Switchgrass Contains Two Cinnamyl Alcohol Dehydrogenases Involved in Lignin Formation
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Switchgrass Contains Two Cinnamyl Alcohol Dehydrogenases Involved in Lignin Formation Aaron J. Saathoff & Christian M. Tobias & Scott E. Sattler & Eric J. Haas & Paul Twigg & Gautam Sarath
Published online: 28 September 2010 # Springer Science+Business Media, LLC. (outside the USA) 2010
Abstract Lignin content of switchgrass (Panicum virgatum L.), a bioenergy species, is a critical determinant of biomass quality since it can negatively impact conversion of biomass into liquid fuels via biochemical platforms. Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. Here, we have shown that cv. Kanlow switchgrass contains at least two closely related CAD genes (PviCAD1 and PviCAD2) that code for proteins containing highly conserved domains and residues that identify them as bona fide CADs. Both recombinant proteins displayed substrate kinetics consistent with their presumed role in cell wall lignification. Proteomic and immunoblotting detected CAD containing spots in interA. J. Saathoff : S. E. Sattler : G. Sarath (*) Grain, Forage, and Bioenergy Research Unit, USDA-ARS, University of Nebraska, 137 Keim Hall, Lincoln, NE 68583-0937, USA e-mail: [email protected] A. J. Saathoff : S. E. Sattler : G. Sarath Department of Agronomy and Horticulture, University of Nebraska, 137 Keim Hall, Lincoln, NE 68583, USA C. M. Tobias Genomics and Gene Discovery Unit, USDA-ARS, 800 Buchanan St, Albany, CA 94710, USA E. J. Haas Department of Chemistry, Creighton University, 2500 California Plaza, Omaha, NE 68178, USA P. Twigg Department of Biology, University of Nebraska-Kearney, Kearney, NE 68849, USA
node protein extracts, and proteomic analyses demonstrated that both CADs were expressed. In planta CAD activity, CAD protein levels were observed at all stages of tiller development. A real-time qPCR analysis of the two CADs and one CAD-like sequence indicated that transcripts coding for PviCAD1 were present in greater abundance than those coding for PviCAD2. Transcripts for a third CAD-like sequence (PviAroADH) were present at intermediate levels as compared to PviCAD1 and CAD2. The predicted protein sequence of PviAroADH indicated that it was an enzyme unrelated to lignification based on phylogenetic and protein modeling data. Keywords Cinnamyl alcohol dehydrogenase . Internodes . Panicum virgatum L. . Proteomic identification . Recombinant enzyme characterization . Tillers . Switchgrass
Introduction Second generation biofuels, if properly developed, are likely to play a part in alleviating carbon emissions while at the same time improving energy security [3]. In the US, switchgrass (Panicum virgatum L.) is being developed as a potential feedstock for these purposes, and intensive efforts are underway towards developing cultivars to serve as dedicated feedstocks. Perennial plants, such as switchgrass, have the advantage of requiring fewer chemical inputs than row crops, enhance soil organic carbon, and can be grown on marginal land. On-farm field research has also reported a highly positive overall ener
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