Progress 06/01/00 to 05/31/05
Outputs
During this project two major areas of research concerning inositol phosphates in plants were addressed. Genes encoding proteins required for myo-inositol synthesis were cloned from the model plant, Arabidopsis thaliana. These genes allowed for the examination of the genetic controls of inositol synthesis in plants. We found that the myo-inositol phosphate synthase (MIPS) enzyme is encoded by three separate genes in Arabidopsis. An antisera that recognizes the MIPS proteins was produced and used to characterize the encoded proteins. We also found that the myo-inositol phosphate phosphatase (IMP) protein is encoded by multiple genes in Arabidopsis. Antisense suppression of IMP in tomato resulted in a decrease in mass inositol levels, and also a reduction in ascorbic acid. This finding led to the discovery that inositol and ascorbic acid synthesis are linked in plants. The promoter of the tomato IMP-2 gene was cloned, characterized, and fused to a reporter gene to
deterimine its regulation. We found a very restricted pattern of expression of this gene, which indicates a spatial control of IMP gene expression. With regards to the second area of research, we identified and cloned 15 genes that encode different isoforms of the myo-inositol polyphosphate 5-phosphatase enzyme (5-PTases). The function of these enzymes is to hydrolyze the 5-phosphate from a variety of inositol-containing substrates, such as the second messenger, inositol (1,4,5)P3 (InsP3). Overexpression of the 5PTase1 gene resulted in a 3-fold reduction of InsP3 levels in transgenic plants, and altered ABA signaling and stomatal function. Examination of different Arabidopsis 5PTase enzymes allowed us to define four categories of enzymes that hydrolyze subsets of different substrates.
Impacts
Since inositol hexakisphosphate (InsP6) is an environemental pollutant in many places, including the Chesapeake Bay watershed, understanding how plants control synthesis of InsP6 is important. All plants start with myo-inositol in their synthesis of InsP6. Our work on the genes encoding enzymes required for myo-inositol synthesis will allow researchers to design better strategies to combat InsP6 pollution. Our work on InsP3 and the genes that control its breakdown also can impact the InsP6 problem. Another area of concern that these studies impact is the control of plant stress repsonses. Since InsP3 is used by plants to respond to salt, drought and cold, our findings that show that 5PTase enzymes can be altered to control InsP3 levels open a new avenue for controlling plant response to stress via genetic engineering of this pathway.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
To accomplish our goal of understanding inositol synthesis in plants we have completed a genomic analysis of the genes required by the model plant, Arabidopsis thaliana, to synthesize inositol. Three families of genes were identified. Each multiple gene family encodes distinct, but highly conserved proteins utilized in inositol synthesis. We have focused biochemical characterization efforts on the inositol monophosphatase proteins. Recombinant proteins, produced in bacteria are being utilized to determine the substrate specificity of these enzymes. Putative loss-of-function mutants for each gene in the three gene families have been identified. Inositol monophosphatase mutants are the focus of mutant characterization and will be used to show the impact of gene disruption on inositol synthesis.
Impacts
Phosphate pollution of the Chesapeake Bay is a major environmental issue. Much of the phosphate comes from the accumulation of undigested plant phosphate in soil from animal manure. This plant phosphate, originally synthesized as an energy source for plants (IP6), exists in a form complexed with the polyol, inositol. By understanding how plants synthesize inositol and IP6, we may be able to control and limit phosphate pollution. To do this, we must first identify and characterize the plant genes responsible for inositol and inositol phosphate synthesis, and determine the effects on altering individual genes in this pathway.
Publications
- Torabinejad, J. and Gillaspy, G.E. (2004) Functional Genomics of Inositol Metabolism. Subcellular Biochemistry, Volume 39: Biology of Inositols and Phosphoinositide, editors: Biswas and Majumder, in press.
