Progress 10/01/16 to 09/30/17
Outputs
Target Audience:During the present reporting period, results were disseminated via poster presentations to attendees at four local (University of California, Riverside campus) and two regional (Southern California or statewide) research symposia. Changes/Problems:As detailed in the Accomplishments section of this progress report, the principal problem encountered in the current reporting period was that transgenic expression of MT1 and MT6 in E. COLI led to the moss proteins being deposited in inclusion bodies in an inactive form that could not be refolded to active form. To address this problem, the expression system is being changed from E. COLI bacteria to the Eukaryotic yeast, PICHIA PASTORIS. What opportunities for training and professional development has the project provided?Aside from of work of the principal investigator and a senior collaborating staff member, all of the work in the current reporting period of the project was conducted by undergraduate students who were on hourly payroll, who earned academic credit, who were supported by programs focused on under-represented or economically disadvantaged students, or who simply volunteered to gain research experience. A total of 20 undergraduates worked on the project during this reporting period. How have the results been disseminated to communities of interest?The results obtained in this reporting period of the project are not yet ready for journal publication but have been disseminated to attendees at four local (University of California, Riverside campus) and two regional (Southern California or statewide) research symposia. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, work will continue on the stated specific objectives of the project. Greatest effort will continue to be directed towards objective (4), with the focus on expression of the MT1 and MT6 genes in PICHIA, after expression in E. COLI did not lead to active proteins in test-tube biochemical assays for methyltransferase activity. A strong effort will also be directed towards objective (2), with work already underway on generating knockout PHYSCOMITRELLA plants in which the MT1 or MT6 gene is disabled.
Impacts
What was accomplished under these goals?
The long-range goal of this project is to tailor the structure of plant cell walls in ways that lead to greater yield of biofuels per ton of biomass. The approach is focused on identifying genes that could be manipulated to alter the abundance of methyl-ether groups on sugar residues in cell wall polymers. Lower methyl-ether content might enhance the yield of bioethanol, while higher methyl-ether content might enhance the yield of synthetic diesel fuel. Success in reaching this goal would benefit persons who directly or indirectly use portable, high-energy-density fuels, whether that use be in agricultural fields, on highways, on railways, in airways, or in shipping lanes. Thus, nearly the entire U.S. population could potentially benefit from this project. Past reports outlined how the project began with specific objective (1): Identify the gene that encodes an O-methyltransferase that produces 3-O-methyl rhamnosyl residues in the arabinogalactan proteins of the moss PHYSCOMITRELLA PATENS. The direction of the project turned when instead of discovering a gene that causes synthesis of 3-O-methyl rhamnosyl residues, two PHYSCOMITRELLA genes, MT1 and MT6, were discovered that cause synthesis of 3-O-methyl galactosyl residues when transgenically expressed in tobacco. Subsequent work focused primarily on specific objective (3): Generate, and characterize the cell wall properties of, transgenic NICOTIANA TABACUM plants that express the PHYSCOMITRELLA galactosyl-3-O-methyltransferase genes (modified to the context of MT1 and MT6). With specific objectives (1) and (3) essentially completed, work towards the end of the last reporting period began to focus on specific objective (4): Biochemically characterize the action of the galactosyl-3-O-methyltransferase enzymes from PHYSCOMITRELLA (modified to the context of MT1 and MT6). The importance of this objective increased with the unexpected finding that MT1 and MT6 cause synthesis of 3-O-methylgalactosyl residues in transgenic tobacco, which was curious because the moss makes abundant 3-O-methylrhamnosyl residues, but the abundance, if any, of 3-O-methylgalactosyl residues in moss is not above noise level. A hypothesis formulated to address this curious finding holds that the MT1 and MT6 methyltransferases have an active site that is not entirely specific. If so, then MT1 and/or MT6 might be able to synthesize both 3-O-methylrhamnosyl residues and 3-O-methylgalactosyl residues, the former being favored in the conditions of moss cells and the latter being favored in the conditions of tobacco cells. To test this hypothesis, the MT1 and MT6 moss genes were