Progress 01/01/05 to 12/31/05
Outputs
Plant defense mechanisms against invading pathogens often include rapid programmed cell death known as the hypersensitive response (HR). Correlative evidence has implicated kinases in regulating HR cell death based on pharmacological studies using kinase inhibitors. Recent genetic studies demonstrated the involvement of a specific mitogen-activated protein kinase (MAPK) cascade consisting of two MAPKs, SIPK, and WIPK, and their upstream MAPKK, NtMEK2. We found that the activation of NtMEK2-SIPK/WIPK pathway induces cell death with phenotypes identical to pathogen-induced HR at macroscopic, microscopic and physiological levels, including loss of membrane potential, electrolyte leakage, and rapid dehydration. Loss of membrane potential in NtMEK2DD plants is associated with the generation of reactive oxygen species (ROS), which is preceded by imbalanced activities in chloroplasts and mitochondria. We observed rapid shutdown of carbon fixation in chloroplasts after
SIPK/WIPK activation, which leads to over-reduction of the electron transport chain and the generation of ROS in chloroplasts under illumination. In plants kept in the dark, we observed a decrease in ATP generation, but an increase in respiration, suggesting that the generation of reducing equivalents and oxidative phosphorylation in mitochondria are uncoupled. Consistent with a role of chloroplast-generated ROS in MAPK-mediated cell death, plants kept in the dark do not accumulate H2O2 in chloroplasts after SIPK/WIPK activation and cell death is significantly delayed. Similar light dependency was observed in HR cell death induced by TMV, which is known to activate the NtMEK2-SIPK/WIPK pathway in an N-gene dependent manner. These results suggest that the activation of the SIPK/WIPK cascade by pathogen actively promotes the generation of ROS in chloroplasts, which play important roles in the signaling and/or execution of HR cell death in plants.
Impacts
Our research will impact several important areas of plant research, including 1) the regulation of plant hormone biosynthesis, 2) the regulation of plant HR cell death and pathogen resistance, and 3) the regulation of energy flow and ROS generation in plants under stress. During evolution, plant MAPKs adopted unique substrates, despite the fact that the structure of MAPK cascade itself is highly conserved. Understanding the regulation of energy flow in plants under stress, and mechanism of HR cell death/pathogen resistance will eventually allow the generation of crops with enhanced yield under stress conditions. This is important for agriculture to produce enough food to sustain the increasing world population by the use of marginal land for crop production.
Publications
- No paper was published in 2005. Several manuscritps are in review or in preparation.
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Progress 07/01/99 to 06/30/05
Outputs
During the project peroid, my lab successfully identified NtMEK2, a tobacco MAPKK, as the upstream kinase of both SIPK and WIPK based on in vitro and in vivo evidence. This is the first MAPKK gene that was functionally placed upstream of a MAPK in plants. The use of a conditional gain-of-function NtMEK2DD transgenic system allowed us to demonstrate that this MAPK cascade regulates several defense responses, including defense gene activation, ethylene biosynthesis, phytoalexin biosynthesis, and HR-like cell death. To take advantage of the fully sequenced genome and the mutant collections, and to use genetic, genomic, and proteomic approaches, we also developed an Arabidopsis system similar to that of tobacco. Based on phylogenetic analysis, MPK6, MPK3, and MKK4/MKK5 are orthologs of tobacco SIPK, WIPK, and NtMEK2, respectively. The available mutants allowed us to obtain loss-of-function data of these two MAPKs. Recently, we identified the first plant MAPK substrate
[First plant MAP kinase substrate. http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2004/263/tw4 47]. Arabidopsis 1-aminocyclopropane-1-carboxylic acid synthase 2 (ACS2) and ACS6 are substrates of stress-responsive MPK3/MPK6. Phosphorylation of ACS2/ACS6 by MAPK leads to the accumulation of ACS proteins (the rate-limiting enzyme catalyzing the committing step of ethylene biosynthesis) in vivo, which elevates the ethylene production. This research revealed an important signaling pathway that regulates ethylene induction in plants under stress.