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Progress 10/01/02 to 09/30/03
Outputs
To accomplish our overall goal of understanding how plants respond to the environment, we have focused on two types of plant enzymes involved in second messenger breakdown. The first type, the Arabidopsis inositol 5-phosphatase (5PTase) enzymes, have the ability to degrade the second messenger, inositol (1,4,5)P3. In the past year we have shown that transgenic plants overexpressing the At5PTase1 gene have an increased capacity to degrade the second messenger, inositol (1,4,5)P3, and that stomates from these plants are insensitive to the drought-induced hormone, abscisic acid. We have also identified a potential loss-of-function mutant in the At5PTase1 gene. From our previous work, we expect that this mutant may contain elevated levels of IP3 which may lead to beneficial plant responses during drought conditions. We have also characterized a second Arabidopsis 5PTase gene, called At5PTase11. This gene encodes a smaller, active 5PTase enzyme which acts on
phosphatidylinositol (PI)(4,5)P2 and PI(3,4,5)P3 substrates. Since PIP2 has been hypothesized to play a role in several basic plant cellular phenomema, studies on this gene are expected to reveal the role PIP2 second messenger plays within the plant. We have also identified a loss-of-function mutant for the At5PTase11 gene. The second class of enzymes are called the inositol monophosphatase (IMP) enzymes. In the past year, we have examined transgenic plants which contain either a decrease or increase in functional IMP protein. The preliminary results from these studies indicate that levels of IMP correlate with the levels of ascorbic acid in plants, linking these two pathways.
Impacts
All organisms require the ability to respond to their environment in order to adapt and survive. Second messengers are molecules that allow individual cells within the organism to respond to signals generated outside of the cell. They are produced or degraded within the cell in response to signals and allow for amplification of environmental signals. Inositol phosphatases are enzymes that can break down second messengers. Through our experiments we have defined the molecules required for response to dry or drought conditions. By understanding how these molecules effect physiological changes in plants, we maybe able to engineer transgenic plants with increased tolerance to drought and other adverse conditions.
Publications
- Styer, J., Spence, J., Keddie, J. and Gillaspy, G. (2003) Genomic organization and regulation of the LeIMP-1 and LeIMP-2 genes encoding myo-inositol monophosphatase in tomato. Gene, in press.
- Gillaspy, G.E., Ercetin, M.E., and Burnette, R.N. (2003) Inositol Metabolism In Plant Cells: A Genomics Perspective. In Advances in Plant Physiology, volume VII; in press.
- Burnette, R.N., Gunesekera, B. and Gillaspy, G.E. (2003) An IP3 Signal Terminating Gene From Arabidopsis Can Alter ABA Signaling. Plant Physiology 132:1101-1109.
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Progress 10/01/01 to 09/30/02
Outputs
To accomplish our goal of understanding inositol synthesis in plants we have completed a genomic analysis of the genes required by the model plant, Arabidopsis thaliana, to synthesize inositol. Three families of genes were identified. Each multiple gene family encodes distinct, but highly conserved proteins utilized in inositol synthesis. We also completed the analysis of the partial loss-of-function of one of the gene families, the inositol monophosphatase (IMP) genes. In tomato plants, loss of IMP protein resulted in lower inositol levels and a surprising reduction in ascorbatic acid synthesis. We have opportunity to characterize the connection between inositol synthesis and ascorbatic acid synthesis in plants.
Impacts
Phosphate pollution of the Chesapeake Bay is a major environmental issue. Much of the phosphate comes from the accumulation of undigested plant phosphate in soil from animal manure. This plant phosphate, originally synthesized as an energy source for plants (IP6), exists in a form complexed with the polyol, inositol. By understanding how plants synthesize IP6, we may be able to control and limit phosphate pollution. To do this, we must first identify and characterize the plant genes responsible for inositol and inositol phosphate synthesis.
Publications
- No publications reported this period
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Progress 10/01/00 to 09/30/01
Outputs
Our overall objective is to understand and manipulate inositol phosphate metabolism in plants. To accomplish this goal we continued studies on the tomato inositol monophosphatase (IMP) gene and lithium inhibition of growth in tomato. Preliminary work indicates that antisense suppression of IMP alters meiosis such that progeny plants become tetraploid. We have also focused on the signal-terminating inositol 5-phosphatase (5PTase) enzymes and the genes that encode these enzymes in Arabidopsis. In the past year we have shown that transgenic plants overexpressing the At5PTase1 gene, have an increased capacity to degrade inositol-(1,4,5) -triphosphate (IP3), and that this gene is expressed at low levels in a variety of plant tissues. We also completed the characterization of the 14 other 5PTase genes from Arabidopsis and analyzed the phylogenetic relationship of all 15 At5PTases. To determine if alterations in IP3 are correlated with alterations in signaling, we have
examined the response of transgenic plants overexpressing the At5PTase1 gene to abscisic acid (ABA). Wild type plants responded to ABA by increasing IP3 levels at 1 minute and 30 minutes post-stimulation and by closing their stomata. Transgenic plants had lower basal IP3 levels that increased only slightly when ABA was added and their stomata were insensitive to ABA. Taken together, we conclude that an increase in IP3 breakdown decreases the ability of a plant to respond to ABA. To examine At5PTase1 protein, we have developed an antibody specific for this protein. Preliminary work indicates that At5PTase1 protein levels are rapidly regulated by light, ABA and cold stimuli. Within hours after these stimuli, At5PTase1 protein levels return to unstimulated levels. Signals such as light, ABA and cold may increase the production of signal terminators that then act to stop signaling. Comparison of the time courses induction of At5PTase1 protein and IP3 accumulation after ABA addition
support this idea.
