Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: My work addresses Objective 3 (Mechanisms regulating photosynthate partitioning) within this multistate project. We are investigating whether genes that control synthesis and breakdown of a specific molecule called inositol are required for responses to low energy. We examined genes encoding the myo-inositol phosphate synthases (MIPS) and myo-inositol oxygenases (MIOX 2 and 4. We looked at whether expression of these genes is regulated by energy/nutrient conditions. We found that expression of both MIOX genes is suppressed by high energy conditions in the shoot, but not in the root. Both MIOX genes were abundantly expressed during low energy conditions. Plants defective in MIPS or MIOX genes contain alterations in inositol levels. MIPS mutants indicated the importance of inositol to control plant growth and to limit cell death. Growth changes in MIOX mutant roots indicate that plants may sense levels of inositol to regulate energy status. Together, these data suggest that inositol may act as either a direct or indirect indicator of energy status in plants. PARTICIPANTS: Glenda Gillaspy: Principal investigator. Role: directed research. Janet Donahue : Research technician. Role: performed biochemistry experiments and constructed and analyzed transgenic plants. Aida Nourbakhksh: PhD student. Role: constructed and analyzed transgenic plants and performed cell imaging experiments. Juin Yen: PhD student. Role: constructed and analyzed transgenic plants. Padma Rangarajan: MS student who characterized energy sensor expression in plants. Sarah Williams: PhD student. Role: subcellular localization of proteins. Elitsa Ananieva. Role: PhD student who identified signaling mutants. (8) Shannon Alford. Role: Physiological studies on mutants. Joonho Park. Role: Postdoctoral fellow who cloned genes. Mihir Mandal. Role: Postdoctoral fellow who cloned genes and studied functions of proteins. Sunyoung Byon: Undergraduate researcher. Role: examined genetic mutants. Caitlin OConnell: Undergraduate researcher. Role: examined and analyzed mutant and transgenic plants. Tyler Stewart. Undergraduate researcher. Role: examined and analyzed mutant and transgenic plants. Jenna Hess. MS student. Role: examined gene expression. Training and professional development. Three undergraduates, seven graduate students and two postdocs received training. Outreach to K-12 classrooms engaged three teachers and exposed high school students to plant science. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Plants sense and utilize energy from the sun and convert this energy into a usable form of carbon-containing compounds. These reactions sustain life on our planet. We study how plants use inositol as delineating the energy sensing pathways of plants is likely to lead to new approaches for maximizing energy use in our environment. Our studies on the MIPS and MIOX genes are useful in that we can now use the MIOX2 gene as a read-out for energy status in plants. Thus this project provides a tool for other laboratories, besides advancing our knowledge of energy sensing and providing training for new scientists.
Publications
- Ananieva, EA, Gillaspy, GE, Ely, A, Burnette, RN, and Erickson, FL (2008) Interaction of the WD40 domain of a myo-inositol polyphosphate 5-phosphatase with snrk1 links inositol, sugar, and stress signaling. Plant Physiology 148:1868-82.
- Gunesekera B, Torabinejad, J, Robinson, JY, and Gillaspy, GE (2007) The inositol polyphosphate5-phosphatases are required for regulating seedling growth. Plant Physiology 143:1408-1417.
- Donahue, J, Ercetin, M and Gillaspy, G (2012) Assaying Inositol and Phosphoinositide Phosphatase Enzymes In Plant Lipid Signaling Protocols (Munnik T. and Heilmann I. Eds) Humana Press (Springer), In press.
- Alford, S, Rangarajan, P, Williams, S, and Gillaspy, G (2012) Myo-inositol oxygenase is required for responses to low energy conditions in Arabidopsis thaliana, Frontiers in Plant Physiology 3:69.
- Gillaspy, G (2012) The cellular language of myo-inositol signaling, New Phytol 192, 823-839.
- Torrens-Spence, MP, Gillaspy, G, Zhao, B, Harich, K, White, RH and Li, J (2012) Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme, Biochemical and biophysical research communications 418, 211-216.
- Kaye Y, Golani Y, Singer Y, Leshem Y, Cohen G, Ercetin M, Gillaspy G, Levine A. (2011) Inositol polyphosphate 5-phosphatase7 regulates production of reactive oxygen species and salt tolerance in Arabidopsis. Plant Physiology. In press.
