Members of the Poaceae include many of the most economically important plants, such as maize, rice, wheat, and sorghum. These grasses not only account for approximately half of the human diet, they are also a likely source of renewable bioenergy. An impediment to using biomass for the production of biofuels and other produces is the recalcitrance of the plant cell to enzymatic digestion. The cell wall is vital for plant strength in that it allows the plant to grow upright, and acts as protection against pests and the environment. Grasses have a unique cell wall that contain glucuronoarabinoxylan instead of xyloglucan and has a large amount of esterified hydroxycinnamic acid. Hydroxycinnamates are derived from phenylalanine in the phenylpropanoid pathway and serve many roles in the plant, including cell wall biosynthesis and specialized metabolism. One of these hydroxycinnamates, namely ferulate, can modify the arabinose side-chain of xylan. These feruloylated arabinoxylan moieties are able to dimerize via radical coupling to form diferulates, resulting in cross-links between xylan polymers and between xylan and lignin. Cross-linking in monocots likely contributes to cell wall strength, but the lack of identified genes involved in this pathway limit our understanding of this monocot-specific modification. The enzyme that adds ferulate to arabinoxylan is currently unidentified preventing alteration of the amount of ferulate in the wall by reverse genetic methods. In order to better understand ferulate-mediated cross-linking in grasses, our goal has been to find the gene encoding the enzyme responsible for adding ferulate onto arabinose residues in arabinoxylan, namely the arabinoxylan ferulate acyltransferase (AraFAT). To accomplish this goal we selected candidates from a grass-specific BAHD acyltransferase clade that were highly differentially expressed in monocots compared to dicots, produced proteins from these genes, and then assayed these proteins with arabinose-containing substrates and feruloyl-CoA. We also explored wheat seedling protein extractions as a method of determining a testable assay for AraFAT. During our search for the AraFAT gene, we discovered that one of our candidate genes, Bradi1g36980, is able to add ferulate to phenylamines to create phenylamides or hydroxycinnamic acid amide conjugates. These phenylamides have several functions in plants that include defense responses to pathogens and wounding. Phenylamides also play an important role in the plant cell wall, providing crosslinks between polymers that add rigidity and strength. The Bradi1g36980 enzyme has activity with donor substrate feruloyl-CoA and the acceptors tyramine, 2-phenyl-ethylamine, tryptamine, and serotonin. To our knowledge, this is the first time a BAHD acyl-transferase has been shown to use tryptamine or serotonin as a substrate. The true substrates of this enzymes have not been established by mutational analysis. Because these hydroxycinnamic acid amides are known to increase in plants exposed to jasmonate, a hormone involved in wound response pathways, we subjected Brachypodium seedlings to methyl-jasmonate and measured the relative expression level of Bradi1g36980 at various time points. We observed a slight increase in Bradi1g36980 expression occurred from methyl-jasmonate exposure, and are investigating the role of the phytohormone ethylene in its regulation.
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