Progress 09/01/08 to 08/31/12
Outputs
OUTPUTS: The principal outputs of this project during the reporting period were activities including conducting and analyzing experiments, and teaching and mentoring of students. The project is focused on O-methylation of hydroxyl groups in cell wall polysaccharides and exploits our finding that arabinogalactan proteins (AGPs) in the moss PHYSCOMITRELLA PATENS contain about 15 mole percent of 3-O-methyl-L-rhamnopyranosyl residues (3-O-Me-Rha). The overall strategy is to exploit this model system to gain a foothold from which a broader attack on O-methylation of cell wall polysaccharides in agricultural plants can be based. Several types of experiments were conducted and analyzed in the present reporting period. The central experiments were focused on analyses of lines of PHYSCOMITRELLA PATENS that were generated in prior reporting periods and contained disabled, or knockout, forms of certain genes that were considered candidates to be the gene encoding the O-methyltransferase responsible for methylating rhamnosyl residues to produce 3-O-Me-Rha in the PHYSCOMITRELLA AGPs. Based on a research publication that appeared in mid-2012, another such candidate gene was selected and similarly investigated. The principal test of function was to determine if the level of 3-O-Me-Rha in AGPs was reduced in the knockout lines. When one of the candidate genes was annotated in databases as a lignin (caffeoyl CoA) methyltransferase, the tests of function were expanded to include analysis of lignin-like content in PHYSCOMITRELLA cell walls. To enable more certain conclusions regarding function, three of the most promising candidate genes were inserted into tobacco plants. The expectation is that if one of the three candidate genes does encode the target O-methyltransferase, then the transgenic tobacco plants will be able to synthesize AGPs containing 3-O-Me-Rha, something that wild-type tobacco cannot do. Other experiments conducted during the reporting period continued prior investigations of whether methylation might occur before the rhamnosyl residues are incorporated into the AGPs, as would be indicated by the presence of nucleotide diphosphate methylated sugar. Finally, still other experiments continued prior efforts to detect the target O-methyltransferase at the level of enzyme activity. The experiments were conducted by one graduate student and 19 undergraduate students. The teaching and mentoring these students, particularly the undergraduate students, in the biochemical and chemical methods of analysis of complex carbohydrates was a significant output. The graduate student was the first author of a poster at a national meeting where some of the results of the project were disseminated. Undergraduates were first authors of six local presentations within California where portions of the research results were disseminated. PARTICIPANTS: Eugene A. Nothnagel-principal investigator/project director (guided all participants and conducted some of the experiments himself). Training included one fifth-year graduate student, Lei Zhu (generated and characterized candidate gene knockouts, generated constructs for transgenic tobacco), and 19 undergraduate students. These undergraduate students measured 3-O-Me-Rha/Rha ratios in PHYSCOMITRELLA knockout lines, measured lignin-like content in knockout lines, conducted enzyme assays on wild-type PHYSCOMITRELLA, prepared nucleotide diphosphate sugars from PHYSCOMITRELLA, and conducted preliminary AGP isolation and characterization from wild-type tobacco in preparation for upcoming analyses of transgenic tobacco plants. These undergraduates fell into five categories. Wynter Hernandez, William Moore, Candace Fajardo, and Robbin Melo were upper division undergraduates who were paid lab assistants supported by work-study funds, or by funds from a campus Hispanic-Serving Institution-College Cost Reduction and Access Act-STEM Pathway grant. Jane Kim and Megan Riley were upper-division students supported by UC LEADS or CEPCEB-REU programs. Yu-Chien Tsai, Jessica Kim, Thinn Zaw, and Rutaraj Kanase were undergraduate volunteers. Jacob Moreno was a freshman lab assistant supported by work-study funds. Diana Bui, Steven Chrysafides, Vincent Chen, Timothy Nguyen, Janki Sheth, Nita Baliga, Sarah Grace, and Gabriela Mamani were students in a one-quarter research seminar course and conducted research on this project in the context of that course. Faculty collaborators in this department included Professor Thomas Girke (bioinformatics work), Professor Linda Walling (Southern blot analysis), and Dr. Martha Orozco-Cardenas (transgenic tobacco generation). TARGET AUDIENCES: During the present reporting period, results were disseminated to attendees at one national and five local (University of California campuses) research symposia. The broader audience of plant biologists throughout the world will be the target of journal publications that result from this project. PROJECT MODIFICATIONS: During the course of the work in 2012, the one of the candidate genes under study became annotated in at least one database as a lignin (caffeoyl CoA) methyltransferase. Although mosses are not considered to produce polymeric lignin, they do produce lignin monomers and dimers called lignans. This annotation stimulated an expansion of the project to include analysis of lignin-like content in some of the knockout lines. Similarly in mid-2012, a highly relevant article appeared reporting the discovery of a gene encoding an O-methyltransferase that forms 4-O-methyl-glucuronic acid in ARABIDOPSIS hemicellulose, the first O-methyltransferase demonstrated to produce an O-methyl ether on a sugar in a plant cell wall polysaccharide. A phylogenetic analysis in that publication found only one similar gene (called here KO-11) in PHYSCOMITRELLA. Because PHYSCOMITRELLA does not make 4-O-methyl-glucuronic acid, KO-11 might instead make 3-O-Me-Rha, and thus KO-11 became of great interest and was added to the group of candidate genes being investigated in this project.
Impacts
Change in knowledge was the principal outcome/impact. The central goal is to identify the O-methyltransferase that methylates rhamnosyl residues to produce 3-O-Me-Rha in PHYSCOMITRELLA. Previously, knockout cassettes were inserted into PHYSCOMITRELLA for 10 genes considered to be candidates to encode the target O-methyltransferase. Survival in 3 rounds of section on antibiotic medium and analyses by polymerase chain reaction indicated that stable knockout lines were obtained for 8 of the 10 candidates (KO-1, 2, 4, 5, 6, 7, 9, 10). Some KO-3 knockouts survived 3 rounds of selection but seemed to be diploid, possibly indicating a haploid lethal. The KO-8 knockout cultures had no survivors at 3 rounds of selection, possibly indicating a lethal. The primary test of the knockouts is to determine whether 3-O-Me-Rha in AGPs is reduced. None of the knockouts have zero 3-O-Me-Rha content. Two knockouts exhibit modest reductions in 3-O-Me-Rha/Rha ratio that are statistically very significant (P=0.0031 for KO-1, P=0.0027 for KO-9). KO-1 also shows reduced rhizoid growth. In 2012, the KO-9 gene was annotated in a database as lignin (caffeoyl CoA) methyltransferase. Although mosses do not make polymeric lignin, they do make lignin monomers and dimers called lignans. Cell walls were prepared from wild-type, KO-1, and KO-9 PHYSCOMITRELLA and analyzed for lignan. The lignin-like content of KO-1 was modestly reduced relative to the wild-type (P<0.001), while KO-9 was not significantly different from the wild-type. This finding seems inconsistent with the annotation of KO-9 as lignin methyltransferase. In mid-2012, an article reported the discovery of a gene encoding an O-methyltransferase that forms 4-O-methyl-glucuronic acid in ARABIDOPSIS hemicellulose. Phylogenetic analysis found only one similar gene (called here KO-11) in PHYSCOMITRELLA. The first attempt to make a KO-11 knockout in PHYSCOMITRELLA resulted in no survivors, possibly indicating a lethal. Additional knockout attempts are currently underway. To enable more certain conclusions regarding function, the KO-1, KO-9, and KO-11 genes have been recently inserted into tobacco plants. When the transgenic plants grow large enough, AGPs will be analyzed for the presence of 3-O-Me-Rha, which wild-type tobacco cannot make. Other continuing experiments were aimed at testing whether methylation to form 3-O-Me-Rha occurs at the level of uridine diphosphate-rhamnose, but the results remain inconclusive. Because the ARABIDOPSIS O-methyltransferase that synthesizes 4-O-methyl-glucuronic acid requires divalent cobalt ion, this ion was tested in enzyme assays with PHYSCOMITRELLA extracts, but no formation of 3-O-Me-Rha was detected. The present results are progress towards understanding the biosynthesis of methylated sugars in plant cell walls. Control of biosynthesis of methylated polysaccharides might lead to production of plant polymers that would be valuable industrial emulsifiers. Manipulating methylation of cell wall polysaccharides might lead to biomass with improved yields in conversion to biofuels.