- Fleet, CM, Ercetin, ME, and Gillaspy, GE (2009) Inositol phosphate signaling and gibberellic acid. Plant Signaling &Behavior 4:73-74.
- Ananieva, EA, Gillaspy, GE (2009) Switches in nutrient and inositol signaling. Plant Signaling & Behavior 4: 304-306.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Two new genes have been identified and their gene products have been characterized. Genetic mutants have been characterized. A biochemical understanding of the encoded enzymes has been determined. For one of these genes, this completes our understanding of the histidine synthetic pathway.The work has been disseminated at local scientific meetings and is being prepared for publication. PARTICIPANTS: Participants: (1) Glenda Gillaspy: Principal investigator. Role: directed research. (2) Janet Donahue: Research technician. Role: constructed transgenic plants and performed biochemical assays. TARGET AUDIENCES: Scientific Community. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
To gain insight into the mechanisms that regulate photosynthate partitioning into pathways of biosynthesis and use of sugar alcohols, my laboratory has completed work on the inositol monophosphatase (IMP) enzymes. This family of enzymes removes a phosphate from inositol monophosphate substrates, producing molecules which are important for stress protection and cell communication. In the model plant Arabidopsis thaliana there are three genes that encode conserved IMPs. We have previously examined the substrate preference of AtIMP. We found this enzyme is bifunctional and acts in both the inositol synthesis and ascorbic acid synthesis pathways. The two other IMP genes are more similar to IMPs from bacteria, and we hypothesized that these proteins played a different role in plants. The AtIMPL proteins are located in the chloroplast. We have been determining how these AtIMPL enzymes contribute to inositol synthesis and/or cell communication; both enzymes appear to have evolved unique properties. Foremost, we found that the AtIMPL2 enzyme acts in the histidine synthesis pathway, which was unexpected. We identified and characterized a mutant in the AtIMPL2 gene that severely disrupts growth. In contrast, AtIMPL1 acts on a different substrate and does not participate in histidine synthesis. It also appears to be different from the AtIMP enzyme, thus AtIMPL1 has unique properties. Since the AtIMPL1 substrate is linked to use of cell communication molecules, our results suggest a role for this enzyme in communication between the chloroplast and interior of the cell.The major impact of our studies is that support for a role for inositol in the chloroplast has been gained. In addition, our studies have provided information about the histidine synthesis pathway in plants, which is a fundamental and required pathway.
Publications
- Gillaspy, GE (2011) The cellular language of myo-Inositol signaling. New Phytologist. DOI: 10.1111/j.1469-8137.2011.03939.x
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: To gain insight into the mechanisms that regulate photosynthate partitioning into pathways of biosynthesis and use of sugar alcohols, my laboratory has completed work on the rate limiting and first step in myo-inositol synthesis. The sugar alcohol myo-inositol has been linked to osmoprotective functions, signal transduction, phosphate storage and ascorbic acid synthesis. We characterized the myo-inositol phosphate synthase (MIPS) genes and encoded enzymes from the model plant, Arabidopsis thaliana. The main question we addressed is how the three MIPS genes in Arabidopsis are regulated and provide myo-inositol synthesis to meet various metabolic demands. We found that the three MIPS genes encode similar enzymes with similar kinetic constants. However, the three genes are spatially regulated at the level of transcription resulting in MIPS1 expression in a broader range of cell types. In contrast MIPS2 and MIPS3 are expressed only in vascular cells. Analysis of genetic mutants indicates MIPS1 has a larger impact on myo-inositol synthesis than MIPS2 or MIPS3. In addition, MIPS1 mutants have uncontrolled cell death that is associated with reduced phosphatidylinositol and increased ceramide levels. This unexpected connection shows that maintenance of phosphatidylinositol is critical for regulating sphingolipid synthesis. PARTICIPANTS: (1) Glenda Gillaspy: Principal investigator. Role: directed research and performed genetic crosses. (2) Janet Donahue : Research technician. Role: performed biochemistry experiments and constructed and analyzed transgenic plants. (3) Aida Nourbakhksh: PhD student. Role: constructed and analyzed transgenic plants and performed cell imaging experiments. (4) Juin Yen: PhD student. Role: constructed and analyzed transgenic plants. (5) Rachel Logan: Undergraduate researcher. Role: examined transgenic reporter plants. (6) Solange Parades: Undergraduate researcher. Role: examined and analyzed mutant and transgenic plants. Training and professional development. Three undergraduates and two graduate students received training. Outreach to K-12 classrooms engaged four teachers and exposed both elementary and high school students to plant science. TARGET AUDIENCES: Results have been disseminated by integrating aspects of the research into an outreach based pre-college education program (the Partnership for Research and Education using Plants). A new focus in 2010 was on increasing critical thinking. Over the past year, more than 200 high school students in Virginia performed their own inquiry-based experiments with mutants generated from this project. During 2010 preparations were made for students to work with a new mutant and the P.I. participated in creating instructional materials (i.e. a film, primers, and mutant seed) for this work. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our studies focused on mechanisms that regulate photosynthate partitioning and developmental and environmental limitations to photosynthesis support a role for inositol and phosphatidylinositol signaling in several important plant processes. These include regulation of growth, and responses to stress and nutrient limitations. These important processes may be a valid target for genetic engineering to increase plant health and/or biomass in the future.
