Progress 09/01/08 to 08/31/12
Outputs
OUTPUTS: Activities: 1. We developed an arabinogalactan-protein (AGP)-fucosyltransferase (FUT) enzyme assay. 2. We expressed His-tagged FUT4 and FUT6 genes in tobacco BY2 cells. 3. We tested enzymatic activity of HIS-tagged FUT4 and FUT6 protein with our assay. 4. We isolated and characterized AGPs from culture media of tobacco BY2 cells and tobacco BY2 cells expressing His-tagged FUT4 and FUT6 protein. 5. We analyzed phenotypes of fut4, fut6, and fut4/fut6 double mutants in Arabidopsis. 6. We optimized a dye-based purification method for FUTs. 7. We extracted cell wall Alcohol Insoluble Residues (AIR) from leaves and roots of fut4, fut6 and fut4/fut6 double mutants for glycome profiling. 8. We conducted phenotypic analysis of fut4, fut6 and fut4/fut6 double mutants. 9. We analyzed monosaccharide composition of leaf and root AGPs from fut4, fut6 and fut4/fut6 double mutants. 10. We conducted expression analysis of FUT4 and FUT6 genes in Arabidopsis under nitrogen-deficient conditions. 11. We identified putative AGP-FUT genes in crop plants (grasses). 12. We obtained additional, allelic fut4 and fut6 mutant lines. 13. Bioinformatics analysis identified 16 Brachypodium putative FUTs (bdFUTs), 10 wheat putative FUTs (TaFUTs), and 16 rice putative FUTs (OsFUTs). Expression profiling methods (RT-PCR and microarray) were used to prioritize candidates from Brachypodium and wheat. 14. We cloned the most promising candidates for AGP- and XyG-FUTs, two TaFUT and four bdFUT genes (Bradi1g46020, Bradi1g46030, Bradi1g46040, Bradi3g58040). 15. We expressed His-tagged versions of these genes in Pichia pastoris, and microsomal membranes were prepared from transformed yeast cells for enzyme testing. 16. We conducted glycome profile analyses of root and leaf tissues from wheat and Brachypodium seedlings. Events: The following conferences were attended by one or more project personnel: Molecular and Cellular Biology (MCB) Program Retreat, Athens, OH, Oct. 10, 2008; Ohio Plant Biotechnology Consortium (OPBC) Annual Meeting, Columbus, OH, Nov. 15, 2008; Gordon Research Conference (GRC) on Plant Cell Walls, Smithfield, RI, Aug. 2-7, 2009; National Research Initiative Project Directors Meeting for the Plant Biology Programs (NRIPDMPBP), Washington, DC, May 7, 2010; XII Plant Cell Wall Meeting, Porto, Portugal, July 25-30, 2010; American Society of Plant Biologists (ASPB) Annual Meeting, Montreal, Canada, July 31-Aug. 4, 2010; MCB Program Retreat, Athens, OH, Oct. 23, 2010; OPBC Annual Meeting, Columbus, OH, Nov.20, 2010; ASPB Midwestern Section Annual Meeting, West Lafayette, IN, March 19-20, 2011; NRIPDMPBP, Washington, DC, May 24, 2011; XVIII International Botanical Congress, Melbourne, Australia, July 23-30, 2011; ASPB Annual Meeting, Minneapolis, MN, Aug. 6-10, 2011; OPBC Annual Meeting, Columbus, OH, Oct. 8, 2011; OPBC Annual Meeting, Columbus, OH, Nov. 17, 2012; MCB Program Retreat, Athens, OH, Nov. 17, 2012; GRC on Plant Cell Walls, Waterville, ME, Aug. 5-10, 2012. Products: A collaboration was fostered with Dr. Michael Hahn at the Complex Carbohydrate Research Center at the University of Georgia to conduct glycome profile analyses and carbohydrate analyses. PARTICIPANTS: Individuals: 1. Allan Showalter (PI): Dr Showalter was responsible for overseeing and training of Yan Liang (Ph.D. student), Rebecca Vondrell (undergraduate), Shawna Callaghan (undergraduate) and Wen-liang Xu (visiting scientist) in the phenotypic characterization of fut4, fut6, and fut4/fut6 double mutants. Dr. Showalter was also responsible for overseeing and training of Yan Liang (Ph.D. student), Debarati Basu (Ph.D. student) and Alexendra Venetos (undergraduate) for sugar composition analysis, genetic screening for allelic fut4 and fut6 mutant lines, and biochemical and phenotypic characterization of these mutants. 