Brachypodium distachyon has been developed as a monocot model plant for temperate grasses and bioenergy crops. Although B. distachyon research is moving forward rapidly, the study of photoresponses has not been explored. To extend our knowledge of responses to light in monocots, we performed photoresponse analysis of B. distachyon using two inbred lines, Bd21 and Bd21-3. In this study, we first compared growing phenotypes between the two lines and investigated coleoptile and primary leaf growths under dark, far-red, red, and white light conditions. The results showed that the growth of the two lines were similar until tillering stage, but other developmental stages from heading to senescence were much delayed in Bd21-3, which resulted in increased height and tiller numbers. Under different light conditions, primary leaf lengths were kept increasing during the growth period, whereas the coleoptile extension was inhibited 4 to 7 days after growth depending on the light conditions applied. These results suggest that the responses to light in B. distachyon can be examined by measuring coleoptile lengths approximately 7 days after seedling growth. Moreover, we selected light-responsive genes known in Arabidopsis thaliana, such as chlorophyll A/B binding protein (CAB), light-harvesting chlorophyll binding protein (Lhcb) and chalcone synthase (CHS), and confirmed their light-induced gene expression in B. distachyon. Therefore, the present study suggests that the inhibition of coleoptile growth can be used as the parameter to analyze photoresponses in the monocot model plant, and also provide the reference genes whose expression is induced by far-red and red light treatment.

Space has many distinguishable characteristics from earth such as strong cosmic radiation, microgravity, supervaccum and weak magnetic field. For this reason, space environments can be used an efficient mutagen for plant breeding nowadays. To identify the affected genes by condition in space with outer space, Brachypodium seeds were placed in the Russia Segment (RS) Biorisk module of International Space Station (ISS). Brachypodium distachyon is a model system for temperature grass, because they represent the characteristics for annual winter grass. Seeds and organs of plants carried by satellite or spacecraft to space can be genetically mutated by exposing space environment. We performed a duplicated RNA sequencing to profile the differentially expressed genes. As a results, about 700 genes were upregulated and 250 genes were downregulated by cosmic environments, respectively. In the molecular function category, protein kinase and transcription activity related genes were upregulated. Among the many transcription factors (TFs), stress related TFs such as ERF, NAC and WRKY were differentially expressed in space exposed samples. In the future, their expression will be identified by using qRT_PCR.

It is necessary to alleviate environmental and economic disadvantages of fossil fuels for global warming. Among the conceivable options, the use of plant biomass for the production of bioethanol is considered as a potential alternative for fossil fuels. Plant biomass that contains lignocellulose for bioethanol production has recently emerged as biofuel feedstock because of its sustainable and environment-friendly properties. However, lignin inhibits the hydrolysis process and the lignin recalcitrance in ethanol conversion remains in a problem. The attempt for down-regulating enzymes involved in lignin biosynthesis is one of attractive strategy to reduce the lignin contents. Recently, Brachypodium distachyon has been proposed as an alternative monocotyledon model species. The close phylogenetic relationship of Brachypodium with other grasses suggests that the Brachypodium may be useful for structural and functional genomic studies in these species. Brachypodium, standard line Bd21, was subjected to irradiation at doses of 50, 100, 150, 200, and 250 Gy. Phenotypes were investigated using M0:2 population. Through histochemical analysis using phloroglucinol, 25 M2 putative lignin deficient mutants were selected. Depend on the phenotypic and histochemical data, mutants were selected and used for measuring lignin content. Total lignin content was measured using the acetyl bromide (AcBr). Mutant #142-3-1 contains 16.9 (mg/g dry cell wall) of total lignin and the lignin level was significantly reduced (87.9%) compared to wild-type (19.23 mg/g dry cell wall). Additionally, Mutant line #2259-1-2 reduced lignin level at 94.4% (18.15 mg/g dry cell wall) in comparison to wild-type. The enzymatic hydrolyses in lignin deficient lines have been performing with the time courses. Lignin composition, cell wall carbohydrates, and genetic analysis in mutant lines will be discussed.

