Project description:APETALA2/ethylene-responsive element binding protein (AP2/EREBP) transcription factors constitute one of the largest and most conserved gene families in plant, and play essential roles in growth, development and stress response. Except a few members, the AP2/EREBP family has not been characterized in Brachypodium distachyon, a model plant of Poaceae. We performed a genome-wide study of this family in B. distachyon by phylogenetic analyses, transactivation assays and transcript profiling. A total of 149 AP2/EREBP genes were identified and divided into four subfamilies, i.e., ERF (ethylene responsive factor), DREB (dehydration responsive element binding gene), RAV (related to ABI3/VP) and AP2. Tandem duplication was a major force in expanding B. distachyon AP2/EREBP (BdAP2/EREBP) family. Despite a significant expansion, genomic organizations of BdAP2/EREBPs were monotonous as the majority of them, except those of AP2 subfamily, had no intron. An analysis of transcription activities of several closely related and duplicated BdDREB genes showed their functional divergence and redundancy in evolution. The expression of BdAP2/EREBPs in different tissues and the expression of DREB/ERF subfamilies in B. distachyon, wheat and rice under abiotic stresses were investigated by next-generation sequencing and microarray profiling. Our results are valuable for further function analysis of stress tolerant AP2/EREBP genes in B. distachyon.
Project description:Brachypodium distachyon is a new model plant closely related to wheat and other cereals. In this study, we performed a comprehensive analysis of hormone-regulated genes in Brachypodium distachyon using RNA sequencing technology. Brachypodium distachyon seedlings were treated with eight phytohormones (auxin, cytokinine, brassinosteroid, gibberelline, abscisic acid, ethylene, jasmonate and salicylic acid) and two inhibitors, Brz220 (brassinosteroid biosynthesis inhibitor) and prohexadione (gibberelline biosynthesis inhibitor). The expressions of 1807 genes were regulated in a phytohormone-dependent manner. We compared the data with the phytohormone responses that have reported in rice. Transcriptional responses to hormones are conserved between Bracypodium and rice. Transcriptional regulation by brassinosteroid, gibberellin and ethylene was relatively weaker than those by other hormones. This is consistent with the data obtained from comprehensive analysis of hormone responses reported in Arabidopsis. Brachypodium and Arabidopsis also shared some common transcriptional responses to phytohormones. Alternatively, unique transcriptional responses to phytohormones were observed in Brachypodium. For example, the expressions of ACC synthase genes were up-regulated by auxin treatment in rice and Arabidopsis, but no orthologous ACC synthase gene was up-regulated in Brachypodium. Our results provide information useful to understand the diversity and similarity of hormone-regulated transcriptional responses between eudicots and monocots.
Project description:MADS-box genes are important transcription factors for plant development, especially floral organogenesis. Brachypodium distachyon is a model for biofuel plants and temperate grasses such as wheat and barley, but a comprehensive analysis of MADS-box family proteins in Brachypodium is still missing. We report here a genome-wide analysis of the MADS-box gene family in Brachypodium distachyon. We identified 57 MADS-box genes and classified them into 32 MIKC(c)-type, 7 MIKC*-type, 9 M?, 7 M? and 2 M? MADS-box genes according to their phylogenetic relationships to the Arabidopsis and rice MADS-box genes. Detailed gene structure and motif distribution were then studied. Investigation of their chromosomal localizations revealed that Brachypodium MADS-box genes distributed evenly across five chromosomes. In addition, five pairs of type II MADS-box genes were found on synteny blocks derived from whole genome duplication blocks. We then performed a systematic expression analysis of Brachypodium MADS-box genes in various tissues, particular floral organs. Further detection under salt, drought, and low-temperature conditions showed that some MADS-box genes may also be involved in abiotic stress responses, including type I genes. Comparative studies of MADS-box genes among Brachypodium, rice and Arabidopsis showed that Brachypodium had fewer gene duplication events. Taken together, this work provides useful data for further functional studies of MADS-box genes in Brachypodium distachyon.
Project description:BACKGROUND: A complete assembled genome sequence of wheat is not yet available. Therefore, model plant systems for wheat are very valuable. Brachypodium distachyon (Brachypodium) is such a system. The WRKY family of transcription factors is one of the most important families of plant transcriptional regulators with members regulating important agronomic traits. Studies of WRKY transcription factors in Brachypodium and wheat therefore promise to lead to new strategies for wheat improvement. RESULTS: We have identified and manually curated the WRKY transcription factor family from Brachypodium using a pipeline designed to identify all potential WRKY genes. 86 WRKY transcription factors were found, a total higher than all other current databases. We therefore propose that our numbering system (BdWRKY1-BdWRKY86) becomes the standard nomenclature. In the JGI v1.0 assembly of Brachypodium with the MIPS/JGI v1.0 annotation, nine of the transcription factors have no gene model and eleven gene models are probably incorrectly predicted. In total, twenty WRKY transcription factors (23.3%) do not appear to have accurate gene models. To facilitate use of our data, we have produced The Database of Brachypodium distachyon WRKY Transcription Factors. Each WRKY transcription factor has a gene page that includes predicted protein domains from MEME analyses. These conserved protein domains reflect possible input and output domains in signaling. The database also contains a BLAST search function where a large dataset of WRKY transcription factors, published genes, and an extensive set of wheat ESTs can be searched. We also produced a phylogram containing the WRKY transcription factor families from Brachypodium, rice, Arabidopsis, soybean, and Physcomitrella patens, together with published WRKY transcription factors from wheat. This phylogenetic tree provides evidence for orthologues, co-orthologues, and paralogues of Brachypodium WRKY transcription factors. CONCLUSIONS: The description of the WRKY transcription factor family in Brachypodium that we report here provides a framework for functional genomics studies in an important model system. Our database is a resource for both Brachypodium and wheat studies and ultimately projects aimed at improving wheat through manipulation of WRKY transcription factors.
