While prokaryotic pan-genomes have been shown to contain many more genes than any individual organism, the prevalence and functional significance of differentially present genes in eukaryotes remains ...poorly understood. Whole-genome de novo assembly and annotation of 54 lines of the grass Brachypodium distachyon yield a pan-genome containing nearly twice the number of genes found in any individual genome. Genes present in all lines are enriched for essential biological functions, while genes present in only some lines are enriched for conditionally beneficial functions (e.g., defense and development), display faster evolutionary rates, lie closer to transposable elements and are less likely to be syntenic with orthologous genes in other grasses. Our data suggest that differentially present genes contribute substantially to phenotypic variation within a eukaryote species, these genes have a major influence in population genetics, and transposable elements play a key role in pan-genome evolution.
The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA ...proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6. AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways.
RGA (repressor of ga1-3) and GAI (gibberellin insensitive) are negative regulators of plant hormone gibberellin (GA) signaling in Arabidopsis. The GA-deficient mutant ga1-3 is a nongerminating, ...extreme dwarf that flowers late and produces male-sterile flowers. The rga and gai null alleles interact synergistically to rescue vegetative growth and floral initiation in ga1-3, indicating that RGA and GAI are major repressors for these processes. However, rga and gai in combination cannot rescue seed germination or floral development in ga1-3. RGA and GAI belong to the DELLA subfamily within the GRAS family of plant regulatory proteins. Three additional DELLA proteins RGL1, RGL2, and RGL3 are present in Arabidopsis. Previous studies provided evidence that RGL2 and possibly RGL1 control seed germination. To investigate further the function of the RGL genes, we examined the expression profiles of all 5 DELLA protein genes by real-time PCR. RGA and, to a lesser extent, GAI mRNAs were expressed ubiquitously in all tissues, whereas RGL1, 2, and 3 transcripts were present at high levels only in germinating seeds and/or flowers and siliques. Using the newly isolated rgl1, rgl2, and rgl3 T-DNA insertion mutants, we demonstrated that RGL2 is the major repressor in seed germination. We further provided evidence that RGA, RGL1, and RGL2 are all involved in modulating floral development. Interestingly, RGL2 expression is regulated not only at the transcript level. We showed that RGL2 protein in imbibed seeds is rapidly degraded by GA treatment and that the F-box protein SLY1 is required for RGL2 degradation to occur.
Glycoside hydrolases cleave the bond between a carbohydrate and another carbohydrate, a protein, lipid or other moiety. Genes encoding glycoside hydrolases are found in a wide range of organisms, ...from archea to animals, and are relatively abundant in plant genomes. In plants, these enzymes are involved in diverse processes, including starch metabolism, defense, and cell-wall remodeling. Glycoside hydrolase genes have been previously cataloged for Oryza sativa (rice), the model dicotyledonous plant Arabidopsis thaliana, and the fast-growing tree Populus trichocarpa (poplar). To improve our understanding of glycoside hydrolases in plants generally and in grasses specifically, we annotated the glycoside hydrolase genes in the grasses Brachypodium distachyon (an emerging monocotyledonous model) and Sorghum bicolor (sorghum). We then compared the glycoside hydrolases across species, at the levels of the whole genome and individual glycoside hydrolase families.
We identified 356 glycoside hydrolase genes in Brachypodium and 404 in sorghum. The corresponding proteins fell into the same 34 families that are represented in rice, Arabidopsis, and poplar, helping to define a glycoside hydrolase family profile which may be common to flowering plants. For several glycoside hydrolase familes (GH5, GH13, GH18, GH19, GH28, and GH51), we present a detailed literature review together with an examination of the family structures. This analysis of individual families revealed both similarities and distinctions between monocots and eudicots, as well as between species. Shared evolutionary histories appear to be modified by lineage-specific expansions or deletions. Within GH families, the Brachypodium and sorghum proteins generally cluster with those from other monocots.
This work provides the foundation for further comparative and functional analyses of plant glycoside hydrolases. Defining the Brachypodium glycoside hydrolases sets the stage for Brachypodium to be a grass model for investigations of these enzymes and their diverse roles in planta. Insights gained from Brachypodium will inform translational research studies, with applications for the improvement of cereal crops and bioenergy grasses.
Brachypodium distachyon is small annual grass that has been adopted as a model for the grasses. Its small genome, high‐quality reference genome, large germplasm collection, and selfing nature make it ...an excellent subject for studies of natural variation. We sequenced six divergent lines to identify a comprehensive set of polymorphisms and analyze their distribution and concordance with gene expression. Multiple methods and controls were utilized to identify polymorphisms and validate their quality. mRNA‐Seq experiments under control and simulated drought‐stress conditions, identified 300 genes with a genotype‐dependent treatment response. We showed that large‐scale sequence variants had extremely high concordance with altered expression of hundreds of genes, including many with genotype‐dependent treatment responses. We generated a deep mRNA‐Seq dataset for the most divergent line and created a de novo transcriptome assembly. This led to the discovery of >2400 previously unannotated transcripts and hundreds of genes not present in the reference genome. We built a public database for visualization and investigation of sequence variants among these widely used inbred lines.
Core Ideas
Genotyping diverse Brachypodium accessions expands research tools for grasses.
The B. hybridum genome is a mosaic of B. distachyon‐ and B. stacei‐like sequences.
Three distinct, ...genetically defined populations of B. distachyon were identified.
Flowering time, more than geography, distinguishes B. distachyon populations.
Results support the feasibility of genome‐wide association studies in a model grass.