Impacts
Identification of important genes in regulating plant growth, development, and stress response/adaptation may lead to the generation of crops with enhanced yield and/or stress tolerance, therefore, allow the use of marginal land for crop production. This is important for U.S. agriculture to produce enough food to sustain the increasing population as the freshwater used for irrigation is becoming increasingly scarce.
Publications
- Hamel, L.-P., Nicole, M.-C., Sritubtim, S., Morency, M.-J., Ellis, M., Ehlting, J., Beaudoin, N., Barbazuk, B., Klessig, D., Lee, J., Martin, G., Mundy, J., Ohashi, Y., Scheel, D., Sheen, J., Xing, T., Zhang, S., Seguin, A., and Ellis, B.E. (2006) Ancient signals: comparative genomics of plant MAPK and MAPKK gene families. Trends Plant Sci. 11:192-198.
- Wang, H., Ngwenyama, N., Liu, Y., Walker, J., and Zhang, S. (2006) Stomata development is regulated by environmentally responsive MAP kinases. Plant Cell, in press.
- Mino, M., Kubota, M., Nogi, T., Zhang, S., and Inoue, M, (2006) Death of interspecific Nicotiana hybrid involves MAP-kinases signaling. Plant Biology, in press.
- Suarez-Rodriguez, M.C., Adams-Phillips, L., Liu, Y., Wang, H., Su, S.-H., Jester, P.J., Zhang, S., Bent, A.F., and Krysan, P.J. (2006) MEKK1 is required for flg22-induced MPK4 activation in Arabidopsis plants. Plant Physiol. Published on December 1, 2006; 10.1104/pp.106.091389.
- Ren, D., Yang, K.-Y., Li, G., Liu, Y., and Zhang, S. (2006) Activation of Ntf4, a tobacco MAPK, during plant defense response and its involvement in hypersensitive response-like cell death. Plant Physiol. 141: 1482-1493.
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Progress 01/01/04 to 12/31/04
Outputs
Our long-term research goal is to elucidate the functions and mechanisms of actions of MAPKs in plant defense responses, which includes identifying the components (MAPKKK-MAPKK-MAPK) in stress-activated MAPK cascades, the upstream signaling molecules involved in turning on the MAPK cascades, the substrates/downstream effectors of MAPKs, and the phosphatases that negatively regulate this MAPK cascade. More importantly, we want to understand how MAPK activation leads to a particular phenotype at biochemical, physiological and cellular levels. Specific activities this year: We identified a subset of ACS family members as the substrates of MPK6, one of the stress-responsive MAPKs, which links the activation of this MAPK directly to the induction of ethylene in plants under stress. MAPKs are implicated in regulating plant growth, development, and response to the environment. However, the underlying mechanisms are unknown due to the lack of information about their
substrates. The identification of the first plant MAPK substrate revealed one mechanism by which MPK6 regulates plant stress responses. Equally important, it advanced our knowledge about the signaling pathways leading to ethylene induction in plants under stress. It also demonstrated that plant MAPKs adopted unique substrates during evolution, despite the fact that the structure of MAPK cascade itself is highly conserved. The paper was featured in the Plant Cell In this issue and the Science STKE website [First plant MAPK substrate. http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2004/263/tw4 47].
Impacts
Our research will impact several important areas of plant research, including 1) the regulation of plant hormone biosynthesis, 2) the regulation of plant HR cell death and pathogen resistance, and 3) the regulation of energy flow and ROS generation in plants under stress. During evolution, plant MAPKs adopted unique substrates, despite the fact that the structure of MAPK cascade itself is highly conserved. Understanding the regulation of energy flow in plants under stress, and mechanism of HR cell death/pathogen resistance will eventually allow the generation of crops with enhanced yield under stress conditions. This is important for agriculture to produce enough food to sustain the increasing world population by the use of marginal land for crop production.
Publications
- Kim, C.Y., and Zhang, S. (2004) Activation of a mitogen-activated protein kinase cascade induces WRKY family of transcription factors and defense genes in tobacco. Plant J. 38:142-151.
