Genome-wide profiling of soybean WRINKLED1 transcription factor binding sites provides insight into seed storage lipid biosynthesis.

Understanding the regulatory mechanisms controlling storage lipid accumulation will inform strategies to enhance seed oil quality and quantity in crop plants. The WRINKLED1 transcription factor (WRI1 TF) is a central regulator of lipid biosynthesis. We characterized the genome-wide binding profile of soybean (Gm)WRI1 and show that the TF directly regulates genes encoding numerous enzymes and proteins in the fatty acid and triacylglycerol biosynthetic pathways. GmWRI1 binds primarily to regions downstream of target gene transcription start sites. We showed that GmWRI1-bound regions are enriched for the canonical WRI1 DNA binding element, the ACTIVATOR of Spomin::LUC1/WRI1 (AW) Box (CNTNGNNNNNNNCG), and another DNA motif, the CNC Box (CNCCNCC). Functional assays showed that both DNA elements mediate transcriptional activation by GmWRI1. We also show that GmWRI1 works in concert with other TFs to establish a regulatory state that promotes fatty acid and triacylglycerol biosynthesis. In particular, comparison of genes targeted directly by GmWRI1 and by GmLEC1, a central regulator of the maturation phase of seed development, reveals that the two TFs act in a positive feedback subcircuit to control fatty acid and triacylglycerol biosynthesis. Together, our results provide unique insights into the genetic circuitry in which GmWRI1 participates to regulate storage lipid accumulation during seed development.

Comparative analysis of embryo proper and suspensor transcriptomes in plant embryos with different morphologies.

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.

PRACTITIONER UPDATE.

OBJECTIVE: To review the development and implementation of prescription formularies by managed care organizations, identify their current applications, and recognize future trends in the managed care pharmacy environment. DATA SOURCES: Current journal articles and texts regarding the use of formularies and the managed care environment. DATA SYNTHESIS: Not applicable. CONCLUSION: Formulary systems have proven to be a valuable means to control the pharmacy benefit and can be expected to expand in both scope and sophistication.

Ensuring the Availability of Clinical Clerkships at U.S. Hospitals: A Tale of 2 States.

U.S. medical schools are facing growing competition for limited clinical training resources, notably slots for the core clerkships that students most often complete in the third year of their undergraduate medical education. In particular, medical schools in the Caribbean (often referred to as offshore medical schools) are buying clerkship slots at U.S. hospitals for their students, most of whom will be U.S. citizen international medical graduates. For hospitals, especially those that are financially stressed, these payments are an attractive source of revenue. Yet, this practice has put pressure on U.S. medical schools to provide similar remuneration for clerkship slots for their students or to find new clinical training sites.In this Perspective, the authors outline the scope of the challenge facing U.S. medical schools and the U.S. medical education system. They outline legislative strategies implemented in 2 states (New York and Texas) to address this issue and propose the passage of similar legislation in other states to ensure that students at U.S. medical schools can access the clerkships they need to obtain the requisite clinical experience before entering residency. Such legislation would preserve the availability of clerkships for U.S. medical students and the educational quality of these clinical training experiences and, therefore, preserve the quantity and quality of the future physician workforce in the United States.

Combinatorial interactions of the LEC1 transcription factor specify diverse developmental programs during soybean seed development.

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.

Using Giant Scarlet Runner Bean (Phaseolus coccineus) Embryos to Dissect the Early Events in Plant Embryogenesis.

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.

Seed genome hypomethylated regions are enriched in transcription factor genes.

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 <5% or <0.4% bulk methylation in CG, CHG, and CHH contexts over all developmental stages. We found that DMVs represent extensive portions of seed genomes, range in size from 5-76 kb, are scattered throughout all chromosomes, and are hypomethylated throughout the plant life cycle. Significantly, DMVs are enriched greatly in transcription factor (TF) genes and other developmental genes that play critical roles in seed formation. Many DMV genes are regulated with respect to seed stage, region, and tissue, and contain H3K4me3, H3K27me3, or bivalent marks that fluctuate during development. Our results indicate that DMVs are a unique regulatory feature of both plant and animal genomes, and that a large number of seed genes are regulated in the absence of methylation changes during development, probably by the action of specific TFs and epigenetic events at the chromatin level.

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 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.

Similarity between soybean and Arabidopsis seed methylomes and loss of non-CG methylation does not affect seed development.

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.

LEC1 sequentially regulates the transcription of genes involved in diverse developmental processes during seed development.

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.

A naïve Bayesian classifier for identifying plant microRNAs.

MicroRNAs (miRNAs) are important regulatory molecules in eukaryotic organisms. Existing methods for the identification of mature miRNA sequences in plants rely extensively on the search for stem-loop structures, leading to high false negative rates. Here, we describe a probabilistic method for ranking putative plant miRNAs using a naïve Bayes classifier and its publicly available implementation. We use a number of properties to construct the classifier, including sequence length, number of observations, existence of detectable predicted miRNA* sequences, the distribution of nearby reads and mapping multiplicity. We apply the method to small RNA sequence data from soybean, peach, Arabidopsis and rice and provide experimental validation of several predictions in soybean. The approach performs well overall and strongly enriches for known miRNAs over other types of sequences. By utilizing a Bayesian approach to rank putative miRNAs, our method is able to score miRNAs that would be eliminated by other methods, such as those that have low counts or lack detectable miRNA* sequences. As a result, we are able to detect several soybean miRNA candidates, including some that are 24 nucleotides long, a class that is almost universally eliminated by other methods.

