Sunday, April 29, 2007

In situ visualization of cellulases in maize mutants with enhanced biomass conversion properties

Wilfred Vermerris1, Jianfei Zhao2, Michael R. Ladisch3, and Nathan S. Mosier3. (1) Agronomy, University of Florida Genetics Institute, 1376 Mowry Road, Gainesville, FL 32610-3610, (2) Plant Mol. Cell. Biology, University of Florida Genetics Institute, 1376 Mowry Road, Gainesville, FL 32610-3610, (3) LORRE/ABE, Purdue University, Potter Engineering Center, 500 Central Drive, West Lafayette, IN 47907

We have recently shown that modification of lignin subunit composition can significantly increase the yield of fermentable sugars obtained from enzymatic saccharifiaction of maize stover.  The brown midrib1 (bm1) and bm3 mutations each increase the yield of glucose per gram dry stover by 50% relative to the wild-type control (inbred A619).  When combined in a near-isogenic bm1-bm3 double mutant, the two mutations act in an additive manner, resulting in a doubling of the yield of glucose.  Even though there was no apparent increase in cellulose content, based on kinetic studies both the rate of hydrolysis and the overall yield of glucose increased as a result of the mutations.  In order to be able to generalize our results, we are investigating if this increased yield is consistent in different genetic backgrounds.  In addition, we are investigating what the basis is of the enhanced hydrolysis in these bm mutants by in situ visualization of cellulases.  We have designed recombinant proteins consisting of the cellulose binding domain (CBD) isolated from Trichoderma reesei endoglucanases labeled with green-fluorescent protein (GFP) to study how changes in cell wall composition and architecture impact the distribution of cellulolytic enzymes.  These analyses will be performed in intact plant tissue as well as in ground stover using UV fluorescence microscopy.  The resulting information will be valuable for designing plant cell wall composition in such a way that agronomic properties and biomass conversion are optimally balanced.