Phylogenetic, Molecular and Biochemical Characterization of Caffeic Acid O-methyltransferase Gene Family in Brachypodium

Some grasses such as Switchgrass and Miscanthus have been recently recognized as important herbaceous energy crops. However, due to their large and complicated genomes as well as difficulties to develop useful mutants, genomic studies and map-based cloning of target genes to improve interesting traits remain as a great challenge. Therefore, Brachypodium distachyon (L) P. Beauv, with its compact genome size, small physical stature and short propagation life cycle has become a more attractive model system after the recent release of the completed genome sequence. Lignin is essential in plant cell wall construction and serves as the hardest components to be digested to release polysaccrides for biofeul industry. Caffeic acid O-methyltransferase (COMT) is one of the important enzymes controlling lignin monomer production. We found that Brachypodium contains multicopies of COMT genes. Comparative sequence analysis with rice, sorghum and maize revealed that besides the conserved COMT ortholog on Chr. 3, Brachypodium contains additional COMT genes on different chromosomes. We discuss detailed evolution of Brachypodium COMTs based on phylogenetic analysis. To address whether they are functioning in the same pathway, we cloned four putative Brachypodium genes in-frame into pQE-7 protein expression vectors, purified each target protein and characterized enzyme activities on five substrates. We found that variations in biochemical properties were observed among these enzymes and in comparison with the rice COMT. Moreover, RNAi mutants of a Brachypodium COMT gene has been generated to further understand the role of COMT genes in lignin biosynthesis in plants.