Publications
- Zhu, L., Girke, T., and Nothnagel, E.A. 2012. Knock-out analysis of candidate methyltransferase genes that might synthesize 3-O-methyl-L-rhamnosyl residues in arabinogalactan proteins of PHYSCOMITRELLA PATENS. http://www.acs.org/meetings, Technical Program Archive, 243rd American Chemical Society National Meeting Spring 2012, Division of Carbohydrate Chemistry, General Posters, Pub #84. San Diego, CA. March 27, 2012.
- Hernandez, W.D., Moore, W.M., and Nothnagel, E.A. 2012. Formation of 3-O-methyl-rhamnose in PHYSCOMITRELLA PATENS. University of California Louis Stokes Alliance for Minority Participation 2012 Statewide Undergraduate Research Symposium Program Book, p. 43, University of California, Irvine. February 11, 2012.
- Zaw, T., Fajardo, C., and Nothnagel, E.A. 2012. Analyzing for 3-O-methyl-rhamnosyl residues in targeted knock-out lines of PHYSCOMITRELLA PATENS. Symposium for Undergraduate Research, Scholarship and Creative Activity Program Book, p. 66, University of California, Riverside. May 3, 2012.
- Moore, W., and Nothnagel, E.A. 2012. Separation of UDP-3-O-methyl-L-rhamnose from a nucleotide diphosphate sugar fraction taken from PHYSCOMITRELLA PATENS. Symposium for Undergraduate Research, Scholarship and Creative Activity Program Book, p. 51, University of California, Riverside. May 3, 2012.
- Mello, R.R., Zhu, L., and Nothnagel, E.A. 2012. Knock-out of candidate methyltransferase reduces lignin in PHYSCOMITRELLA PATENS. 2012 Research in Science and Engineering Symposium Program Book, p. 22, University of California, Riverside. August 29, 2012.
- Kim, J., Riley, M., Zhu, L., and Nothnagel, E.A. 2012. Purification of arabinogalactan proteins containing methylated rhamnosyl sugar residues from PHYSCOMITRELLA PATENS and the effects of methylated sugars in wound healing properties. 25th Annual Mentoring Summer Research Internship Program Symposium Book, p. V-11, University of California, Riverside. August 15, 2012.
- Riley, M., Kim, J., Zhu, L., and Nothnagel, E.A. 2012. Characterization of a candidate rhamnosyl methyltransferase in PHYSCOMITRELLA. Center for Plant Cell Biology-Research Experience for Undergraduates Program, University of California, Riverside. August 17, 2012.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Activities including conducting and analyzing experiments, and teaching and mentoring of students were the principal outputs of this project during the reporting period. The project is focused on O-methylation of hydroxyl groups in cell wall polysaccharides and exploits our finding that arabinogalactan proteins (AGPs) in the moss PHYSCOMITRELLA PATENS contain about 15 mole percent of 3-O-methyl-L-rhamnopyranosyl residues (3-O-Me-L-Rha). The overall strategy is to exploit this model system to gain a foothold from which a broader attack on O-methylation of cell wall polysaccharides in agricultural plants can be based. Several types of experiments were conducted and analyzed in the present reporting period. The central experiments involved a bioinformatics/gene knockout approach to identifying the gene encoding the O-methyltransferase responsible for methylating rhamnosyl residues to produce 3-O-Me-L-Rha in PHYSCOMITRELLA AGPs. In these experiments, candidate PHYSCOMITRELLA genes, selected on the basis of sequence similarity to O-methyltransferase genes from other organisms, were tested for function by disabling their expression in the moss. The principal test of function was to determine if the level of 3-O-Me-L-Rha in AGPs was reduced in the knockout lines. Experiments were conducted to develop a microscale method for making this determination, and then this method was applied to begin analysis of the knockout lines. Other experiments conducted during the reporting period were aimed at testing a hypotheses about biosynthesis of the 3-O-Me-L-Rha residues, in particular whether the methylation occurs before or after the rhamnosyl residues are incorporated into the AGPs. These experiments were focused on developing a chromatographic method for separating nucleotide diphosphate sugars to enable determination of whether or not rhamnose might be present in methylated form at the nucleotide diphosphate sugar level. Experiments were also conducted in a different approach to this