Impacts
All organisms require the ability to respond to their environment in order to adapt and survive. Second messengers are molecules that allow individual cells within the organism to respond to signals generated outside of the cell. Plants in the field respond to a variety of signals such as drought, pests and pathogens. This work will help identify the molecular machinery that plants use to respond to these signals. The identification of new targets for modulating signal transduction may permit the engineering of transgenic plants with altered physiological responses.
Publications
- Berdy, S., Kudla, J. Gruissem, W. and Gillaspy, G. 2001. Molecular Characterization of At5PTase1, an Inositol Phosphatase Capable of Terminating IP3 Signaling. Plant Physiology , 126:801-810.
- Gillaspy, G. and Gruissem, W. 2001. Li+ Induces Hypertrophy and Down Regulation of Myo-Inositol Monophosphatase in Tomato. Journal of Plant Growth Regulation, 20:78-86.
- Strahl, E.D., Gillaspy, G. E., and Falkingham, J.O. 2001. Fluorescent acid-fast microscopy for measuring phagocytosis of Myocobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth. App. Env. Microbiology, 67:4432-4439.
- Gillaspy G. 2001. A change of heart. finding the right balance. Plant Physiol, 127:377-8.
- Burnette, R., Gunesekera, B., Ecertin, M., Berdy, S. and Gillaspy, G. 2001. A Signal Terminating Gene From Arabidopsis Can Alter ABA Signaling. 12th International Conference on Arabidopsis Research Proceedings, 373.
- Burnette, R., Gunesekera, B., Berdy, S. and Gillaspy, G. 2001. Characterization of Inositol 5'-Phosphatase 1 (5PTase1) in the IP3 Signaling Pathway in Arabidopsis thaliana. Proceedings of the 2001 Experimental Biology Meeting.
- Hubbard, K., Holbrook, E., Styer, J. and Gillaspy, G. 2001. Alterations in Myo-Inositol Synthesis Affect Plant Growth and Development. Proceedings of the 2001 Experimental Biology Meeting.
- Storrie, B., Lederman, M., Wong, E., Walker, R. and Gillaspy, G. 2000. Working With Molecular Cell Biology: A Study Companion, ed: W.H. Freeman and Co., New York, NY.
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Progress 10/01/99 to 09/30/00
Outputs
To address how plants use inositol-containing second messengers, fifteen genes that encode putative signal terminators from the model plant Arabidopsis thaliana have been identified. We have analyzed the genomic sequences of these genes and from this can predict that each gene is regulated by different environmental signals. To test whether this is true, we are measuring mRNA levels of these genes in plants exposed to darkness, cold and anerobic conditions. To test the function of one of these genes, At5PTase1, we have constructed transgenic plants that overexpress this gene. We have shown that these transgenic plants contain elevated ability to degrade IP3 second messenger. We are currently testing the response of these transgenic plants to various environmental stimuli to determine whether they have altered responses.
Impacts
All organisms require the ability to respond to their environment in order to adapt and survive. Second messengers are molecules that allow individual cells within the organism to respond to signals generated outside of the cell. Plants in the field respond to a variety of signals such as drought, pests and pathogens. This work will help identify the molecular machinery that plants use to respond to these signals. The identification of new targets for modulating signal transduction may permit the engineering of transgenic plants with altered physiological responses.
Publications
- Holbrook, B., Styer, J., Saucier, N.and Gillaspy, G. 2000. Expression Of Myo-Inositol Phosphate Synthase Isoforms In Arabidopsis Thaliana. Virginia Journal of Science 51 (2) addendum.
- Berdy, S., Goley, M., Gruissem, W. and Gillaspy, G. 2000. Ectopic and antisense expression of gene products that hydrolyze IP3. Proceedings of the 13th Annual Penn State Plant Physiology Symposium. p.13.
- Styer, J. 2000. Molecular genetics of inositol synthesis in plants. M.S. Thesis, Virginia Polytechnic Institute and State University, Blacksburg, VA.
- Goley, M. 2000. Transgenic alteration of inositol monophosphatase in tomato. M.S. Thesis, Bucknell University, Lewisburg, PA.
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