In Arabidopsis, development during flowering is coordinated by transport of the hormone auxin mediated by polar-localized PIN-FORMED1 (AtPIN1). However Arabidopsis has lost a PIN clade sister to AtPIN1, Sister-of-PIN1 (SoPIN1), which is conserved in flowering plants. We previously proposed that the AtPIN1 organ initiation and vein patterning functions are split between the SoPIN1 and PIN1 clades in grasses. Here we show that in the grass Brachypodium sopin1 mutants have organ initiation defects similar to Arabidopsis atpin1, while loss of PIN1 function in Brachypodium has little effect on organ initiation but alters stem growth. Heterologous expression of Brachypodium SoPIN1 and PIN1b in Arabidopsis provides further evidence of functional specificity. SoPIN1 but not PIN1b can mediate flower formation in null atpin1 mutants, although both can complement a missense allele. The behavior of SoPIN1 and PIN1b in Arabidopsis illustrates how membrane and tissue-level accumulation, transport activity, and interaction contribute to PIN functional specificity.

Due to a large and growing collection of genomic and experimental resources, Brachypodium distachyon has emerged as a powerful experimental model for the grasses. To add to these resources we sequenced 21 165 T-DNA lines, 15 569 of which were produced in this study. This increased the number of unique insertion sites in the T-DNA collection by 21 078, bringing the overall total to 26 112. Thirty-seven per cent (9754) of these insertion sites are within genes (including untranslated regions and introns) and 28% (7217) are within 500 bp of a gene. Approximately 31% of the genes in the v.2.1 annotation have been tagged in this population. To demonstrate the utility of this collection, we phenotypically characterized six T-DNA lines with insertions in genes previously shown in other systems to be involved in cellulose biosynthesis, hemicellulose biosynthesis, secondary cell wall development, DNA damage repair, wax biosynthesis and chloroplast synthesis. In all cases, the phenotypes observed supported previous studies, demonstrating the utility of this collection for plant functional genomics. The Brachypodium T-DNA collection can be accessed at http://jgi.doe.gov/our-science/science-programs/plant-genomics/brachypodium/brachypodium-t-dna-collection/.

Background Cereal diseases cause tens of billions of dollars of losses annually and have devastating humanitarian consequences in the developing world. Increased understanding of the molecular basis of cereal host-pathogen interactions should facilitate development of novel resistance strategies. However, achieving this in most cereals can be challenging due to large and complex genomes, long generation times and large plant size, as well as quarantine and intellectual property issues that may constrain the development and use of community resources. Brachypodium distachyon (brachypodium) with its small, diploid and sequenced genome, short generation time, high transformability and rapidly expanding community resources is emerging as a tractable cereal model.Scope Recent research reviewed here has demonstrated that brachypodium is either susceptible or partially susceptible to many of the major cereal pathogens. Thus, the study of brachypodium-pathogen interactions appears to hold great potential to improve understanding of cereal disease resistance, and to guide approaches to enhance this resistance. This paper reviews brachypodium experimental pathosystems for the study of fungal, bacterial and viral cereal pathogens; the current status of the use of brachypodium for functional analysis of cereal disease resistance; and comparative genomic approaches undertaken using brachypodium to assist characterization of cereal resistance genes. Additionally, it explores future prospects for brachypodium as a model to study cereal-pathogen interactions.Conclusions The study of brachypodium-pathogen interactions appears to be a productive strategy for understanding mechanisms of disease resistance in cereal species. Knowledge obtained from this model interaction has strong potential to be exploited for crop improvement.

Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in a severe phenotype. Heterozygous parents produce progeny that segregate 3:1 for wild-type:growtharrested seedlings. Homozygous T-DNA insertion lines are recovered for BRIZ1 and BRIZ2 after introduction of a transgene containing the respective coding sequence, demonstrating that disruption of BRIZ1 or BRIZ2 in the T-DNA insertion lines is responsible for the observed phenotype. Both proteins have multiple predicted domains in addition to the RING domain as follows: a BRAP2 (BRCA1-Associated Protein 2), a ZnF UBP (Zinc Finger Ubiquitin Binding protein), and a coiled-coil domain. In vitro, both BRIZ1 and BRIZ2 are active as E3 ligases but only BRIZ2 binds ubiquitin. In vitro synthesized and purified recombinant BRIZ1 and BRIZ2 preferentially form heterooligomers rather than homo-oligomers, and the coiled-coil domain is necessary and sufficient for this interaction. BRIZ1 and BRIZ2 co-purify after expression in tobacco leaves, which also requires the coiled-coil domain. BRIZ1 and BRIZ2 coding regions with substitutions in the RING domain are inactive in vitro and, after introduction, fail to complement their respective mutant lines. In our current model, BRIZ1 and BRIZ2 together are required for formation of a functional ubiquitin E3 ligase in vivo, and this complex is required for germination and early seedling growth.The ubiquitin pathway is a protein modification pathway that regulates many processes such as cell cycle, signal transduction, translational regulation, and chromatin remodeling (1-4). This pathway utilizes three enzymes for attachment of ubiquitin to its target. An E1 (ubiquitin-activating enzyme) forms a thioester bond with ubiquitin in an ATP-dependent manner. E1 then passes the attached ubiquitin to an E2 (ubiquitin-conjugating enzyme), which also forms a thioester bond with ubiquitin. Subsequently, the E3, also called a ubiquitin ligase, either accepts this activated ubiquitin before transfer to the substrate or serves as a scaffold to bring the substrate and E2-ubiquitin together. After ubiquitin is linked to the substrate, additional ubiquitin moieties can be conjugated to the previously attached ubiquitin, forming polyubiquitin chains. Ubiquitinated proteins have a number of fates, with the most characterized being degradation by the proteasome (5-7). Additionally, nonproteasomal fates have been described (8 -10).In the ubiquitin pathway, the E3 is an important contributor to substrate specificity. Currently, there are ϳ1300 E3 ligases predicted in the annotated proteome of Arabidopsis thaliana, which includes ϳ480 RING-type E3 ligases (11-13). The RING domain, consisting of eight conserved cysteine or histidine residues...

