Transcriptional Regulatory Logic of Valve Margin Patterning in Arabidopsis.

SEHRA, BHUPINDER. Transcriptional Regulatory Logic of Valve Margin Patterning in Arabidopsis (Under the direction of Robert G. Franks.) In flowering plants the gynoecium (seedpod) is critical for reproductive success. The mature seedpod protects the seeds until maturation and dehiscence (pod shatter) occurs allowing for seed dispersal. The valve margin (VM) undergoes a specific developmental program to form the dehiscence zone (DZ), longitudinal furrows in the walls of the seedpod containing lignified and cell separation layers, which are required for pod shatter. The MADS-domain containing protein paralogs SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) are essential for VM and DZ formation. This is evidenced by the loss of VM formation and the indehiscent phenotype of the shp1 shp2 double mutant seed pod. During VM development SHP2 expression within the lateral domains of the gynoecium is repressed by MADS protein FRUITFULL (FUL) expressed in the valve and BELL-like homeodomain protein REPLUMLESS (RPL) expressed in the replum. Phylogenetic analysis of the SHP2 promoter-enhancer highlights regions of high sequence similarity: a 1kb region and 700bp enhancer region. Detailed analysis indicates a previously identified 2.2kb region upstream from the SHP2 translation start site (TSS) provides early SHP2 reporter expression in the medial portion of the gynoecium, VM and DZ expression. A 1kb region upstream from the SHP2 TSS and a 700bp both provide VM and DZ expression highlighting two independent cis-regulatory modules that provide VM and DZ expression. Three closely positioned MADS proteins binding sites (CArG boxes) are located within the 1kb region. Functional analysis of these binding sites by mutagenesis in the context of the 2.2kb reporter results in a moderate de-repression of SHP2 within the valve domain, suggesting a role for these CArG boxes in SHP2 repression. Crossing this CArG boxdeficient reporter into a ful mutant background produces significant additional de-repression of the reporter. This action of FUL on the CArG-box deficient reporter suggests that either additional non-consensus CArG boxes can be used to mediate FUL repression of SHP2 or that the repression of SHP2 by FUL is in part CArG box independent. We have also identified a cluster of GA-rich regions (GAGA elements) within 1kb of the translation start site, which may be potential binding sites for BASIC PENTACYSTEINE PROTEINS (BPC), a family of transcription factors that have been shown to transcriptionally repress SEEDSTICK (STK), a paralog of SHP2. Mutagenesis of specific GAGA elements in the context of 2.2kb reporter produced no change in expression in planta when compared to an unmutagenized version of the reporter, suggesting redundant elements may function in place of the mutated GAGA elements. The 2.2kb and 1kb SHP2 reporters in bpc1-1 bpc2 bpc3-1 triple mutants gave consistently weak VM expression, unlike the expression observed in a WT background. The effects of loss of BPC activity on the SHP2 reporter expression suggests that additional regulatory mechanisms may be affecting SHP2 expression in the bpc1-1 bpc2 bpc3-1 mutant background. We also describe ongoing efforts to functionally analyze three regions of high sequence similarity identified from phylogenetic analysis of the SHP2 5’ regulatory region. © Copyright 2017 Bhupinder Sehra All Rights Reserved Transcriptional Regulatory Logic of Valve Margin Patterning in Arabidopsis