Jennifer N. Bragg

Overexpression of the maize Corngrass1 microRNA prevents flowering, improves digestibility, and increases starch content of switchgrass

Biofuels developed from biomass crops have the potential to supply a significant portion of our transportation fuel needs. To achieve this potential, however, it will be necessary to develop improved plant germplasm specifically tailored to serve as energy crops. Liquid transportation fuel can be created from the sugars locked inside plant cell walls. Unfortunately, these sugars are inherently resistant to hydrolytic release because they are contained in polysaccharides embedded in lignin. Overcoming this obstacle is a major objective toward developing sustainable bioenergy crop plants. The maize Corngrass1 (Cg1) gene encodes a microRNA that promotes juvenile cell wall identities and morphology. To test the hypothesis that juvenile biomass has superior qualities as a potential biofuel feedstock, the Cg1 gene was transferred into several other plants, including the bioenergy crop Panicum virgatum (switchgrass). Such plants were found to have up to 250% more starch, resulting in higher glucose release from saccharification assays with or without biomass pretreatment. In addition, a complete inhibition of flowering was observed in both greenhouse and field grown plants. These results point to the potential utility of this approach, both for the domestication of new biofuel crops, and for the limitation of transgene flow into native plant species.

Generation and characterization of the Western Regional Research Center Brachypodium T-DNA insertional mutant collection.

The model grass Brachypodium distachyon (Brachypodium) is an excellent system for studying the basic biology underlying traits relevant to the use of grasses as food, forage and energy crops. To add to the growing collection of Brachypodium resources available to plant scientists, we further optimized our Agrobacterium tumefaciens-mediated high-efficiency transformation method and generated 8,491 Brachypodium T-DNA lines. We used inverse PCR to sequence the DNA flanking the insertion sites in the mutants. Using these flanking sequence tags (FSTs) we were able to assign 7,389 FSTs from 4,402 T-DNA mutants to 5,285 specific insertion sites (ISs) in the Brachypodium genome. More than 29% of the assigned ISs are supported by multiple FSTs. T-DNA insertions span the entire genome with an average of 19.3 insertions/Mb. The distribution of T-DNA insertions is non-uniform with a larger number of insertions at the distal ends compared to the centromeric regions of the chromosomes. Insertions are correlated with genic regions, but are biased toward UTRs and non-coding regions within 1 kb of genes over exons and intron regions. More than 1,300 unique genes have been tagged in this population. Information about the Western Regional Research Center Brachypodium insertional mutant population is available on a searchable website (http://brachypodium.pw.usda.gov) designed to provide researchers with a means to order T-DNA lines with mutations in genes of interest.

Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines

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.

A Division in PIN-Mediated Auxin Patterning during Organ Initiation in Grasses

The hormone auxin plays a crucial role in plant morphogenesis. In the shoot apical meristem, the PIN-FORMED1 (PIN1) efflux carrier concentrates auxin into local maxima in the epidermis, which position incipient leaf or floral primordia. From these maxima, PIN1 transports auxin into internal tissues along emergent paths that pattern leaf and stem vasculature. In Arabidopsis thaliana, these functions are attributed to a single PIN1 protein. Using phylogenetic and gene synteny analysis we identified an angiosperm PIN clade sister to PIN1, here termed Sister-of-PIN1 (SoPIN1), which is present in all sampled angiosperms except for Brassicaceae, including Arabidopsis. Additionally, we identified a conserved duplication of PIN1 in the grasses: PIN1a and PIN1b. In Brachypodium distachyon, SoPIN1 is highly expressed in the epidermis and is consistently polarized toward regions of high expression of the DR5 auxin-signaling reporter, which suggests that SoPIN1 functions in the localization of new primordia. In contrast, PIN1a and PIN1b are highly expressed in internal tissues, suggesting a role in vascular patterning. PIN1b is expressed in broad regions spanning the space between new primordia and previously formed vasculature, suggesting a role in connecting new organs to auxin sinks in the older tissues. Within these regions, PIN1a forms narrow canals that likely pattern future veins. Using a computer model, we reproduced the observed spatio-temporal expression and localization patterns of these proteins by assuming that SoPIN1 is polarized up the auxin gradient, and PIN1a and PIN1b are polarized to different degrees with the auxin flux. Our results suggest that examination and modeling of PIN dynamics in plants outside of Brassicaceae will offer insights into auxin-driven patterning obscured by the loss of the SoPIN1 clade in Brassicaceae.

Brachypodium distachyon, a New Model for the Triticeae

Brachypodium distachyon (Brachypodium) is a small annual grass with biological, physical and genomic attributes (e.g. rapid cycling, small stature, inbreeding, small genome, diploid accessions) suitable for use as a modern model system. In pursuit of this goal, researchers have made rapid progress in developing genomic resources that will transform Brachypodium into a powerful model system including: facile Agrobacterium-mediated transformation methods, BAC libraries, physical maps, genetic maps, and germplasm resources. In addition, a preliminary 4x draft of the entire genome has been released, and completion of the final 8x assembly is anticipated in 2009. This chapter provides an overview of the advantages of Brachypodium as a model system and surveys the use and potential applications of this system to aid wheat, barley and Lolium research.

Fine mapping of the Bsr1 barley stripe mosaic virus resistance gene in the model grass Brachypodium distachyon.

