Investigating Partners of OsMADS1 Transcription Factor and functions for some associated factors for roles in rice inflorescence and floral development
Title: Investigating Partners of OsMADS1 Transcription Factor and functions for some associated factors for roles in rice inflorescence and floral development The classic ABCDE model for floral organ identity show that the combinatorial action of MADS domain transcription factors directs floral organ identity and their normal development. While floral MADS-box transcription factors are in general conserved among diverse flowering plants, several studies suggest emergence of distinct functions for some of these conserved factors. Rice E- or SEP-class genes, when compared to Arabidopsis SEP-class, are only partly conserved with rice homologous genes which show some distinct functional diversifications. Rice genes of this class form two clades: LOFSEP- (OsMADS1, OsMADS34, OsMADS5) and SEP3 (OsMADS7, OsMADS8) clades. Unlike Arabidopsis SEP-class genes that are redundant, just loss of function of OsMADS1 alone leads to severe defects in the development of all floral organs. Extensive molecular genetic studies, including from our laboratory, showed OsMADS1 is a master transcription regulator for floral meristem determinacy, floral organ specification and identity. The gene regulatory network controlled by OsMADS1 including other transcription factor targets, and phytohormone auxin and cytokinin pathways. Here we probed for the interacting partners of OsMADS1 to get a better understanding of its function in higher protein complexes during the broad program of floral development. In addition, we took up functional genomic studies of OsMADS1 associated factors for studies on roles for downstream factors controlling rice inflorescence and floral development. Part I: Investigating interacting partners of OsMADS1 transcription factor While interactions between MADS transcription factors are well described, interactions with other classes of factors that can influence its regulatory functions in higher protein complexes are not studied yet. We adopted two methods to identify OsMADS1 interacting factors. First, we predicted interactors of OsMADS1 based on the previous report from our group that identified the co-occurrence of cis DNA binding motifs in the genome-wide OsMADS1ChIP-Seq dataset (Khanday et al., 2016). This was combined with co-expression analysis of transcription factors that could bind these cis-elements based using publicly available rice transcriptome databases. A small set of putative deduced from the above approaches were examined by yeast-two hybrid (Y2H) assays and the interactions noted were further validated by the in-planta Bimolecular fluorescence complementation (BiFC) assay. Y2H assay suggests OsbZIP47 and ERF68 are strong interactors of OsMADS1. Further, we note RFL as a weak interactor, whereas the eudicot Arabidopsis homologs LEAFY and SEP3 have a strong association. In the second approach, we carried out pulldown of endogenous OsMADS1 associated protein complexes from maturing 3-5cm inflorescence tissue lysates using OsMADS1specific antibodies. By mass spectroscopy analysis we identify several classes of proteins that demonstrate association of OsMADS1 function with transcriptional corepressors, meristem transition regulators of floral meristem determinacy, organ identity and also indicate interactions with factors in auxin and Brassinosteroid signaling pathways. Part II: Functional studies on the predicted meristem regulator OsbZIP47, a downstream target and a partner of OsMADS1 OsbZIP47 is a predicted rice homolog for the Arabidopsis and maize factors, PERIANTHIA (PAN) and FASCIATED EAR4 (FEA4) respectively that regulate meristem and floral organs in those model species. Here we aimed to understand functional relevance and pathways controlled by OsbZIP47 for normal rice inflorescence development. Transgenic plants with RNAi based knockdown of OsbZIP47 were raised and they showed pleiotropic effects on both vegetative and reproductive phases of growth including meristem and floral organ defects. Whereas transgenics with overexpression (Ox) of OsbZIP47 did not show any phenotypic defects. Importantly, similar to the PAN protein, we observe that