inserted into E. COLI bacteria with affinity tags to enable purification of the MT1 and MT6 proteins. The aim was to perform test-tube biochemical enzyme assays with various substrates to shed light on the substrate specificity of the enzymes. At the last report, abundant expression of polypeptides with MT1 and MT6 molecular weight were detected in the bacteria, but work was still underway to solve problems encountered in extracting the MT1 and MT6 proteins from the bacteria. Work in the current reporting period focused on specific objectives (4), (5), and (2). In work on objective (4), which consumed the most time, it became apparent that the bacteria were putting MT1 and MT6 in inclusion bodies. Because this problem had been anticipated, the membrane-spanning domain near the N-terminus of MT1 and MT6 was deleted when the vector was constructed for expression in E. COLI. Also, the expression vector included a targeting sequence to send the MT1 and MT6 proteins to the periplasmic space. Both of these precautions were taken to reduce the likelihood of an inclusion body problem, but the problem occurred anyway. Having found MT1 and MT6 in inclusion bodies, considerable effort was put into purifying the inclusion bodies from the bacteria, testing various harsh buffers to resolubilize the proteins out of the inclusion bodies, and then testing various mild buffers to encourage proper refolding of the solubilized protein. A procedure that gave satisfactory results in electrophoretic analysis of the presumably refolded proteins was found, but no methyltransferase activity could be detected. Because the methyltransferase likely requires a divalent cation as an inorganic cofactor, seven different divalent cations (all present in the moss culture medium) were tested, but none resulted in detectable methyltransferase activity. The enzyme assay was also attempted in the presence of membrane vesicles, to test whether membrane association had an effect, but again no methyltransferase activity could be detected. Finally, several alternative polysaccharide substrates were developed to have fewer side chains and thereby be more accessible to the methyltransferase than the wild-type tobacco wall polysaccharide that was usually used as substrate, but these alternative substrates also yielded no methyltransferase activity. Clearly, the MT1 and MT6 proteins made by the bacteria have some fundamental problem that leaves them inactive as enzymes. This outcome sometimes occurs when expressing Eukaryotic proteins in bacteria, since bacteria do not readily make disulfide bonds and cannot glycosylate proteins as Eukaryotes can. Continuing work on specific objective (4) is now focused on expressing the MT1 and MT6 proteins in the yeast PICHIA PASTORIS. PICHIA is one of the most popular Eukaryotic cell types for transgenic expression, particularly among scientists working with plant cell wall-active enzymes. Considerable progress has been made on specific objective (5): Use the PHYSCOMITRELLA galactosyl-3-O-methyltransferase sequences as probes in bioinformatic searches to find other cell wall methyltransferases in model angiosperms; in seed crops that produce considerable residual biomass, such as maize, wheat, barley, sorghum, rice, and other cereals; and in proposed bioenergy crops that produce high biomass yield such as POPULUS, switchgrass, and MISCANTHUS. Both the nucleotide and peptide sequences for MT1 and MT6 were loaded into Basic Local Alignment Search Tools hosted by the National Center for Biotechnology Information (NCBI), Phytozome, and other databases to find sequences from other species that are similar to either MT1 or MT6. The found sequences were then loaded into the conserved domain search tool at NCBI to identify those that contained the same conserved domain as MT1 and MT6. Sequences with the conserved domain were then loaded into the Molecular Evolutionary Genetics Analysis (MEGA7) program or the CDTree Program (NCBI) to construct phylogenetic trees. Through this analysis more than 75 plant species, including both eudicots and monocots, were found to contain proteins similar to MT1 and MT6. Towards the end of the current reporting period, work also got underway on specific objective (2): Generate, and characterize the cell wall properties of, knockout PHYSCOMITRELLA plants in which one or both galactosyl-3-O-methyltransferase genes are disabled. Knockout cassettes have been designed for both MT1 and MT6, and the cassette for MT1 has been constructed. Work on constructing the MT6 knockout cassette is underway. When both cassettes are ready, generation of knockout PHYSCOMITRELLA lines will be attempted. Research performed in the current period with the goal of testing the substrate specificities of MT1 and MT6 was considerably slowed by inclusion body problems encountered with E. COLI but is now moving forward with PICHIA. The bioinformatic finding of more than 75 plant species with genes similar to MT1 and MT6 strongly suggests that research on the PHYCOMITRELLA model system will be applicable to other plants with greater promise towards biofuel production and towards lower net carbon dioxide release into the atmosphere, thereby benefiting the U.S. population.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Higuera, M., and Nothnagel, E.A. 2016. Methylated sugar residues in arabinogalactan-proteins of the moss PHYSCOMITRELLA PATENS. Southern California Conferences for Undergraduate Research Program, University of California, Riverside. November 12, 2016. http://www.sccur.org/sccur/fall_2016_conference/posters
