p-Coumarates acylate grass lignins. A transferase, p-coumaroyl-CoA:Monolignol Transferase (PMT), was recently identified from rice, and proposed to be a biosynthetic pathway enzyme responsible for the acylating monolignols with p-coumarate. Collaborators have been misregulating the PMT gene in Brachypodium distachyon plants to establish the veracity of its role in grass lignification. We therefore needed to establish a means to identify, and quantitate, the relative levels of lignin p-coumaroylation in the up- and down-regulated transgenics, and in a mutant derived from an INRA tilling collection. The plant samples were derivatized to the corresponding acetylated a-bromides via dissolution in AcBr, then subjected to two-electron reductive cleavage, and finally acetylate, yielding the (acetylated) monolignol p-coumarate ester conjugates that were the lignin precursors. [In fact the p-coumarate becomes hydrogenated during the method, so strictly we release monolignol dihydro-p-coumarates]. The conjugates are quantitatively analyzed using GC-MS. As shown here and in a companion paper dealing more extensively with the Brachypodium lines themselves (�Misregulation of p-Coumaroyl-CoA:Monolignol Transferase in Brachypodium distachyon�), DFRC analysis established that the PMT enzyme acted solely in the lignin pathway, with p-coumarate levels on lignin decreasing substantially in down-regulated and mutant Brachypodium, and increasing substantially in over-expressedtransgenic lines compared to the wild type. The method is currently also being used to assess the p-coumaroylation of a series of monocots in collaboration with New Zealand collaborators (Heather Free, Philip Harris, Bronwen Smith).

Quantitative DFRC method for determining monolignol <i>p</i>-Coumarates released from grass lignin

247th National Meeting (2014)

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Quantitative DFRC method for determining monolignol p-Coumarates released from grass lignin