequent moist chilling, a treatment effective in breaking dormancy. In contrast, the protein was preserved (albeit in lower amounts) in seeds subjected to a control treatment (12 weeks in warm, moist conditions) that is ineffective in breaking dormancy. A decline in CnABI3 gene transcripts was also positively correlated with dormancy breakage, but did not occur during moist chilling itself, but rather during subsequent germination, indicating potential control at the post-transcriptional level.