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An important question is what genes govern the differentiation of plant embryos into suspensor and embryo proper regions following fertilization and division of the zygote. We compared embryo proper and suspensor transcriptomes of four plants that vary in embryo morphology within the suspensor region. We determined that genes encoding enzymes in several metabolic pathways leading to the formation of hormones, such as gibberellic acid, and other metabolites are up-regulated in giant scarlet runner bean and common bean suspensors. Genes involved in transport and Golgi body organization are up-regulated within the suspensors of these plants as well, strengthening the view that giant specialized suspensors serve as a hormone factory and a conduit for transferring substances to the developing embryo proper. By contrast, genes controlling transcriptional regulation, development, and cell division are up-regulated primarily within the embryo proper. Transcriptomes from less specialized soybean and Arabidopsis suspensors demonstrated that fewer genes encoding metabolic enzymes and hormones are up-regulated. Genes active in the embryo proper, however, are functionally similar to those active in scarlet runner bean and common bean embryo proper regions. We uncovered a set of suspensor- and embryo proper-specific transcription factors (TFs) that are shared by all embryos irrespective of morphology, suggesting that they are involved in early differentiation processes common to all plants. Chromatin immunoprecipitation sequencing (ChIP-Seq) experiments with scarlet runner bean and soybean WOX9, an up-regulated suspensor TF, gained entry into a regulatory network important for suspensor development irrespective of morphology.

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.

An important question is what genes govern the differentiation of plant embryos into suspensor and embryo proper regions following fertilization and division of the zygote. We compared embryo proper and suspensor transcriptomes of four plants that vary in embryo morphology within the suspensor region. We determined that genes encoding enzymes in several metabolic pathways leading to the formation of hormones, such as gibberellic acid, and other metabolites are up-regulated in giant scarlet runner bean and common bean suspensors. Genes involved in transport and Golgi body organization are up-regulated within the suspensors of these plants as well, strengthening the view that giant specialized suspensors serve as a hormone factory and a conduit for transferring substances to the developing embryo proper. By contrast, genes controlling transcriptional regulation, development, and cell division are up-regulated primarily within the embryo proper. Transcriptomes from less specialized soybean and Arabidopsis suspensors demonstrated that fewer genes encoding metabolic enzymes and hormones are up-regulated. Genes active in the embryo proper, however, are functionally similar to those active in scarlet runner bean and common bean embryo proper regions. We uncovered a set of suspensor- and embryo proper-specific transcription factors (TFs) that are shared by all embryos irrespective of morphology, suggesting that they are involved in early differentiation processes common to all plants. Chromatin immunoprecipitation sequencing (ChIP-Seq) experiments with scarlet runner bean and soybean WOX9, an up-regulated suspensor TF, gained entry into a regulatory network important for suspensor development irrespective of morphology. [ABSTRACT FROM AUTHOR]

Pollination initiates a suite of postpollination developmental events in orchid flowers. Ethylene plays a key role in coordinating these developmental changes. We examined the temporal-spatial patterns of expression of genes encoding 1-aminocyclopropane 1-carboxylate acid (ACC) synthase and ACC oxidase following pollination and associated hormonal treatments using quantitative procedures. We present the first in situ hybridization analysis of ACC synthase and an earlier time course of ACC oxidase gene expression. In immature ovaries, critical morphological changes involving cell differentiation and growth were affected by pollination and various hormone and inhibitor treatments. Patterns of ACC synthase and ACC oxidase mRNA accumulation were determined by RNA blot hybridization analysis. Gene expression analysis using real-time quantitative polymerase chain reaction indicated that the unpollinated ovary had very low levels of ACC oxidase expression at 6 h and that pollination caused near-maximal levels in this organ by 12 h. Quantitative mRNA analysis showed that auxin could mimic pollination by inducing a similar pattern of organ-specific expression, but earlier and to higher levels. Ethylene had the most dramatic effect by inducing gene activity to its highest levels at all time points and in all floral organs. The early induction in situ hybridization analyses contribute new findings that ACC synthase and ACC oxidase transcripts were localized sequentially in epidermal, phloem, and parenchyma cells of the stigma and that by 8-12 h postpollination, saturation had occurred throughout the stigma. Finally, phylogenetic analysis of orchid ACC synthase proteins leads to the conclusion that at least a subgroup of the angiosperm ACC synthase gene family is induced by auxin but may not be posttranscriptionally regulated, indicating regulatory sequence but not functional divergence of orchid genes with other seed plant ACC synthase proteins. [ABSTRACT FROM AUTHOR]

The giant embryo of the scarlet runner bean (Phaseolus coccineus) has been used historically to investigate the molecular and developmental processes that control the early events of plant embryo development. In more recent years, our laboratory has been using scarlet runner bean embryos to uncover the genes and regulatory events that control embryo proper and suspensor region differentiation shortly after fertilization. In this chapter we describe methods that we have developed to isolate scarlet runner bean embryos at the globular stage of development, and capture embryo proper and suspensor regions by either hand dissection or laser capture microdissection (LCM) for use in downstream genomic analysis. These methods are also applicable for use in investigating the early events of common bean (Phaseolus vulgaris) embryo development, a close relative of scarlet runner bean, which also has a giant embryo in addition to a sequenced genome.