Offshore Medical Schools Are Buying Clinical Clerkships in U.S. Hospitals: The Problem and Potential Solutions.

U.S. medical education faces a threat from for-profit Caribbean medical schools which purchase clinical rotation slots for their students at U.S. hospitals. These offshore schools are monetizing a system that was previously characterized as a duty-the duty of the current generation of physicians to educate their successors. Offshore schools purchase clinical rotation slots using funds largely derived from federally subsidized student loans. This leads to pressure on U.S. schools to pay for clinical clerkships and is forcing some of them to find new clinical training sites.For-profit Caribbean schools largely escape the type of scrutiny that U.S. schools face from U.S. national accreditation organizations. They also enroll large classes of students with lower undergraduate GPAs and Medical College Admission Test scores than those of students at U.S. medical schools; their students take and pass Step 1 of the United States Medical Licensing Examination at a substantially lower rate than that of U.S. medical students; and their students match for residencies at a fraction of the rate of U.S. medical school graduates.Among the potential solutions proposed by the authors are passing laws to hold for-profit Caribbean schools to standards for board passage rates, placing restrictions on federal student loans, monitoring attrition rates, and denying offshore schools access to U.S. clinical training sites unless they meet accreditation standards equivalent to those of U.S. medical schools.

Down-Regulating the Expression of 53 Soybean Transcription Factor Genes Uncovers a Role for SPEECHLESS in Initiating Stomatal Cell Lineages during Embryo Development.

We used an RNA interference screen to assay the function of 53 transcription factor messenger RNAs (mRNAs) that accumulate specifically within soybean (Glycine max) seed regions, subregions, and tissues during development. We show that basic helix-loop-helix (bHLH) transcription factor genes represented by Glyma04g41710 and its paralogs are required for the formation of stoma in leaves and stomatal precursor complexes in mature embryo cotyledons. Phylogenetic analysis indicates that these bHLH transcription factor genes are orthologous to Arabidopsis (Arabidopsis thaliana) SPEECHLESS (SPCH) that initiate asymmetric cell divisions in the leaf protoderm layer and establish stomatal cell lineages. Soybean SPCH (GmSPCH) mRNAs accumulate primarily in embryo, seedling, and leaf epidermal layers. Expression of Glyma04g41710 under the control of the SPCH promoter rescues the Arabidopsis spch mutant, indicating that Glyma04g41710 is a functional ortholog of SPCH. Developing soybean embryos do not form mature stoma, and stomatal differentiation is arrested at the guard mother cell stage. We analyzed the accumulation of GmSPCH mRNAs during soybean seed development and mRNAs orthologous to MUTE, FAMA, and inducer of C-repeat/dehydration responsive element-binding factor expression1/scream2 that are required for stoma formation in Arabidopsis. The mRNA accumulation patterns provide a potential explanation for guard mother cell dormancy in soybean embryos. Our results suggest that variation in the timing of bHLH transcription factor gene expression can explain the diversity of stomatal forms observed during plant development.

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements.

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.

Using giant scarlet runner bean embryos to uncover regulatory networks controlling suspensor gene activity.

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.

Transcriptome atlas of the Arabidopsis funiculus--a study of maternal seed subregions.

The funiculus anchors the structurally complex seed to the maternal plant, and is the only direct route of transport for nutrients and maternal signals to the seed. While our understanding of seed development is becoming clearer, current understanding of the genetics and cellular mechanisms that contribute to funiculus development is limited. Using laser microdissection combined with global RNA-profiling experiments we compared the genetic profiles of all maternal and zygotic regions and subregions during seed development. We found that the funiculus is a dynamic region of the seed that is enriched for mRNAs associated with hormone metabolism, molecular transport, and metabolic activities corresponding to biological processes that have yet to be described in this maternal seed structure. We complemented our genetic data with a complete histological analysis of the funiculus from the earliest stages of development through to seed maturation at the light and electron microscopy levels. The anatomy revealed signs of photosynthesis, the endomembrane system, cellular respiration, and transport within the funiculus, all of which supported data from the transcriptional analysis. Finally, we studied the transcriptional programming of the funiculus compared to other seed subregions throughout seed development. Using newly designed in silico algorithms, we identified a number of transcriptional networks hypothesized to be responsible for biological processes like auxin response and glucosinolate biosynthesis found specifically within the funiculus. Taken together, patterns of gene activity and histological observations reveal putative functions of the understudied funiculus region and identify predictive transcriptional circuits underlying these biological processes in space and time.

Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed.

Seeds are complex structures that consist of the embryo, endosperm, and seed-coat regions that are of different ontogenetic origins, and each region can be further divided into morphologically distinct subregions. Despite the importance of seeds for food, fiber, and fuel globally, little is known of the cellular processes that characterize each subregion or how these processes are integrated to permit the coordinated development of the seed. We profiled gene activity genome-wide in every organ, tissue, and cell type of Arabidopsis seeds from fertilization through maturity. The resulting mRNA datasets offer the most comprehensive description of gene activity in seeds with high spatial and temporal resolution,providing unique insights into the function of understudied seed regions. Global comparisons of mRNA populations reveal unexpected overlaps in the functional identities of seed subregions. Analyses of coexpressed gene sets suggest that processes that regulate seed size and filling are coordinated across several subregions. Predictions of gene regulatory networks based on the association of transcription factors with enriched DNA sequence motifs upstream of coexpressed genes identify regulators of seed development. These studies emphasize the utility of these data sets as an essential resource for the study of seed biology.