Progress 01/01/13 to 12/31/13
Outputs
Target Audience: The target audience is other scientists interested in the topic of plant cell wall biosynthesis. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? One manuscript was published in the Plant Journal and another has been accepted for publication in Plant Molecular Biology. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the past year, we made progress in identifying proteins required for mannan synthesis as well as transcription factors that are involved in regulating the produciton of mannan.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Wang Y, Mortimer JC, Davis J, DuPree P, Keegstra K (2013) Identification of an additional protein involved in mannan biosynthesis. Plant J, 73:105-117.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Kim W-C, Reca I-B, Kim Y, Park S Thomashow MF, Keegstra K, Han, K-Y (2014) Transcription factors that directly regulate the expression of CSLA9 encoding mannan synthase in Arabidopsis thaliana. Plant Mol Biol, in press.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The results of this project has been and will be published in scientific journals and books. Four manuscripts have been published during the last year (see publications) and two more manuscripts are in preparation and will be published next year. PARTICIPANTS: Yan Wang, a postdoctoral associate in our lab was the main person from our research group involved in the studies on mannan biosynthesis. In one case, he cooperated with Curt Wilkerson, a faculty member in the Department of Plant Biology to perform these studies. In the other case, he collaborated with Paul Dupree and his colleague, Jenny Mortimer, from Cambridge University in England. The work on xylosyl addition to xyloglucan was done by David Cavalier, a postdoctoral associate in our lab and Linda Danhof, a technician in our group. In this case, we collaborated with colleagues at Iowa State University (Olga Zabotina and Y-H Chou) as well as colleagues at the University of Georgia (Michael Hahn, S Pattahil and S Eberhard). These efforts provided research training for David Cavalier and Yan Wang. TARGET AUDIENCES: The scientific community interested in plant cell walls. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We reported progress on the identification and characterization of the enzymes and genes required for the biosynthesis of two different hemicellulosic polysaccharides. The publication by Jensen et al., identified a new protein required for the addition of a galactosyl residue to xyloglucan. The publication by Zabotina et al., reports on more complete studies on the genes required for the addition of xylosyl residues to xyloglucan. The two manuscripts by Wang et al report on the genes and proteins required for galactomannan biosynthesis.
Publications
- Jensen JK, Schultink A, Keegstra K, Wilkerson CG, Pauly M (2012) RNA-Seq analysis of developing nasturtium seeds (Tropaeolum majus): Identification and characterization of an additional galactosyltransferase involved in xyloglucan biosynthesis. Mol Plant 5: 984-992.
- Wang Y, Alonso AP, Wilkerson CG, Keegstra K (2012) Deep EST profiling of developing fenugreek endosperm to investigate glactomannan biosynthesis and its regulation. Plant Mol Biol 79: 243-258.
- Zabotina OA, Avci U, Cavalier D, Pattathil S, Chou Y-H, Eberhard S, Danhof, L, Keegstra K, Hahn MG (2012) Mutations in multiple XXT genes of Arabidopsis reveal the complexity of xyloglucan biosynthesis. Plant Physiol 159: 1367-1384.
- Wang Y, Mortimer JC, Davis J, DuPree P, Keegstra K (2012) Identification of an additional protein involved in mannan biosynthesis. Plant J, doi:10.1111/tpj.12019.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The results of this project has been and will be published in scientific journals and books. One book chapter was published this year (see publications) and one manuscript will be submitted before the year end. PARTICIPANTS: Work this year has been conducted by David Cavalier, a postdoctoral associate. This work is being done in collaboration with colleagues at Iowa State University and the University of Georgia. It is part of a larger effort to understand the deposition of hemicellulosic polysaccharides that is supported by the Great Lakes Bioenergy Research Center. TARGET AUDIENCES: This work is of interest to others working the structure, function and biosynthesis of plant cell wall components. These components have received renewed attention in recent years because of the interest in using plant biomass to produce liquid transportation fuels. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Xylosyltransferases are enzymes that add xylosyl residues to the backbone of xyloglucan, a major hemicellulose in the primary walls of plant cells. We have performed a genetic analysis of the various genes encoding the multiple xylosyltransferases that are involved in xyloglucan biosynthesis. The major conclusion of our series of studies is that three different genes are involved in the process. They encode three different proteins required for xyloglucan biosynthesis. Our genetic analysis has allowed us to deduce new insights into the role of each of these proteins.
