Redundant CArG Box Cis-motif Activity Mediates SHATTERPROOF2 Transcriptional Regulation during Arabidopsis thaliana Gynoecium Development.

Bhupinder Sehra,Robert G Franks

doi:10.3389/fpls.2017.01712

Abstract

In the Arabidopsis thaliana seed pod, pod shatter and seed dispersal properties are in part determined by the development of a longitudinally orientated dehiscence zone (DZ) that derives from cells of the gynoecial valve margin (VM). Transcriptional regulation of the MADS protein encoding transcription factors genes SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) are critical for proper VM identity specification and later on for DZ development. Current models of SHP1 and SHP2 regulation indicate that the transcription factors FRUITFULL (FUL) and REPLUMLESS (RPL) repress these SHP genes in the developing valve and replum domains, respectively. Thus the expression of the SHP genes is restricted to the VM. FUL encodes a MADS-box containing transcription factor that is predicted to act through CArG-box containing cis-regulatory motifs. Here we delimit functional modules within the SHP2 cis-regulatory region and examine the functional importance of CArG box motifs within these regulatory regions. We have characterized a 2.2kb region upstream of the SHP2 translation start site that drives early and late medial domain expression in the gynoecium, as well as expression within the VM and DZ. We identified two separable, independent cis-regulatory modules, a 1kb promoter region and a 700bp enhancer region, that are capable of giving VM and DZ expression. Our results argue for multiple independent cis-regulatory modules that support SHP2 expression during VM development and may contribute to the robustness of SHP2 expression in this tissue. Additionally, three closely positioned CArG box motifs located in the SHP2 upstream regulatory region were mutated in the context of the 2.2kb reporter construct. Mutating simultaneously all three CArG boxes caused a moderate de-repression of the SHP2 reporter that was detected within the valve domain, suggesting that these CArG boxes are involved in SHP2 repression in the valve.

Highlights

Dehiscence in plants is a process that involves controlled developmental programs that result in the formation of specialized tissues to aid cell separation (Spence et al, 1996; Dong and Wang, 2015)
In this study we demonstrate a functional role of CArG boxes within the SHP2 cis-regulatory regions for repression of SHP2 promoter activity in the valves
A 2.2kb region covering −2168 to +1 relative to the SHP2 translation start site was shown to be sufficient for expression of a reporter gene within the valve margins (VM) and the early medial domain (Figure 2 – region A; Roeder et al, 2003; Larsson et al, 2014; Villarino et al, 2016)

Summary

Introduction

Dehiscence in plants is a process that involves controlled developmental programs that result in the formation of specialized tissues to aid cell separation (Spence et al, 1996; Dong and Wang, 2015). In the dry fruits of the Brassicaceae family, including Canola (Brassica napus) and the model plant Arabidopsis thaliana, dehiscence zones (DZs) in the seedpod form to facilitate seed dispersal through pod shatter. The mature Arabidopsis seedpod, or silique, is mainly comprised of the ovary that contains the seeds (Figure 1) (Sessions and Zambryski, 1995, 1997; Ferrándiz et al, 1999). The VM undergoes a specific developmental program to later form the DZ and is critical for dehiscence and seed dispersal

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Results

Conclusion