The small, annual grass Brachypodium distachyon (L.) Beauv., a close relative of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), is a powerful model system for cereals and bioenergy grasses. Genome‐wide association studies (GWAS) of natural variation can elucidate the genetic basis of complex traits but have been so far limited in B. distachyon by the lack of large numbers of well‐characterized and sufficiently diverse accessions. Here, we report on genotyping‐by‐sequencing (GBS) of 84 B. distachyon, seven B. hybridum, and three B. stacei accessions with diverse geographic origins including Albania, Armenia, Georgia, Italy, Spain, and Turkey. Over 90,000 high‐quality single‐nucleotide polymorphisms (SNPs) distributed across the Bd21 reference genome were identified. Our results confirm the hybrid nature of the B. hybridum genome, which appears as a mosaic of B. distachyon‐like and B. stacei‐like sequences. Analysis of more than 50,000 SNPs for the B. distachyon accessions revealed three distinct, genetically defined populations. Surprisingly, these genomic profiles are associated with differences in flowering time rather than with broad geographic origin. High levels of differentiation in loci associated with floral development support the differences in flowering phenology between B. distachyon populations. Genome‐wide association studies combining genotypic and phenotypic data also suggest the presence of one or more photoperiodism, circadian clock, and vernalization genes in loci associated with flowering time variation within B. distachyon populations. Our characterization elucidates genes underlying population differences, expands the germplasm resources available for Brachypodium, and illustrates the feasibility and limitations of GWAS in this model grass.
Assessment of student learning is a cornerstone of biochemistry and molecular biology (BMB) education. Yet few BMB instructors receive meaningful professional development in best practices in ...assessment. To promote such training in the BMB education community, we conducted a workshop on writing questions for summative assessment in the summer of 2020. Recognizing the individual and institutional homogeneity in our BMB education community, we recruited workshop participants primarily from the Southeastern United States due to the multitude of Historically Black Colleges and Universities and Hispanic‐Serving Institutions in the region. 28 participants were selected to provide diversity in individual and institutional demographics. Led by six members of the ASBMB Accreditation and Certification Exam Team, the online workshop, spread across nine days, featured whole‐group discussions interspersed with small‐group activities. Post‐workshop surveys revealed significant gains in self‐reported professional development in assessment creation. Surveys also highlighted the inclusive environment of the workshop. 43% of the workshop participants were subsequently recruited into the ASBMB Certification Exam Team, providing fresh voices and perspective to the group. This workshop is an excellent example of how the intentional partnership with individuals and institutions from historically marginalized populations can begin a movement toward necessary and meaningful change in the BMB education community.
Each year, volunteers from the biochemistry and molecular (BMB) community come together to construct and score the American Society for Biochemistry and Molecular Biology (ASBMB) Certification Exam, ...a tool provided to ASBMB‐accredited programs for assessing key competencies in the discipline. Grounded in a community consensus about core BMB concept and skill areas, the annual exam is designed to evaluate undergraduate students’ competence in the areas of energy and metabolism; information storage and transfer; macromolecular structure, function, and assembly; and skills including analytical and quantitative reasoning. First offered in 2014, this assessment tool is now representative of a relatively mature exam development process. The one‐hour exam consists of 12 questions, with a mix of multiple‐select/multiple‐choice and constructed‐response formats, balanced across the four concept and skill areas and addressing both lower‐ and higher‐order cognitive processing levels. Each constructed response is scored by a team of at least three BMB scientist‐educators, and inter‐rater reliability is calculated to ensure consistency. All questions are piloted and revised before they contribute to students’ exam scores. To date, nearly 5,000 students from over 70 colleges and universities have taken the ASBMB exam. Of these, an average of approximately 42% of students per year have scored sufficiently well to earn “Certification” of their exam performance by ASBMB; approximately 13% of students have earned “Certification with Distinction”. Upwards of 120 scientist‐educators – with professional affiliations ranging from small, primarily undergraduate institutions to large research universities – have contributed to question development and/or scoring. Many volunteers have participated over multiple years; for example, 97% of the 2020 scorers plan to score again in 2021. Future directions include building the community of volunteers, expanding the question bank, delivering the exam online (in spring of 2021), and investigating the potential of the ASBMB Certification Exam to inform programmatic assessment.
Assessment is a critical feature of effective instruction. Beyond assigning a grade to student performance, regular assessment can provide instructors, departments, and institutions with ongoing ...feedback on instructor, course, and program effectiveness. Despite its importance, designing assessments that are accessible to all students and measure relevant learning outcomes can be challenging for many biochemistry and molecular biology (BMB) instructors because they receive little, if any, formal training in assessment practices. Consequently, we ‐ a team of writers for the ASBMB Certification Examination ‐ have launched ICABL, an inclusive community for the assessment of BMB learning. With funding from an NSF grant (Award No. 2018204), we piloted a virtual workshop on summative assessment in Summer 2020. The 28 participants discussed best practices in summative assessment, including Bloom’s taxonomy, backward design using the ASBMB Foundational Concepts and Skills, and rubrics development as tools for informing instructors on the effectiveness of instruction. An explicit objective of the workshop was to broaden representation in the ASBMB education community. Accordingly, 68% of participants taught at minority‐serving institutions, as compared with less than 10% of current ASBMB membership. Both immediate and six‐month follow‐up surveys highlighted the efficacy of this virtual workshop in supporting participants’ professional development. In addition, we have subsequently recruited many of the participants to write and score for the ASBMB Certification Exam as one step in our goal to develop an examination team that better represents and can more effectively address the diversity of individuals in our BMB classrooms. With continued support from the NSF (Award No. 2120673), over the next three years we will be offering a series of additional workshops, both in the United States and overseas, addressing summative, formative, and alternative assessment.
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