Kelli F. Henry, Anh thu Q. Bui, Tomokazu Kawashima, and Robert B. Goldberg

PNAS June 19, 2018. 115 (25) E5824-E5833

Significance                                

Little 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.

Abstract

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 KF, et al. (2015) Plant Mol Biol 88:207–217; Kawashima T, et al. (2009) Proc Natl Acad Sci USA 106:3627–3632]. 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 coexpressed 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′ 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.

See http://www.pnas.org/content/115/25/E5824

Figure 1: Localization of GA biosynthesis pathway enzyme mRNAs in SRB and Common Bean embryos. (A) SRB flower. (B) Plastic section of SRB globular-stage embryo. (C) Common Bean flower. (D) Paraffin section of Common Bean globular-stage embryo. (E) Localization of G564 mRNA in a SRB globular-stage embryo. (F–K) In situ localization of GA biosynthesis enzyme mRNAs in globular-stage SRB seeds: ent-kaurene synthase A (F), ent-kaurene synthase B (G), ent-kaurene oxidase (H), ent-kaurenoic acid hydroxylase (I), GA 20-oxidase (J), and GA 3-oxidase (K). Photographs were taken using dark-field microscopy. (L–Q) Genome browser views of RNA-seq coverage of ent-kaurene synthase A (L), ent-kaurene synthase B (M), ent-kaurene oxidase (N), ent-kaurenoic acid hydroxylase (O), GA 20-oxidase (P), and GA 3-oxidase (Q) in SRB and Common Bean globular-stage suspensor and embryo proper regions. RNA-seq data were taken from GEO accession no. GSE57537. Numbers indicate average reads per kilobase per million of two biological replicates. Each panel depicts an 8-kb window including the gene structure. Black boxes represent exons. Black lines represent UTRs and introns. Arrows indicate the transcription start site. bc, basal cells; ep, embryo proper; h, hypophysis region; Pc, P. coccineus; Pv, P. vulgaris; s, suspensor. (Scale bars: 50 μm.) The images in B and D are reproduced from ref. 10. The image in E is reproduced from ref. 12. Copyright American Society of Plant Biologists, www.plantcell.org.