Publications
- Keegstra K, Cavalier, D (2011) Glycosyltransferase of the GT34 and GT37 families. Annual Plant Reviews. Plant Polysaccharides: Biosynthesis and Bioengineering, Vol 41, pgs 235-249.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The research performed in this project was published in scientific journals and as part of a doctoral dissertation of one student who was supported in part by this project. PARTICIPANTS: This project provided a training opportunity for Jonathan Davis. He completed his doctoral dissertation in the fall of 2010 and successfully defended his dissertation on December 3, 2010. TARGET AUDIENCES: The scientific community intersted in plant cell walls. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The glycan synthases that produce the backbones of hemicellulosic polysaccharides are polytopic membrane proteins that span the Golgi membrane multiple times. While these proteins, and the genes that encode them have been identified from work in our lab, and other labs, in recent years, many important questions remain to be answered about how they operate to form the hemicellulosic polysaccharides. One such question is the topology of these proteins in the Golgi membrane. Protease protection assays were used to investigate the topology of the Arabidopsis mannan synthase and xyloglucan glucan synthase. The topology of the mannan synthase is characterized by an odd number of transmembrane domains (probably five) and an active site that faces the Golgi lumen. In contrast, the xyloglucan glucan synthase has an even number of transmembrane domains (probably six) and an active site that faces the cytosol. These new conclusions have important consequences for the biochemical mechanisms involved in polysaccharide biosynthesis.
Publications
- Jonathan Davis, Federica Brandizzi, Aaron H. Liepman, and Kenneth Keegstra. Arabidopsis mannan synthase CSLA9 and glucan synthase CSLC4 have opposite orientations in the Golgi membrane. The Plant Journal 64: 1028-1037 (2010).
- Markus Pauly and Kenneth Keegstra. Plant cell wall polymers as precursors for biofuels. Current Opinion in Plant Biology 13: 305-312 (2010).
- Kenneth Keegstra. Plant Cell Walls. Plant Physiology 154: 483-486 (2010).
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The results of our studies have been presented at scientific conferences around the world. For example, all three project investigators made presentations at the Gordon Research Conference on Plant Cell Walls that was held in August of 2009. In addition, we and the students and postdocs from our labs have made oral presentations and poster presentations at other scientific conferences. Finally, we have presented seminars at universities in the US and sometimes abroad. PARTICIPANTS: This project is being conducted in collaboration with support from the DOE via the Great Lakes Bioenergy Research Center. Funding from that center provided support for three postdoctoral associates and two technicians and a programmer who are performing this work. This work is contributing to their professional development. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We are working with three seed systems that produce large amounts of hemicellulose during a very short time period. Each system produces a different hemicellulose and in each system the tissue type that accumulates the hemicellulose is different. We used next-generation sequencing platforms to obtain deep transcriptional profiles of each seed system during developmental stages where hemicellulose is accumulating. The major results from these sequencing efforts were pesented last year. We have continued to work on new genes whose functions were not previously know. For example, in fenugreek, we identified a gene that is highly expressed and therefore is a candidate for involvement in mannan biosynthesis. We have confirmed that the encoded protein is located in the Golgi apparatus and are attempting to define the biochemical function of the protein via reverse genetics by knocking out the homologs in Arabidopsis. The functions of other genes are being explored via heterologus expression and biochemical assays of the resulting proteins.
Publications
- Pauly M, Keegstra K (2008) Cell wall carbohydrates and their modification as a resource for biofuels. Plant J, 54:559-568.
- Cavalier, DM, Lerouxel, O, Neumetzler, L, Yamauchi, K, Reinecke, A, Freshour, G, Zabotina, O, Hahn, MG, Burgert, I, Pauly, M, Raikhel NV, Keegstra, K (2008) Disruption of two Arabidopsis thaliana alpha-xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component. Plant Cell 20: 1519-1537.
- Bir Singh, Utku Avci, Sarah E. Eichler Inwood, Mark J. Grimson, Jeff Landgraf, Debra Mohnen, Iben Sorensen, Curtis G. Wilkerson, William G.T. Willats and Candace H. Haigler (2009) A Specialized Outer Layer of the Primary Cell Wall Joins Elongating Cotton Fibers into Tissue-Like Bundles. Plant Physiol, 150:684-699.
