Disentanglement of functional complexity associated with plant mitogen-activated protein kinase (MAPK) signaling has benefited from transcriptomic, proteomic, phosphoproteomic, and genetic studies. Published transcriptomic analysis of a double homozygous recessive anp2anp3 mutant of two MAPK kinase kinase (MAPKKK) genes called Arabidopsis thaliana Homologues of Nucleus- and Phragmoplast-localized Kinase 2 (ANP2) and 3 (ANP3) showed the upregulation of stress-related genes. In this study, a comparative proteomic analysis of anp2anp3 mutant against its respective Wassilevskaja ecotype (Ws) wild type background is provided. Such differential proteomic analysis revealed overabundance of core enzymes such as FeSOD1, MnSOD, DHAR1, and FeSOD1-associated regulatory protein CPN20, which are involved in the detoxification of reactive oxygen species in the anp2anp3 mutant. The proteomic results were validated at the level of single protein abundance by Western blot analyses and by quantitative biochemical determination of antioxidant enzymatic activities. Finally, the functional network of proteins involved in antioxidant defense in the anp2anp3 mutant was physiologically linked with the increased resistance of mutant seedlings against paraquat treatment.

Centre of the Region Haná for Biotechnological and Agricultural Research Department of Cell Biology Faculty of Science Palacký University Šlechtitelů 11 CZ 783 71 Olomouc Czech Republic

Zobrazit více v PubMed

Rodriguez MCS, Petersen M, Mundy J. Mitogen-activated protein kinase signaling in plants. Annu. Rev. Plant Biol. 2010;61:621–649. PubMed

Šamajová O, Plíhal O, Al-Yousif M, Hirt H, Šamaj J. Improvement of stress tolerance in plants by genetic manipulation of mitogen-activated protein kinases. Biotechnol. Adv. 2013;31:118–128. PubMed

Andreasson E, Ellis B. Convergence and specificity in the Arabidopsis MAPK nexus. Trends Plant Sci. 2010;15:106–113. PubMed

Krysan PJ, Jester PJ, Gottwald JR, Sussman M. R An Arabidopsis mitogen-activated protein kinase kinase kinase gene family encodes essential positive regulators of cytokinesis. Plant Cell. 2002;14:1109–1120. PubMed PMC

Sasabe M, Soyano T, Takahashi Y, Sonobe S, Igarashi H, Itoh TJ, Hidaka M, Machida Y. Phosphorylation of NtMAP6S–l by a MAP kinase down-regulates its activity of microtubule bundling and stimulates progression of cytokinesis of tobacco cells. Genes Dev. 2006;20:1004–1014. PubMed PMC

Takahashi Y, Soyano T, Kosetsu K, Sasabe M, Machida Y. HINKEL kinesin, ANP MAPKKKs and MKK6/ANQ MAPKK, which phosphorylates and activates MPK4 MAPK, constitute a pathway that is required for cytokinesis in Arabidopsis thaliana . Plant Cell Physiol. 2010;51:1766–1776. PubMed PMC

Beck M, Komis G, Muller J, Menzel D, Šamaj J. Arabidopsis homologs of nucleus- and phragmoplast-localized kinase 2 and 3 and mitogen-activated protein kinase 4 are essential for microtubule organization. Plant Cell. 2010;22:755–771. PubMed PMC

Sasabe M, Machida Y. Regulation of organization and function of microtubules by the mitogen-activated protein kinase cascade during plant cytokinesis. Cytoskeleton (Hoboken) 2012;69:913–918. PubMed

Kovtun Y, Chiu WL, Tena G, Sheen J. Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc. Natl. Acad. Sci. U. S. A. 2000;97:2940–2945. PubMed PMC

Savatin DV, Bisceglia NG, Marti L, Fabbri C, Cervone F, Lorenzo GD. The Arabidopsis Nucleus- And Phragmoplast-Localized Kinasel-Related Protein Kinases Are Required for Elicitor-Induced Oxidative Burst and Immunity. Plant Physiol. 2014;165:1188–1202. PubMed PMC

Gawroński P, Witoń D, Vashutina K, Bederska M, Betliński B, Rusaczonek A, Karpiński S. Mitogen-Activated Protein Kinase 4 Is a Salicylic Acid-Independent Regulator of Growth But Not of Photosynthesis in Arabidopsis. Mol. Plant. 2014;7:1151–1166. PubMed

