Functional analysis of genes support roles for and as master regulators of sex determination.

The Salicaceae family is of growing interest in the study of dioecy in plants because the sex determination region (SDR) has been shown to be highly dynamic, with differing locations and heterogametic systems between species. Without the ability to transform and regenerate in tissue culture, previous studies investigating the mechanisms regulating sex in the genus have been limited to genome resequencing and differential gene expression, which are mostly descriptive in nature, and functional validation of candidate sex determination genes has not yet been conducted. Here, we used Arabidopsis to functionally characterize a suite of previously identified candidate genes involved in sex determination and sex dimorphism in the bioenergy shrub willow . Six candidate master regulator genes for sex determination were heterologously expressed in Arabidopsis, followed by floral proteome analysis. In addition, 11 transcription factors with predicted roles in mediating sex dimorphism downstream of the SDR were tested using DAP-Seq in both male and female DNA. The results of this study provide further evidence to support models for the roles of and as master regulator genes of sex determination in , contributing to a regulatory system that is notably different from that of its sister genus . Evidence was also obtained for the roles of two transcription factors, an / family gene and a homeodomain-like transcription factor, in downstream regulation of sex dimorphism.