Progress 08/15/04 to 08/14/08
Outputs
OUTPUTS: Water stress is one of the devastating environmental problems causing severe loss in agriculture. Abscisic acid (ABA) plays an essential role in protection of plants from a variety of environmental stresses such as drought, salt, and cold. Reactive oxygen species (ROS) have been suggested to function in guard cell ABA signaling. Based on our preliminary results, we hypothesized that (i) A MAPK pathway mediates ABA-induced ROS signaling in guard cells and that (ii) phosphorylation of NADPH oxidases by protein kinases is important for the enzymatic activity and for the regulation of ABA signaling. Our pharmacological study with the MAPKK inhibitor PD98059 shows that MAPK cascade(s) functions downstream of ROS in ABA signaling in guard cells. To identify and characterize MAPK cascades mediating guard cell ABA/ROS signaling, we identified two MAPK genes, MPK9 and MPK12. RT-PCR and GUS reporter gene analysis shows that these two MPK genes are preferentially and highly expressed in guard cells. To provide direct genetic evidence, RNAi-based gene silencing plant lines were generated in which both genes are silenced. In parallel, Arabidopsis single and double mutants carrying deleterious point mutations in these genes were identified. ABA-induced stomatal closure was strongly impaired in the RNAi lines in which both MPK9 and MPK12 transcripts were significantly silenced. Consistent with this result, the mpk9-1/mpk12-1 double mutants showed an enhanced transpirational water loss and ABA- and H2O2- insensitive response in stomatal movement assays, whereas mutants carrying a mutation in one of these genes did not show any altered phenotype. An MPK12-YFP fusion construct rescued the double mutant phenotype in ABA-induced stomatal movements, demonstrating that the mutations in these genes caused the phenotype. MPK12 appears to be localized to the cytosol and the nucleus, and ABA does not affect its protein localization, suggesting that there might be a corresponding MAPKK that translocates to the nucleus to phosphorylate MPK12 upon stimulus. Together, these results provide genetic evidence that MPK9 and MPK12 function downstream of ROS to positively regulate guard cell ABA signaling. The OST1 protein kinase was previously shown to function upstream of ROS in guard cell ABA signaling. AtrbohF NADPH oxidase is responsible for ABA-triggered ROS production in ABA signaling in guard cells. Thus, we hypothesized that AtrbohF could be a direct target of OST1. Our in vitro phosphorylation assays clearly show that AtrbohF is phosphorylated in its N-terminus by OST1. Ser174 was predicted to be phosphorylate by OST1. In vitro protein kinase assays show that the phosphorylation signal from the mutant AtrbohF, in which Ser174 was mutated to Ala by site directed mutagenesis, was reduced to 50-70% of the signal obtained from WT AtrbohF, demonstrating that Ser174 is one of amino acid residues that are phoshorylated by OST1. Mass Spectrometry analysis combined with in vitro protein kinase assay confirmed that Ser174 is phoshorylated by OST1 and identified another Ser residue that is phosphorylated by OST1. PARTICIPANTS: Dongjin Shin (postdoc) Department of Cell Biology and Molecular Genetics, University of Maryland, College Park (Tel: 301-405-9727; Email: djshin99@umd.edu) Dan Gu (postdoc) Department of Cell Biology and Molecular Genetics, University of Maryland, College Park (Tel: 301-405-9727; Email: dangu@umd.edu) Koji Takeda, postdoc, Department of Cell Biology and Molecular Genetics, University of Maryland, College Park (Tel: 301-405-9727; Email: ktakeda@umd.edu) Sangmee Lee, technician, Department of Cell Biology and Molecular Genetics, University of Maryland, College Park Youngsook Lee, Collaborator, Division of Molecular Life Sciences, POSTECH, Pohang, 790-784, Korea (Tel: +82-54-279-2296 : Email : ylee@postech.ac.kr) Brian Ellis (Collaborator), Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada (Tel: 604.822.3451; Email: bee@msl.ubc.ca) Sylvain Merlot (Collaborator), ISV-CNRS UPR 2355, Bat23, piece 245, 1 avenue de la Terrasse, 91190 Gif sur Yvette, France (Tel: +33-1-69-82-3598; Email: sylvain.merlot@isv.cnrs-gif.fr) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Agricultural productivity largely depends on the ability of crops to successfully grow in harsh environments. Drought causes severe damage and reduced yields for crops and floricultural plants, which costs the US $6 to 8 billion annually. The cost of the worst drought occurred in 1988 was estimated at about $61.6 billion total costs, including losses in agriculture, energy, water, ecosystems, and other sectors of the economy, making it the second most expensive natural disaster ever to affect the nation. Abscisic acid (ABA) plays a central role in plant adaptation to drought, cold, and salt stresses. Understanding the molecular mechanisms by ABA signaling modulates the plant response is a vital component to moderating water loss from plants and allowing plants to more effectively tolerate water stress. During the funding period, we have generated the double MAP kinase mutants, RNAi transgenic plants, and many molecular constructs, all of which will allow us to further genetically dissect ROS-mediated ABA signaling networks in guard cells. Currently, we are isolating proteins physically interacting with the two MAP kinase proteins and AtRbohF NADPH oxidases. Furthermore, the mutant and transgenic plants are being transformed with the calcium indicator protein yellow cameleon, which will provide us with more precise location of the MAP kinase in the signaling cascade. Therefore, the knowledge and information that came from our research will lead to the identification of novel molecular elements in the network involved in ABA signaling and water stress response, contributing to development of new approaches to agricultural plants with increased productivity and water stress tolerance.