hypothesis. An enzyme assay method was developed to test for methyltransferase activity acting on a nucleotide disphosphate sugar to produce a nucleotide diphosphate methylated sugar. The bioinformatics/gene knockout experiments were conducted principally by a graduate student, while all of the other experiments were conducted with strong participation by undergraduate students. Thus, the teaching and mentoring these students in biochemical and chemical methods of analysis of complex carbohydrates was a significant output. Some of the results of the project were disseminated through poster presentations at two research symposia. PARTICIPANTS: Eugene A. Nothnagel-principal investigator/project director (guided all participants and conducted some of the experiments himself). Training included one fourth-year graduate student, Lei Zhu (paid by this grant, generated and characterized candidate gene knockouts), and 13 undergraduate students. These undergraduate students worked on preparation of nucleotide diphosphate sugars from PHYSCOMITRELLA, developed a chromatographic method for further purification of nucleotide diphosphate sugars, developed a method for analysis of 3-O-Me-L-Rha in PHYSCOMITRELLA AGPs when starting with very small amounts (500 mg or less) of tissue, and measured 3-O-Me-L-Rha/Rha ratios in several knockout lines. These undergraduates fell into four categories. Apolonio Huerta, Wynter Hernandez, Lisa Thai, William Moore, Jessica Toth, and Candace Fajardo were upper division undergraduates who were paid lab assistants supported by work-study funds, or by funds from a campus Hispanic-Serving Institution-College Cost Reduction and Access Act-STEM Pathway grant. Jane Kim and Yu-Chien Tsai were freshmen who started in a research seminar class, became summer interns paid by college funds, and then continued as volunteers in the lab. Jessica Kim, Thinn Zaw, and Rutaraj Kanase were students in the research seminar class and then continued as volunteers in the lab. David Nguyen and Ryan Brown were students in the research seminar class only. Collaborators in this department included Professor Thomas Girke (bioinformatics work) Professor Jian-kang Zhu (gene knockout work), and Professor Linda Walling (Southern blot analysis). TARGET AUDIENCES: During the present reporting period, results were disseminated to attendees at two undergraduate research symposia. The broader audience of plant biologists throughout the world will be the target of journal publications that result from this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The principal outcome/impact was change in knowledge. The central goal is to identify the O-methyltransferase that methylates rhamnosyl residues to produce 3-O-Me-L-Rha in PHYSCOMITRELLA AGPs. Based on a gene sequence for a microbial O-methyltransferase that produces 3-O-Me-L-Rha, and on sequences for other O-methyltransferases, homology searches were conducted in PHYSCOMITRELLA and yielded 10 genes as candidates to encode the O-methyltransferase that produces 3-O-Me-L-Rha. Knockout cassettes were designed for all 10 candidate genes and used in transformations. Knockout culture lines for 8 of the 10 candidates (KO-1, 2, 3, 4, 6, 7, 9, 10) have survived 3 rounds of selection on antibiotic medium. These knock-out lines appear to be stable, although KO-3 might be lethal because the survivors seem to be diploid. Knockout lines for the other 2 genes (KO-5, 8) have survived only 2 rounds of antibiotic selection thus far and might also be lethals. The presence of the antibiotic resistance gene has been confirmed by polymerase chain reaction for 5 of the genes (KO-1, 2, 3, 7, 9), and targeting of the insert has been confirmed by sequencing for 3 of the genes (KO-1, 3, 9). The phenotype test of the knockout lines is to determine whether the abundance of 3-O-Me-L-Rha in AGPs is reduced. Microscale methods were developed for preparation of an AGP-rich fraction and quantitation of its 3-O-Me-Rha/Rha content ratio. The 4 knockouts (KO-1, 2, 7, 9) tested thus far all contain some amount of 3-O-Me-Rha, indicating that the candidate gene either does not encode a rhamnosyl 3-O-methyltransferase or encodes one member of a family of such methyltransferases. The latter seems possible for KO-1, where 3 separate lines