- Liu, Y. and Zhang, S. (2004) Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis. Plant Cell 16: 3386-3399. (Featured in the Plant Cell In this issue and Science STKE website)
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Progress 01/01/03 to 12/31/03
Outputs
Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryotic signaling modules downstream of sensors/receptors that transduce extracellular stimuli into intracellular responses. Our long-term research goal is to elucidate the functions and mechanisms of actions of two plant stress-responsive MAPKs, represented by tobacco SIPK and WIPK, and their Arabidopsis orthologs MPK6 and MPK3. We recently identified the first plant MAPK substrate, which revealed the signaling pathway leading to ethylene induction in plants under stress. Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), the rate-limiting enzyme of ethylene biosynthesis, by SIPK/MPK6 stabilizes the ACS proteins in vivo, which results in elevated cellular ACS activity, ethylene production, and ethylene-induced responses. As an important plant hormone (also known as plant stress hormone), the biosynthetic pathway of ethylene has been elucidated and a number of downstream components
have been identified. However, the signaling pathway leading to the ethylene induction, which is the first step of ethylene-regulated processes, remained largely unknown. One of our research focuses along this line is to investigate how phosphorylation of ACS proteins by MPK6 leads to their stabilization in vivo.
Impacts
Our research will impact several important areas of plant research, including 1) the regulation of plant hormone biosynthesis, 2) the regulation of plant HR cell death and pathogen resistance, and 3) the regulation of energy flow and ROS generation in plants under stress. During evolution, plant MAPKs adopted unique substrates, despite the fact that the structure of MAPK cascade itself is highly conserved. Understanding the regulation of energy flow in plants under stress, and mechanism of HR cell death/pathogen resistance will eventually allow the generation of crops with enhanced yield under stress conditions. This is important for agriculture to produce enough food to sustain the increasing world population by the use of marginal land for crop production.
Publications
- Wan, J., Zhang, S. and Stacey, G. (2004). Activation of a mitogen-activated protein kinase pathway in Arabidopsis by chitin. Mol. Plant Pathol., in press.
- Jin, H., Kim, C.-Y., Liu, Y., Yang, K.-Y., Baker, B., and Zhang, S. (2003) Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco. Plant J. 33:719-731.
- Liu, Y., Jin, H., Yang, K.-Y., Kim, C.-Y., Baker, B., and Zhang, S. (2003) Interaction between two mitogen-activated protein kinases during plant defense signaling. Plant J. 34:149-160.
- Kim, C.Y., Liu, Y., Thorne, E.T., Yang, H., Fukushige, H., Gassmann, W., Hildebrand, D., Sharp, R.E., and Zhang, S. (2003) Activation of a stress-responsive mitogen-activated protein kinase cascade induces the biosynthesis of ethylene in plants. Plant Cell 15:2707-2718.
- Kim, C.Y., and Zhang, S. (2004) Activation of a mitogen-activated protein kinase cascade induces WRKY family of transcription factors and defense genes in tobacco. Plant J. 38:142-151.
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Progress 01/01/02 to 12/31/02
Outputs
Our long-term research goal is to elucidate the functions and mechanisms of actions of MAPKs in plant defense responses, which includes identifying the components (MAPKKK-MAPKK-MAPK) in stress-activated MAPK cascades, the upstream signaling molecules involved in turning on the MAPK cascades, the substrates/downstream effectors of MAPKs, and the phosphatases that negatively regulate this MAPK cascade. More importantly, we want to understand how MAPK activation leads to a particular phenotype at biochemical, physiological and cellular levels. We made important progress in several areas in 2002, which includes: i) demonstrated the roles of avirulent pathogen-activated MAPK cascades in plant disease resistance; and ii) identify the targets of NtMEK2-SIPK/WIPK pathway at the organellar level in hypersensitive response, a type of plant programmed cell death that plays a critical role in plant disease resistance.