Pitzschke A, Djamei A, Bitton F, Hirt H. A major role of the MEKK1-MKK1/2-MPK4 pathway in ROS signalling. Mol. Plant. 2009;2:120–137. PubMed PMC

Nakagami H, Soukupová H, Schikora A, Zárský V, Hirt H. A Mtogen-activated protein kinase kinase kinase mediates reactive oxygen species homeostasis in Arabidopsis . J. Biol. Chem. 2006;281:38697–38704. PubMed

Xing Y, Cao Q, Zhang Q, Qin L, Jia W, Zhang J. MKK5 regulates high light-induced gene expression of Cu/Zn superoxide dismutase 1 and 2 in Arabidopsis . Plant Cell Physiol. 2013;54:1217–1227. PubMed

Xing Y, Jia W, Zhang J. AtMEK1 mediates stress-induced gene expression of CAT1 Catalase by triggering H2O2 production in Arabidopsis . J. Exp. Bot. 2007;58:2969–2981. PubMed

Xing Y, Jia W, Zhang J. AtMKKl mediates ABA-induced CAT1 expression and H2O2 production via AtMPK6-coupled signaling in Arabidopsis . Plant J. Cell Mol. Biol. 2008;54:440–451. PubMed

Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 1962;15:473–497.

Takáč T, Pechan T, Richter H, Müller J, Eck C, Böhm N, Obert B, Ren H, Niehaus K, Šamaj J. Proteomics on brefeldin A-treated Arabidopsis roots reveals profilin 2 as a new protein involved in the cross-talk between vesicular trafficking and the actin cytoskeleton. J. Proteome Res. 2011;10:488–501. PubMed

Hurkman WJ, Tanaka C. K Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 1986;81:802–806. PubMed PMC

Bridges SM, Magee GB, Wang N, Williams WP, Burgess SG, Nanduri B. ProtQuant: a tool for the label-free quantification of MudPIT proteomics data. BMC Bioinformatics. 2007;8(Suppl 7):S24. PubMed PMC

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248–254. PubMed

Pan SM, Yau YY. Characterization of superoxide dismutase in Arabidopsis . Plant Cell Physiol. 1998;37:58–66.

Beauchamp G, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 1971;44:276–87. PubMed

Amako K, Chen GX, Asada K. Separate assays specific for ascorbate peroxidase and guaiacol peroxidase and for the chloroplastic and cytosolic isozymes of ascorbate peroxidase in plants. Plant Cell Physiol. 1994;35:497–504.

Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 1981;22:867–880.

Gillespie KM, Ainsworth EA. Measurement of reduced, oxidized and total ascorbate content in plants. Nat. Protoc. 2007;2:871–874. PubMed

Sagi M, Fluhr R. Superoxide production by plant homologues of the gp91 (phox) NADPH oxidase. Modulation of activity by calcium and by tobacco mosaic virus infection. Plant Physiol. 2001;126:1281–1290. PubMed PMC

Able AJ, Guest DI, Sutherland MW. Use of a new tetrazolium-based assay to study the production of superoxide radicals by tobacco cell cultures challenged with avirulent zoospores of Phytophthora parasitica var nicotianae . Plant Physiol. 1998;117:491–499. PubMed PMC

Cheeseman JM. Hydrogen peroxide concentrations in leaves under natural conditions. J. Exp. Bot. 2006;57:2435–2444. PubMed

Ramel F, Sulmon C, Bogard M, Couée I, Gouesbet G. Differential patterns of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets. BMC Plant Biol. 2009;9:28. PubMed PMC

Daudi A, Cheng Z, O’Brien JA, Mammarella N, Khan S, Ausubel FM, Bolwell GP. The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity. Plant Cell. 2012;24:275–287. PubMed PMC

Oxborough K, Baker N. R Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components-calculation of qP and Fv′/Fm′ without measuring Fo′. Photosynth. Res. 1997;54:135–142.

