Deciphering different layers of regulation of FLOWERING LOCUS D, a bZIP transcription factor that promotes flowering of Arabidopsis

ended

Floral transition denotes the initiation of plant reproduction. Several genetic pathways regulate this important transition and many of them converge on the regulation of FLOWERING LOCUS T (FT), the systemic flowering signal that moves from leaves to the shoot apex. FT reprograms transcription at the shoot apex by contributing to Florigen Activation Complex (FAC). A central component of this complex is the bZIP transcription factor FLOWERING LOCUS D (FD), which is responsible for DNA binding and gene specific regulation by the complex. I will employ single-cell RNA sequencing to capture the transient cell state where the FAC complex and two different target genes, APETALA1 and SOC1, are coexpressed. Defining the transcriptomic network of FD at the single-cell level will provide a deeper understanding of this dynamic process. Phosphorylation of a threonine residue in the SAP motif at the C terminus of FD is a key step in formation of the complex. Phosphomimetic or non-phosphorylatable FD SAP motif versions have opposite effects on FD function, being able or not, respectively, to complement the late flowering phenotype of fd-3. I will investigate whether SAP motif phosphorylation affects FD protein stability or impairs its protein-protein interactions using confocal microscopy and protein mass spectrometry. In addition, other bZIPs closely related to FD are phosphorylated at other sites, but no analysis of FD phosphorylation sites other than the SAP motif has been presented. I will map post-translational modifications of FD, which may reveal new regulatory steps in controlling FD activity. I will explore the ability of FD to form protein complexes with other transcription factors by employing yeast-two hybrid, protein pull-downs and interaction studies using immunoprecipitation-mass spectrometry and biotin-based proximity labeling. The identification of new FD protein interactors will start reverse genetic and biochemical approaches to better understand FD function.