Impacts
Plant disease has a major negative impact on crops. It has been estimated that up to 40% of plant productivity in Africa and Asia, and about 20% in the developed world, is lost to pests and pathogens. A number of strategies/methods have been proposed/used to fight against plant diseases. One of the attractive strategies is to enhance the plant's own defense system by engineering regulatory components in resistance signaling pathways. Such an approach will lead to the generation of crops with resistance against a broad spectrum of pathogens, because similar defense responses are elicited in plants by viral, bacterial and fungal pathogens, suggesting shared signaling pathway(s). Identification and characterization of the key regulatory component(s) in the shared pathways that are suitable for genetic engineering, therefore, becomes critical. Our research is focused on one such potential pathway.
Publications
- Ren, D., Yang, H., and Zhang, S. (2002) Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis. J. Biol. Chem. 277: 559-565.
- MAPK group. (2002) Mitogen-activated-protein-kinase cascades in plants: a new nomenclature. Trends Plant Sci. 7: 301-308.
- Yang, K.-Y., Blee, K.A., Zhang, S., and Anderson, A.J. (2002) OxycomTM treatment suppresses Pseudomonas syringae infection and activates a mitogen-activated protein kinase pathway in tobacco. Physiol. Mol. Plant Pathol. 61: 249-256.
- Jin, H., Axtell, M.J., Dahlbeck, D., Ekwenna, O., Zhang, S., Staskawicz, B., and Baker, B. (2002). NPK1, an MEKK1-like mitogen-activated protein kinase kinase kinase, regulates innate immunity and development in plants. Dev. Cell 3, 291-297.
- Hailing Jin, Yidong Liu, Kwang-Yeol Yang, Cha Young Kim, Barbara Baker, and Shuqun Zhang (2003) Function of a Mitogen-Activated Protein Kinase Pathway in N Gene-Mediated Resistance in Tobacco. Plant J. 33, 719-731.
- Yidong Liu, Hailing Jin, Kwang-Yeol Yang, Cha Young Kim, Barbara Baker, and Shuqun Zhang. (2003) Interaction between two mitogen-activated protein kinases during tobacco defense signaling. Plant J. in press.
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Progress 01/01/01 to 12/31/01
Outputs
We have made several important progresses last year, which include: i) identified the upstream kinase of WIPK and SIPK, two MAP kinase involved in plant disease resistance; ii) demonstrated that WIPK gene activation by stress stimuli, including pathogen infection is controlled by SIPK; and iii) provided both loss- and gain-of-function evidence for the NtMEK2-SIPK/WIPK pathway in plant disease resistance.
Impacts
Plant disease has a major negative impact on crops. It has been estimated that up to 40% of plant productivity in Africa and Asia, and about 20% in the developed world, is lost to pests and pathogens. A number of strategies/methods have been proposed/used to fight against plant diseases. One of the attractive strategies is to enhance the plant's own defense system by engineering regulatory components in resistance signaling pathways. Such an approach will lead to the generation of crops with resistance against a broad spectrum of pathogens, because similar defense responses are elicited in plants by viral, bacterial and fungal pathogens, suggesting shared signaling pathway(s). Identification and characterization of the key regulatory component(s) in the shared pathways that are suitable for genetic engineering, therefore, becomes critical. Our research is focused on one such potential pathway.
Publications
- Yang, K.-Y., Liu, Y., and Zhang, S. (2001) Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco. Proc. Natl. Acad. Sci. USA 98: 741-746.
- Zhang, S., and Klessig, D.F. (2001) MAPK cascades in plant defense signaling. Trends Plant Sci. 6: 520-527.
- Zhang, S., and Liu, Y. (2001) Activation of salicylic acid-induced protein kinase, a mitogen-activated protein kinase, induces multiple defense responses in tobacco. Plant Cell 13: 1877-1889.