The ND18 strain of Barley stripe mosaic virus (BSMV) infects several lines of Brachypodium distachyon, a recently developed model system for genomics research in cereals. Among the inbred lines tested, Bd3-1 is highly resistant at 20 to 25°C, whereas Bd21 is susceptible and infection results in an intense mosaic phenotype accompanied by high levels of replicating virus. We generated an F6∶7 recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21 and used the RILs, and an F2 population of a second Bd21 × Bd3-1 cross to evaluate the inheritance of resistance. The results indicate that resistance segregates as expected for a single dominant gene, which we have designated Barley stripe mosaic virus resistance 1 (Bsr1). We constructed a genetic linkage map of the RIL population using SNP markers to map this gene to within 705 Kb of the distal end of the top of chromosome 3. Additional CAPS and Indel markers were used to fine map Bsr1 to a 23 Kb interval containing five putative genes. Our study demonstrates the power of using RILs to rapidly map the genetic determinants of BSMV resistance in Brachypodium. Moreover, the RILs and their associated genetic map, when combined with the complete genomic sequence of Brachypodium, provide new resources for genetic analyses of many other traits.

Annotation and comparative analysis of the glycoside hydrolase genes in Brachypodium distachyon

BackgroundGlycoside 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 Populustrichocarpa (poplar). To improve our understanding of glycoside hydrolases in plants generally and in grasses specifically, we annotated the glycoside hydrolase genes in the grasses Brachypodiumdistachyon (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.ResultsWe 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.ConclusionsThis 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.

Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to- cell movement and eliminates long-distance virus movement

Cell-to-cell movement of potexviruses requires coordinated action of the coat protein and triple gene block (TGB) proteins. The structural properties of Alternanthera mosaic virus (AltMV) TGB3 were examined by methods differentiating between signal peptides and transmembrane domains, and its subcellular localization was studied by Agrobacterium-mediated transient expression and confocal microscopy. Unlike potato virus X (PVX) TGB3, AltMV TGB3 was not associated with the endoplasmic reticulum, and accumulated preferentially in mesophyll cells. Deletion and site-specific mutagenesis revealed an internal signal VL(17,18) of TGB3 essential for chloroplast localization, and either deletion of the TGB3 start codon or alteration of the chloroplast-localization signal limited cell-to-cell movement to the epidermis, yielding a virus that was unable to move into the mesophyll layer. Overexpression of AltMV TGB3 from either AltMV or PVX infectious clones resulted in veinal necrosis and vesiculation at the chloroplast membrane, a cytopathology not observed in wild-type infections. The distinctive mesophyll and chloroplast localization of AltMV TGB3 highlights the critical role played by mesophyll targeting in virus long-distance movement within plants.

Brachypodium sylvaticum, a model for perennial grasses: transformation and inbred line development.

Perennial species offer significant advantages as crops including reduced soil erosion, lower energy inputs after the first year, deeper root systems that access more soil moisture, and decreased fertilizer inputs due to the remobilization of nutrients at the end of the growing season. These advantages are particularly relevant for emerging biomass crops and it is projected that perennial grasses will be among the most important dedicated biomass crops. The advantages offered by perennial crops could also prove favorable for incorporation into annual grain crops like wheat, rice, sorghum and barley, especially under the dryer and more variable climate conditions projected for many grain-producing regions. Thus, it would be useful to have a perennial model system to test biotechnological approaches to crop improvement and for fundamental research. The perennial grass Brachypodium sylvaticum is a candidate for such a model because it is diploid, has a small genome, is self-fertile, has a modest stature, and short generation time. Its close relationship to the annual model Brachypodium distachyon will facilitate comparative studies and allow researchers to leverage the resources developed for B . distachyon . Here we report on the development of two keystone resources that are essential for a model plant: high-efficiency transformation and inbred lines. Using Agrobacterium tumefaciens-mediated transformation we achieved an average transformation efficiency of 67%. We also surveyed the genetic diversity of 19 accessions from the National Plant Germplasm System using SSR markers and created 15 inbred lines.

Phylogenetic, Molecular, and Biochemical Characterization of Caffeic Acid o-Methyltransferase Gene Family in Brachypodium distachyon

Caffeic acid o-methyltransferase (COMT) is one of the important enzymes controlling lignin monomer production in plant cell wall synthesis. Analysis of the genome sequence of the new grass model Brachypodium distachyon identified four COMT gene homologs, designated as BdCOMT1, BdCOMT2, BdCOMT3, and BdCOMT4. Phylogenetic analysis suggested that they belong to the COMT gene family, whereas syntenic analysis through comparisons with rice and sorghum revealed that BdCOMT4 on Chromosome 3 is the orthologous copy of the COMT genes well characterized in other grass species. The other three COMT genes are unique to Brachypodium since orthologous copies are not found in the collinear regions of rice and sorghum genomes. Expression studies indicated that all four Brachypodium COMT genes are transcribed but with distinct patterns of tissue specificity. Full-length cDNAs were cloned in frame into the pQE-T7 expression vector for the purification of recombinant Brachypodium COMT proteins. Biochemical characterization of enzyme activity and substrate specificity showed that BdCOMT4 has significant effect on a broad range of substrates with the highest preference for caffeic acid. The other three COMTs had low or no effect on these substrates, suggesting that a diversified evolution occurred on these duplicate genes that not only impacted their pattern of expression, but also altered their biochemical properties.