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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 LEAFY COTYLEDON1 (LEC1) transcription factor is a central regulator of seed development, because it controls diverse biological programs during seed development, such as embryo morphogenesis, photosynthesis, and seed maturation. To understand how LEC1 regulates different gene sets during development, we explored the possibility that LEC1 acts in combination with other transcription factors. We identified and compared genes that are directly transcriptionally regulated by ABA-RESPONSIVE ELEMENT BINDING PROTEIN3 (AREB3), BASIC LEUCINE ZIPPER67 (bZIP67), and ABA INSENSITIVE3 (ABI3) with those regulated by LEC1. We showed that LEC1 operates with specific sets of transcription factors to regulate different gene sets and, therefore, distinct developmental processes. Thus, LEC1 controls diverse processes through its combinatorial interactions with other transcription factors. DNA binding sites for the transcription factors are closely clustered in genomic regions upstream of target genes, defining cis-regulatory modules that are enriched for DNA sequence motifs that resemble sequences known to be bound by these transcription factors. Moreover, cis-regulatory modules for genes regulated by distinct transcription factor combinations are enriched for different sets of DNA motifs. Expression assays with embryo cells indicate that the enriched DNA motifs are functional cis elements that regulate transcription. Together, the results suggest that combinatorial interactions between LEC1 and other transcription factors are mediated by cis-regulatory modules containing clustered cis elements and by physical interactions that are documented to occur between the transcription factors.

The precise mechanisms that control gene activity during seed development remain largely unknown. Previously, we showed that several genes essential for seed development, including those encoding storage proteins, fatty acid biosynthesis enzymes, and transcriptional regulators (e.g., ABI3, FUS3) are located within hypomethylated regions of the soybean genome. These hypomethylated regions are similar to the DNA methylation valleys (DMVs), or canyons, found in mammalian cells. Here, we address the question of the extent to which DMVs are present within seed genomes and what role they might play in seed development. We scanned soybean and Arabidopsis seed genomes from postfertilization through dormancy and germination for regions that contain

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.

We profiled soybean and Arabidopsis methylomes from the globular stage through dormancy and germination to understand the role of methylation in seed formation. CHH methylation increases significantly during development throughout the entire seed, targets primarily transposable elements (TEs), is maintained during endoreduplication, and drops precipitously within the germinating seedling. By contrast, no significant global changes in CG- and CHG-context methylation occur during the same developmental period. An Arabidopsis ddcc mutant lacking CHH and CHG methylation does not affect seed development, germination, or major patterns of gene expression, implying that CHH and CHG methylation does not play a significant role in seed development or in regulating seed gene activity. By contrast, over 100 TEs are transcriptionally de-repressed in ddcc seeds, suggesting that the increase in CHH-context methylation may be a failsafe mechanism to reinforce transposon silencing. Many genes encoding important classes of seed proteins, such as storage proteins, oil biosynthesis enzymes, and transcription factors, reside in genomic regions devoid of methylation at any stage of seed development. Many other genes in these classes have similar methylation patterns, whether the genes are active or repressed. Our results suggest that methylation does not play a significant role in regulating large numbers of genes important for programming seed development in both soybean and Arabidopsis. We conclude that understanding the mechanisms controlling seed development will require determining how cis-regulatory elements and their cognate transcription factors are organized in genetic regulatory networks.

LEAFY COTYLEDON1 (LEC1), an atypical subunit of the nuclear transcription factor Y (NF-Y) CCAAT-binding transcription factor, is a central regulator that controls many aspects of seed development including the maturation phase during which seeds accumulate storage macromolecules and embryos acquire the ability to withstand desiccation. To define the gene networks and developmental processes controlled by LEC1, genes regulated directly by and downstream of LEC1 were identified. We compared the mRNA profiles of wild-type and lec1-null mutant seeds at several stages of development to define genes that are down-regulated or up-regulated by the lec1 mutation. We used ChIP and differential gene-expression analyses in Arabidopsis seedlings overexpressing LEC1 and in developing Arabidopsis and soybean seeds to identify globally the target genes that are transcriptionally regulated by LEC1 in planta Collectively, our results show that LEC1 controls distinct gene sets at different developmental stages, including those that mediate the temporal transition between photosynthesis and chloroplast biogenesis early in seed development and seed maturation late in development. Analyses of enriched DNA sequence motifs that may act as cis-regulatory elements in the promoters of LEC1 target genes suggest that LEC1 may interact with other transcription factors to regulate distinct gene sets at different stages of seed development. Moreover, our results demonstrate strong conservation in the developmental processes and gene networks regulated by LEC1 in two dicotyledonous plants that diverged ∼92 Mya.

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean (Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we use site-directed mutagenesis experiments in transgenic tobacco globular-stage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. A homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.

One of the major unsolved issues in plant development is understanding the regulatory networks that control the differential gene activity that is required for the specification and development of the two major embryonic regions, the embryo proper and suspensor. Historically, the giant embryo of scarlet runner bean (SRB), Phaseolus coccineus, has been used as a model system to investigate the physiological events that occur early in embryogenesis-focusing on the question of what role the suspensor region plays. A major feature distinguishing SRB embryos from those of other plants is a highly enlarged suspensor containing at least 200 cells that synthesize growth regulators required for subsequent embryonic development. Recent studies have exploited the giant size of the SRB embryo to micro-dissect the embryo proper and suspensor regions in order to use genomics-based approaches to identify regulatory genes that may be involved in controlling suspensor and embryo proper differentiation, as well as the cellular processes that may be unique to each embryonic region. Here we review the current genomics resources that make SRB embryos a compelling model system for studying the early events required to program embryo development.

Arabidopsis APETALA2 (AP2) controls seed mass maternally, with ap2 mutants producing larger seeds than wild type. Here, we show that AP2 influences development of the three major seed compartments: embryo, endosperm, and seed coat. AP2 appears to have a significant effect on endosperm development. ap2 mutant seeds undergo an extended period of rapid endosperm growth early in development relative to wild type. This early expanded growth period in ap2 seeds is associated with delayed endosperm cellularization and overgrowth of the endosperm central vacuole. The subsequent period of moderate endosperm growth is also extended in ap2 seeds largely due to persistent cell divisions at the endosperm periphery. The effect of AP2 on endosperm development is mediated by different mechanisms than parent-of-origin effects on seed size observed in interploidy crosses. Seed coat development is affected; integument cells of ap2 mutants are more elongated than wild type. We conclude that endosperm overgrowth and/or integument cell elongation create a larger postfertilization embryo sac into which the ap2 embryo can grow. Morphological development of the embryo is initially delayed in ap2 compared with wild-type seeds, but ap2 embryos become larger than wild type after the bent-cotyledon stage of development. ap2 embryos are able to fill the enlarged postfertilization embryo sac, because they undergo extended periods of cell proliferation and seed filling. We discuss potential mechanisms by which maternally acting AP2 influences development of the zygotic embryo and endosperm to repress seed size.