Molecular biology of flower development in Viola pubescens, a species with the chasmogamous-cleistogamous mixed breeding system

Plants with both open, out-crossing chasmogamous and closed, self-pollinating cleistogamous flowers are distributed over 50 angiosperm families. The chasmogamous-cleistogamous mixed breeding system is a successful reproductive strategy because it can ensure seed output over a range of environmental and pollinator conditions and maintain sexually produced progeny even in the absence of pollination agents. Many ecologists and biologists have conducted research on the mixed breeding system, but the underlying molecular mechanisms remain largely unknown. The research presented here examined the molecular biology of flower development in a widespread North American violet, Viola pubescens. The plant growth regulator gibberellic acid (GA) was reported to induce chasmogamous flowers from cleistogamous ones in some species. Two genes, VGA20ox and VGA3ox, which encode key enzymes (GA20 oxidase and GA3 oxidase) in the biosynthesis of GA, were identified by reverse transcriptase polymerase chain reactions (RT-PCR) from V. pubescens. Semi-quantitative RT-PCR indicated that both genes exhibited increased expression in chasmogamous flowers compared to cleistogamous flowers. Exogenous application of GA3, however, did not induce cleistogamous flowers to open. Thus, GA is involved in the mixed breeding system, but it is not sufficient to convert the closed status of cleistogamous flowers in V. pubescens. GA was demonstrated to target the floral meristem identity gene LEAFY ( LFY) in Arabidopsis. Two LFY orthologs (VLFY1 and VLFY2) were obtained by screening a genomic library of V. pubescens. Semi-quantitative RT-PCR suggested the two LFY orthologs function together in the early stages of development of both flower types. Analysis of their promoters indicated they might be differentially regulated. The ABCE floral organ identity genes come into play once the floral meristems are determined. Two A class genes (VAP1 and VAP2), two B class genes (VAP3 and VPI), one C class gene ( VAG) and one E class gene (VSEP3) were identified by RT-PCR. The differential expression patterns of the ABCE floral genes between the chasmogamous and cleistogamous flowers were consistent with their morphological differences. Taken together, most of the floral genes examined here in V. pubescens were expressed differently between the two flower types, which might be a response to the different environmental conditions under which the two flower types were produced, contributing to the production of both chasmogamous and cleistogamous flowers.