Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon

Background

The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using parallel analysis of RNA ends (PARE) data is lacking in B. distachyon.

Results

B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset for analyzing small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner.

Conclusions

B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants.     

Genome-wide exploration of the molecular evolution and regulatory network of mitogen-activated protein kinase cascades upon multiple stresses in Brachypodium distachyon

Background

Brachypodium distachyon is emerging as a widely recognized model plant that has very close relations with several economically important Poaceae species. MAPK cascade is known to be an evolutionarily conserved signaling module involved in multiple stresses. Although the gene sequences of MAPK and MAPKK family have been fully identified in B. distachyon, the information related to the upstream MAPKKK gene family especially the regulatory network among MAPKs, MAPKKs and MAPKKKs upon multiple stresses remains to be understood.

Results

In this study, we have identified MAPKKKs which belong to the biggest gene family of MAPK cascade kinases. We have systematically investigated the evolution of whole MAPK cascade kinase gene family in terms of gene structures, protein structural organization, chromosomal localization, orthologs construction and gene duplication analysis. Our results showed that most BdMAPK cascade kinases were located at the low-CpG-density region, and the clustered members in each group shared similar structures of the genes and proteins. Synteny analysis showed that 62 or 21 pairs of duplicated orthologs were present between B. distachyon and Oryza sativa, or between B. distachyon and Arabidopsis thaliana respectively. Gene expression data revealed that BdMAPK cascade kinases were rapidly regulated by stresses and phytohormones. Importantly, we have constructed a regulation network based on co-expression patterns of the expression profiles upon multiple stresses performed in this study.

Conclusions

BdMAPK cascade kinases were involved in the signaling pathways of multiple stresses in B. distachyon. The network of co-expression regulation showed the most of duplicated BdMAPK cascade kinase gene orthologs demonstrated their convergent function, whereas few of them developed divergent function in the evolutionary process. The molecular evolution analysis of identified MAPK family genes and the constructed MAPK cascade regulation network under multiple stresses provide valuable information for further investigation of the functions of BdMAPK cascade kinase genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1452-1) contains supplementary material, which is available to authorized users.     

An efficient method for transient gene expression in monocots applied to modify the Brachypodium distachyon cell wall

Background

Agrobacterium-mediated transformation is widely used to produce insertions into plant genomes. There are a number of well-developed Agrobacterium-mediated transformation methods for dicotyledonous plants, but there are few for monocotyledonous plants.

Methods

Three hydrolase genes were transiently expressed in Brachypodium distachyon plants using specially designed vectors that express the gene product of interest and target it to the plant cell wall. Expression of functional hydrolases in genotyped plants was confirmed using western blotting, activity assays, cell wall compositional analysis and digestibility tests.

Key Results

An efficient, new, Agrobacterium-mediated approach was developed for transient gene expression in the grass B. distachyon, using co-cultivation of mature seeds with bacterial cells. This method allows transformed tissues to be obtained rapidly, within 3–4 weeks after co-cultivation. Also, the plants carried transgenic tissue and maintained transgenic protein expression throughout plant maturation. The efficiency of transformation was estimated at around 5 % of initially co-cultivated seeds. Application of this approach to express three Aspergillus nidulans hydrolases in the Brachypodium cell wall successfully confirmed its utility and resulted in the expected expression of active microbial proteins and alterations of cell wall composition. Cell wall modifications caused by expression of A. nidulans α-arabinofuranosidase and α-galactosidase increased the biodegradability of plant biomass.

Conclusions

This newly developed approach is a quick and efficient technique for expressing genes of interest in Brachypodium plants, which express the gene product throughout development. In the future, this could be used for broad functional genomics studies of monocots and for biotechnological applications, such as plant biomass modification for biofuel production.     

Characterization of the caleosin gene family in the Triticeae

Background

The caleosin genes encode proteins with a single conserved EF hand calcium-binding domain and comprise small gene families found in a wide range of plant species. Some members of the gene family have been shown to be upregulated by environmental stresses including low water availability and high salinity. Caleosin 3 from wheat has been shown to interact with the α-subunit of the heterotrimeric G proteins, and to act as a GTPase activating protein (GAP). This study characterizes the size and diversity of the gene family in wheat and related species and characterizes the differential tissue-specific expression of members of the gene family.

Results

A total of 34 gene family members that belong to eleven paralogous groups of caleosins were identified in the hexaploid bread wheat, T. aestivum. Each group was represented by three homeologous copies of the gene located on corresponding homeologous chromosomes, except the caleosin 10, which has four gene copies. Ten gene family members were identified in diploid barley, Hordeum vulgare, and in rye, Secale cereale, seven in Brachypodium distachyon, and six in rice, Oryza sativa. The analysis of gene expression was assayed in triticale and rye by RNA-Seq analysis of 454 sequence sets and members of the gene family were found to have diverse patterns of gene expression in the different tissues that were sampled in rye and in triticale, the hybrid hexaploid species derived from wheat and rye. Expression of the gene family in wheat and barley was also previously determined by microarray analysis, and changes in expression during development and in response to environmental stresses are presented.

Conclusions

The caleosin gene family had a greater degree of expansion in the Triticeae than in the other monocot species, Brachypodium and rice. The prior implication of one member of the gene family in the stress response and heterotrimeric G protein signaling, points to the potential importance of the caleosin gene family. The complexity of the family and differential expression in various tissues and under conditions of abiotic stress suggests the possibility that caleosin family members may play diverse roles in signaling and development that warrants further investigation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-239) contains supplementary material, which is available to authorized users.     