Plant cells are surrounded and protected by cell walls, which consist primarily of polymers such as cellulose, hemicelluloses, pectic polysaccharides, and the polyphenol lignin. One of the major hemicelluloses in the primary cell wall is xyloglucan (XyG), which consists of a b-1,4-glucan backbone containing xylosyl substituents. XyG attaches and partially covers the surface of cellulose microfibrils and is therefore thought to act as a spacer between microfibrils, reducing cellulose aggregation (Pauly et al., 2013).

17In Arabidopsis, development during flowering is coordinated by transport of the 18 hormone auxin mediated by polar-localized PIN-FORMED1 (AtPIN1). However 19Arabidopsis has lost a PIN clade sister to AtPIN1, Sister-of-PIN1 (SoPIN1), which is 20 conserved in flowering plants. We previously proposed that the AtPIN1 organ initiation 21 and vein patterning functions are split between the SoPIN1 and PIN1 clades in grasses. and interaction contribute to PIN functional specificity. 30

The ubiquitin system is essential for multiple hormone signaling pathways in plants. Here, we show that the Arabidopsis thaliana E3 ligase BRIZ, a heteromeric ligase that consists minimally of BRIZ1 and BRIZ2 proteins, functions in abscisic acid (ABA) signaling or response. briz1 and briz2 homozygous mutants either fail to germinate or emerge later than wild-type seedlings, with little cotyledon expansion or root elongation and no visible greening. Viability staining indicates that briz1 and briz2 embryos are alive but growth-arrested. Germination of briz mutants is improved by addition of the carotenoid biosynthetic inhibitor fluridone or gibberellic acid (GA3), and briz mutants have improved development in backgrounds deficient in ABA synthesis (gin1-3/aba2) or signaling (abi5-7). Endogenous ABA is not higher in briz2 seeds compared to wild-type seeds, and exogenous ABA does not affect BRIZ mRNAs in imbibed seeds. These results indicate that briz embryos are hypersensitive to ABA and that under normal growth conditions, BRIZ acts to suppress ABA signaling or response. ABA signaling and sugar signaling are linked, and we found that briz1 and briz2 mutants excised from seed coats are hypersensitive to sucrose. Although briz single mutants do not grow to maturity, we were able to generate mature briz2-3 abi5-7 double mutant plants that produced seeds. These seeds are more sensitive to exogenous sugar and are larger than seeds from sibling abi5-7 BRIZ2/briz2-3 plants, suggesting that BRIZ has a parental effect on seed development. From these data, we propose a model in which the BRIZ E3 ligase suppresses ABA responses during seed maturation and germination and early seedling establishment.

In Arabidopsis, development during flowering is coordinated by transport of the hormone auxin mediated by polar-localized PIN-FORMED1 (AtPIN1). However Arabidopsis has lost a PIN clade sister to AtPIN1, Sister-of-PIN1 (SoPIN1), which is conserved in flowering plants. We previously proposed that the AtPIN1 organ initiation and vein patterning functions are split between the SoPIN1 and PIN1 clades in grasses. Here we show that in the grass Brachypodium sopin1 mutants have organ initiation defects similar to Arabidopsis atpin1, while loss of PIN1 function in Brachypodium has little effect on organ initiation but alters stem growth. Heterologous expression of Brachypodium SoPIN1 and PIN1b in Arabidopsis provides further evidence of functional specificity. SoPIN1 but not PIN1b can mediate flower formation in null atpin1 mutants, although both can complement a missense allele. The behavior of SoPIN1 and PIN1b in Arabidopsis illustrates how membrane and tissue-level accumulation, transport activity, and interaction contribute to PIN functional specificity.