2. Ahmed Faik (Co-PI): Dr. Faik was responsible for overseeing and training of Yingying Wu (M.S. student who transferred to another university) in developing the AGP-FUT enzymatic assay and expressing and characterizing FUT4 and FUT6 in tobacco BY2 cultured cells. Dr. Faik was also responsible for overseeing and training of Richard Wiemels (M.S. student), Rebekah Whitley (part time research technician) and two undergraduates (Johnny Rader and Jonathan Lucas) in the identification, expression and testing of candidate FUT gene from wheat and Brachypodium in Pichia pastoris. 3. Yan Liang, with Rebecca Vondrell, conducted phenotypic characterization of fut4, fut6, and fut4/fut6 double mutants. Yan was also responsible for AIR extractions of fut4, fut6, and fut4/fut6 double mutants for glycome profiling. Yan, with Alexendra Venetos, conducted genetic screening of allelic fut4 and fut6 mutant lines and biochemical and physiological phenotypic characterization of fut4, fut6, and fut4/fut6 double mutants. 4. Yingying Wu developed the AGP-FUT enzymatic assay and expressed and characterized FUT4 and FUT6 in tobacco BY2 cultured cells. 5. Wen-liang Xu, with Shawna Callaghan and Yan, respectively, conducted histological analysis of fut4, fut6, and fut4/fut6 double mutants and expression analysis of FUT4 and FUT6 under nitrogen-deficient conditions. 6. Richard Wiemels identified and examined putative AGP-FUT genes in grasses, specifically in wheat and Brachypodium. Richard, with help from Rebekah Whitley, Johnny Rader and Jonathan Lucas, was responsible for cloning six candidate FUT genes, and testing them for AGP- and XyG-FUT activities. 7. Debarati Basu conducted monosaccharide composition analysis in this project. Training and professional development: Dr. Showalter was responsible for training and development of Yan Liang (Ph.D. graduate student who completed her PhD in 2012), Rebecca Vondrell (an undergraduate), Shawna Callaghan (an undergraduate), Wen-liang Xu (a visiting scientist), Debarati Basu (Ph.D. graduate student) and Alexendra Venetos (an undergraduate). Dr. Faik was primarily responsible for training and development of Yingying Wu (M.S. graduate student), Richard Wiemels (M.S. graduate student who will complete his M.S. in January 2013), Rebekah Whitley (part time research technician), and two undergraduates (Johnny Rader and Jonathan Lucas). Collaborators: Dr. Michael Hahn at the Complex Carbohydrate Research Center at the University of Georgia conducted glycome profile analyses and carbohydrate analyses for this project. TARGET AUDIENCES: This project provided training opportunities for graduate students and undergraduate students in a geographic location with atypical and disadvantaged segments of the population. Ohio University is located in the foothills of the Appalachian Mountains in southeast Ohio, a predominantly rural setting, and draws 25% of its undergraduate population from the surrounding Appalachian region of Ohio and West Virginia. According to the U.S. Census Bureau, Ohios poverty rate jumped 1.8% in 2009, ranking Ohio number 24th in the nation, with Southeastern Ohio having the highest poverty rate. The PIs actively participated every year of the grant in Southern Ohios Women in Science and Engineering Day during which Middle School girls are bussed to Ohio Universitys campus for a day of activities to encourage participation in science. The PIs also participated every year of the grant in the Southeast District Science Day to judge interdisciplinary projects developed by students in grades 5 to 12. In addition, Dr. Showalter has made science presentations at local K-12 schools and to various student groups at Ohio University in which he mentioned research conducted in this project and acknowledged research support by the USDA. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