Publications
- Mori, IC, Murata, Y, Yang, Y, Munemasa, S, Wang, Y-F, Andreoli, S, Tiriac, H, Alonso, J, Harper, JF, Ecker, JR, Kwak, JM and Schroeder, JI (2006). Calcium-dependent protein kinases function in S-type anion- and Ca2+ permeable-channel regulation in Arabidopsis guard cells. PLoS Biology, 4:1749-1762.
- Kwak, J. M., Maser, P. and Schroeder, J. I. (2008) The Clickable Guard Cell, Version II: Interactive model of guard cell signal transduction mechanisms and pathways. In The Arabidopsis Book. Chang, C., Graham, I., Last, R., Leyser, O., McClung, M. and Weinig, C. eds., American Society of Plant Biologists. In press.
- Cho, S. K., Ryu, M. Y., Song, C., Kwak, J. M. and Kim, W. T. (2008) Two highly homologous U-Box E3 ubiquitin ligase AtPUB22/23 play a role in the response to drought stress in Arabidopsis. Plant Cell, 20: 1899-1914.
- Jeon, B. W., Hwang, J.-U., Fu, Y., Li, G., Bao, F., Cho, D., Kwak, J. M., Yang, Z. and Lee, Y. (2008). Rop2 is activated by light in guard cells and inhibits light-induced stomatal opening. Plant Cell, 20: 75-87.
- Song, C. J., Shin, D. J., Takeda, K., Lee, S., Cho, D. S., Giordo, R., Leonhardt, N., Sritubtim, S., Ellis, B. and Kwak, J. M. (2008) Guard cell-expressed MPK9 and MPK12 positively regulate ABA signaling in guard cells. In prep.
- Sirichandra, C., Gu, D. J., Djaoui, M., Lee, S., Leung, J., Merlot, S. and Kwak, J. M. (2008) AtrbohF NADPH oxidase is phosphorylated by the guard cell protein kinase OST1. In prep.
- Lee, KH, Piao, HL, Kim, H-Y, Choi, SM, Jiang, F, Hartung, W, Hwang, I, Kwak, JM, Lee, I-J and Hwang, I (2006). Activation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid. Cell, 126: 1109-120.
- Kwak, JM, Ngyuen, V and Schroeder, JI (2006) The role of reactive oxygen species in hormonal responses. Plant Physiol., 141: 323-329.
