Collection and Analysis of Expressed Sequence Tags Derived from Laser Capture Microdissected Switchgrass (Panicum virgatum L. Alamo) Vascular Tissues
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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.
KeywordsExpressed sequence tags Laser-capture microdissection Lignin Secondary cell wall Switchgrass Vascular tissue
We thank Drs Zengyu Wang and Jiyi Zhang for critical reading of the manuscript, and Dr. Ji He, for assistance with the EST data analysis. The research described in this paper was carried out as part of the BESC (The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science US Department of Energy) and also funded by the Samuel Roberts Noble Foundation. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of the authors expressed herein do not necessarily reflect those of the United States Government or any agency thereof.
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