of KO-1 all show reduced 3-O-Me-Rha/Rha ratio and reduced rhizoids. Analyses of 3-O-Me-Rha/Rha ratio are in progress on the other 6 knockout lines. Other experiments were aimed at testing the hypothesis that methylation to form 3-O-Me-L-Rha occurs at the level of uridine diphosphate-rhamnose (UDP-Rha), instead of the anticipated methylation after the incorporation of rhamnose into the AGP polymer. Some experiments focused on developing a chromatographic method for separating components in a total nucleotide diphosphate-sugar fraction obtained from PHYSCOMITRELLA, to enable determination of whether UDP-3-O-Me-L-Rha is present. Other experiments focused on developing an enzyme assay to test for methyltransferase action on a nucleotide disphosphate sugar to produce a nucleotide diphosphate methylated sugar. Because UDP-Rha is not commercially available, this enzyme assay was developed for LYCOPODIUM LUCIDULUM, which contains considerable 3-O-methyl-galactose (3-O-Me-Gal) in its cell wall and for which UDP-Gal is available. Overall, the present results are progress towards understanding the biosynthesis of methylated sugars in plant cell walls. Knowledge and control of biosynthesis of methylated polysaccharides might lead to production of plant polymers that would be valuable industrial emulsifiers. Manipulating methylation of cell wall polysaccharides might lead to biomass with improved yields in conversion to biofuels.
Publications
- Moore, W., Hernandez, W., Barakat, A., Chung, B., Jain, S., Noche, K., and Nothnagel, E.A. 2011. Analyzing for 3-O-methyl-rhamnose in a nucleotide diphosphate sugar fraction. Symposium for Undergraduate Research, Scholarship and Creative Activity Program Book, University of California, Riverside. May 5, 2011.
- Kim, J., Zhu, L., Toth, J., Tsai, Y., Thai, L., and Nothnagel, E.A. 2011. Biosynthesis of 3-O-methyl-rhamnosyl residues in PHYSCOMITRELLA PATENS. Research in Science and Engineering Symposium Program Book, University of California, Riverside. August 25, 2011.
- Tsai, Y., Kim, J., Thai, L., and Nothnagel, E.A. 2011. Development of biochemical assay for galactosyl methyltransferase activity. Research in Science and Engineering Symposium Program Book, University of California, Riverside. August 25, 2011.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: The outputs of this project during the reporting period were principally activities that included conducting and analyzing experiments, and teaching and mentoring of students. The project is focused on O-methylation of hydroxyl groups in cell wall polysaccharides and exploits our finding that arabinogalactan proteins (AGPs) in the moss PHYSCOMITRELLA PATENS contain about 15 mole percent of 3-O-methyl-L-rhamnopyranosyl residues (3-O-Me-L-Rha). The overall strategy is to exploit this model system to gain a foothold from which a broader attack on O-methylation of cell wall polysaccharides in agricultural plants can be based. In the present reporting period, several types of experiments were conducted and analyzed. With regard to use of financial resources, the most important type of experiment conducted during the reporting period and still ongoing is a bioinformatics/gene knockout approach to identifying the gene encoding the O-methyltransferase responsible for methylating rhamnosyl residues to produce 3-O-Me-L-Rha in PHYSCOMITRELLA AGPs. In these experiments, candidate PHYSCOMITRELLA genes, selected on the basis of sequence similarity to O-methyltransferase genes from other organisms, are tested for function by disabling their expression in the moss. Other experiments conducted during the reporting period were focused on developing microscale methods for screening the candidate knock-out lines for the presence or absence of 3-O-Me-L-Rha in AGPs. Still another set of experiments aimed to test a hypotheses about biosynthesis of the 3-O-Me-L-Rha residues, in particular whether the methylation occurs before or after the rhamnosyl residues are incorporated into the AGPs. In a broader view of cell walls, some experiments were conducted to survey the presence of all types of singly-methylated sugars in the pectin, hemicellulose, and cellulose fractions of the cell wall from a lycopodiophyte plant found to contain 3-O-Me-L-Rha, and still other