1. Developed an in vitro enzyme assay to monitor AGP fucosylation. 2. Expressed and purified two putative AGP fucosyltransferases (FUT4 and FUT6) as His-tagged proteins from tobacco BY2 cells. 3. Demonstrated that FUT4 and FUT6 are indeed fucosyltransferases by their ability to fucosylate endogenous AGPs from tobacco BY2 cells both in vivo and in vitro. 4. Phenotypic analysis of fut4, fut6, and fut4/fut6 double mutants showed these mutants to be identical to wild type plants with few exceptions as noted below. Eel lectin staining, which indicates the presence of terminal fucose residues, was reduced in fut6 and fut4/fut6 double mutants compared to the fut4 mutant and wild type plants. Carbohydrate analysis indicated a reduction in fucose of AGPs in the mutants compared to wild type. 5. Roots of fut6 and fut4/fut6 double mutants showed different glycome profiles compared to roots of wild type and fut4 mutant plants. AIR extracts from fut6 and fut4/fut6 double mutant roots showed lower signal intensities when reacted with antibodies against hemicelluloses, rhamnogalacturonans and arabinogalactans, indicating that fucosyl residues on AGPs may regulate intermolecular interactions between AGPs and certain wall components (i.e., hemicelluloses and rhamnogalacturonans). 6. The fut4, fut6 and fut4/fut6 double mutants did not show anomalies at the tissue and cellular levels. 7. Monosaccharide composition analysis of AGPs showed the absence of fucose in roots of fut4/fut6 double mutant plants. 8. We showed expression of FUT4 and FUT6 is induced under nitrogen-deficient conditions, consistent with public microarray data from Genevestigator. 9. We identified putative AGP-FUT genes in grasses, specifically in wheat and Brachypodium. 10. Fucose is present in AGPs in the roots of wild type plants but absent in the roots of fut4/fut6 double mutant plants. 11. Two new mutant lines for fut6 and one new mutant line for fut4 were obtained. 12. Bioinformatics analysis demonstrated that grasses have candidate FUTs for AGPs and XyG. Biochemical assays for these activities have been optimized and are ready for testing these candidates. 13. Yan Liang completed her Ph.D. degree, and Richard Wiemels will complete his M.S. degree in January 2013. 14. Wheat and Brachypodium protein sequences were analyzed; all proteins have one transmembrane domain at the N-terminal end (type II membrane protein), and contained FUT motifs. 15. These genes were introduced into Pichia cells and transgenic cells producing wheat or Brachypodium protein of expected sizes were identified by western blots using anti-His-tag antibody. 16. At least five colonies per gene were tested on tamarind xyloglucan (TXyG) and on AGPs using our FUT assays. So far, none of the wheat genes and two Brachypodium genes (Bradi1g46020 and Bradi3g58040) showed FUT activity. We are now testing cell wall extracts (1M and 4M KOH) from wheat roots, which were defucosylated with an α(1,2)fucosidase, as substrate acceptors. We expect to observe [3H]fucose incorporation into one of these extracts as glycome profiling results showed that wheat XyG is fucosylated in 1M and 4M KOH extracts.
Publications
- Wiemels, R. and Faik, A. (2008) Optimization of a dye-based purification method for glycosyltransferases involved in plant cell wall polysaccharides biosynthesis. Ohio Plant Biotechnology Consortium (OPBC) annual meeting, Columbus, Ohio. November 15, 2008.
- Wu, Y., Showalter, A.M., and Faik, A. (2009) Characterization of two Arabidopsis fucosyltransferases acting on arabinogalactan-proteins. Gordon Research Conference on Plant Cell Walls, Smithfield, Rhode Island. August, 2-7, 2009.
- Xu, W., Liang Y., Wu, Y., Faik, A., and Showalter, A.M. (2009) Biochemical and functional analysis of AtFUT4 and AtFUT6 in Arabidopsis, Gordon Research Conference on Plant Cell Walls, Smithfield, Rhode Island. August, 2-7, 2009.
- Showalter, A. M. (2010) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, National Research Initiative Project Directors meeting for the Plant Biology Programs- Program and Abstract Book, Washington, DC. May 2010.
- Showalter, A. M. (2010) Structure, biosynthesis and functions of the arabinogalactan-proteins in plant cell walls, Ohio Plant Biotechnology Consortium (OPBC) annual meeting-Program and Abstract Book, Columbus, Ohio. November 20, 2010.
