Functional Characterization of a SAND–Domain–containing Factor OsULTRAPETALA1 – a Direct Downstream Target of OsMADS1

Abstract

Studies on meristem identity regulators in Rice (Oryza sativa), a model plant for cereal crops have revealed how meristem identity and transitions are controlled, bearing implications for crop yield improvement. We were interested in exploring the function of OsULTRAPETALA1 (OsULT1), whose Arabidopsis homolog is a TrxG Factor, in rice inflorescence and spikelet/floret organ development. OsULT1 is a direct downstream target of floret meristem identity and development transcriptional regulator OsMADS1 (Khanday et al., 2016). The aim of this thesis is to functionally characterize OsULT1 by raising transgenic rice plants with ubiquitous knockdown and other lines that are overexpressors of OsULT1. In the dsRNAi-OsULT1 knockdown transgenics, we observed reduced plant height, panicle branching, and delay in flowering time. Interestingly, the pUbi-OsULT1 overexpression transgenics showed a converse phenotype of precocious flowering. Histological studies on young branching inflorescence meristem from the dsRNAi-OsULT1 knockdown transgenics and wild-type plants, was done to understand the onset of developmental abnormalities during panicle development. We observed an increase in the number of lateral organs made from the spikelet meristem; this could be due to a delay in SM to FM transition. These data suggest OsULT1 to be a heterochronic factor regulating meristem progression. Histological analyses of young spikelets showed a homeotic conversion of sterile lemma to a lemma-like organ. In floral meristem of knockdown plants, reduced palea and altered stamen number were noted. We quantified expression levels of some selected well-studied spikelet meristem regulators. qRT-PCR done on RNA from pooled dsRNAi-OsULT1 panicle tissues (Early-stage: 0.1 to 0.3cm and Late-stage: 0.4 to 2cm) and compared to similarly staged wild-type panicles. We found OsMADS1 and OsIDS1 transcript levels to be up-regulated and down-regulated respectively in the dsRNAi-OsULT1 transgenics compared to the wild-type. This could relate to the sterile lemma and rudimentary glume phenotypes observed in the affected knockdown spikelets. Since chromatin modifiers lacking DNA-binding domain recruit TF’s to target genes, we tested the possibility of protein interaction between OsULT1 and OsMADS1 using the yeast two-hybrid assay. This assay confirmed the interaction between OsULT1 and OsMADS1, thus raising the prospect of such a TF–Chromatin factor complex regulating downstream target gene expression by modulation of the histone modification status of the gene loci relevant for SM and FM development. We surveyed the abundance of repressive and activating histone marks in two developmental stages in wild-type panicle tissues as an attempt to correlate histone marks with transcript abundance. We observed as expected an inverse correlation between the expression levels of OsMADS34 and OsMADS22 and the abundance of the H3K27me3 mark at these two loci. We then evaluated the chromatin status at the genes which are differentially expressed in the dsRNAi-OsULT transgenics like OsMADS1, OsIG1, and OsIDS1. The results of the ChIP-qPCR analysis to assess the abundance of histone marks indicate a complex relationship between chromatin marks and transcript abundance. For future in-depth studies of gene targets in specific stages of SM and FM, we have standardized Laser Capture Micro-dissection of specific rice wild-type panicle meristems for transcriptomic studies in these recessed difficult to access tissues. Preliminary data indicate sets of transcripts that could be specific to Primary Branch Meristem (PBM), Secondary Branch Meristem (SBM), and Floral Meristem (FM). Overall, we have used reverse genetics tools to elucidate the functions of a predicted Trithorax-Group factor OsULT1 in spikelet meristem transient maintenance, its lateral organ development, and effects on floret organ numbers. The implications of the studies support the published hypothesis that ancestral rice species had three-fertile floret per spikelet (Ren et al., 2020; Zhang et al., 2017), instead of the one floret per spikelet in seen in extant species. The work in this thesis highlights the important role of chromatin modifiers like the Trithorax factors in rice panicle development.