- Song, CJ, Steinebrunner, I, Wang, X, Stout, SC and Roux, SJ (2006) Extracellular ATP induces the accumulation of superoxide via NADPH oxidases in Arabidopsis. Plant Physiol., 140: 1222-1232.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Water stress is one of the devastating environmental problems causing severe loss in agriculture. Abscisic acid (ABA) plays a central role in the protection of plants from various environmental stresses. In stomatal guard cells, reactive oxygen species (ROS) have been suggested to function in ABA signaling. Based on our preliminary results, we hypothesized that (i) A MAPK pathway mediates ABA-induced ROS signaling in guard cells and that (ii) phosphorylation of NADPH oxidases by protein kinases is important for the enzymatic activity and for the regulation of ABA signaling. In order to identify and characterize MAPK cascades mediating guard cell ABA/ROS signaling, we identified two MAPK genes, GCMPK3 and GCMPK4, that are preferentially and highly expressed in guard cells. To provide direct genetic evidence, RNAi-based gene silencing plant lines were generated in which both genes are silenced. In parallel, Arabidopsis single and double mutants carrying deleterious point
mutations in these genes were identified. ABA-induced stomatal closure was strongly impaired in the RNAi lines in which both GCMPK3 and GCMPK4 transcripts were significantly silenced. Consistent with this result, gcmpk3/4 mutants carrying point mutations in both genes showed an enhanced transpirational water loss, and ABA- and H2O2- insensitive response in stomatal movement assays, whereas mutants carrying a mutation in one of these genes did not show any altered phenotype. Our preliminary results also showed that gcmpk3/4 double mutants are highly sensitive to drought compared to wild type plants. A GCMPK4-YFP fusion construct rescued the gcmpk3/4 double mutant phenotype in ABA-induced stomatal movements, demonstrating that the mutations in these genes caused the phenotype. Together, these results provide genetic evidence that GCMPK3 and GCMPK4 function in guard cell ABA signaling, and there is functional redundancy in these genes. Furthermore, to investigate the role of
phosphorylation in the regulation of NADPH oxidases in guard cell ABA signaling, using a biochemical approach, we identified a guard cell protein kinase that phosphorylates the AtrbohF NADPH oxidase in its N-terminus by the OST1 protein kinase. Moreover, the phosphorylation signal from a mutant AtrbohF one of which Ser residues was replaced with Ala was reduced to 70% of the signal obtained from WT AtrbohF, demonstrating that the Ser residue participates in the phosphorylational regulation.
PARTICIPANTS: Koji Takeda (postdoc) Department of Cell Biology and Molecular Genetics, University of Maryland, College Park (Tel: 301-405-9726; Email: ktakeda@umd.edu) Sangmee Lee (technician) Department of Cell Biology and Molecular Genetics, University of Maryland, College Park Youngsook Lee, Collaborator, Division of Molecular Life Sciences, POSTECH, Pohang, 790-784, Korea (Tel: +82-54-279-2296 : Email : ylee@postech.ac.kr) Brian Ellis (Collaborator), Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada (Tel: 604.822.3451; Email: bee@msl.ubc.ca) Sylvain Merlot (Collaborator), ISV-CNRS UPR 2355, Bat23, piece 245, 1 avenue de la Terrasse, 91190 Gif sur Yvette, France (Tel: +33-1-69-82-3598; Email: sylvain.merlot@isv.cnrs-gif.fr)
Impacts
Agricultural productivity largely depends on the ability of crops to successfully grow in harsh environments. Drought causes severe damage and reduced yields for crops and floricultural plants, which costs the US $6 to 8 billion annually. The cost of the worst drought occurred in 1988 was estimated at about $61.6 billion total costs (normalized to 2002 dollars), including losses in agriculture, energy, water, ecosystems, and other sectors of the economy, making it the second most expensive natural disaster ever to affect the nation. The phytohormone abscisic acid (ABA) plays a central role in plant adaptation to drought, cold, and salt stresses. Understanding the molecular mechanisms by ABA signaling modulates the plant response is a vital component to moderating water loss from plants and allowing plants to more effectively tolerate water stress. Therefore, the knowledge and information that come from the proposed research will lead to the identification of novel
molecular elements in the network involved in ABA signaling and water stress response, contributing to development of new approaches to agricultural plants with increased productivity and water stress tolerance. Furthermore, our research addresses two CSREES goals, "Enhance protection and safety of the nation's agriculture and food supply. Protect and enhance the nation's natural resource base and environment."
Publications
- Jeon, B. W., Hwang, J.-U., Fu, Y., Li, G., Bao, F., Cho, D., Kwak, J. M., Yang, Z. and Lee, Y. (2007). Rop2 is activated by light in guard cells and inhibits light-induced stomatal opening. Plant Cell, In press.
- Takeda, K., Song, C. J., Lee, S., Cho, D. S., Giordo, R., Garg, A., Leonhardt, N., Sritubtim, S., Ellis, B. and Kwak, J. M. (2008) Two guard cell-expressed MAPK genes function as positive regulators of ABA signaling in guard cells. In prep.
- Cho, S. K., Ryu, M. Y., Song, C., Kwak, J. M. and Kim, W. T. (2008) Two highly homologous U-Box E3 ubiquitin ligase AtPUB22/23 play a role in the response to drought stress in Arabidopsis. In prep.