experiments focused on the sugar composition of cell walls in a plant mutated in a cellulose synthase-like gene. The bioinformatics/gene knockout experiments were conducted principally by a graduate student, while all of the other experiments were conducted by undergraduate students. Thus, the teaching and mentoring these students in biochemical and chemical methods of analysis of complex carbohydrates was a significant output. Some of the results of the project were disseminated through poster presentations at a research symposium. PARTICIPANTS: Eugene A. Nothnagel-principal investigator/project director (guided all participants and conducted some of the experiments himself). Training included one third-year graduate student, Lei Zhu (paid by this grant, worked on candidate gene knockout), and 13 undergraduate students. These undergraduate students worked on preparation of nucleotide diphosphate sugars from PHYSCOMITRELLA, developed a chromatography method for further purification of nucleotide diphosphate sugars, and developed a method for analysis of 3-O-Me-L-Rha in PHYSCOMITRELLA AGPs when starting with very small amounts (500 mg) of tissue. These undergraduates fell into three categories. Apolonio Huerta, Viet Nguyen, Wynter Hernandez, and Lisa Thai were upper division undergraduates who were paid lab assistants supported by college and/or work-study funds. William Moore, Persiah Bakhtiari, Gabriella Laussu, and Mollie Mackenzie were lower division undergraduates who started in a research seminar class and then became paid lab assistants. Ayeh Barekat, Shika Jain, Kathleen Noche, Brittany Chung, and Ashni De Alwis were students in the research seminar class only. Collaborators in this department included Professor Thomas Girke (bioinformatics work) and Professor Jian-kang Zhu (gene knockout work). TARGET AUDIENCES: During the present reporting period, results were disseminated to attendees at an undergraduate research symposium. The broader audience of plant biologists throughout the world will be the target of journal publications that result from this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge was the principal outcome/impact during this period. Most available resources were put into identifying the gene encoding the O-methyltransferase responsible for methylating rhamnosyl residues to produce 3-O-Me-L-Rha in PHYSCOMITRELLA AGPs. Sequence information is available for a microbial O-methyltransferase that produces 3-O-Me-L-Rha. Starting from this microbial sequence, searches against the PHYSCOMITRELLA predicted proteome yielded seven candidate genes. Starting with all predicted O-methyltransferases in PHYSCOMITRELLA yielded an additional three candidate genes with similarity to the microbial O-methyltransferase. Knockout cassettes have been designed for all ten candidate PHYSCOMITRELLA genes and used in protoplast transformation experiments. Apparently unstable transformants have required multiple rounds of selection, and this additional handling has sometimes led to contamination problems. This work has progressed much slower than anticipated and is ongoing. Screening of these lines involves isolation of AGPs and analysis for the presence or absence of 3-O-Me-L-Rha. Our laboratory routinely performs this work on tissue samples of 10 to 100 grams, but we have recently scaled the analysis down to tissue samples of 500 milligrams, as needed for screening knockout lines available only in small amounts. Other experiments tested the hypothesis that methylation to form 3-O-Me-L-Rha occurs at the level of uridine diphosphate-rhamnose (UDP-Rha), instead of the anticipated methylation after the incorporation of rhamnose into the AGP polymer. A total nucleotide diphosphate (NDP)-sugar fraction was obtained from PHYSCOMITRELLA and analyzed to determine whether 3-O-Me-L-Rha was present. We have repeatedly detected 3-O-Me-L-Rha in the total NDP-sugar fraction, and in one experiment, exact mass spectrometry detected a very low abundance ion at m/z=563.0678, a match to the calculated m/z=563.0679 of UDP-3-O-Me-L-Rha. Because many other NDP-sugars were present in this fraction, it was impossible to completely rule out occurrence of a chemical reaction in the mass spectrometer that generates the m/z=563 ion. To eliminate this