- Nicolai Obel, Veronika Erben, Tatjana Schwarz, Stefan Kuhnel, Andrea Fodor and Markus Pauly (2009) Microanalysis of Plant Cell Wall Polysaccharides. Mol Plant 2: 922-932.
- Sascha Gille, Ulrike Hansel,Mark Ziemann, and Markus Pauly (2009) Identification of plant cell wall mutants by means of a forward chemical genetic approach using hydrolases. PNAS 106: 14699-14704.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: We are working with three seed systems that produce large amounts of hemicellulose during a very short time period. Each system produces a different hemicellulose and in each system the tissue type that accumulates the hemicellulose is different. We used next-generation sequencing platforms to obtain deep transcriptional profiles of each seed system during developmental stages where hemicellulose is accumulating. Four cDNA populations were sequenced from psyllium resulting in over 900,000 ESTs, three cDNA populations were sequenced from fenugreek resulting in over 1,500,000 ESTs and four cDNA populations were sequenced from nasturtium resulting in over 1,250,000 ESTs. We have developed a clustering pipeline that effectively clusters these large datasets. We have developed software that allows the user to efficiently identify genes likely to be involved in hemicellulose biosynthesis. The software also allows the user to add annotation and metadata to the clusters and share this information with other users. Using the above datasets and analysis software we have been able to verify that genes known to function in the synthesis of particular hemicelluloses are very abundant in the mRNA populations we chose to analyze. In particular the biosynthetic enzymes for the synthesis of GDP-mannose and UDP-galactose required for galactomannan biosynthesis are some of the most highly expressed genes in fenugreek endosperm tissues. Similarly, the biosynthetic enzymes for UDP-xylose and UDP-arabinose are among the top 100 highest expressed genes in psyllium. The presences of these genes at such high levels in the EST datasets suggest other genes involved in the synthesis of these hemicelluloses will also be among the most highly expressed sequences. We have used this information along with bioinformatics analyses to identify a number of candidate genes for both a xylosyltransferase and an arabinosyltransferase from psyllium. Both of these acivities are required for arabinoxylan-biosynthesis and nither of these activities have been identified in any plant species. In the nasturtium dataset we have confirmed that CSL C genes are the most abundant CSL gene family member present in stages of nasturtium depositing xyloglucan. The pattern of expression of CSL C gene and its abundance closely match those of a galatosyltransferase involved in xyloglucan biosynthesis. Similarly, we have confirmed that the CSLA (mannan synthase) and galactosyltransferase genes know to be responsible for galactomannan biosynthesis are among the most highly expressed genes in fenugreek. Athough several genes involved in xyloglucan and mannan biosynthesis have been identified, many gaps in our knowledge still exist. Thus, it is important to note that in addition to confirming the expression of known genes, we have identified over 15 candidate genes of unknown function that are strong candidates for involvement in hemicellulose biosynthesis. PARTICIPANTS: This project is being conducted in collaboration with support from the DOE via the Great Lakes Bioenergy Research Center. Funding from that center provided support for three postdoctoral associates and two technicians and a programmer who are performing this work. This work is contributing to their professional development and we expect several publications to be submitted in the coming year. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We are taking several approaches to validate candidate genes. For biosynthetic genes we are expressing the genes in both E. coli and Pichia pastoris. We have generated a number of such constructs for xylan synthase and arabinosyltransferase candidates and have isolated cell lines expressing these proteins. We have developed assays for these activities using fluorescently labeled oligo-xylan acceptors. We have had UDP-arbinofuranose (which is not commercially available) synthesized for the arabinosyltransferase assay. We have obtained t-DNA knock out lines for the arabidopsis orthologues of these candidate genes. Due to gene redundancy in arabidopsis we are in the process of making double knockout lines for both of these gene families. These lines will be assayed using a novel screening technique developed in the Pauly lab that uses specific antibodies to hemicelluloses and dot blotting. This method is both sensitive and specific. The development of this assay is a significant advance in our ability to rapidly process our knockout lines.
Publications
- No publications reported this period
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