The efficient production of second-generation lignocellulose biofuels largely depends on the identification of the factors underlying biomass recalcitrance, which necessarily involves the characterization of the molecular mechanisms controlling secondary cell wall (SCW) content and composition. Despite recent advances, most of our current knowledge of cell wall formation and lignin metabolism is primarily derived from dicotyledoneous species. However, the molecular mechanisms controlling different aspects of secondary cell wall deposition in grasses, such as transcriptional regulation, biosynthesis and polymerization of the different components, remain largely unknown, despite of the economic importance of this group of plants. One of the reasons for such a scenario is the lack of model systems that would allow the proper identification and characterization of grass-specific features. Several reports have demonstrated that the content and composition of the different components of SCW are regulated in a spatio-temporal fashion and, in grasses, this pattern is also observed along a single elongating internode. Therefore, the aim of this project is to develop a model system based on a single elongating internode to study secondary cell wall deposition in sorghum. More precisely, we aim to analyses the changes in cell wall biochemical compositions along an elongating internode and correlate them with mRNA levels for genes encoding enzymes involved in cell wall metabolism. For this purpose, a single elongating internode will be cut into 8 fragments in order to separate the four developmental zones and to ensure differential secondary wall deposition among fragments. These samples will be analyzed for cellulose, hemicellulose and lignin content and composition, using both wet chemistry techniques and histochemical analysis. Moreover, RT-qPCR will be used to evaluate the changes in the expression of SCW genes along the 8 fragments of the elongating internode. In order to evaluate a possible compensation mechanism among biosynthetic pathways, brown midrib (bmr) mutants affected in lignin biosynthesis will also be used. Noteworthy, this project is part of the BIOEN Young Investigator Award Project (Fapesp n° 2015/02527-1) and will be subsequently used in high-throughput transcriptomic analysis (RNAseq) for the identification of candidate genes that potentially play a role in secondary cell wall formation and lignin biosynthesis in grasses.
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