Abstract 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. 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. Significance How plant embryos are differentiated into embryo proper and suspensor regions following fertilization is a major unanswered question. The suspensor is unique because it can vary in morphology in different plant species. We hypothesized that regulatory genes controlling the specification of embryo proper and suspensor regions should be shared by all plants irrespective of embryo morphology. We compared embryo proper and suspensor transcriptomes of plants with distinct suspensor morphologies. Scarlet Runner Bean and Common Bean have highly specialized giant suspensor regions, whereas soybean and Arabidopsis suspensors are smaller and less specialized. We uncovered a small set of embryo-proper- and suspensor-specific transcription factors shared by all embryos irrespective of morphology, suggesting that they play an important role in early embryo differentiation.
Significance How plant embryos are differentiated into embryo proper and suspensor regions following fertilization is a major unanswered question. The suspensor is unique because it can vary in morphology in different plant species. We hypothesized that regulatory genes controlling the specification of embryo proper and suspensor regions should be shared by all plants irrespective of embryo morphology. We compared embryo proper and suspensor transcriptomes of plants with distinct suspensor morphologies. Scarlet runner bean and common bean have highly specialized giant suspensor regions; soybean and Arabidopsis suspensors are smaller and less specialized. We uncovered a small set of embryo proper– and suspensor-specific transcription factors shared by all embryos irrespective of morphology, suggesting that they play an important role in early embryo differentiation.
Significance We describe the spatial and temporal profiles of soybean and Arabidopsis seed methylomes during development. CHH methylation increases globally from fertilization through dormancy in all seed parts, decreases following germination, and targets primarily transposons. By contrast, CG- and CHG-context methylation remains constant throughout seed development. Mutant seeds lacking non-CG methylation develop normally, but have a set of up-regulated transposon RNAs suggesting that the CHH methylation increase may be a failsafe mechanism to reinforce transposon silencing. Major classes of seed genes have similar methylation profiles, whether they are active or not. Our results suggest that soybean and Arabidopsis seed methylomes are similar, and that DNA methylation does not play a significant role in regulating many genes important for seed development.
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.
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, 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. 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.
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.
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 to understand how genes are activated specifically within the suspensor during early embryo development. Previously, we showed that the G564 upstream region has a block of tandem repeats, which contain a conserved 10-bp motif (GAAAAG(C)/(T)GAA), and that deletion of these repeats results in a loss of suspensor transcription. Here, we use gain-of-function (GOF) experiments with transgenic globular-stage tobacco embryos to show that only 1 of the 5 tandem repeats is required to drive suspensor-specific transcription. Fine-scale deletion and scanning mutagenesis experiments with 1 tandem repeat uncovered a 54-bp region that contains all of the sequences required to activate transcription in the suspensor, including the 10-bp motif (GAAAAGCGAA) and a similar 10-bp-like motif (GAAAAACGAA). Site-directed mutagenesis and GOF experiments indicated that both the 10-bp and 10-bp-like motifs are necessary, but not sufficient to activate transcription in the suspensor, and that a sequence (TTGGT) between the 10-bp and the 10-bp-like motifs is also necessary for suspensor transcription. Together, these data identify sequences that are required to activate transcription in the suspensor of a plant embryo after fertilization.
Most of the transcription factors (TFs) responsible for controlling seed development are not yet known. To identify TF genes expressed at specific stages of seed development, including those unique to seeds, we used Affymetrix GeneChips to profile Arabidopsis genes active in seeds from fertilization through maturation and at other times of the plant life cycle. Seed gene sets were compared with those expressed in prefertilization ovules, germinating seedlings, and leaves, roots, stems, and floral buds of the mature plant. Most genes active in seeds are shared by all stages of seed development, although significant quantitative changes in gene activity occur. Each stage of seed development has a small gene set that is either specific at the level of the GeneChip or up-regulated with respect to genes active at other stages, including those that encode TFs. We identified 289 seed-specific genes, including 48 that encode TFs. Seven of the seed-specific TF genes are known regulators of seed development and include the LEAFY COTYLEDON (LEC) genes LEC1, LEC1-LIKE, LEC2, and FUS3. The rest represent different classes of TFs with unknown roles in seed development. Promoter-beta-glucuronidase (GUS) fusion experiments and seed mRNA localization GeneChip datasets showed that the seed-specific TF genes are active in different compartments and tissues of the seed at unique times of development. Collectively, these seed-specific TF genes should facilitate the identification of regulatory networks that are important for programming seed development.
The prediction of operons in Mycobacterium tuberculosis (MTB) is a first step toward understanding the regulatory network of this pathogen. Here we apply a statistical model using logistic regression to predict operons in MTB. As predictors, our model incorporates intergenic distance and the correlation of gene expression calculated for adjacent gene pairs from over 474 microarray experiments with MTB RNA. We validate our findings with known examples from the literature and experimentation. From this model, we rank each potential operon pair by the strength of evidence for cotranscription, choose a classification threshold with a true positive rate of over 90% at a false positive rate of 9.1%, and use it to construct an operon map for the MTB genome.
Author(s): Henry, Kelli Frances | Advisor(s): Goldberg, Robert B | Abstract: Seed crops, such as corn and soybean, are a major source of food for human and animal consumption. Understanding how genes are regulated in seeds is essential for the future development of genetically engineered seed crops that could significantly augment the food supply available for a rapidly growing human population. Given the importance of understanding the processes controlling seed development, it is surprising that the gene regulatory networks operating in seeds remain largely unknown. I have been using scarlet runner bean (SRB; Phaseolus coccineus), a close relative of soybean, to characterize a gene regulatory network active during early embryo development. Specifically, I have focused on gene activity in the suspensor, a specialized embryonic region involved in synthesizing and transporting nutrients to the growing embryo. To identify suspensor cis-regulatory sequences, I performed promoter dissection experiments on the SRB G564 gene, which is expressed in the suspensor early in embryo development. A 54-bp DNA fragment within the G564 upstream region is sufficient for suspensor-specific transcription in transgenic tobacco and Arabidopsis, indicating the suspensor transcriptional machinery is conserved in flowering plants. Mutagenesis of the 54- bp fragment identified five suspensor cis-regulatory elements: (i) three 10-bp motifs with the consensus 5'-GAAAAGCGAA-3', (ii) a Region 2 sequence 5'-TTG(A/G)(A/G/T)AAT-3' and (iii) a Fifth motif 5'-(A/G)AGTTA-3'. The Fifth motif sequence is a predicted MYB transcription factor binding site. A yeast one-hybrid screen identified three MYB transcription factors that bind to the 54-bp fragment and are expressed in the suspensor of Arabidopsis. Promoter deletion and mutagenesis experiments uncovered that sequences similar to these three types of suspensor motifs also activate suspensor transcription in the SRB GA 20-oxidase gene, which encodes an enzyme required for synthesis of the phytohormone gibberellic acid. The SRB G564 and GA 20-oxidase genes are activated by the same suspensor cis-regulatory elements and thus comprise a suspensor gene regulatory network, which is activated shortly after fertilization by transcriptional machinery that is conserved in the suspensors of flowering plants.
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