Publications
- Donahue, JL, Alford, SR, Torabinejad, J, Kerwin, R, Nourbakhsh, A, Ray, WK, Lyons, B, Hein PP, and Gillaspy, GE. (2010) The Arabidopsis thaliana Myo-Inositol 1-Phosphate Synthase1 Gene Is Required for Maintenance of Myo-inositol Synthesis and Suppression of Cell Death. The Plant Cell 22: 888-903.
- Gillaspy, GE (2010) The Polyphosphoinositide Phosphatases in Lipid Signaling in Plants. Springer; ed: T. Munnik.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: To gain insight into the mechanisms that regulate photosynthate partitioning into pathways of biosynthesis and use of sugar alcohols, my laboratory has been continuing studies on the synthesis and metabolism of myo-inositol. The sugar alcohol myo-inositol has been linked to osmoprotective functions, signal transduction, and ascorbic acid synthesis. We characterized the myo-inositol monophosphatase (IMP) enzyme and reported that the plant IMP (also called VTC-4) has a similar Km for D-inositol 3-P, a precursor to myo-inositol, and L-galactose-1-P, a substrate used for ascorbic acid synthesis in the GDP-mannose (Smirnoff-Wheeler) pathway. Mutants in IMP exhibit reductions in both myo-inositol and ascorbic acid, therefore IMP impacts both pathways. Two other IMP-like (IMPL) genes may also impact both pathways. The IMP gene is most similar to other eukaryotic IMPs, while IMPL1 and IMPL2 encode proteins most similar to prokaryotic IMPs. We reported this year that recombinant IMPL2 had a similar substrate preference as IMP, however, IMPL1 was found to prefer a D-inositol 1-P substrate, indicating it might play a role in recycling inositol second messengers. We also continued to examine loss of function mutants in both the IMPL1 and IMPL2 genes in Arabidopsis. The study of these gene families will allow us to genetically dissect this important synthesis pathway. PARTICIPANTS: (1) Glenda Gillaspy: Principal investigator. Role: directed research and performed cell biological experiments to localize proteins. (2) Janet Donahue : Research technician. Role: performed biochemistry experiments and constructed transgenic plants. (3) Shannon Alford: PhD student. Role: constructed and analyzed transgenic plants and performed metabolite measurements. (4) Aida Nourbakhksh: PhD student. Role: constructed and analyzed transgenic plants and performed cell imaging experiments. (5) Matthew Allen-Daniels: Undergraduate researcher. Role: complemented imp mutants and performed cold physiology experiments. (6) Rachel Logan: Undergraduate researcher. Role: examined transgenic reporter plants. (7) Blair Lyons: Undergraduate researcher. Role: constructed and examined transgenic reporter plants. Regarding training and professional development: Three undergraduates and two graduate students received training. Outreach to K-12 classrooms engaged four teachers and exposed both elementary and high school students to plant science. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
One outcome of this project is that we have determined the role of the IMP protein in contributing to both myo-inositol an ascorbate production in plants. Since we found that the IMP protein is bifunctional in hydrolyzing both D inositol-1-P and L-galactose-1-P, this indicates that both molecules could serve as substrates for the IMP enzyme. A second outcome is that we found that an IMP loss of function affects both the ascorbic acid level (from loss of L-galactose-1-P hydrolysis) and the myo-inositol level (from loss of the D- inositol 1-P hydrolysis). Together with our biochemical results, this further strengthens our conclusion that the plant IMP enzyme is bifunctional. New data this year supports a role for other genes, such as the IMPL genes, in myo-inositol homeostasis as well. The impact of this work is on agricultural productivity. The myo-inositol synthesis pathway impacts several crucial plant physiological processes that are important for plant vigor and agricultural yield. For example, production of Vitamin C by plants is critical to sustain human nutrition. Our work contributes to the general body of knowledge on this pathway, as myo-inositol alterations have been linked to changes in plant Vitamin C content.