- Wu, Y., Williams, M., Bernard, S., Driouich, A., Showalter, A. M., and Faik, A. (2010). Functional identification of two nonredundant Arabidopsis α(1,2)fucosyltransferases specific to arabinogalactan-proteins, J. Biol. Chem, 285, 13638-13645.
- Liang, Y., Wu, Y. Y., Pattathil, S., Xu, W. L., Faik, A., Hahn, M. G., and Showalter, A. M. (2011) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, American Society of Plant Biologists (ASPB) - Midwestern Section annual meeting- Program and Abstract Book, West Lafayette, Indiana. March 2011.
- Liang, Y., Wu, Y. Y., Pattathil, S., Xu, W. L., Faik, A., Hahn, M. G., and Showalter, A. M. (2011) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, Ohio University Student Research and Creative Activity Fair, Athens, Ohio. May 2011 [Sigma Xi Excellence Award Winner].
- Showalter, A. M. (2011) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, National Research Initiative Project Directors meeting for the Plant Biology Programs- Program and Abstract Book, Washington, DC. May 2011.
- Showalter, A. M. (2011) Identification and characterization of fucosyltransferases and galactosyltransferases involved in the biosynthesis of arabinogalactan-proteins, XVIII International Botanical Congress-Program and Abstract Book, Melbourne, Australia. July 23, 2011.
- Liang, Y., Wu, Y. Y., Pattathil, S., Xu, W. L., Faik, A., Hahn, M. G., and Showalter, A. M. (2011) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, American Society of Plant Biologists (ASPB) annual meeting-Program and Abstract Book, Minneapolis, Minnesota. August 2011.
- Showalter, A. M. (2011) Identification and characterization of fucosyltransferases and galactosyltransferases involved in the biosynthesis of arabinogalactan-proteins, University of Georgia, Department of Plant Biology, Athens, Georgia. September 26, 2011.
- Liang, Y., Wu, Y. Y., Pattathil, S., Xu, W. L., Faik, A., Hahn, M. G., and Showalter, A. M. (2011) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, Ohio Plant Biotechnology Consortium (OPBC) annual meeting-Program and Abstract Book, Columbus, Ohio. October 2011.
- Liang, Y., Wu, Y. Y., Pattathil, S., Xu, W. L., Faik, A., Hahn, M. G., and Showalter, A. M. (2011) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, Molecular and Cellular Biology (MCB) Program Retreat-Program and Abstract Book, Athens, Ohio. November 2011.
- Liang, Y., Pattathil, S., Xu, W. L., Basu, B., Venetos, A., Faik, A., Hahn, M. G., and Showalter, A. M. (2012) Biochemical and physiological characterization of fut4 and fut6 mutants defective in arabinogalactan-protein fucosylation in Arabidopsis. Gordon Research Conference on Plant Cell Walls, Waterville, ME, Aug. 5-10, 2012.
- Tan L., A.M. Showalter, J. Egelund, A. Hernandez-Sanchez, M.S. Doblin, A. Bacic. (2012) Arabinogalactan-proteins and the research challenges for these enigmatic plant cell surface proteoglycans. Frontiers in Plant Science 3, 1-10.
