Studies on Molecular Targets and Pathways Regulated by Rice RFL for Flowering Transition and Panicle Development
LFY of Arabidopsis is a member of a unique plant specific transcription factor family. It is involved in giving meristem a determinate floral fate by the activation of floral organ identity genes and preventing inflorescence meristem identity. RFL is a homolog of FLO/LFY in rice. Studies from our lab on rice RFL, based on the effects of knockdown or overexpression, showed its major functions are in timing the conversion of SAM to IM and to prevent the premature conversion of branch meristem to spikelets. Additionally roles in vegetative axillary meristem specification have been also been identified in laboratory. Here, we attempt to delineate molecular pathways directly regulated by RFL as a transcription factor controlling inflorescence and floral development in rice. Part I: Identification of global target genes bound by RFL in developing rice inflorescences We carried out ChIP sequencing of the DNA bound by RFL in panicles (01.-0.3cm stage) using anti-RFL antibody. DNA sequences in one library pool were analyses by the MACS algorithm (FDR<0.01), to find 8000 binding sites while the SPP algorithm identified 5000 enriched peaks. These mapped to 2500 or 2800 gene-associated loci respectively, 617 of which were common loci to both pipelines. Several RFL bound gene loci were homologs of Arabidopsis thaliana LFY gene targets. Such gene targets underscore conserved downstream targets for LFY-proteins in evolutionarily very distinct species. AtLFY is known to bind variants of CCANT/G cis element classified as primary, inflorescence or seedling type. We scanned for these three types of cis elements at 123 RFL bound genes with likely functions in flowering. For a few of these 123 rice loci we find one of these cis motifs (p-value<0.001) in RFL bound ChIP-seq data. To validate these targets of RFL, we adopted in vitro DNA-protein binding assays with bacterially purified RFL protein. We confirm RFL target interactions with some genes implicated in flowering time, others in photoperiod triggered flowering, circadian rhythm, gibberellin hormone pathway, inflorescence development and branching. The in vitro experiments hint different RFL-DNA binding properties as compared to Arabidopsis LFY. We report binding to sequences at rice gene loci that are unique targets. Part II: Pathways regulated by RFL for reproductive transition and panicle development To co-relate DNA binding of RFL to target loci with changes in their gene expression, expression studies were taken up for selected set of genes implicated in rice flowering transition and panicle architecture. To study in planta and tissue specific gene regulation by RFL we raised RFL dsRNAi transgenics. Comparative transcript analysis in these RFL partial knockdown lines and matched wild type tissues reveal that RFL is an activator for some genes and repressor for other gene targets. We also examined if the gene expression effects of RFL knockdown can be reversed by induced complementation with an RFL-GR protein. We raised transgenics plants with a T-DNA ubi:RFL-GR, 35S CaMV:amiR RFL for these experiments. In planta target gene transcript levels were assessed in various conditions conditions. These studies validate rice RFL as an activator of some panicle architecture genes. Part III: Analysis of endogenous RFL protein in WT rice tissues Studies in Arabidopsis and in petunia with LFY and AFL, respectively, implicate these some abnormal mobility as compared to their predicted molecular weight when overexpressed. We studied endogenous RFL protein abundance in planta, adopting western analysis with anti-RFL antibody. We consistently identify two prominent cross reacting bands in different tissues which can be also be pulled-down from whole nuclear extracts of panicle and axillary meristem tissues. We speculate on likely modifications and possible functions for the same.