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Gomez, M., and Nothnagel, E.A. 2017. Methylated sugar residues in arabinogalactan-proteins of the moss PHYSCOMITRELLA PATENS. 2017 CAMP Statewide Undergraduate Research Symposium Program Book, p. 58, University of California, Irvine. February 4, 2017.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Yen, J.J., Aguilar, C., Orozco-C�rdenas, M.L., and Nothnagel, E.A. 2017. Expression of plant cell wall methyltransferases in bacteria. 11th Annual Undergraduate Research, Scholarship, and Creative Activity Symposium Program Book, p. 9, University of California, Riverside. May 3, 2017.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Masters, H.M., Huang, A., Orozco-C�rdenas, M.L., and Nothnagel, E.A. 2017. Testing for heterodimerization of moss methyltransferases in progeny of genetically crossed transgenic tobacco plants. 11th Annual Undergraduate Research, Scholarship, and Creative Activity Symposium Program Book, p. 25, University of California, Riverside. May 4, 2017.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Gomez, M., Theodory, B.G., Eulgem, T., and Nothnagel, E.A. 2017. Methods development for analysis of persistence and fate of synthetic plant defense elicitors. 30th Annual UCR Graduate Division Summer Research Program Symposium Book, p. C5, University of California, Riverside. August 18, 2017.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Yen, J.J, Aguilar, C., Tom, L.Y., Orozco-C�rdenas, M.L., and Nothnagel, E.A. 2017. Solubilization and renaturation of plant cell wall methyltransferases expressed in E. coli. Center for Plant Cell Biology-Research Experience for Undergraduates Program, University of California, Riverside. August 23, 2017. http://cepceb.ucr.edu/reu/2017.html
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Darden, N., and Nothnagel, E.A. 2017. Bioinformatic discovery of cell wall methyltransferases using a moss prototype. 2017 Research in Science and Engineering Symposium Program Book, p. 37, University of California, Riverside. August 29, 2017.
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:During the present reporting period, results were disseminated via poster presentations to attendees at three local (University of California, Riverside campus) research symposia. Changes/Problems:As detailed in the Accomplishments section of this progress report, the unanticipated and very fortuitous discovery of two moss genes encoding galactosyl-3-O-methyltransferases has caused a shift which amounts to us focusing on the same stated goals of the project, but in the context of 3-O-methylgalactosyl residues rather than 3-O-methylrhamnosyl residues. What opportunities for training and professional development has the project provided?Aside from of work of the principal investigator and a senior collaborating staff member, all of the work in the current reporting period of the project was conducted by undergraduate students who were on hourly payroll, who earned academic credit, who were supported by programs focused on under-represented or economically disadvantaged students, or who simply volunteered to gain research experience. A total of 15 undergraduates worked on the project during this reporting period. How have the results been disseminated to communities of interest?The results obtained in this reporting period of the project are not quite ready for journal publication but have been disseminated to attendees at three local (University of California, Riverside campus) research symposia. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, work will continue to be focused on the stated goals of the project, but mostly in the context of 3-O-methylgalactosyl residues rather than 3-O-methylrhamnosyl residues. Highest priority will be focused on test-tube biochemical enzyme assays with the MT1 and MT6 proteins synthesized in bacteria. These experiments will be directly relevant to goal (4) of the original project proposal. Planning is also underway to generate knockout PHYSCOMITRELLA plants in which the MT1 or MT6 gene is disabled. These experiments will be directly relevant to goal (2) of the original project proposal.