- Liang, Y., Pattathil, S., Xu, W. L., Basu, B., Venetos, A., Faik, A., Hahn, M. G., and Showalter, A. M. (2012) Biochemical and physiological characterization of fut4 and fut6 mutants defective in arabinogalactan-protein fucosylation in Arabidopsis. Plant Physiology, revised manuscript submitted and pending.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Activities: 1. We have established standard procedures for sample preparation and data analysis for monosaccharide composition analysis with our new GS-MS instrument. We isolated arabinogalactan-protein samples from leaves and roots of wild type, fut4, fut6, fut4/fut6 Arabidopsis plants and performed monosaccharide composition analysis of these samples with the in-house GC-MS system. An identical set of samples was provided to our collaborator, Dr. Michael Hahn, at the Complex Carbohydrate Research Center at the University of Georgia to corroborate our monosaccharide analyses. 2. We obtained Arabidopsis seeds from the Arabidopsis Biological Research Center in order to find additional (allelic) fut4 and fut6 T-DNA mutant lines. These seeds were planted and screened for homozygous T-DNA insertion plants. The flanking regions of the T-DNA insert of each line were sequenced to determine the exact insertion site. 3. Bioinformatics analysis identified 16 Brachypodium (Brachypodium distachyon) putative FUTs (bdFUTs), 10 wheat (Triticum aestivum) putative FUTs (TaFUTs), and 16 rice (Oryza sativa) putative FUTs (OsFUTs). Phylogenetic analysis grouped these candidates into three main groups. The group that contains the three characterized Arabidopsis FUTs (one XyG-FUT and two AGP-FUTs) contains also eight OsFUTs, six TaFUTs, and eight bdFUTs. These are promising candidates for AGP- and XyG-FUTs. Expression profiling methods (RT-PCR and microarray) were used to prioritize the candidates from Brachypodium and wheat. We are currently in process of cloning the most promising candidates (four bdFUTs and two TaFUTs). Since the genomes of these species are GC-rich, several cloning strategies are being tested and acquiring sophisticated PCR machines (with touchdown PCR capability) is required. PARTICIPANTS: Individuals: 1. Allan Showalter (PI): Dr. Showalter was responsible for overseeing and training of Yan Liang (Ph.D. graduate student), Debarati Basu (Ph.D. graduate student) and Alexendra Venetos (a female undergraduate) in the method development for sugar composition analysis, genetic screening for allelic fut4 and fut6 mutant lines, and biochemical and phenotypic characterization of fut4 and fut6 null mutants as well as the fut4/fut6 double mutant and wild type (control) plants. 2. Ahmed Faik (Co-PI): Dr. Faik was responsible for overseeing and developing the AGP-FUT enzymatic assay and for expressing and characterizing FUT4 and FUT6 in tobacco BY2 cultured cells. Dr. Faik trained Richard Wiemels (M.S. graduate student), Rebekah Whitley (part time research technician) and two undergraduates students (Johnny Rader and Jonathan Lucas). 3. Yan Liang (Ph.D. graduate student): Ms. Liang with Alexendra Venetos (an undergraduate) was responsible for genetic screening of fut4 and fut6 mutant lines and biochemical and physiological phenotypic characterization of fut4 and fut6 null mutants as well as the fut4/fut6 double mutant. 4. Debarati Basu (Ph.D. graduate student): Ms. Basu was trained with respect to sugar composition analysis techniques with the in-house GC-MS instrument. 5. Richard Wiemels (M.S. graduate student): Mr. Wiemels has begun to look for putative AGP-FUT genes in grasses, specifically in wheat and Brachypodium. Richard, with the help of Rebekah, Johnny, and Jonathan, was responsible for (i) the cloning of six genes, (ii) preparation of microsomal fractions from BY2, wheat, and Brachypodium, (iii) solubilization/purification of AGP- and XyG-FUT activities, (iv) analysis of the products from AGP and XyG fucosyl transfer reactions. Because the departure of Yingying Wu (M.S. graduate student), extra work was added to other members of Faik's lab. Training and professional development: Dr. Showalter was primarily responsible for the training and development of Yan Liang (Ph.D. graduate student), Debarati Basu (Ph.D. graduate student) and Alexendra Venetos (an undergraduate). Dr. Faik was primarily responsible for the training and development of Richard Wiemels (M.S. graduate student), Rebekah Whitley (part time research technician), and two undergraduate students (Johnny Rader and Jonathan Lucas). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Outcomes: 1. Our GC-MS data is consistent with our previous result that fucose is present in arabinogalactan-proteins in the roots of wild type plants but absent in the roots of fut4/fut6 double mutant plants. 2. Two new T-DNA mutant lines for fut6 and one new T-DNA mutant line for fut4 were obtained. The T-DNA insertion site in these mutants was confirmed by comparing our DNA sequencing result with published data on the FUT4 and FUT6 gene sequences. These additional mutant lines will now need to be confirmed as fut4 or fut6 knock-out/knock-down mutants at the transcriptional level and then used for mutant analysis. The result of the allelic mutant analysis is expected to be consistent with the results obtained from our current fut4 and fut6 mutant lines and will insure that the observed phenotypes are attributed to the mutated FUT4 or FUT6 genes. 3. Our bioinformatics analysis demonstrated that grasses have candidate FUTs for AGPs and XyG. The biochemical assays for these activities have been optimized in our labs and is ready for testing these candidates. The only bottleneck is the cloning of GC-rich genes (65-70%) from wheat and Brachypodium. We have shown from previous work that microsomal preparations from wheat and Brachypodium have XyG-FUT, which has never been documented before. Therefore, we expect to identify promising TaFUT and bdFUT candidates enzymes for AGPs and XyG.
