Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis

Brachypodium distachyon is a promising model system for the structural and functional genomics of temperate grasses because of its physical, genetic and genome attributes. The sequencing of the inbred line Bd21 ( http://www.brachypodium.org ) started in 2007. However, a transformation method remains to be developed for the community standard line Bd21. In this article, a facile, efficient and rapid transformation system for Bd21 is described using Agrobacterium -mediated transformation of compact embryogenic calli (CEC) derived from immature embryos. Key features of this system include: (i) the use of the green fluorescent protein (GFP) associated with hygromycin selection for rapid identification of transgenic calli and plants; (ii) the desiccation of CEC after inoculation with Agrobacterium ; (iii) the utilization of Bd21 plants regenerated from tissue culture as a source of immature embryos; (iv) the control of the duration of the selection process; and (v) the supplementation of culture media with CuSO₄ prior to and during the regeneration of transgenic plants. Approximately 17% of CEC produced transgenic plants, enabling the generation of hundreds of T-DNA insertion lines per experiment. GFP expression was observed in primary transformed Bd21 plants (T₀) and their progeny (T₁). The Mendelian inheritance of the transgenes was confirmed. An adaptor-anchor strategy was developed for efficient retrieval of flanking sequence tags (FSTs) of T-DNA inserts, and the resulting sequences are available in public databases. The production of T-DNA insertion lines and the retrieval of associated FSTs reported here for the reference inbred line Bd21 will facilitate large-scale functional genomics research in this model system.     

A T-DNA mutation in the RNA helicase eIF4A confers a dose-dependent dwarfing phenotype in Brachypodium distachyon

In a survey of the BrachyTAG mutant population of Brachypodium distachyon, we identified a line carrying a T-DNA insertion in one of the two eukaryotic initiation factor 4A (eIF4A) genes present in the nuclear genome. The eif4a homozygous mutant plants were slow-growing, and exhibited reduced final plant stature due to a decrease in both cell number and cell size, consistent with roles for eIF4A in both cell division and cell growth. Hemizygous plants displayed a semi-dwarfing phenotype, in which stem length was reduced but leaf length was normal. Linkage between the insertion site and phenotype was confirmed, and we show that the level of eIF4A protein is strongly reduced in the mutant. Transformation of the Brachypodium homozygous mutant with a genomic copy of the Arabidopsis eIF4A-1 gene partially complemented the growth phenotype, indicating that gene function is conserved between mono- and dicotyledonous species. This study identifies eIF4A as a novel dose-dependent regulator of stem elongation, and demonstrates the utility of Brachypodium as a model for grass and cereals research.     

Cytochemistry of pollen development in Brachypodium distachyon

Brachypodium distachyon is a widely recognized model plant belonging to subfamily Pooideae with a sequenced genome. To gain a better understanding of the male reproductive development in B. distachyon we examined pollen morphology and cytochemical changes of microspore cytoplasm from pollen mother cell stage to mature pollen using light, fluorescent and scanning electron microscopy. Our results show that B. distachyon exhibits a typical monocot-type pollen ontogeny. Meiosis in the pollen mother cells is accomplished by successive cytokinesis generating isobilateral tetrads. Cytochemical examination indicated that microspore cytoplasm contains variable amounts of insoluble carbohydrates and proteins at different developmental stages. Deposition of starch in the cytoplasm of microspores starts at the bicellular stage and continues till the mature pollen stage. The formation of the exine wall progresses by the deposition of sporopollenin from the tapetum layer of the anther. The mature pollen is trinucleate, spheroidal in shape and possesses a single pore with an annulus and operculum. The exine pattern is smooth and of granular type.     

Natural variation of drought response in Brachypodium distachyon

Brachypodium distachyon (Brachypodium) is a temperate wild grass species and is a powerful model system for studying grain, energy, forage and turf grasses. Exploring the natural variation in the drought response of Brachypodium provides an important basis for dissecting the genetic network of drought tolerance. Two experiments were conducted in a greenhouse to assess the drought tolerance of 57 natural populations of Brachypodium. Principle component analysis revealed that reductions in chlorophyll fluorescence (Fv/Fm) and leaf water content (LWC) under drought stress explained most of the phenotypic variation, which was used to classify the tolerant and susceptible accessions. Four groups of accessions differing in drought tolerance were identified, with 3 tolerant, 16 moderately tolerant, 32 susceptible and 6 most susceptible accessions. The tolerant group had little leaf wilting and fewer reductions in Fv/Fm and LWC, while the most susceptible groups showed severe leaf wilting and more reductions in Fv/Fm and LWC. Drought stress increased total water soluble sugar (WSS) concentration, but no differences in the increased WSS were found among different groups of accessions. The large phenotypic variation of Brachypodium in response to drought stress can be used to identify genes and alleles important for the complex trait of drought tolerance.     

