Gibberellins are required for dimorphic flower development in Viola philippica.

Photoperiod is a major determinant of chasmogamous (CH)-cleistogamous (CL) dimorphic flower development in Viola philippica, and only long-day (LD) conditions induce CL flowers. In this study, it was found that the active gibberellin (GA) content in CL floral buds was higher than in CH floral buds formed under short-day (SD) conditions, suggesting that the biosynthesis of active GAs is enhanced by a longer photoperiod and may be associated with dimorphic flower development. Thus, the next step was to molecularly characterize the key V. philippica GA synthesis genes GA 20-oxidase (VpGA20ox) and GA 3-oxidase (VpGA3ox). In terms of the expression of VpGA20ox and VpGA3ox, it was found that the active GAs could be upregulated in developing pistils under both LD and SD conditions to develop functional pistils, and GAs could also accumulate in the stamens under SD conditions. The anthers and the adjacent petals were well developed under SD conditions. In contrast, the above-mentioned floral organs displayed low GA contents under LD conditions and were poorly developed. Although the application of paclobutrazol, an inhibitor of GA synthesis, did not reverse CL development under LD conditions, exogenous GAs could partially trigger the transition from CH to CL flowers under relative SD conditions (≤12 h daylight). This was coupled with the downregulation of B-class MADS-box genes, thereby restraining stamen and petal development. Both VpGA20ox and VpGA3ox exhibited similar expression profiles with B-class MADS-box genes in the development of the stamens and petals. Therefore, in response to photoperiod, GA signaling could affect the expression of B-class homeotic genes and regulate dimorphic flower development in Viola. As a compensation for poorly-developed nectaries, anthers, and petals, filament elongation, style shortness, and inward bending could ensure self-pollination in CL flowers. This work provides new insights into the regulation of CH-CL floral development and the evolutionary significance of the formation of dimorphic flowers.