A shared cis-regulatory module activates transcription in the suspensor of plant embryos

The mechanisms controlling the transcription of gene sets in specific regions of a plant embryo shortly after fertilization remain unknown. Previously, we showed that G564 mRNA, encoding a protein of unknown function, accumulates to high levels in the giant suspensor of both Scarlet Runner Bean (SRB) and Common Bean embryos, and a cis-regulatory module containing three unique DNA sequences, designated as the 10-bp, Region 2, and Fifth motifs, is required for G564 suspensor-specific transcription [Henry, K. F. et al., Plant Mol. Biol. 88(3):207-217 (2015); Kawashima, T. et al., Proc. Natl. Acad. Sci USA 106(9):3627-3632 (2009)]. We tested the hypothesis that these motifs are also required for transcription of the SRB GA 20-oxidase gene, which encodes a gibberellic acid hormone biosynthesis enzyme and is co-expressed with G564 at a high level in giant bean suspensors. We used deletion and gain-of-function experiments in transgenic tobacco embryos to show that two GA 20-oxidase DNA regions are required for suspensor-specific transcription - one in the 5 untranslated region (UTR) (+119 to +205) and another in the 5 upstream region (-341 to -316). Mutagenesis of sequences in these two regions determined that the cis-regulatory motifs required for G564 suspensor transcription are also required for GA 20-oxidase transcription within the suspensor, although the motif arrangement differs. Our results demonstrate the flexibility of motif positioning within a cis-regulatory module that activates gene transcription within giant bean suspensors, and suggest that G564 and GA 20-oxidase comprise part of a suspensor gene regulatory network.\n\nSignificanceLittle is known about how genes are expressed in different plant embryo regions. We tested the hypothesis that shared cis-regulatory motifs control the transcription of genes specifically in the suspensor. We carried out functional studies with the Scarlet Runner Bean (SRB) GA 20-oxidase gene that encodes a gibberellic acid (GA) hormone biosynthesis enzyme, and is expressed specifically within the suspensor. We show that cis-regulatory motifs required for GA 20-oxidase transcription within the suspensor are the same as those required for suspensor-specific transcription of the SRB G564 gene, although motif number, spacing and order differ. These cis-elements constitute a control module that is required to activate genes in the SRB suspensor and may form part of a suspensor regulatory network.