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Progress 10/01/05 to 09/30/06
Outputs
Water stress is one of the devastating environmental problems causing severe loss in agriculture. Abscisic acid (ABA) plays a central role in the protection of plants from various environmental stresses. In stomatal guard cells, reactive oxygen species (ROS) have been suggested to function in ABA signaling. Based on our preliminary results, we hypothesized that (i) A MAPK pathway mediates ABA-induced ROS signaling in guard cells and that (ii) phosphorylation of NADPH oxidases by protein kinases is important for the enzymatic activity and for the regulation of ABA signaling. In order to identify and characterize MAPK cascades mediating guard cell ABA/ROS signaling, we identified two MAPK genes, GCMAPK3 and GCMAPK4, that are preferentially and highly expressed in guard cells. To provide direct genetic evidence, RNAi-based gene silencing plant lines were generated in which either each of the genes or both are specifically silenced. In parallel, Arabidopsis single and
double mutants carrying deleterious point mutations in these genes were generated. Interestingly, ABA-induced stomatal closure was strongly impaired in the RNAi lines in which both GCMAPK3 and GCMAPK4 transcripts were significantly silenced. Consistent with this result, the Arabidopsis mutants carrying point mutations in both genes showed a strong ABA insensitive response in stomatal movement assays. Together, these results provide genetic evidence that GCMAPK3 and GCMAPK4 function in guard cell ABA signaling, and there is functional redundancy in these genes. Currently, we are trying to further determine functional contribution of these genes to the ABA/ROS signaling and stress response network. Furthermore, to investigate the role of phosphorylation in the regulation of NADPH oxidases in guard cell ABA signaling, an N-terminal cytosolic domain and full-length AtrbohF were expressed in E. coli. Our in vitro data show that AtrbohF is phosphorylated by a protein kinase that is highly
expressed in guard cells.
Impacts
Agricultural productivity depends on the ability of crops to successfully grow in changing environments with which plants constantly interact. Drought causes severe damage and reduced yields for crops and floricultural plants, which costs the US $6 to 8 billion annually. The phytohormone abscisic acid (ABA) plays a central role in plant adaptation to drought, cold, and salt stresses. The knowledge and information that come from the proposed research will lead to the identification of novel molecular elements in the network involved in ABA signaling and water stress response, contributing to development of new approaches to agricultural plants with increased productivity and water stress tolerance. Furthermore, The proposed research addresses two of the five CSREES goals, (i) Enhance economic opportunities for agricultural producers. (ii) Protect and enhance the Nation's natural resource base and environment.
Publications
- Lee, KH, Piao, HL, Kim, H-Y, Choi, SM, Jiang, F, Hartung, W, Hwang, I, Kwak, JM, Lee, I-J and Hwang, I (2006). Activation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid. Cell, 126: 1109-120.
- Mori, IC, Murata, Y, Yang, Y, Munemasa, S, Wang, Y-F, Andreoli, S, Tiriac, H, Alonso, J, Harper, JF, Ecker, JR, Kwak, JM and Schroeder, JI (2006). Calcium-dependent protein kinases function in S-type anion- and Ca2+ permeable-channel regulation in Arabidopsis guard cells. PLoS Biology, 4:1749-1762.
- Kwak, JM, Ngyuen, V and Schroeder, JI (2006) The role of reactive oxygen species in hormonal responses. Plant Physiol., 141: 323-329.
- Song, CJ, Steinebrunner, I, Wang, X, Stout, SC and Roux, SJ (2006) Extracellular ATP induces the accumulation of superoxide via NADPH oxidases in Arabidopsis. Plant Physiol., 140: 1222-1232.
- Lee, Y, Kim, Y-W, Jeon, B, Park, K-Y, Suh, SJ, Seo, J, Kwak, JM, Martinoia, E, Hwang, I and Lee, Y (2006) Phosphatidylinositol 4,5 bis-phosphate is important for stomatal opening. Plant J., Submitted.
- Song, CJ, Cho, D, Garg, A, Kwak, JM (2006) Two guard cell-expressed MAPK genes function as positive regulators of ABA signaling in guard cells. In preparation.