Impacts
What was accomplished under these goals?
The major goal of this project is to find ways to cause plants to synthesize modified plant cell walls that result in improved yield of biofuels from cell wall biomass. The approach is focused on altering the content of methyl-ether groups on sugar residues in cell wall polymers, the idea being that lower methyl-ether content might enhance the yield of bioethanol while higher methyl-ether content might enhance the yield of synthetic diesel fuel. Success inreaching this goal would benefit persons who directly or indirectly use transportation fuels, whether that use be in agricultural fields, on highways, on railways,in airways, or on ships. Thus, nearly the entire U.S. population could be potentially impacted. The initiation our research focus on O-methyl-ether-carrying sugar residues in plant cell wall polymers followed from our 2007 report that arabinogalactan proteins in the moss PHYSCOMITRELLA PATENS contain up to approximately 15 mol% of 3-O-methyl-L-rhamnosyl residues. The top priority in this research, and goal (1) in the present project, is to identify the gene that encodes an O-methyltransferase that produces these 3-O-methylrhamnosyl residues in the moss arabinogalactan proteins. By the end of the prior reporting period of the present project, the gene encoding the moss O-methyltransferase that produces 3-O-methylrhamnosyl residues had not been found. We had discovered, however, two moss genes, named MT1 and MT6, that when transgenically expressed in tobacco caused synthesis of 3-O-methylgalactosyl residues in polymers of the pectin and hemicellulosic fractions of the tobacco cell wall. Wild-type tobacco does not contain detectable levels of either 3-O-methylrhamnosyl residues or 3-O-methylgalactosyl residues, but approximately 10% of the galactosyl residues became methylated in each of the organs of the transgenic tobacco plants. Work during the prior reporting period thus focused on goals (1) and (3), but in the context of 3-O-methylgalactosyl residues instead of 3-O-methylrhamnosyl residues. Among the most interesting findings was that leaf tissue from either MT1 or MT6 transgenic tobacco plants, which contained 3-O-methylgalactosyl residues, was more tolerant of desiccation stress than leaf tissue from wild-type tobacco plants, which contained no detectable 3-O-methylgalactosyl residues. The research performed in the current reporting period was relevant to goals (4) and (1) of the original project. The finding of two moss genes, MT1 and MT6, that cause synthesis of 3-O-methylgalactosyl residues in transgenic tobacco is curious because the moss makes quite a lot of 3-O-methylrhamnosyl residues, but the level of 3-O-methylgalactosyl residues in moss, as in wild-type tobacco, is no more than noise level and might be zero. In research during the current reporting period, we formulated several hypotheses that might be consistent with this curious result. One hypothesis is that synthesis of 3-O-methylgalactosyl residues is not expressed in the leafy gametophyte phase of the moss life cycle, but is expressed in a different phase of the life cycle. The leafy gametophyte is the dominant phase of the life cycle in moss, and the phase that we routinely analyze. To test this hypothesis, protonemal cultures, representing the filamentous gametophyte phase of the moss life cycle, were grown and analyzed. The results showed that the level of 3-O-methylgalactosyl residues in filamentous protonemal gametophyte tissue, like in leafy gametophyte tissue, was no more than noise level and might be zero. Remaining as uncharacterized in this aspect is moss sporophyte tissue. In PHYSCOMITRELLA, the sporophyte phase of the life cycle is very small and is embedded in gametophyte tissue. Dissection of the moss to separate enough sporophyte tissue for analysis by our methods of gas chromatography-mass spectrometry would be extremely challenging. More feasible would be to test for 3-O-methylgalactosyl residues in the sporophyte by using an immunocytochemical approach with a monoclonal antibody specific for 3-O-methylgalactosyl residues, but to our knowledge no such monoclonal antibody has been developed. Another hypothesis regarding the curious discovery of moss genes MT1 and MT6 that cause synthesis of 3-O-methylgalactosyl residues in transgenic tobacco holds that when the MT1 and MT6 polypeptides, which are 76% identical at the aminoacyl level, are