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Title:   Author  item
                                            Verdonk, Julianitem
                                            Hatfield, Ronalditem
                                            Sullivan, Michael
  Submitted to: Frontiers in Plant Science
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 11/26/2012
                                    Publication  Date: 12/13/2012
                                    Citation: Verdonk, J.C., Hatfield, R.D., Sullivan, M.L. 2012. Proteomic analysis of cell walls of two developmental stages of alfalfa stems. Frontiers in Plant Science. DOI: 10.3389/fpls.2012.00279.Interpretive Summary: Cell walls are important for the growth and development of all plants. They are also valuable resources for feed and fiber, and more recently as a potential feedstock for bioenergy production. Cell walls are composed primarily of the complex carbohydrates cellulose, hemicelluloses, and pectin. Proteins comprise a smaller fraction of the cell wall (about 10-20% by weight) and likely play regulatory, enzymatic, and structural roles. Chemical interactions of cell wall components provide structure and rigidity to the wall, but restrict digestibility of the complex carbohydrates, limiting available energy in both animal and bioenergy production systems. Research has focused on manipulation of lignin, whose crosslinking to other cell wall components is associated with limiting digestibility. Another approach to improve cell wall digestibility might be manipulation of proteins present in the cell wall (known as the cell wall proteome), either to alter enzymatic activities associated with component crosslinking or to reduce levels of structural proteins, which may also be cross-linked to other cell wall components. Most studies of plant cell wall proteomes have focused on the model plant Arabidopsis, which lacks true stems. Only one study, carried out nearly a decade ago, used the agriculturally important forage crop plant, alfalfa. This, along with recent advances in cell wall protein extraction methodology and improved technology for proteomic analyses, prompted the need for additional study. An analysis was conducted on the cell wall proteins present in two maturities of alfalfa stems: apical stems (less mature, more digestible) and basal stems (more mature, less digestible). We identified 224 proteins in the alfalfa stem cell wall proteome, 102 of which had not previously been identified in cell wall proteomic analyses. Most of the proteins are predicted to be secreted and have a hypothetical function in the cell wall. The most abundant class of proteins in apical stems was proteins with interacting domains (suggesting roles in protein-protein interactions or regulatory processes). In the basal stem, the most abundant protein classes were proteins predicted to function in the biosynthesis or polymerization of carbohydrates and proteins predicted to be oxidoreductases (which could facilitate cell wall polymer crosslinking). These data will be useful for scientists studying plant cell wall biology and should provide targets for the modification of plant cell walls to improve their digestibility and thus, enhance their value in animal and bioenergy production systems.
                                Technical Abstract: Cell walls are important for the growth and development of all plants. They are also valuable resources for feed and fiber, and more recently as a potential feedstock for bioenergy production. Cell wall proteins comprise only a fraction of the cell wall, but play important roles in establishing the walls and in the chemical interactions (e.g., crosslinking) of cell wall components. This crosslinking provides structure, but restricts digestibility of cell wall complex carbohydrates, limiting available energy in animal and bioenergy production systems. Manipulation of cell wall proteins could be a strategy to improve digestibility. An analysis of the cell wall proteome of apical alfalfa stems (less mature, more digestible) and basal alfalfa stems (more mature, less digestible) was conducted using a recently developed low-salt/density gradient method for the isolation of cell walls. Walls were subsequently subjected to a modified extraction utilizing EGTA to remove pectins, followed by a LiCl extraction to isolate more tightly bound proteins. Recovered proteins were identified using shotgun proteomics. We identified 221 proteins in the alfalfa stem cell wall proteome, 110 of which had not previously been identified in cell wall proteomic analyses. Nearly 70% percent of the identified proteins were predicted to be secreted, as would be expected for most cell wall proteins, an improvement over previously published studies using traditional cell wall isolation methods. A comparison of our and several other cell wall proteomic studies indicated little overlap in identified proteins among them, which may be largely due to differences in the tissues used, as well as differences in experimental approach.


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