Proteomic and metabolic disturbances in lignin-modified Brachypodium distachyon

Lignin biosynthesis begins with the deamination of phenylalanine and tyrosine (Tyr) as a key branch point between primary and secondary metabolism in land plants. Here, we used a systems biology approach to investigate the global metabolic responses to lignin pathway perturbations in the model grass Brachypodium distachyon. We identified the lignin biosynthetic protein families and found that ammonia-lyases (ALs) are among the most abundant proteins in lignifying tissues in grasses. Integrated metabolomic and proteomic data support a link between lignin biosynthesis and primary metabolism mediated by the ammonia released from ALs that is recycled for the synthesis of amino acids via glutamine. RNA interference knockdown of lignin genes confirmed that the route of the canonical pathway using shikimate ester intermediates is not essential for lignin formation in Brachypodium, and there is an alternative pathway from Tyr via sinapic acid for the synthesis of syringyl lignin involving yet uncharacterized enzymatic steps. Our findings support a model in which plant ALs play a central role in coordinating the allocation of carbon for lignin synthesis and the nitrogen available for plant growth. Collectively, these data also emphasize the value of integrative multiomic analyses to advance our understanding of plant metabolism.

Ammonia-lyases play a key role in coordinating the allocation of carbon for lignin synthesis and nitrogen availability for plant growth.     

Plant regeneration and micropropagation of Brachypodium distachyon

Brachypodium distachyon (L.) P. Beauv. has several features of its genome and growth habit reminiscent of Arabidopsis thaliana (L.) Heyn. that may allow it to be developed as a model molecular genetic system representative of the temperate grasses. In order for B. distachyon to be exploited in this way, it will be necessary to develop tissue culture procedures. This report details initial studies of the characteristics of mature seed-derived callus and the production of fertile plants from callus of three ecotypes of B. distachyon. Optimum development of embryogenic callus occurred on LS (Linsmaier & Skoog 1965) and N6 (Chu et al. 1975) media containing 3.0% w/v sucrose and 11.25 M (2.5 mg l^-1) 2,4-dichlorophenoxyacetic acid. Plants were recovered at a high frequency from embryogenic callus of ecotype B200 maintained on growth regulator-free N6 medium and were easy to establish in compost. A method was also developed for the in vitro clonal propagation of shoots using MS (Murashige & Skoog 1962) medium supplemented with 4 to 13 M (1.0 to 3.0 mg l^-1) benzyladenine. It was concluded that B. distachyon performed well in tissue culture and was suitable for further studies aimed at genetic transformation and its continued development as a model molecular genetic system.Abbreviations BA benzyladenine - 2,4-d dichlorophenoxyacetic acid - LS Linsmaier and Skoog (1965) - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid - MSO growth regulator-free Murashige & Skoog (1962)     

以色列二穗短柄草遗传多样性分析

采用ISJ(Intron-Splice Junction,内含子切接点引物)分子标记方法对29份源自以色列的二穗短柄草(Brachypodium distachyon)及7份其他地区的二穗短柄草材料进行遗传多样性分析.结果表明:8个ISJ引物和10个引物组合共扩增出稳定条带145条,其中93条为多态性条带,多态性比率为64.58%.聚类分析将36份供试材料分成3大类,二倍体类型的二穗短柄草,六倍体类型源自以色列以外地区的二穗短柄草及来自以色列的六倍体二穗短柄草且以色列二穗短柄草材料进一步聚成不同生态环境下的两个亚类.主坐标分析结果与聚类分析结果基本吻合.研究结果为二穗短柄草的起源与分布提供了一定的理论依据,并为分子标记技术在二穗短柄草种质资源收集、评价和鉴定等方面的利用奠定了基础.     

Globulins are the main seed storage proteins in Brachypodium distachyon

Brachypodium distachyon is being developed as a model system to study temperate cereals and forage grasses. We have begun to investigate its utility to understand seed development and grain filling by identifying the major seed storage proteins in a diploid accession Bd21. With the use of ID SDS-PAGE and mass spectrometry we detected seven major storage protein bands, six of which were identified as globulins. A subset of the major seed proteins isolated from three hexaploid accessions, Bd4, Bd14 and Bd17 were also identified as globulins. Several Brachypodium cDNAs clones encoding globulin were completely sequenced. Two types of globulin genes were identified, Bd.glo1 and Bd.glo2, which are similar to maize 7S and oat 12S globulins, respectively. The derived polypeptide sequences of the globulins contain a typical signal peptide sequence in their polypeptide N-termini and two cupin domains. Bd.glo1 is encoded by a single copy gene, whereas, Bd.glo2 belongs to a gene family.     