both present, such as what might occur in the moss, they form a heterodimer enzyme with a conformation that results in synthesis of 3-O-methylrhamnosyl residues. When only one of MT1 or MT6 is present, however, as occurs in the transgenic tobacco, the conformation of the monomer is such that it results in synthesis of 3-O-methylgalactosyl residues. This hypothesis was tested by means of a classical genetic cross of T1 generation MT1 and MT6 transgenic tobacco plants to produce T2 generation plants that have both the MT1 and MT6 genes. The result of this set of experiments was that none of the T2 plants contained detectable 3-O-methylrhamnosyl residues, but instead all contained 3-O-methylgalactosyl residues, like their T1 parents. While this result does not support the heterodimer hypothesis, it does not disprove it either. Most likely, MT1 and MT6 are targeted the Golgi body in the moss, but it remains possible that targeting in tobacco is slightly different with the result that MT1 and MT6 are localized in different Golgi cisternae and thus cannot get together to form a heterodimer. Still another hypothesis holds that the MT1 and MT6 methyltransferases have an active site that is not entirely specific In this case, MT1 and/or MT6 might be able to synthesize both 3-O-methylrhamnosyl residues and 3-O-methylgalactosyl residues, the former being favored in the conditions of moss cells and the latter being favored in the conditions of tobacco cells. To test this hypothesis, the MT1 and MT6 moss genes have been separately inserted into E. COLI bacteria with affinity tags incorporated to enable ready purification of the MT1 and MT6 proteins. At the end of the current reporting period, expression in the bacteria had been detected, but work was still underway to solve problems encountered in extracting the MT1 and MT6 proteins from the bacteria. The aim is to purify the proteins and then perform test-tube biochemical enzyme assays with various substrates that will shed light on the substrate specificity of the enzymes and will also enable direct mixing of the MT1 and MT6 proteins in the same test-tube assay as another test of the heterodimer hypothesis. The research performed in the current reporting period has provided further characterization of the MT1 and MT6 methyltransferases and thus has the potential for direct impact on the general goal of finding ways to cause plants to synthesize modified plant cell walls that result in improved yield of biofuels from cell wall biomass. If the MT1 and MT6 enzymes prove to be dual functional with regard to acceptable substrates, then their impact on the general goal might be magnified.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Masters, H.M., Theodory, B.G., Orozco-Cardenas, M., and Nothnagel, E.A. 2016. Testing for heterodimerization of moss methyltransferases through genetic crossing of transgenic tobacco plants. 10th Annual Undergraduate Research, Scholarship, and Creative Activity Symposium Program Book, p. 16, University of California, Riverside. April 20, 2016.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Tavernier, E.-K.G., and Nothnagel, E.A. 2016. Analysis and discovery of singly methylated sugars in wild type and transgenic tobacco cell walls. Center for Plant Cell Biology-Research Experience for Undergraduates Program, University of California, Riverside. August 19, 2016. http://cepceb.ucr.edu/reu/2016.html
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Aguilar, C., Jr., Yen, J., Orozco-Cardenas, M., and Nothnagel, E.A. 2016. Bacterial expression of cell wall methyltransferase genes from moss. 2016 Research in Science and Engineering Symposium Program Book, p. 28, University of California, Riverside. August 24, 2016.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Higuera, M., and Nothnagel, E.A. 2016. Relictual sugar residues in arabinogalactan proteins of PHYSCOMITRELLA PATENS. 2016 Research in Science and Engineering Symposium Program Book, p. 33, University of California, Riverside. August 24, 2016.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Theodory, B.G., Lee, J., Cid, A., Bol, S., Rolshausen, P.E, and Nothnagel, E.A. 2016. Glycosyl composition of polymeric components of gums exuded from uninfected and NEOFUSICOCCUM-infected almond trees. 10th Annual Undergraduate Research, Scholarship, and Creative Activity Symposium Program Book, p. 18, University of California, Riverside. April 20, 2016.
