A plastid-localized glycogen synthase kinase 3 modulates stress tolerance and carbohydrate metabolism

Glycogen synthase kinase 3 (GSK-3) was originally identified as a regulator of glycogen synthesis in mammals. Like starch in plants, glycogen is a polymer of glucose, and serves as an energy and carbon store. Starch is the main carbohydrate store in plants. Regulation of starch metabolism, in particular in response to environmental cues, is of primary importance for carbon and energy flow in plants but is still obscure. Here, we provide evidence that MsK4, a novel Medicago sativa GSK-3-like kinase, connects stress signalling with carbon metabolism. MsK4 was found to be a plastid-localized protein kinase that is associated with starch granules. High-salt stress rapidly induced the in vivo kinase activity of MsK4. Metabolic profiling of MsK4 over-expressor lines revealed changes in sugar metabolism, including increased amounts of maltose, the main degradation product of starch in leaves. Plants over-expressing MsK4 showed improved tolerance to salt stress. Moreover, under high-salinity conditions, MsK4-over-expressing plants accumulated significantly more starch and showed modified carbohydrate content compared with wild-type plants. Overall, these data indicate that MsK4 is an important regulator that adjusts carbohydrate metabolism to environmental stress

Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense

Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3l isoform (GRF6), a previously reported DBP1 interactor, and MAP kinase (MAPK) MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-offunction mutants showed altered response to infection by the potyvirus Plum pox virus (PPV), and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.This work was supported by the Spanish MICINN (Grants BFU2009-09771, EUI2009-04009 to PV), Generalitat Valenciana (Prometeo2010/020 to PV) and the German DFG (SCHE 235/15-1 to DS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Carrasco Jiménez, JL.; Castelló Llopis, MJ.; Naumann, K.; Lassowskat, I.; Navarrete Gomez, ML.; Scheel, D.; Vera Vera, P. (2014). Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense. PLoS ONE. 9:1-10. https://doi.org/10.1371/journal.pone.0090734S1109Carrasco, J. L. (2003). A novel transcription factor involved in plant defense endowed with protein phosphatase activity. The EMBO Journal, 22(13), 3376-3384. doi:10.1093/emboj/cdg323Carrasco, J. L., Ancillo, G., Castelló, M. J., & Vera, P. (2005). A Novel DNA-Binding Motif, Hallmark of a New Family of Plant Transcription Factors. Plant Physiology, 137(2), 602-606. doi:10.1104/pp.104.056002Castelló, M. J., Carrasco, J. L., & Vera, P. (2010). DNA-Binding Protein Phosphatase AtDBP1 Mediates Susceptibility to Two Potyviruses in Arabidopsis. Plant Physiology, 153(4), 1521-1525. doi:10.1104/pp.110.158923Castelló, M. J., Carrasco, J. L., Navarrete-Gómez, M., Daniel, J., Granot, D., & Vera, P. (2011). A Plant Small Polypeptide Is a Novel Component of DNA-Binding Protein Phosphatase 1-Mediated Resistance to Plum pox virus in Arabidopsis. Plant Physiology, 157(4), 2206-2215. doi:10.1104/pp.111.188953Denison, F. C., Paul, A.-L., Zupanska, A. K., & Ferl, R. J. (2011). 14-3-3 proteins in plant physiology. Seminars in Cell & Developmental Biology, 22(7), 720-727. doi:10.1016/j.semcdb.2011.08.006Carrasco, J. L., Castelló, M. J., & Vera, P. (2006). 14-3-3 Mediates Transcriptional Regulation by Modulating Nucleocytoplasmic Shuttling of Tobacco DNA-binding Protein Phosphatase-1. Journal of Biological Chemistry, 281(32), 22875-22881. doi:10.1074/jbc.m512611200Colcombet, J., & Hirt, H. (2008). ArabidopsisMAPKs: a complex signalling network involved in multiple biological processes. Biochemical Journal, 413(2), 217-226. doi:10.1042/bj20080625Kiegerl, S., Cardinale, F., Siligan, C., Gross, A., Baudouin, E., Liwosz, A., … Meskiene, I. (2000). SIMKK, a Mitogen-Activated Protein Kinase (MAPK) Kinase, Is a Specific Activator of the Salt Stress–Induced MAPK, SIMK. The Plant Cell, 12(11), 2247-2258. doi:10.1105/tpc.12.11.2247CAMPS, M., NICHOLS, A., & ARKINSTALL, S. (2000). Dual specificity phosphatases: a gene family for control of MAP kinase function. The FASEB Journal, 14(1), 6-16. doi:10.1096/fasebj.14.1.6Bethke, G., Pecher, P., Eschen-Lippold, L., Tsuda, K., Katagiri, F., Glazebrook, J., … Lee, J. (2012). Activation of the Arabidopsis thaliana Mitogen-Activated Protein Kinase MPK11 by the Flagellin-Derived Elicitor Peptide, flg22. Molecular Plant-Microbe Interactions, 25(4), 471-480. doi:10.1094/mpmi-11-11-0281Wolschin, F., Wienkoop, S., & Weckwerth, W. (2005). Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide/hydroxide affinity chromatography (MOAC). PROTEOMICS, 5(17), 4389-4397. doi:10.1002/pmic.200402049Petersen, M., Brodersen, P., Naested, H., Andreasson, E., Lindhart, U., Johansen, B., … Mundy, J. (2000). Arabidopsis MAP Kinase 4 Negatively Regulates Systemic Acquired Resistance. Cell, 103(7), 1111-1120. doi:10.1016/s0092-8674(00)00213-0Asai, T., Tena, G., Plotnikova, J., Willmann, M. R., Chiu, W.-L., Gomez-Gomez, L., … Sheen, J. (2002). MAP kinase signalling cascade in Arabidopsis innate immunity. Nature, 415(6875), 977-983. doi:10.1038/415977aKosetsu, K., Matsunaga, S., Nakagami, H., Colcombet, J., Sasabe, M., Soyano, T., … Machida, Y. (2010). The MAP Kinase MPK4 Is Required for Cytokinesis in Arabidopsis thaliana. The Plant Cell, 22(11), 3778-3790. doi:10.1105/tpc.110.077164Koroleva, O. A., Tomlinson, M. L., Leader, D., Shaw, P., & Doonan, J. H. (2004). High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions. The Plant Journal, 41(1), 162-174. doi:10.1111/j.1365-313x.2004.02281.xVierstra, R. D. (2009). The ubiquitin–26S proteasome system at the nexus of plant biology. Nature Reviews Molecular Cell Biology, 10(6), 385-397. doi:10.1038/nrm2688Gökirmak, T., Paul, A.-L., & Ferl, R. J. (2010). Plant phosphopeptide-binding proteins as signaling mediators. Current Opinion in Plant Biology, 13(5), 527-532. doi:10.1016/j.pbi.2010.06.001Keyse, S. M. (2000). Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Current Opinion in Cell Biology, 12(2), 186-192. doi:10.1016/s0955-0674(99)00075-7Gupta, R., & Luan, S. (2003). Redox Control of Protein Tyrosine Phosphatases and Mitogen-Activated Protein Kinases in Plants. Plant Physiology, 132(3), 1149-1152. doi:10.1104/pp.103.020792Katou, S., Karita, E., Yamakawa, H., Seo, S., Mitsuhara, I., Kuchitsu, K., & Ohashi, Y. (2005). Catalytic Activation of the Plant MAPK Phosphatase NtMKP1 by Its Physiological Substrate Salicylic Acid-induced Protein Kinase but Not by Calmodulins. Journal of Biological Chemistry, 280(47), 39569-39581. doi:10.1074/jbc.m508115200Schweighofer, A., Kazanaviciute, V., Scheikl, E., Teige, M., Doczi, R., Hirt, H., … Meskiene, I. (2007). The PP2C-Type Phosphatase AP2C1, Which Negatively Regulates MPK4 and MPK6, Modulates Innate Immunity, Jasmonic Acid, and Ethylene Levels in Arabidopsis. 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SIMKK, a Mitogen-Activated Protein Kinase (MAPK) Kinase, Is a Specific Activator of the Salt Stress–Induced MAPK, SIMK