possible artifact, the putative UDP-3-O-Me-L-Rha must be purified away from the other NDP-sugars. Development of suitable chromatography is challenging but underway in the lab. Smaller experiments in the lab showed that methylated sugars occur predominantly in the pectic and hemicellulosic fractions of the cell wall of LYCOPODIUM LUCIDULUM, and that mutation of a cellulose synthase-like gene required for osmotic stress resistance in ARABIDOPSIS causes only small changes in cell wall composition, these being mostly in the pectic fraction. Overall, the results from this reporting period represent progress towards understanding the biosynthesis and occurrence of methylated sugars in plant cell walls. Knowledge and control of biosynthesis of methylated polysaccharides might lead to production of plant polymers that would be valuable industrial emulsifiers. Manipulating methylation of cell wall polysaccharides might lead to biomass with improved yields in conversion to biofuels.
Publications
- Zhu, J., Lee, B.-H., Dellinger, M., Cui, X., Zhang, C., Wu, S., Nothnagel, E.A., and Zhu, J.-K. 2010. A cellulose synthase-like protein is required for osmotic stress tolerance in ARABIDOPSIS. Plant J. 63: 128-140.
- Hernandez, W., and Nothnagel, E.A. 2010. Analysis of methylated sugars in the pectin, hemicellulose, and cellulose plant cell wall fractions of LYCOPODIUM LUCIDULUM. California Alliance for Minority Participation in Science, Engineering, and Mathematics; Statewide Undergraduate Research Symposium Program Book, University of California, Irvine. Feb. 27, 2010.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Outputs during the current reporting period were principally activities including conducting and analyzing experiments, and teaching and mentoring of students. The focus of this project is on O-methylation of hydroxyl groups on sugars in plant cell wall proteoglycans and polysaccharides. The approach takes advantage of the high 15 percent abundance of 3-O-methyl-L-rhamnopyranosyl residues (3-O-Me-L-Rha) in arabinogalactan proteins in the moss PHYSCOMITRELLA PATENS, with the aim being to exploit this model system to gain a foothold from which a broader attack on O-methylation of cell wall polysaccharides in agricultural plants will be based. In the present reporting period, several types of experiments were conducted and analyzed. One type of experiment aimed to test hypotheses about biosynthesis of the 3-O-Me-L-Rha residues, in particular whether the methylation occurs before or after the rhamnosyl residues are incorporated into the arabinogalactan proteins. Linked with this type of experiment was the teaching and mentoring of a postdoctoral associate who learned chromatography and other methods required for purification and analysis of nucleotide diphosphate sugars. A second type of experiment was conducted to assess the occurrence of 3-O-Me-L-Rha residues, as well as other methylated sugars, in plant cell walls across a wide variety of plants ranging from the most primitive (algae) to the most advanced (angiosperms, or flowering plants). These experiments were conducted entirely by undergraduate students, and the teaching and mentoring these students in the analysis of methylated sugars by gas chromatography-mass spectrometry was a significant output. The third type of experiments, and the most important as ranked by expenditures of grant funds, involved identifying candidate rhamnosyl 3-O-methyltransferase genes in PHYCOMITRELLA by bioinformatics approaches, and then testing the function of one or more of these genes by disabling their expression in PHYSCOMITRELLA. These experiments were undertaken by a second-year graduate student with no prior experience in designing and generating knockout cassettes. Thus, teaching and mentoring of this graduate student was another significant output. Some of the results of the project were disseminated through a poster presentation by two of the undergraduate students and the principal investigator at a research conference in January, 2009. PARTICIPANTS: Eugene A. Nothnagel-principal investigator/project director (guided all participants and conducted some of the experiments himself). Training included one second-year graduate student, Lei Zhu (paid by this grant, worked on candidate