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:During the present reporting period, results were presented to a world-wide audience through publication of three articles in professional journals. Results were also disseminated to attendees at one Pacific regional, one state-level, and three local (University of California campus) research symposia. Changes/Problems:As detailed in the Accomplishments section of this progress report, the unanticipated and very fortuitous discovery of a moss gene encoding a galactosyl-3-O-methyltransferase has caused a shift which amounts to us focusing on the same stated objectives of the project, but in the context of 3-O-methyl galactosyl residues rather than 3-O-methyl rhamnosyl residues. The discovery of the galactosyl-3-O-methyltransferase gene has also caused us to broaden the project a bit to consider the effects of methylation of cell wall sugar residues on desiccation tolerance. What opportunities for training and professional development has the project provided?One postdoctoral student worked on the project as a staff research associate for approximately five months while awaiting the start date of her postdoctoral associate position at another institution. Limited financial resources have prevented hiring another person to replace this skilled scientist who has move on to her new position. Much of the work of the project was conducted by undergraduates who earned academic credit, who were supported by programs focused on under-represented or economically disadvantaged students, or who simply volunteered to gain research experience. A total of 11 undergraduates worked on the project during this initial reporting period. How have the results been disseminated to communities of interest?The results obtained in this initial reporting period are not quite ready for journal publication but have been disseminated to attendees at one Pacific regional and three local (University of California campus) research symposia. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, work will continue to be focused on the stated objectives of the project, but in the context of 3-O-methyl galactosyl residues rather than 3-O-methyl rhamnosyl residues. Work will also continue on the problem of identifying the originally targeted rhamnosyl-3-O-methyltransferase gene. In this context it is noteworthy that the level of 3-O-methyl galactosyl residues in the moss cell walls is barely detectable, far less than the level of 3-O-methyl rhamnosyl residues. This observation has caused us to wonder if the proteins encoded by MT1 and the related gene might be dual functional, producing 3-O-methyl galactosyl residues in the environment of tobacco cells but producing 3-O-methyl rhamnosyl residues in the environment of moss cells. This and related hypotheses will also be investigated in the upcoming reporting periods.
Impacts
What was accomplished under these goals?
The major goal of this project is to modify plant cell walls in ways that will result in improved yield of biofuels from cell wall biomass. The approach is focused on altering the content of methyl-ether groups on sugar residues in cell wall polymers, the idea being that lower methyl-ether content might enhance the yield of bioethanol while higher methyl-ether content might enhance the yield of synthetic diesel fuel. Success in this reaching this goal would benefit all persons who directly or indirectly use transportation fuels, whether that use be on highways, in airways, on ships, or in agricultural fields. Thus, the potential impact would be nearly universal among the U.S. population. The crux of the project is the identification of plant genes that encode methyltransferase enzymes that add methyl-ether groups to sugar residues in cell wall polymers. If methyltransferase encoding genes can be identified, then a variety of fairly standard methods are available to increase or decrease the expression of these genes, thereby potentially altering the methyl-ether content of the cell walls. At the start of this project, only one gene encoding a methyltransferase that adds methyl ethers to cell wall sugar residues has been identified, that producing 4-O-methylglucuronic acid residues in hemicellulosic xylans in the model plant Arabidopsis. The goals of the present project hinge on the identification of a gene that encodes a methyltransferase that produces 3-O-methyl rhamnosyl residues in the arabinogalactan proteins in the cell wall of the moss PHYSCOMITRELLA PATENS. In the prior project, 11 moss genes were investigated as candidates to encode the target