such as small plant size, small genome, short life cycle, self-fertility, and etc. Moreover, Brachypodium standard line Bd21 had already been sequenced and the data of which now have been available to the public. The development of next-generation sequencing technologies has allowed the discovery of large numbers of genome-wide DNA polymorphisms. Brachypodium standard line Bd21 was exposed to chronic gamma radiation. M2 1376-1 line was less stained with phloroglucinol compared to wild-type, which indicated reduced lingin content. Also, it’s filial generations showed dwarf phenotype. Genomic DNA was extracted from the M3 plant and was used to construct a whole-genome re-sequencing library for using Illumina HiSeq2500. The trimmed reads were aligned to the Brachypodium reference genome sequence (http:// www.phytozome.net). SICKLE, BWA, and Picard were used for accurate variant detection. More than 110 M reads were generated and 96.53% of them were mapped. This represents ~35 fold coverage. As a result, mutant specific SNVs, Insert/Deletions, and non-synonymous mutations were obtained. Moreover, non-synonymous mutations were identified from 5 lignin biosynthesis genes (Bradi1g31320, Bradi3g52350, Bradi5g21550, Bradi3g22980, Bradi5g14720). The obtained results will be incorporated in development of biofuel crops.

Brachypodium distachyon is a temperate annual grass that has a short life cycle, a small genome size, self fertility, and a small physical stature. The relationship with major cereal crop including wheat, Brachypodium is considered as a monocot model plant. Recently, the cell wall composition of Brachypodium is reported closely related with maize and Miscanthus giganteus. Therefore, Brachypodium is emrging as a powerful model plant for bioethanol production. Here, Brachypodium was chronically irradiated with the doses of 50 Gy, 100 Gy, 150 Gy, 200Gy, 250Gy, and 300 Gy. Plant height and fresh weight were observed dosage-dependent negative effect. However, tiller number and internode diameter were found to be increased their value as compared to control. The cell wall yield showed a decreased tendency with dosage-dependent negative, but cell wall yield of 50 Gy and 200 Gy were detected higher than control. The lignin content of irradiated Brachypodium stem was reduced with dosage incease The ratios of lignin content to control were 97.6% (50 Gy), 91.9 (100 Gy), 87.3% (150 Gy), 89.4% (200 Gy), 81.6% (250 Gy), 85.2% (300 Gy). SEM image analysis demonstrated that cell size of 300 Gy plant was decreased by 45% of control. RT-PCR was performed to analyze transcript accumulation of lignin pathway related genes with irradiated Brachypodium stem. CCR, PAL, C4H, and 4CL were detected at least 2 times higher expression than control at 150 Gy, 200 Gy, 250 Gy. The preteatment and enzyme hydrolysis will be discussed for bioethanol production.

Brachypodium has been focused as new model plant for grass species. Like small size, small room requirement, and fast growth, Brachypodium shows numerous advantages as a model plant. Brachypodium is a typical grass at the genome level, which also exhibits an overall similarity of gene content and gene families when compared with rice (Oryza sativa) and sorghum (Sorghum bicolor) genomes. Brachypodium is an excellent material for structural and functional genomic studies in grass species. Targeting-Induced Local Lesions IN Genomes (TILLING) is a high-throughput technique and an approach for reverse genetics study. Moreover, it has been wildly utilized to find induced mutation. Bradi3g45515 is orthologue of the cellulose synthase-like HvCslF8 in barley. For TILLING library construction, 384 M2 Brachypodium mutants induced by chronic-gamma irradiation were used. Single nucleotide polymorphysm (SNP) and small deletion in Bradi3g45515 were searched through TILLING analysis. Template DNA for PCR reaction were prepared according to two dimensional pooling (eightfold) strategy. Heteroduplex DNA was digested by SURVEYOR nuclease (TRANSGENOMIC) and the DNA fragment was detected using polyacrylamide gel electrophoresis. Positive signal appeared at polyacrylamide gel from more than 4 lines and their Bradi3g45515 region were sequenced. SNP(s) were identified in 509-2 and 677-3 mutant line. Cellulose content and/or cell wall materials content will be measured using these mutants.