Project description:The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.
Project description:BACKGROUND:Little is known about the potential of Brachypodium distachyon as a model for low temperature stress responses in Pooideae. The ice recrystallization inhibition protein (IRIP) genes, fructosyltransferase (FST) genes, and many C-repeat binding factor (CBF) genes are Pooideae specific and important in low temperature responses. Here we used comparative analyses to study conservation and evolution of these gene families in B. distachyon to better understand its potential as a model species for agriculturally important temperate grasses. RESULTS:Brachypodium distachyon contains cold responsive IRIP genes which have evolved through Brachypodium specific gene family expansions. A large cold responsive CBF3 subfamily was identified in B. distachyon, while CBF4 homologs are absent from the genome. No B. distachyon FST gene homologs encode typical core Pooideae FST-motifs and low temperature induced fructan accumulation was dramatically different in B. distachyon compared to core Pooideae species. CONCLUSIONS:We conclude that B. distachyon can serve as an interesting model for specific molecular mechanisms involved in low temperature responses in core Pooideae species. However, the evolutionary history of key genes involved in low temperature responses has been different in Brachypodium and core Pooideae species. These differences limit the use of B. distachyon as a model for holistic studies relevant for agricultural core Pooideae species.
Project description:The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticum. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. tumefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.
Project description:BACKGROUND AND AIMS: Cold is a major constraint for cereal cultivation under temperate climates. Winter-hardy plants interpret seasonal changes and can acquire the ability to resist sub-zero temperatures. This cold acclimation process is associated with physiological, biochemical and molecular alterations in cereals. Brachypodium distachyon is considered a powerful model system to study the response of temperate cereals to adverse environmental conditions. To date, little is known about the cold acclimation and freezing tolerance capacities of Brachypodium. The main objective of this study was to evaluate the cold hardiness of seven diploid Brachypodium accessions. METHODS: An integrated approach, involving monitoring of phenological indicators along with expression profiling of the major vernalization regulator VRN1 orthologue, was followed. In parallel, soluble sugars and proline contents were determined along with expression profiles of two COR genes in plants exposed to low temperatures. Finally, whole-plant freezing tests were performed to evaluate the freezing tolerance capacity of Brachypodium. KEY RESULTS: Cold treatment accelerated the transition from the vegetative to the reproductive phase in all diploid Brachypodium accessions tested. In addition, low temperature exposure triggered the gradual accumulation of BradiVRN1 transcripts in all accessions tested. These accessions exhibited a clear cold acclimation response by progressively accumulating proline, sugars and COR gene transcripts. However, whole-plant freezing tests revealed that these seven diploid accessions only have a limited capacity to develop freezing tolerance when compared with winter varieties of temperate cereals such as wheat and barley. Furthermore, little difference in terms of survival was observed among the accessions tested despite their previous classification as either spring or winter genotypes. CONCLUSIONS: This study is the first to characterize the freezing tolerance capacities of B. distachyon and provides strong evidence that some diploid accessions such as Bd21 have a facultative growth habit.
Project description:Members of plant WRKY gene family are ancient transcription factors that function in plant growth and development and respond to biotic and abiotic stresses. In our present study, we have investigated WRKY family genes in Brachypodium distachyon, a new model plant of family Poaceae. We identified a total of 86 WRKY genes from B. distachyon and explored their chromosomal distribution and evolution, domain alignment, promoter cis-elements, and expression profiles. Combining the analysis of phylogenetic tree of BdWRKY genes and the result of expression profiling, results showed that most of clustered gene pairs had higher similarities in the WRKY domain, suggesting that they might be functionally redundant. Neighbour-joining analysis of 301 WRKY domains from Oryza sativa, Arabidopsis thaliana, and B. distachyon suggested that BdWRKY domains are evolutionarily more closely related to O. sativa WRKY domains than those of A. thaliana. Moreover, tissue-specific expression profile of BdWRKY genes and their responses to phytohormones and several biotic or abiotic stresses were analysed by quantitative real-time PCR. The results showed that the expression of BdWRKY genes was rapidly regulated by stresses and phytohormones, and there was a strong correlation between promoter cis-elements and the phytohormones-induced BdWRKY gene expression.