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Progress 01/02/00 to 12/31/00
Outputs
Hypersensitive response (HR), a form of programmed cell death is frequently associated with plant disease resistance. It has been proposed that mitogen-activated protein kinase (MAPK) cascades regulate HR cell death based on pharmacological studies using kinase inhibitors. However, direct evidence is lacking. Here we demonstrate that NtMEK2, a novel MAPK kinase (MAPKK) is upstream of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco MAPKs that are activated by various pathogens or pathogen-derived elicitors. Ectopic expression of a constitutively active mutant of NtMEK2 induces HR-like cell death in tobacco, which is preceded by the activation of endogenous SIPK and WIPK. In addition, NtMEK2-SIPK/WIPK cascade appears to control the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and L-phenylalanine ammonia lyase (PAL), two defense genes encoding key enzymes in the phytoalexin and salicylic acid
biosynthesis pathways. These results demonstrate that a plant MAPK cascade controls multiple defense responses against pathogen invasion. To further analyze the role(s) that SIPK and WIPK each plays in the process, we transiently transformed tobacco leaves with SIPK under the control of a steroid-inducible promoter. The induction of SIPK expression after the application of dexamethasone, a steroid leads to the elevation of SIPK activity. The increase of SIPK activity is dependent on phosphorylation of newly synthesized SIPK by its endogenous upstream kinase. In contrast, expression of WIPK under the same condition fails to elevate its activity, even though the protein accumulates to a similar level. Studies using chimeras of SIPK and WIPK demonstrated that the C-terminus of SIPK contains the molecular determinant for its activation, which is rather surprising as the N-termini of the SIPK and WIPK are more divergent. SIPK has been previously implicated in the regulation of both plant
defense gene activation and HR-cell death based on evidence from pharmacological studies using kinase inhibitors. This gain-of-function study provided more direct evidence for its role in the signaling of multiple defense responses in tobacco.
Impacts
Diseases cause major loss of crop production each year. By understanding the mechanism how resistant plants defend themself against pathogens, we could potentially improve crops and make them more resistant. Our studies identified a key player in the signaling pathway that is involved in the activation of plant defense responses.
Publications
- Yang, K.-Y., Liu, Y., and Zhang, S. (2001). Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco. Proc. Natl. Acad. Sci. USA, 98: 741-746.
- Hoyos, M. E., and Zhang, S. (2000). Calcium-independent activation of salicylic acid-induced protein kinase and a 40-kilodalton protein kinase by hyperosmotic stress. Plant Physiol. 122: 1355-1363.
- Zhang, S., Liu, Y., and Klessig, D. F. (2000). Multiple levels of tobacco WIPK activation during the induction of cell death by fungal elicitins. Plant J. 23: 339-347.
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Progress 01/01/99 to 12/31/99
Outputs
Our researches are focused on four major subjects: 1) identifying other components in plant MAPK cascades, 2) functional analyses of MAPKs in plant defense responses, 3) promoter analysis of MAPK gene, and 4) identification of a novel 40-kD kinase that is responsive to salt/osmotic stress. The first two goals are interlinked. Several genes in the MAPK pathways have been cloned and their functional analyses are in progress. We have already obtained several genomic clones and different deletion constructs of the promoter have been made and ready for transformation. The last project progressed well and a manuscript has been accepted for publication by Plant Physiology.
Impacts
Our research into the function of stress-activated protein kinases in plants is mainly theoretical at this stage. However, identifying new components in the MAPK cascades and understanding the role of pathogen-activated MAPK signaling pathways should provide useful targets for manipulating plant disease resistance.
Publications
- Hoyos, M. E. and Zhang, S. (2000) Calcium-independent activation of SIPK and a 40-kD protein kinase by hyperosmotic stress. Plant Physiol, in press.
- Romeis, T., Piedras, P., Zhang, S., Klessig, D. F., Hirt, H., and Lones, J. (1999). Rapid Avr9- and Cf-9-dependent activation of MAP kinases in tobacco cell cultures and leaves: convergence of resistance gene, elicitor, wound and salicylate responses. Plant Cell 11, 273-287.
- Zhang, S., and Klessig, D. F. (1999). Pathogen-induced MAP kinases in Tobacco. In MAP kinases in Plant Signal Transduction, H. Hirt, ed. (Heidelberg: Springer-Verlag), pp. 65-84.
- Liu, Y., Zhang, S. and Klessig, D. F. (2000) Molecular cloning and characterization of a tobacco MAP kinase kinase that interacts with SIPK. Mol. Plant-Microbe Interaction 13, 118-124.
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