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.

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.

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 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.