rhamnosyl-3-O-methyltransferase, but none of these genes appeared to be the correct gene. At the start of the present project, 5 other moss genes were being investigated as new candidates. These 5 moss genes were being transgenically expressed in tobacco, NICOTIANA TABACUM, a plant that has no detectable 3-O-methyl rhamnosyl residues in its arabinogalactan proteins. The hypothesis was that if the moss rhamnosyl-3-O-methyltransferase gene was transgenically expressed in tobacco, then the transgenic tobacco would produce arabinogalactan proteins containing 3-O-methyl rhamnosyl residues. After the 5 types of transgenic tobacco plants were successfully produced, arabinogalactan proteins were purified from the leaves of the transgenic plants and analyzed by gas chromatography-mass spectrometry for the presence of 3-O-methyl rhamnosyl residues. Unfortunately, none of the 5 moss genes, which we call MT1-MT5, caused production of 3-O-methyl rhamnosyl residues in arabinogalactan proteins of the transgenic tobaccos. Before abandoning these 5 candidate genes, however, we observed that the MT1 transgenic plants exhibited an unanticipated phenotype. Although the plants were always adequately watered, occurrence of wilty lower leaves was observed at times during the growth of some proportion of all 5 types of transgenic plants. For the MT2-MT5 genes, this proportion ranged from 3 of 24 transgenic MT5 plants to 6 of 18 transgenic MT4 plants. With MT1, however, wilting was observed in 16 of 19 transgenic plants, a proportion statistically significantly higher than for any of MT2-MT5. This observed phenotype led us to look at the whole cell wall, rather than just arabinogalactan proteins, and these analyses led to the very fortuitous discovery that the MT1 plants were producing 3-O-methyl galactosyl residues in their cell walls, residues that were not detectable in either MT2-MT5 or wild-type tobacco plants. This discovery that MT1 is apparently a galactosyl-3-O-methyltransferase-encoding gene has caused us to change the direction of this project to focus on this gene, rather than the still unidentified gene that encodes rhamnosyl-3-O-methyltransferase. In particular, work during the remainder of the reporting period focused on specific objectives (1) and (3), but in the context of 3-O-methyl galactosyl residues rather than 3-O-methyl rhamnosyl residues. Analysis of cell walls from the different organs of the MT1 transgenic plants showed that leaves, stems, roots, and flowers all contain 3-O-methyl galactosyl residues amounting to about 10 percent of the total cell wall galactosyl residues. Because the leaf cell walls have slightly higher galactosyl content than the other 3 organs, the leaf cell walls have the highest 3-O-methyl galactosyl content, amounting as much as 1.6 mole percent of all cell wall sugar residues. Fractionation of leaf cell walls by sequential extraction and subsequent analyses of the subfractions showed some level of 3-O-methyl galactosyl residues in all subfractions but the highest level in a subfraction rich in pectic and hemicellulosic polysaccharides. Further chemical analysis showed that the 3-O-methyl galactosyl residues produced by MT1 have the absolute configuration 3-O-methyl-D-galactose, rather than 3-O-methyl-L-galactose. The unanticipated wilty-leaf phenotype of the MT1 transgenic tobacco plants prompted us to investigate water stress-related properties of MT1 leaves. Because other investigators have shown that 3-O-methyl galactosyl residues are particularly abundant in the cell walls of desiccation-tolerant plants such as lycophytes, we exposed tobacco leaf strips to high levels of polyethylene glycol to impose desiccation stress. The transgenic tobacco MT1 that expresses galactosyl-3-O-methyltransferase activity had a statistically significant lower percentage of electrolyte leakage after incubation with polyethylene glycol compared to the other transgenic tobacco MT2-MT5 controls. The presence of the methylated sugar had a positive effect on the ability of the leaf cells to withstand the desiccation stress applied from the polyethylene glycol. Further bioinformatics searching in the moss genome led to the finding of another apparent methyltransferase gene that is about 76% identical at the amino acid level to MT1. We hypothesized that this other methyltransferase might encode the rhamnosyl-3-O-methyltransferase that is the original target of this project. Towards the end of the current reporting period, we generated transgenic tobacco plants expressing this other methyltransferase and found that, like MT1, the