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.     

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.     

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: 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.     

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.     

Evolution and taxonomic split of the model grass Brachypodium distachyon

Background and Aims

Brachypodium distachyon is being widely investigated across the world as a model plant for temperate cereals. This annual plant has three cytotypes (2n =  10, 20, 30) that are still regarded as part of a single species. Here, a multidisciplinary study has been conducted on a representative sampling of the three cytotypes to investigate their evolutionary relationships and origins, and to elucidate if they represent separate species.

Methods

Statistical analyses of 15 selected phenotypic traits were conducted in individuals from 36 lines or populations. Cytogenetic analyses were performed through flow cytometry, fluorescence in situ hybridization (FISH) with genomic (GISH) and multiple DNA sequences as probes, and comparative chromosome painting (CCP). Phylogenetic analyses were based on two plastid (ndhF, trnLF) and five nuclear (ITS, ETS, CAL, DGAT, GI) genes from different Brachypodium lineages, whose divergence times and evolutionary rates were estimated.

Key Results

The phenotypic analyses detected significant differences between the three cytotypes and demonstrated stability of characters in natural populations. Genome size estimations, GISH, FISH and CCP confirmed that the 2n = 10 and 2n = 20 cytotypes represent two different diploid taxa, whereas the 2n = 30 cytotype represents the allotetraploid derived from them. Phylogenetic analysis demonstrated that the 2n = 20 and 2n = 10 cytotypes emerged from two independent lineages that were, respectively, the maternal and paternal genome donors of the 2n = 30 cytotype. The 2n = 20 lineage was older and mutated significantly faster than the 2n = 10 lineage and all the core perennial Brachypodium species.

Conclusions

The substantial phenotypic, cytogenetic and molecular differences detected among the three B. distachyon sensu lato cytotypes are indicative of major speciation processes within this complex that allow their taxonomic separation into three distinct species. We have kept the name B. distachyon for the 2n = 10 cytotype and have described two novel species as B. stacei and B. hybridum for, respectively, the 2n = 20 and 2n = 30 cytotypes.     

Lipid profiling of the model temperate grass, Brachypodium distachyon

Lipids are essential metabolites in cells and they fulfil a variety of functions, including structural components of cellular membranes, energy storage, cell signalling, and membrane trafficking. In plants, changes in lipid composition have been observed in diverse responses ranging from abiotic and biotic stress to organogenesis. Knowledge of the lipid composition is an important first step towards understanding the function of lipids in any given biological system. As Brachypodium distachyon is emerging as the model species for temperate grass research, it is therefore fundamentally important to gain insights of its lipid composition. We used HPLC-coupled with tandem mass spectrometry to profile and quantify levels of sphingolipids and glycerophospholipids in shoots and undifferentiated cells in suspension cultures of B. distachyon. A total of 123 lipids belonging to 10 classes were identified and quantified. Our results showed that there are differences in lipid profiles and levels of individual lipid species between shoots and undifferentiated cells in suspension cultures. Additionally, we showed that 4-sphingenine (d18:1^??4) is the main unsaturated dihydroxy-long chain base (LCB) in B. distachyon, and we were unable to detect d18:1^??8, which is the main unsaturated dihydroxy-LCB in the model dicotyledonous species, Arabidopsis thaliana. This work serves as the first step towards a comprehensive characterization of the B. distachyon lipidome that will complement future biochemical studies.     

Natural Variation of Flowering Time and Vernalization Responsiveness in Brachypodium distachyon

Dedicated bioenergy crops require certain characteristics to be economically viable and environmentally sustainable. Perennial grasses, which can provide large amounts of biomass over multiple years, are one option being investigated to grow on marginal agricultural land. Recently, a grass species (Brachypodium distachyon) has been developed as a model to better understand grass physiology and ecology. Here, we report on the flowering time variability of natural Brachypodium accessions in response to temperature and light cues. Changes in both environmental parameters greatly influence when a given accession will flower, and natural Brachypodium accessions broadly group into winter and spring annuals. Similar to what has been discovered in wheat and barley, we find that a portion of the phenotypic variation is associated with changes in expression of orthologs of VRN genes, and thus, VRN genes are a possible target for modifying flowering time in grass family bioenergy crops.