Comparative cytogenetic analysis of the genomes of the model grass Brachypodium distachyon and its close relatives

Background and Aims

Brachypodium is a small genus of temperate grasses that comprises 12–15 species. Brachypodium distachyon is now well established as a model species for temperate cereals and forage grasses. In contrast to B. distachyon, other members of the genus have been poorly investigated at the chromosome level or not at all.

Methods

Twenty accessions comprising six species and two subspecies of Brachypodium were analysed cytogenetically. Measurements of nuclear genome size were made by flow cytometry. Chromosomal localization of 18–5·8–25S rDNA and 5S rDNA loci was performed by dual-colour fluorescence in situ hybridization (FISH) on enzymatically digested root-tip meristematic cells. For comparative phylogenetic analyses genomic in situ hybridization (GISH) applied to somatic chromosome preparations was used.

Key Results

All Brachypodium species examined have rather small genomes and chromosomes. Their chromosome numbers and genome sizes vary from 2n = 10 and 0·631 pg/2C in B. distachyon to 2n = 38 and 2·57 pg/2C in B. retusum, respectively. Genotypes with 18 and 28 chromosomes were found among B. pinnatum accessions. GISH analysis revealed that B. pinnatum with 28 chromosomes is most likely an interspecific hybrid between B. distachyon (2n = 10) and B. pinnatum (2n = 18). Two other species, B. phoenicoides and B. retusum, are also allopolyploids and B. distachyon or a close relative seems to be one of their putative ancestral species. In chromosomes of all species examined the 45S rDNA loci are distally distributed whereas loci for 5S rDNA are pericentromeric.

Conclusions

The increasing significance of B. distachyon as a model grass emphasizes the need to understand the evolutionary relationships in the genus Brachypodium and to ensure consistency in the biological nomenclature of its species. Modern molecular cytogenetic techniques such as FISH and GISH are suitable for comparative phylogenetic analyses and may provide informative chromosome- and/or genome-specific landmarks.     

Alien introgression in the grasses Lolium perenne (perennial ryegrass) and Festuca pratensis (meadow fescue): the development of seven monosomic substitution lines and their molecular and cytological characterization

Background and Aims

To address the issues associated with food security, environmental change and bioenergy in the context of crop plants, the production, identification and evaluation of novel plant phenotypes is fundamental. One of the major routes to this end will be wide hybridization and introgression breeding. The transfer of chromosomes and chromosome segments between related species (chromosome engineering or alien introgression) also provides an important resource for determining the genetic control of target traits. However, the realization of the full potential of chromosome engineering has previously been hampered by the inability to identify and characterize interspecific introgressions accurately.

Methods

Seven monosomic substitution lines have been generated comprising Festuca pratensis as the donor species and Lolium perenne as the recipient. Each of the seven lines has a different L. perenne chromosome replaced by the homoeologous F. pratensis chromosome (13 L. perenne + 1 F. pratensis chromosome). Molecular markers and genomic in situ hybridization (GISH) were used to assign the F. pratensis chromosomes introgressed in each of the monosomic substitutions to a specific linkage group. Cytological observations were also carried out on metaphase I of meiosis in each of the substitution lines.

Results

A significant level of synteny was found at the macro-level between L. perenne and F. pratensis. The observations at metaphase I revealed the presence of a low level of interspecific chromosomal translocations between these species.

Discussion

The isolation of the seven monosomic substitution lines provides a resource for dissecting the genetic control of important traits and for gene isolation. Parallels between the L. perenne/F. pratensis system and the Pooideae cereals such as wheat, barley, rye, oats and the model grass Brachypodium distachyon present opportunities for a comparison across the species in terms of genotype and phenotype.     

Genome-wide analysis of alternative splicing of pre-mRNA under salt stress in Arabidopsis

Background

Alternative splicing (AS) of precursor mRNA (pre-mRNA) is an important gene regulation process that potentially regulates many physiological processes in plants, including the response to abiotic stresses such as salt stress.

Results

To analyze global changes in AS under salt stress, we obtained high-coverage (~200 times) RNA sequencing data from Arabidopsis thaliana seedlings that were treated with different concentrations of NaCl. We detected that ~49% of all intron-containing genes were alternatively spliced under salt stress, 10% of which experienced significant differential alternative splicing (DAS). Furthermore, AS increased significantly under salt stress compared with under unstressed conditions. We demonstrated that most DAS genes were not differentially regulated by salt stress, suggesting that AS may represent an independent layer of gene regulation in response to stress. Our analysis of functional categories suggested that DAS genes were associated with specific functional pathways, such as the pathways for the responses to stresses and RNA splicing. We revealed that serine/arginine-rich (SR) splicing factors were frequently and specifically regulated in AS under salt stresses, suggesting a complex loop in AS regulation for stress adaptation. We also showed that alternative splicing site selection (SS) occurred most frequently at 4 nucleotides upstream or downstream of the dominant sites and that exon skipping tended to link with alternative SS.

Conclusions

Our study provided a comprehensive view of AS under salt stress and revealed novel insights into the potential roles of AS in plant response to salt stress.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-431) contains supplementary material, which is available to authorized users.