oligomerization of the OsbZIP47 protein is sensitive to redox reagents (diamide, S-glutathionylation modifier and DTT). We also detect robust transcriptional activity for a reporter gene in yeast despite the fact that OsbZIP47 lacks the extended N-ter protein domain seen in PAN which is essential for PAN functions in Arabidopsis. We also show that OsMADS1 can positively regulate expression levels of OsbZIP47 and that of the gene encoding its potential glutaredoxin enzyme, OsGRX19 or MICROSPORELESS1 (OsMIL1). This co-ordinated effect may be to maintain homeostasis and enforce their interaction. To reveal OsbZIP47 downstream pathways, global transcriptome profiling (RNA-Seq) of 0.1-0.5cm panicle tissues from OsbZIP47 KD line was compared with similarly staged wild-type tissues and the data suggest ~2800 deregulated genes. Most important inference is that OsbZIP47 regulates meristem characteristics by controlling the canonical WUS-CLV pathway for stem cell homeostasis and the cytokinin-mediated WUS-KNOX (OSH1/STM/KN1) pathway. Floral defects (particularly of stamens and lodicules) seen in OsbZIP47 KD lines can to an extent be explained by the deregulation of OsMADS16 (B-class gene), OsDL (DROOPING LEAF), APO1, OsMADS68, TGA10, Osnop (Oryza sativa no pollen) that are known regulators for proper development of these organs. Taking leads from Arabidopsis and Maize reports we also tested interaction of OsbZIP47 with auxin response factors ETTIN1 and ETTIN 2, and with meristem regulators, KNOXI-OSH1, RFL using the Y2H assay. We report strong interaction of OsbZIP47 with OSH1 and RFL which further add a layer of complexity in molecular mechanisms by which OsbZIP47 can contribute to meristem characteristics. Part III: B-class OsMADS2 gene, a partner and a downstream factor of OsMADS1 controls gene expression for normal lodicule and stamen development In rice, and all grass florets, lodicules are modified organs equivalent to petal and they have a crucial role for efficient fertilization. Rice floret stamens have functions and structure with greater similarity to their eudicot counterparts. OsMADS1 regulates the normal development and identity of all floral organs; its partnership with B-class factors AP3-like OsMADS16, PI-like OsMADS2, and OsMADS4 is known and is critical for lodicule and stamen developmental control. Noteworthy is that B-class genes are expressed in the lodicule and stamen floral primordia but OsMADS1 transcripts are not detected in these primordia. Prior work from our group showed that OsMADS2, a PI-like factor has evolved to have a greater role in lodicule identity and lesser redundant role for stamen identity. Here, using immunolocalization we detect the presence of OsMADS1 protein in lodicule and stamen primordia, hinting at possibility of either intercellular trafficking or other non-cell-autonomous manner of OsMADS1 action in lodicule and stamen primordia. Further, to delineate gene sets that are coregulated by OsMADS1 and OsMADS2 we determined OsMADS2 genome-wide occupancy by ChIP-Seq using 0.3-2cm panicle tissues and OsMADS2 affinity-purified antibodies. After comparing data acquired in this study with the OsMADS1ChIP-Seq dataset (Khanday et al., 2016) we identify a common set of 280 gene targets. Also, OsMADS2 ChIP-Seq shows that homologs of Arabidopsis AP1 and AP2 class genes are enriched. Thus, as known in Arabidopsis the latter factors can explain OsMADS2 functions in lodicule development. Interestingly, OsMADS2 ChIP-PCR and qRT-PCR transcript measurements in transgenic with partial knockdown of OsMADS2 show that OsMADS1 and OsMADS2 are linked by a positive feedback loop. We confirm HECATE/OsbHLH120 as a common target of OsMADS1 (Dr. Grace Lhainekim Thesis, UVR lab) and OsMADS2 (this study). The homolog of OsbHLH120 in Arabidopsis is a meristem and carpel development regulator. Here we perform functional characterization of OsbHLH120 by RNA interference-mediated (dsRNAi) knockdown (KD). The transgenics had majority of their florets with elongated and deformed lodicules, and a minority showed increased stigma/pistil number. The spikelet organ called the sterile lemma was elongated resembling a lemma-like organ. These phenotypes suggest OsbHLH120 regulates spikelet and floral organ development.