Collection and Analysis of Expressed Sequence Tags Derived from Laser Capture Microdissected Switchgrass (Panicum virgatum L. Alamo) Vascular Tissues

Switchgrass is a perennial C4 grass that thrives in a wide range of North American habitats and is an emerging crop for the production of lignocellulosic biofuels. Lignin is an integral component of secondary plant cell walls that provides structural rigidity to the cell wall but it interferes with the conversion of cellulose to fermentable sugars by preventing chemical access to cellulose. Thus, one strategy for improving production of cellulosic ethanol is the down-regulation of lignin in plants. To achieve this goal, it is important to understand the molecular processes involved in vascular tissue development, lignification and secondary wall synthesis. Since active lignification occurs in the vascular system of the plant, we refined a protocol for isolating vascular tissues using laser-capture microdissection (LCM) in an effort to identify transcripts of switchgrass involved in lignification and secondary cell wall synthesis. ESTs (5,734) were sequenced from the cDNA libraries derived from laser microdissected vascular tissues. These Sanger sequences converged into 2,766 unigenes with an average length of 652 bp. Gene ontology of the unigenes indicated that 11% of the sequences were lignin and cell wall related. Several transcription factors involved in lignin and secondary cell wall synthesis and sugar- or vesicle-mediated transporters were also present in this EST data set. In situ hybridization of seven representative genes confirmed the preferential expression of five genes in the vascular tissues. Comparison of our switchgrass vascular tissue derived ESTs with that of other plant species validated our LCM approach. Furthermore, our switchgrass vascular tissue ESTs revealed additional lignin and cell wall related genes that were not present in other existing switchgrass EST collections. Inventory of the switchgrass vascular tissue ESTs presented here provides an important genomic resource for mining genes to reduce recalcitrance in this important bioenergy crop.