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Progress 08/15/04 to 08/14/05
Outputs
The goal of the project is to genetically dissect ROS-mediated ABA signaling in guard cells by testing two hypotheses: (a) A MAPK pathway mediates ABA-induced ROS signaling. (b) NADPH oxidases are phosphorylated by protein kinases acting upstream of ABA-triggered ROS production. Current progress is summarized in each of the specific aims. (i) Identify and characterize MAPK cascades working in guard cell ABA signaling. We first confirmed by RT-PCR that several genes in MAPK cascades that we have identified from microarray results are preferentially expressed in guard cells or up-regulated by ABA in guard cells. In order to investigate roles of two guard cell-preferential MAPK genes, GCMAPK3 and GCMAPK4, we used the RNAi approach and generated 5 different gene-silenced plant lines in which either each of the genes or both are designed to be specifically silenced. Furthermore, we generated TILLING lines for these genes (2 deleterious mutations for GCMAPK3 and 2 for
GCMAPK4), and transgenic Arabidopsis plants overexpressing HA-tagged GCMAPK3 and GCMAPK4 are generated for phenotype analysis. In addition, promoter sequences are currently isolated to determine these genes' spatial and temporal expression. We will pursue detailed cell biological, biochemical, and molecular genetic analyses of these plant lines to characterize functional contribution of these genes to guard cell ABA/ROS signaling and stress response. In parallel, we are testing the contribution of two MAPKK genes to guard cell ABA signaling in collaboration with Dr. Heribert Hirt in Austria. Stomatal movement assays revealed that overexpression plants and knockout mutants for one of these MAPKK genes are likely to have impaired ABA responses, requiring further detailed experiments. We also cloned 7 MAPK, 3 MAPKK, and 8 MAPKKK full-length cDNAs for various experiments, including overexpression in Arabidopsis and protein expression in a heterologous system. For example, GCAMPK3 and
GCMAPK4 are cloned in a yeast two hybrid vector and thus ready for interacting protein screen. (ii) Investigate the role of phosphorylation in the regulation of NADPH oxidases. To achieve this specific aim, a full-length cDNA and N-terminal cytosolic domain of AtrbohF was cloned in the pGEX4T-3 vector, and the protein expression in E. coli was verified. We are now testing whether the OST1 protein kinase can phosphorylate these proteins. Furthermore, the N-terminal cytosolic domain of AtrbohF is also cloned in a yeast two hybrid bait vector. We will carry out yeast two hybrid screen to look for proteins interacting with AtrbohF. In addition to this effort, several manuscripts in which the USDA support is acknowledged are either in press, under revision, or submitted.
Impacts
Agricultural productivity depends on the ability of crops to successfully grow in changing environments with which plants constantly interact. Drought causes severe damage and reduced yields for crops and floricultural plants, which costs the US $6 to 8 billion annually. The phytohormone abscisic acid (ABA) plays a central role in plant adaptation to drought, cold, and salt stresses. The knowledge and information that come from the proposed research will lead to the identification of novel molecular elements in the network involved in ABA signaling and water stress response, contributing to development of new approaches to agricultural plants with increased productivity and water stress tolerance. Furthermore, The proposed research addresses two of the five CSREES goals, (i) Enhance economic opportunities for agricultural producers. (ii) Protect and enhance the Nation's natural resource base and environment.
Publications
- Song, C., Steinebrunner, I., Wang, X., Stout, S. C., and Roux, S. J. (2006) Extracellular ATP induces the accumulation of superoxide via NADPH oxidases in Arabidopsis thaliana. Plant Physiol. In press.
- Lee, K. H., Piao, H. L., Choi, S. M., Hwang, I., Kwak, J. M. and Hwang, I. (2006) Activation of an ER-localized beta-glucosidase, AtBG1, through dehydration-induced polymerization rapidly increases active pools of ABA for physiological responses. Cell, Submitted.
- Mori, I. C., Murata, Y., Mackesy, D. Z., Yang, Y., Alonso, J., Harper, J. F., Ecker, J. P., Kwak, J. M. and Schroeder, J. I. (2006) Calcium-dependent protein kinases function in S-typeanion- and Ca2+ permeable-Channel regulation in Arabidopsis guard cells. Plant Cell, Submitted.
- Jeon, B. W., Hwang, J.-U., Fu, Y., Li, H., Cho, D. S., Kwak, J. M., Yang, Z. and Lee, Y. (2006) Rop2 is activated by light in guard cells and inhibits light-induced stomatal opening. Plant Cell, Submitted.
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