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Title:   Author  item
                                            Gebeyaw, Mesfinitem
                                            Lamb, Joannitem
                                            Samac, Deborah

                                                         - *Debby*item
                                            Gronwald, Johnitem
                                            Jung, Hans Joachim
  Submitted to: Cell Wall Proceedings
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 5/1/2007
                                    Publication  Date: 8/13/2007
                                    Citation: Gebeyaw, M.T., Lamb, J.F., Samac, D.A., Gronwald, J.W., Jung, H.G. 2007. A system for identification of candidate genes controlling cell wall synthesis in alfalfa stems [abstract]. Physiologia Plantarum. 130:259.Interpretive Summary:
                                Technical Abstract: Usefulness of alfalfa for livestock feeding and production of lignocellulose-derived ethanol would be improved by genetic alteration of stem cell wall concentration and composition. This could be accomplished through selective breeding and transgenic technologies. However, development of alfalfa cell walls is a complex and highly integrated process making identification of candidate gene targets for molecular manipulation or as marker genes for breeding difficult. As part of a breeding program to improve forage quality for livestock, we identified alfalfa genotypes with consistent differences in stem cellulose and lignin concentrations under field and greenhouse conditions. We are using these genotypes to rapidly identify candidate genes in alfalfa for further characterization of function and for genetic modification of stem cell wall accumulation and composition. Stem internodes were collected from bud-stage alfalfa plants, from a clone that accumulates high concentrations of cellulose and lignin, immediately above and below the internode that is in transition between the elongation and post-elongation, respectively, phases of development. This sampling protocol takes advantage of the shift in development from growth in cell size and deposition of non-lignified primary walls during stem internode elongation compared to the deposition of xylem tissues (containing secondary walls rich in lignin and cellulose) due to cambium activity in post-elongation internodes. RNA was extracted from these internodes and hybridized to the Affymetrix Medicago GeneChip, which contains more than 50,000 probe sets representing expressed and prediction-based gene sequences from M. truncatula and alfalfa. Transcripts of 903 probe sets were differentially expressed (P < 0.01) between elongating and post-elongation internodes, with transcripts of 585 genes more abundant in elongating internodes and 318 genes more abundant in post-elongation internodes. As expected, genes associated with the phenylpropanoid pathway such as cinnamyl-alcohol dehydrogenase and naringenin-chalcone synthase, as well as some transcription factors, were highly expressed in post-elongation internodes. Additionally, there were 313 and 119 unique genes only detected in elongating or post-elongation internodes, respectively. The 432 genes with more abundant transcript levels or unique to elongating internodes are candidate cell wall synthesis and regulatory genes important for concentration and composition of cell wall material. Our expectation is that comparing gene expression between elongating and post-elongation internodes will allow identification of genes critical to xylem tissue proliferation and secondary wall synthesis in post-elongation internodes. We are currently analyzing microarray data for an alfalfa clone that has low concentrations of both lignin and cellulose. Transcript profiling between contrasting clones should provide insights into genes that regulate quantitative accumulation of cell wall components in alfalfa stems. This system, which combines use of tissues at different stages of development with germplasm of known divergence in cell wall accumulation, provides a powerful method for identifying candidate genes for genetic manipulation of alfalfa stem quality.


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