Highly-expressed polyamine oxidases catalyze polyamine back conversion in Brachypodium distachyon

Journal of Plant Research - To understand the polyamine (PA) catabolic pathways in Brachypodium distachyon, we focused on the flavin-containing polyamine oxidase enzymes (PAO), and characterized...     

Recent Progress in Model Grass Brachypodium distachyon (Poaceae)

Brachypodium distachyon, recently developed model system for temperate grasses, exhibited many traits with cereal crops and proposed to be an experimental system to access the biological approach. These traits have shown a surprised degree of phenotypic variation in many collected accessions. Like some important economical cereals, Bdistachyon also belongs to subfamily Pooideae, which make it become an unquestionable model system to research the economically important crops, such as wheat, barley and several potential biofuel plants. Recently, genome sequence and annotation of Bdistachyon has been finished. Associated with the development of the functional genomics and other experimental resources establishment, Bdistachyon will provide a key resource for improving cereal crops and facilitate the approach of sequence analyze gene expression and functional resources available for a variety of species. In this article we review and assess the current progress of Bdistachyon as a model system and then focus specifically on recent studies of comparative genomics, biological improvement, transformation and T DNA mutations.     

Phylogenetic and expression analysis of histone acetyltransferases in Brachypodium distachyon

Histone acetylation is an important post-translational modification in eukaryotes and is regulated by two antagonistic enzymes, namely histone acetyltransferase (HAT) and histone deacetylase (HDAC). However, little has been done on the HAT superfamily in Brachypodium distachyon (B. distachyon), a new model plant of Poaceae. In this study, eight HATs were identified from B. distachyon and classified into four major families. Subcellular localization analysis showed that a majority of BdHATs were predominantly localized in the nucleus. Syntenic and phylogenetic analysis indicated there may be two common ancestral CREB-binding protein (p300/CBP, HAC) genes prior to the separation of monocots and dicots. Expression analysis revealed that the potential roles of BdHATs in B. distachyon development and responses to four abiotic stresses. Protein-protein network analysis identified some potential interactive genes with BdHATs. Thus, our results will provide solid basis for further study the function of HAT genes in B. distachyon and other monocot plants.     

Brachypodium distachyon: making hay with a wild grass

Brachypodium distachyon is a wild grass with a short life cycle. Although it is related to small grain cereals such as wheat, its genome is only a fraction of the size. A draft genome sequence is currently available, and molecular and genetic tools have been developed for transformation, mutagenesis and gene mapping. Accessions collected from across its ancestral range show a surprising degree of phenotypic variation in many traits, including those implicated in domestication of the cereals. Thus, given its rapid cycling time and ease of cultivation, Brachypodium will be a useful model for investigating problems in grass biology.     

短柄草染色体酶解法制片技术

以六倍体短柄草为研究材料,对短柄草的染色体制片方法进行了优化,建立了一种改进的短柄草染色体酶解制片方法。试验结果表明,以45%醋酸固定液固定根尖、酶解时间2h可以获得最佳的根尖染色体制片。此方法不仅可以得到分散良好的有丝分裂中期分裂相,而且还缩短了酶解的时间,提高了制片的效率。     

Brachypodium distachyon grain: characterization of endosperm cell walls

The wild grass Brachypodium distachyon has been proposed as an alternative model species for temperate cereals. The present paper reports on the characterization of B. distachyon grain, placing emphasis on endosperm cell walls. Brachypodium distachyon is notable for its high cell wall polysaccharide content that accounts for ～52% (w/w) of the endosperm in comparison with 2-7% (w/w) in other cereals. Starch, the typical storage polysaccharide, is low [<10% (w/w)] in the endosperm where the main polysaccharide is (1-3) (1-4)-β-glucan [40% (w/w) of the endosperm], which in all likelihood plays a role as a storage compound. In addition to (1-3) (1-4)-β-glucan, endosperm cells contain cellulose and xylan in significant amounts. Interestingly, the ratio of ferulic acid to arabinoxylan is higher in B. distachyon grain than in other investigated cereals. Feruloylated arabinoxylan is mainly found in the middle lamella and cell junction zones of the storage endosperm, suggesting a potential role in cell-cell adhesion. The present results indicate that B. distachyon grains contain all the cell wall polysaccharides encountered in other cereal grains. Thus, due to its fully sequenced genome, its short life cycle, and the genetic tools available for mutagenesis/transformation, B. distachyon is a good model to investigate cell wall polysaccharide synthesis and function in cereal grains.