Mutant analysis is one of most optimized genome-wide approach towards acquiring utile phenotypes and defining related genes. Gamma-irradiation, an acknowledged way of mutant-generating method, was applied to gain sets of mutant line in Brachypodium distachyon. B. distachyon is a model plant, commonly used in genus of Gramineae for the research of structure genomics and functional genomics. B. distachyon contribute to rapid and easy analysis because of its small size and quick growth. Mutant population was generated by different doses of gamma-irradiation (0, 50, 100, 150, 200, 250 Gy) in the gamma field phytotron. Distance from the source gives same irradiation duration for each plant. Plant growth parameters such as plant height, tiller number, leaf length & width, internode number & diameter, maturity and yield components (ear number biomass) were scored on M0 plants. Plant responses to different doses of radiation are evaluated and the effective radiation dosages to generate mutant using gamma-phytotron are suggested. Chronic irradiation using gamma-phytotron is useful tool to generate mutants for genomic variations such as SNP or INDEL as well as suitable for functional study of genes in Gramineae.

Brachypodium distachyon is rapidly emerged in biological study and has been currently used as a model system for genetics and functional studies for crop improvement and biofuel production. Phosphinothricin (PPT) has been widely used as a selectable agent, which raises ammonium content and induces toxicity in non-transformed plant cells. However PPT selection is not much effective on Brachypodium callus consequently reducing transformation efficiency. In order to identify the efficient conditions of PPT selection, calli obtained from mature seeds of Brachypodium (PI 254867) were cultured on the callus inducing medium (CIM) or regeneration medium (ReM) containing serial dilutions of the PPT (0, 2, 5, 10, and 15 mg/l) in dark or light condition. Callus growth and ammonium content of each treatment were measured 2 weeks after the treatment. Although callus growth and ammonium content did not show much difference in CIM, slow callus growth and increased ammonium accumulation were found in ReM. No significant difference of ammonium accumulation in response to PPT was found between dark and light conditions. In order to identify major factors affecting increased ammonium accumulation, callus was cultured on the media in combined with phytohormones (2,4-D or kinetin) and carbon sources (sucrose or maltose) containing with PPT (5 mg/l). The highest ammonium content in callus was found in the kinetin and maltose media.

Glutamine synthetase (GS) plays important roles in plants like assimilation of ammonium and detoxification of the ammonium released from many metabolic processes such as amino acid degradation or photorespiration. Using ATP, ammonia is combined with glutamate to yield glutamine by the action of GS. Phosphinothricin (PPT) is widely used as a herbicide because it competes with glutamate to bind the active site of GS. PPT has been used to produce transgenic Brachypodium distachyon callus and plants as a selectable agent. PPT treatment raises ammonium content and induces toxicity in non-transformed cells. To find out efficient condition for selecting transformed callus, ammonium content were measured in this study. Non-transformed callus were derived from mature seeds of Brachypodium distachyon (Bd21). The callus were cultured on the callus inducing media (CIM) or regeneration media (RM) containing serial dilutions of the PPT (2, 5, 10 and 15 mg/l) with or without light. Ammonium content was measured 2 weeks after PPT application. Ammonium toxicity associated with PPT treatment was dose-dependent on RM whereas PPT treatment was not significantly influenced on CIM. There is no influence on dark or light condition. Additionally, callus were cultured on the media containing phytohormones combined with PPT (5 mg/l) and the most affecting element causing increased ammonium content has been identified. Acknowledgements: This work was supported by a grant (No. 20070301-034-016-007) from BioGreen 21 Program, RDA, Republic of Korea.