Utilizing Biochemical Adaptations of Plants and Next-Generation Sequencing Instruments to Discover Enzymes and Transcription Factors Involved in Plant Cell Wall Biosynthesis Jacob Kruger Jensen1,2, Jean-Christophe Cocuron1,2, Curtis G. Wilkerson*1,2,3 1 DOE Great Lakes Bioenergy Research Center, 2Department of Plant Biology, 3 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824. Wilker13@msu.edu This research was funded by the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494). One way to discover enzymes and regulatory proteins involved in a biological pathway is to examine the mRNAs of cells actively engaged in the pathway of interest, ideally when the pathway is a major activity for the tissue. There are many such plant tissues that produce very large amounts of product from a single biochemical pathway. As an example, the mucilagous layer of psyllium (Plantago ovata Forsk) seed contains about 60% arabinoxylan by weight (Fischer et al. 2004) and so is a good tissue to use for transcriptional profiling to discover genes involved in arabinoxylan biosynthesis. Similarly, the endosperm tissue of Fenugreek seeds contains 80% galactomannan by weight and hence provides a system to study mannan and galactomannan biosynthesis. The recent development of DNA sequencing instruments that can produce millions of sequences quickly and at moderate cost now allows us to exploit such systems to discover both biosynthetic enzymes from specific pathways and the transcription factors that control their expression. We are interested in using such a strategy to gain a greater understanding of plant cell wall biosynthesis. Our initial work has been to gain a better understanding of the enzymes required for arabinoxylan biosynthesis using the psyllium system. Arabinoxylan is a major component of grass cell walls and as grasses are likely to be important as bioenergy crops such work could have a major impact on bioenegry research. We have successfully generated 4 cDNA libraries from psyllium mucligaous tissue at 6, 8, 10 and 12 days post anthesis (DPA) and have obtained over 850,000 DNA sequences using the Roche GS-FLX sequencer. We have developed an analysis pipeline and query software to allow us to cluster, annotate and search these large datasets. An examination of these sequences using our software revealed that enzymes involved in the biosynthesis of UDP-xylose were highly represented in those cDNA libraries suggesting that these libraries likely are enriched in genes involved in arabinoxylan biosynthesis and its regulation. We find that homologues of the putative xylan synthases IRX10 and IRX10-like are very abundant these tissues. We find at least six genes encoding proteins in the glycosyl transfease family 61 at high abundance. We also find other genes that have been implicated in secondary cell wall biosynthesis that could be involved in xylan biosynthesis as well. One of these genes is related to the Arabidopsis gene At3g50220. The expression of this gene is highly correlated with the expression of IRX10 in Arabidopsis and so may represent a component of the xylan synthase. Since the psyllium mucilagous layer is synthesizing almost exclusively arabinoxylan it is likely that the subset of genes present in this tissue compared to the larger set of genes found in cambial tissues defines a minimal set of genes required to synthesize arabinoxylan. We have also identified a subset of transcription factors known to be up regulated during secondary cell wall biosynthesis. Since psyllium is making only arabinoxylan and not a secondary cell wall likely the trancription factors we have identified are involved in the regulation of arbinoxylan biosynthetic enzymes. We are currently expressing the psyllium versions of these genes in various heterologous systems to establish the function of these proteins.
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