Ultrastructure of microsporogenesis and microgametogenesis in Campsis radicans (L.) Seem. (Bignoniaceae)

In the present study, microsporogenesis, microgametogenesis and pollen wall ontogeny in Campsis radicans (L.) Seem. were studied from sporogenous cell stage to mature pollen using transmission electron microscopy. To observe the ultrastructural changes that occur in sporogenous cells, microspores and pollen through progressive developmental stages, anthers at different stages of development were fixed and embedded in Araldite. Microspore and pollen development in C. radicans follows the basic scheme in angiosperms. Microsporocytes secrete callose wall before meiotic division. Meiocytes undergo meiosis and simultaneous cytokinesis which result in the formation of tetrads mostly with a tetrahedral arrangement. After the development of free and vacuolated microspores, respectively, first mitotic division occurs and two-celled pollen grain is produced. Pollen grains are shed from the anther at two-celled stage. Pollen wall formation in C. radicans starts at tetrad stage by the formation of exine template called primexine. By the accumulation of electron dense material, produced by microspore, in the special places of the primexine, first of all protectum then columellae of exine elements are formed on the reticulate-patterned plasma membrane. After free microspore stage, exine development is completed by the addition of sporopollenin from tapetum. Formation of intine layer of pollen wall starts at the late vacuolated stage of pollen development and continue through the bicellular pollen stage.     

T-DNA mutagenesis in Brachypodium distachyon

During the past decade, Brachypodium distachyon has emerged as an attractive experimental system and genomics model for grass research. Numerous molecular tools and genomics resources have already been developed. Functional genomics resources, including mutant collections, expression/tiling microarray, mapping populations, and genome re-sequencing for natural accessions, are rapidly being developed and made available to the community. In this article, the focus is on the current status of systematic T-DNA mutagenesis in Brachypodium. Large collections of T-DNA-tagged lines are being generated by a community of laboratories in the context of the International Brachypodium Tagging Consortium. To date, >13?000 lines produced by the BrachyTAG programme and USDA-ARS Western Regional Research Center are available by online request. The utility of these mutant collections is illustrated with some examples from the BrachyTAG collection at the John Innes Centre-such as those in the eukaryotic initiation factor 4A (eIF4A) and brassinosteroid insensitive-1 (BRI1) genes. A series of other mutants exhibiting growth phenotypes is also presented. These examples highlight the value of Brachypodium as a model for grass functional genomics.     

Ultrastructure of microsporogenesis and microgametogenesis inArabidopsis thaliana (L.) Heynh. ecotype Wassilewskija (Brassicaceae)

Summary The process of microsporogenesis and microgametogenesis was studied at the ultrastructural level in wild-typeArabidopsis thaliana ecotype Wassilewskija to provide a basis for comparison with nuclear male-sterile mutants of the same ecotype. From the earliest stage studied to mature pollen just prior to anther dehiscence, microsporocyte/microspore/pollen development follows the general pattern seen in most angiosperms. The tapetum is of the secretory type with loss of the tapetal cell walls beginning at about the time of microsporocyte meiosis. Wall loss exhibits polarity with the tapetal protoplasts becoming located at a distance from the inner tangential walls first, followed by an increase in distance from the radial walls beginning at the interior edge and progressing outward. The inner tangential and radial tapetal walls are completely degenerated by the microspore tetrad stage. Unlike other members of the Brassicaceae that have been studied, the tapetal cells ofA. thaliana Wassilewskija also lose their outer tangential walls, and secretion occurs from all sides of the cells. Exine wall precursors are secreted from the tapetal cells in a process that appears to involve dilation of individual endoplasmic reticulum cisternae that fuse with the tapetal cell membrane and release their contents into the locule. Following completion of the exine, the tapetal cell plastids develop membranebound inclusions with osmiophilic and electron-transparent regions. The plastids undergo ultrastructural changes that suggest breakdown of the inclusion membranes followed by release of their contents into the locule prior to the complete degeneration of the tapetal cells.     

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)     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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...