In eukaryotes, mitogen-activated protein kinases (MAPKs) play key roles in the transmission of external signals, such as mitogens, hormones, and different stresses. MAPKs are activated by MAPK kinases through phosphorylation of MAPKs at both the threonine and tyrosine residues of the conserved TXY activation motif. In plants, several MAPKs are involved in signaling of hormones, stresses, cell cycle, and developmental cues. Recently, we showed that salt stress–induced MAPK (SIMK) is activated when alfalfa cells are exposed to hyperosmotic conditions. Here, we report the isolation and characterization of the alfalfa MAPK kinase SIMKK (SIMK kinase). SIMKK encodes an active protein kinase that interacts specifically with SIMK, but not with three other MAPKs, in the yeast two-hybrid system. Recombinant SIMKK specifically activates SIMK by phosphorylating both the threonine and tyrosine residues in the activation loop of SIMK. SIMKK contains a putative MAPK docking site at the N terminus that is conserved in mammalian MAPK kinases, transcription factors, and phosphatases. Removal of the MAPK docking site of SIMKK partially compromises but does not completely abolish interaction with SIMK, suggesting that other domains of SIMKK also are involved in MAPK binding. In transient expression assays, SIMKK specifically activates SIMK but not two other MAPKs. Moreover, SIMKK enhances the salt-induced activation of SIMK. These data suggest that the salt-induced activation of SIMK is mediated by the dual-specificity protein kinase SIMKK