gene knockout), one postdoctoral associate, Huijuan Zhang (salary paid by this grant only during the three-month period of 9/1/08-11/30/08, worked on nucleotide diphosphate sugars), and six undergraduates, including Daranee Yongpradit, Apolonio Huerta, Viet Nguyen, Wynter Hernandez, Lisa Thai, and Jerry Khong (salaries of all undergraduates paid by college and/or work-study funds; worked on survey of methylated sugars in a diversity of plants and on making PHYSCOMITRELLA protoplasts for gene knockout). Collaborators in this department include Professor Thomas Girke (bioinformatics work) and Professor Jian-kang Zhu (gene knockout work). TARGET AUDIENCES: During the present reporting period, results were disseminated to attendees at a research symposium. The broader audience of plant biologists throughout the world will be the target of journal publications that result from this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Outcomes/impacts during the current reporting period were principally changes in knowledge. Experiments conducted to test hypotheses about biosynthesis of the 3-O-Me-L-Rha residues in PHYSCOMITRELLA arabinogalactan proteins began to shed some light on the process, particularly with regard to whether the methylation occurs before or after the rhamnosyl residues are incorporated into the arabinogalactan proteins. If methylation occurs before the residues are incorporated into the polymer, then the methylation would be expected to occur at the level of UDP-rhamnose, the activated nucleotide diphosphate sugar. Thus, a key issue is whether or not UDP-3-O-Me-Rha is present in PHYSCOMITRELLA. Results of extraction and analysis of nucleotide diphosphate sugars from PHYSCOMITRELLA led to tantalizing, although not yet conclusive, results. In particular, liquid chromatography-electrospray mass spectrometry of the nucleotide diphosphate sugar fraction detected the presence of a minor component of mass/charge ratio of 563.0678, which is close enough to be considered a hit to the exact 563.0679 calculated for the free acid form of UDP-3-O-Me-Rha. The same fraction contained a much greater abundance of 565.0488, a hit to the calculated 565.0477 for the free acid form of UDP-hexose. Firming up these results will require further purification to separate away the UDP-hexose. Because funds were not available to continue the employment of the postdoctoral associate doing this work, this set of experiments was paused and is currently awaiting restart. Experiments conducted to assess the occurrence of 3-O-Me-L-Rha residues and other methylated sugars in cell walls across a wide variety of plants involved preparation and analysis of cell walls from 18 species, ranging from an alga to angiosperms. Three well-known methylated sugars, 2-O-methyl-D-xylose, 2-O-methyl-L-fucose, and 4-O-methyl-D-glucuronic acid, were found in most of the samples. Two methylated sugars, 3-O-Me-L-Rha and 3-O-methyl-D-galactose, were found predominantly in the more primitive plants. Two additional methylated sugars, 3-O-methyl-L-arabinose and a 2-O-methyl-hexose (further identification still underway), were found only in a lycophyte and fern, respectively, and seem to be novel discoveries. Experiments focused on functional tests of candidate rhamnosyl 3-O-methyltransferase genes in PHYCOMITRELLA by disabling their expression are currently underway but have not yet yielded useful results. Overall, the results from this reporting period represent progress towards understanding the biosynthesis and occurrence of methylated sugars in plant cell walls. Knowledge and control of biosynthesis of methylated polysaccharides might lead to production of plant polymers that would be valuable emulsifiers in the food, beverage, and other industries. Altering the capacity for methylating polysaccharides in plants through biotechnological manipulations might lead to cell wall polysaccharides with improved yields in biofuel conversion.
Publications
- Yongpradit, D., Nguyen, V., and Nothnagel, E.A. 2009. Methylated sugar residues in cell walls over the course of plant evolution. 25th Symposium in Plant Biology: The Evolution of Plant Development Program, P. Springer, H. Smith, E. Kramer (eds.), Riverside, CA. p.58.
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