transgenic plants do not contain 3-O-methyl rhamnosyl residues but do contain 3-O-methyl galactosyl residues and, like MT1 leaves, the leaves of these transgenic plants are also desiccation tolerant relative to wild-type control leaves. We consider the results obtained in the initial reporting period of this project to be extremely exciting. The discovery of MT1, only the second gene known to cause attachment of methyl-ether groups to sugar residues in plant cell wall polymers, is very significant. In particular, this discovery overcomes a key hurdle that should enable progress on the original objectives of this project as well as expanding the project to the investigation of cell wall modification relative to plant water stress tolerance.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Cid, A.N., Orozco-Cardenas, M., and Nothnagel, E.A. 2015. Effect of 3-O-methylation of cell wall galactosyl residues on desiccation tolerance of leaves from transgenic tobacco plants. 40th Annual West Coast Biological Sciences Undergraduate Research Conference Program & Abstracts Book, p. 56, Point Loma Nazarene University, San Diego, CA. April 25, 2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Ghobadi, R., Sasaninia, B., Cryder, Z., Wube, S., Juloya, G., Weston, B., Seo, S., Lee, J., Pardo, A., Orozco-Cardenas, M., and Nothnagel, E.A. 2015. Expression of a moss methyltransferase that produces 3-O-methyl-galactosyl residues in transgenic tobacco. 9th Annual Undergraduate Research, Scholarship, and Creative Activity Symposium Program Book, p. 5, University of California, Riverside. April 28, 2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Cid, A.N., Orozco-Cardenas, M., and Nothnagel, E.A. 2015. Effect of 3-O-methylation of cell wall galactosyl residues on desiccation tolerance of leaves from transgenic tobacco plants. 9th Annual Undergraduate Research, Scholarship, and Creative Activity Symposium Program Book, p. 14, University of California, Riverside. April 29, 2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Sasaninia, B., Ghobadi, R., Cryder, Z., Wube, S., Juloya, G., Weston, B., Seo, S., Lee, J., Pardo, A., Orozco-Cardenas, M., and Nothnagel, E.A. 2015. Organ localization of a methylated cell wall sugar in transgenic tobacco expressing a moss methyltransferase gene. 9th Annual Undergraduate Research, Scholarship, and Creative Activity Symposium Program Book, p. 14, University of California, Riverside. April 29, 2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Theodory, B.G., Lee, J., Orozco-Cardenas, M., and Nothnagel, E.A. 2015. D-L Analysis of 3-O-methyl-galactosyl residues in walls of transgenic tobacco. 2015 Research in Science and Engineering Symposium Program Book, p. 14, University of California, Riverside. August 26, 2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Sasaninia, B., Parnell, E.A., Sakhon, O.S., Nothnagel, E.A., and Lo, D.D. 2015. Pectic polysaccharide probes with 3-O- methyl-galactosyl residues for investigating uptake activity by microfold cells in the gastrointestinal tract. 28th Annual Mentoring Summer Research Internship Program Symposium Book, p. 37, University of California, Riverside. August 14, 2015.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Dhall, S., Do, D. C., Garcia, M., Kim, J., Mirebrahim, S. H., Lyubovitsky, J., Lonardi, S., Nothnagel, E. A., Schiller, N., and Martins-Green, M. 2014. Generating and reversing chronic wounds in diabetic mice by manipulating wound redox parameters. Journal of Diabetes Research 2014: article ID 562625, 18 pages. doi:10.1155/2014/562625.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Dou, Y., Robles, A., Roy, F., Aruni, A. W., Sandberg, L., Nothnagel, E., and Fletcher, H. M. 2015. The roles of RgpB and Kgp in late onset gingipain activity in the vimA-defective mutant of Porphyromonas gingivalis W83. Mol. Oral Microbiol. 14 pages. doi:10.1111/omi.12098.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Davis, B., Razo, A., Nothnagel, E., Chen, M., and Talbot, P. 2015. Unexpected nicotine in Do-it-Yourself in electronic cigarette flavourings. Tob Control Published Online First: July 27, 2015 doi:10.1136/tobaccocontrol-2015-052468
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Davis, B., Razo, A., Talbot, P., and Nothnagel, E. 2014. Nicotine analysis and quantification in Tasty Puff Do It Yourself flavoring solutions for electronic cigarettes. Southern California Conferences for Undergraduate Research Program, p. 13, California State University, Fullerton. November 22, 2014.
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