Publications
- Liang, Y., Wu, Y. Y., Pattathil, S., Xu, W. L., Faik, A., Hahn, M. G., and Showalter, A. M. (2011) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, Molecular and Cellular Biology (MCB) Fall Retreat, Athens, Ohio. November 2011.
- Liang, Y., Wu, Y. Y., Pattathil, S., Xu, W. L., Faik, A., Hahn, M. G., and Showalter, A. M. (2011) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, Ohio Plant Biotechnology Consortium (OPBC) annual meeting, Columbus, Ohio. October 2011.
- Showalter, A. M. (2011) Identification and characterization of fucosyltransferases and galactosyltransferases involved in the biosynthesis of arabinogalactan-proteins, University of Georgia, Department of Plant Biology, Athens, Georgia. September 26, 2011.
- Liang, Y., Wu, Y. Y., Pattathil, S., Xu, W. L., Faik, A., Hahn, M. G., and Showalter, A. M. (2011) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, American Society of Plant Biologists (ASPB) annual meeting, Minneapolis, Minnesota. August 2011.
- Showalter, A. M. (2011) Identification and characterization of fucosyltransferases and galactosyltransferases involved in the biosynthesis of arabinogalactan-proteins, XVIII International Botanical Congress, Melbourne, Australia. July 23, 2011.
- Showalter, A. M. (2011) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, National Research Initiative Project Directors meeting for the Plant Biology Programs, Washington, DC. May 2011.
- Liang, Y., Wu, Y. Y., Pattathil, S., Xu, W. L., Faik, A., Hahn, M. G., and Showalter, A. M. (2011) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, Ohio University Student Research and Creative Activity Fair, Athens, Ohio. May 2011 [Sigma Xi Excellence Award Winner].
- Liang, Y., Wu, Y. Y., Pattathil, S., Xu, W. L., Faik, A., Hahn, M. G., and Showalter, A. M. (2011) Functional and physiological characterization of two fucosyltransferase genes for arabinogalactan-protein glycosylation in Arabidopsis thaliana, American Society of Plant Biologists (ASPB) - Midwestern Section annual meeting, West Lafayette, Indiana. March 2011.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Activities: 1. We extracted cell wall Alcohol Insoluble Residues (AIR) from the leaves and roots of fut4, fut6 and the fut4/fut6 double mutant in Arabidopsis. The AIR extracts were sent to the Complex Carbohydrate Research Center (CCRC) at the University of Geogia for glycome profiling using carbohydrate monoclonal antibodies. 2. We conducted histological and physiological phenotypic analysis of the fut4, fut6 and the fut4/fut6 double mutant in Arabidopsis. 3. We analyzed the monosaccharide composition of arabinogalactan-proteins (AGPs) of the fut4, fut6 and the fut4/fut6 double mutant in Arabidopsis by total acid hydrolysis of AGPs followed by high performance anion exchange chromatography analysis (HPAEC). 4. We conducted expression analysis of the FUT4 and FUT6 genes in Arabidopsis plants under nitrogen-deficient conditions. 5. We have begun to expand our investigation to involve the identification of putative AGP-FUT genes in crop plants (grasses). Events: The following conferences were attended by one or more of the project personnel: 1. XII Plant Cell Wall Meeting, Porto, Portugal, July 25-30, 2010. 2. The American Society of Plant Biologists Annual Meeting, Montreal, Canada, July 31-August 4, 2010. 3. The Molecular and Cellular Biology (MCB) Program Retreat, Athens, OH, October 23, 2010. 4. The Ohio Plant Biotechnology Consortium (OPBC) Annual Meeting, Columbus, OH, November 20, 2010. PARTICIPANTS: Individuals: 1. Allan Showalter (PI): Dr. Showalter was responsible for overseeing and training of Yan Liang (Ph.D. graduate student), Shawna Callaghan (an undergraduate) and Dr. Wen-liang Xu (a visiting scientist from China) in the biochemical and phenotypic characterization of fut4 and fut6 null mutants as well as the fut4/fut6 double mutant and wild type (control) plants. 