Location: Grain, Forage & Bioenergy Research
Title: A Fluorescent Assay for Plant Caffeic Acid O-methyltransferases Authors
Submitted to: Biotechnology for Fuels and Chemicals Symposium Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: April 19, 2010
Publication Date: April 19, 2010
Citation: Palmer, N.A., Sattler, S.E., Saathoff, A.J., Sarath, G. 2010. A Fluorescent Assay for Plant Caffeic Acid O-methyltransferases. Abstract only - Poster presented at the Biotechnology for Fuels and Chemicals Symposium Proceedings in Clearwater, FL, April 19-22, 2010. Not published elsewhere. Interpretive Summary: We have developed and validated a new enzymatic assay to measure the activity of caffeic acid-O-methyltransferases (COMT) in plants. Using this method, the native activity in several switchgrass plants differing in lignin content has been documented. This method will have utility in routinely querying switchgrass plants for COMT activity within a breeding program.
Technical Abstract: We have developed a facile, sensitive and continuous assay to measure the activities of plant COMTs using s-adenosyl homocysteine hydrolase as a coupling enzyme and and adeonsine a thiol-specific fluor, Thioglo1, as the detecting reagent. This assay was validated using recombinant sorghum COMT (BMR-12) a key enzyme involved in cell wall lignification, and used to analyze COMT activity in maturing tillers from switchgrass plants. Data indicated that the calculated Km, Vmax and Kcat values for the recombinant sorghum COMT using different substrates in the fluorescent assay were similar to published values for COMT enzymes isolated and characterized from other plant species using other methods. For switchgrass, native COMT activity and COMT transcript abundance was greatest in internodes at the top of a tiller and declined in the more basal internodes, although COMT protein as determined by immunoblotting of internode extracts did not appear to change significantly between the internodes along a switchgrass tiller. We have used this assay to determine the level of COMT activity in switchgrass genotypes differing in lignin content. Data on the correlations between activity, protein content and transcript abundance for several switchgrass genotypes will be presented.