Publications
- Torabinejad, J, Donahue, JL, Gunesekera, BN, Allen-Daniels, MJ, Gillaspy, GE (2009) VTC4 is a bifunctional enzyme that affects myo-inositol and ascorbate biosynthesis in plants. Plant Physiology 150:951-61.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: To gain insight into the mechanisms that regulate photosynthate partitioning into pathways of biosynthesis and use of sugar alcohols, my laboratory has been continuing studies on the synthesis and metabolism of myo-inositol, with a focus on identifying and characterizing the genes involved. The sugar alcohol myo-inositol has been linked to growth, osmoprotection and other beneficial plant traits, yet we do not currently understand how levels of inositol are controlled by the cell, and whether different pools of inositol are used for signaling versus metabolic needs. The long-term goal of this project was to understand specifically, how the plant cell coordinates inositol synthesis with its varying needs. We have utilized genome information and the availability of loss-of-function mutants in Arabidopsis thaliana to provide a comprehensive analysis of the architecture of the inositol metabolism pathway in plants. We have characterized the gene family that encodes the first committed step in inositol synthesis, myo-inositol phosphate synthase (MIPS). MIPS is encoded by three genes with nearly identical nucleic acid sequences and the three MIPS proteins are predicted to function in a similar manner, in converting glucose-6-P into myo-inositol-1-P. During the past year we have examined MIPS mutants and found that the three MIPS genes impact plant physiology in diverse and unique ways. Specifically, we have found that a MIPS1, but not MIPS2 or MIPS3 mutant, is defective in control of cell death. Mutants that lack MIPS1 have spontaneous cell death that is linked to a decrease in both inositol and ascorbic acid. Growing MIPS1 mutants with inositol or ascorbic acid-supplemented water "rescues' the effects of the MIPS mutation. This indicates that inositol and ascorbic acid levels are critical for maintaining control of cell death mechanisms. PARTICIPANTS: The PI, Glenda Gillaspy, participated in this project. Ph.D. students who received training from this project are William Slade and Aida Nourbaksh. Undergraduate students Matthew Allen-Daniels, Blair Lyons and Christy Perry also received training and contributed. TARGET AUDIENCES: The target audience for this project are the scientific community, the biotech industry and students. Elementary and high school students were engaged through outreach programs. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The impact of this work is on agricultural productivity. The myo-inositol synthesis pathway impacts several crucial plant physiological processes that are important for plant vigor and agricultural yield. For example, InsP6 is made from myo-inositol and is a critical supplier of energy to germinating seeds. High levels of seed InsP6, however, end up generating phosphate pollution in many US watersheds. Therefore, plant biologists are attempting to lower seed InsP6 levels. A thorough understanding of myo-inositol synthesis, one outcome of this work, is required to refine current InsP6 engineering strategies. As we have shown that reduction of MIPS1 adversely affects cell growth and physiology, this gene should not be used as a target for genetic engineering in plants. Another important impact is the production of Vitamin C by plants. This project contributes to the general body of knowledge on this pathway, as myo-inositol alterations have been linked to changes in plant Vitamin C content. Our work this year shows that alterations in inositol synthesis may impact Vitamin C synthesis, and this aspect should be considered in designing strategies to reduce InsP6.
Publications
- Gillaspy, G. E. 2008. Plant Development and Physiology, pp. 83-111. in Plant Biotechnology editor: N. Stewart; Publisher: John Wiley & Sons, Hoboken, NJ.
- Ercetin, M., Torabinejad, J., Robinson, J., Gillaspy and G. 2008. A Phospholipid-Specific Myo-Inositol Polyphosphate 5-Phosphatase Required for Seedling Growth. Plant Molecular Biology 67:375-388.
- Ananieva, E. A., Gillaspy, G. E., Ely, A., Burnette, R. N. and Erickson, F. L. 2008. Interaction of the WD40 Domain of a Myo-Inositol Polyphosphate 5-Phosphatase with SnRK1 links Inositol, Sugar and Stress Signaling. Plant Physiology, in press.
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