2. Ahmed Faik (Co-PI): Dr. Faik was responsible for overseeing and developing the AGP-FUT enzymatic assay and for expressing and characterizing FUT4 and FUT6 in tobacco BY2 cultured cells. Dr. Faik trained Yingying Wu (M.S. graduate student) who has now transferred to a graduate program at another university and Richard Wiemels (M.S. graduate student). 3. Yan Liang (Ph.D. graduate student): Ms. Liang was responsible for AIR extractions and the biochemical and physiological phenotypic characterization of fut4 and fut6 null mutants as well as the fut4/fut6 double mutant. 4. Dr. Wen-liang Xu (visiting scientist from China): Dr. Xu along with Shawna Callaghan (an undergraduate) and Ms. Liang, respectively, was responsible for histological analysis of fut4 and fut6 null mutants as well as the fut4/fut6 double mutant and for expression analysis of FUT4 and FUT6 under nitrogen-deficient conditions. 5. Richard Wiemels (M.S. graduate student): Mr. Wiemels has begun to look for putative AGP-FUT genes in grasses, specifically in wheat and Brachypodium. Training and professional development: Dr. Showalter was primarily responsible for the training and development of Yan Liang (Ph.D. graduate student), Shawna Callaghan (undergraduate student) and Dr. Wen-liang Xu (visiting scientist from China). Dr. Faik was primarily responsible for the training and development of Yingying Wu (M.S. graduate student) and Richard Wiemels (M.S. graduate student). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1. The roots of fut6 and the fut4/fut6 double mutant showed different glycome profiles compared to the roots of wild type and fut4 mutant plants. The AIR extracts from fut6 and fut4/fut6 double mutant roots showed lower signal intensities when reacted with antibodies against hemicelluloses, rhamnogalacturonans and arabinogalactans, indicating that fucosyl residues on AGPs may regulate intermolecular interactions between AGPs and certain wall components (i.e., hemicelluloses and rhamnogalacturonans). 2. The fut4, fut6 and the fut4/fut6 double mutant in Arabidopsis did not show anomalies at the tissue and cellular levels as indicated by the histological phenotypic analysis. On the physiological level, preliminary results indicate that the presence or absence of certain sugars in the growth media can specifically inhibit growth of the roots of the fut 4, fut6, and the fut4/fut6 double mutant plants. 3. Monosaccharide composition analysis of the AGPs from the mutant Arabidopsis plants showed the absence of fucose in the roots of fut4/fut6 double mutant plants. Confirmation of this result by mass spectrometry is underway. 4. Our preliminary data showed the expression of FUT4 and FUT6 is induced under nitrogen-deficient conditions, which is consistent with public microarray data for FUT4 and FUT6 expression from Genevestigator. The potential roles of FUT4 and FUT6 in response to stress, such as the nitrogen-deficient conditions, are being investigated now. 5. We have begun to expand our investigation to involve the identification of putative AGP-FUT genes in grasses, specifically in wheat and Brachypodium.
Publications
- Showalter, A. M. (2010) Structure, biosynthesis and functions of the arabinogalactan-proteins in plant cell walls, Ohio Plant Biotechnology Consortium (OPBC) annual meeting, Columbus, Ohio. November 20, 2010.
- Wu, Y., Williams, M., Bernard, S., Driouich, A., Showalter, A. M., and Faik, A. (2010). Functional identification of two nonredundant Arabidopsis α(1,2)fucosyltransferases specific to arabinogalactan-proteins, J. Biol. Chem, 285, 13638-13645.
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