Jensen LJ, Kuhn M, Stark M, Chaffron S, Creevey G, Muller J, Doerks T, Julien P, Roth A, Simonovic M, Bork P, von Mering C. STRING 8-a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res. 2009;37:D412–D416. PubMed PMC

McCormack E, Tsai Y-G, Braam J. Handling calcium signaling: Arabidopsis CaMs and CMLs. Trends Plant Sci. 2005;10:383–389. PubMed

Kuo WY, Huang GH, Liu AG, Cheng GP, Li SH, Chang WC, Weiss C, Azem A, Jinn TL. CHAPERONIN 20 mediates iron superoxide dismutase (FeSOD) activity independent of its co-chaperonin role in Arabidopsis chloroplasts. New Phytol. 2013;197:99–110. PubMed

Camacho L, Smertenko AP, Perez-Gomez J, Hussey PJ, Moore I. Arabidopsis Rab-E GTPases Exhibit a Novel Interaction with a Plasma-membrane Phosphatidylinositol-4-phosphate 5-kinase. J. Cell Sci. 2009;122:4383–4392. PubMed

Seltmann MA, Stingl NE, Lautenschlaeger JK, Krischke M, Mueller MJ, Berger S. Differential impact of lipoxygenase 2 and jasmonates on natural and stress-induced senescence in Arabidopsis . Plant Physiol. 2010;152:1940–1950. PubMed PMC

Takahashi F, Yoshida R, Ichimura K, Mzoguchi T, Seo S, Yonezawa M, Maruyama K, Yamaguchi-Shinozaki K, Shinozaki K. The mitogen-activated protein kinase cascade MKK3-MPK6 is an important part of the jasmonate signal transduction pathway in Arabidopsis . Plant Cell. 2007;19:805–818. PubMed PMC

Pilon M, Ravet K, Tapken W. The biogenesis and physiological function of chloroplast superoxide dismutases. Biochim. Biophys. Acta BBA-Bioenerg. 2011;1807:989–998. PubMed

Gallie D. R The Role of L-ascorbic acid recycling in responding to environmental stress and in promoting plant growth. J. Exp. Bot. 2013;64:433–443. PubMed

Smirnoff N. Ascorbate biosynthesis and function in photo-protection. Philos. Trans. R. Soc. London, B: Biol. Sci. 2000;355:1455–1464. PubMed PMC

Wolucka BA, Van Montagu M. GDP-mannose 3′,5′-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants. J. Biol. Chem. 2003;278:47483–47490. PubMed

Valpuesta V, Botella MA. Biosynthesis of L-ascorbic acid in plants: new pathways for an old antioxidant. Trends Plant Sci. 2004;9:573–577. PubMed

Bus JS, Aust SD, Gibson JE. Superoxide- and singlet oxygen-catalyzed lipid peroxidation as a possible mechanism for paraquat (methyl viologen) toxicity. Biochem. Biophys. Res. Commun. 1974;58:749–755. PubMed

Karpiński S, Szechyńska-Hebda M, Wituszyńska W, Burdiak P. Light acclimation, retrograde signalling, cell death and immune defenses in plants. Plant Cell Environ. 2013;36:736–744. PubMed

Pesaresi P, Scharfenberg M, Weigel M, Granlund I, Schroder WP, Finazzi G, Rappaport F, Masiero S, Furini A, Jahns P, Leister D. Mutants, overexpressors, and interactors of Arabidopsis plastocyanin isoforms: revised roles of plastocyanin in photosynthetic electron flow and thylakoid redox state. Mol. Plant. 2009;2:236–248. PubMed

Allahverdiyeva Y, Suorsa M, Rossi F, Pavesi A, Kater MM, Antonacci A, Tadini L, Pribil M, Schneider A, Wanner G, Leister D, Aro EM, Barbato R, Pesaresi P. Arabidopsis plants lacking PsbQ and PsbR subunits of the oxygen-evolving complex show altered PSII super-complex organization and short-term adaptive mechanisms. Plant J. 2013;75:671–684. PubMed

Ihnatowicz A, Pesaresi P, Varotto G, Richly E, Schneider A, Jahns P, Salamini F, Leister D. Mutants for photosystem I subunit D of Arabidopsis thaliana: effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions. Plant J. 2004;37:839–52. PubMed

Sinha AK, Jaggi M, Raghuram B, Tuteja N. Mitogen-activated protein kinase signaling in plants under abiotic stress. Plant Signal. Behav. 2011;6:196–203. PubMed PMC

Li G, Peng X, Xuan H, Wei L, Yang Y, Guo T, Kang G. Proteomic analysis of leaves and roots of common wheat (Triticum aestivum L.) under copper-stress conditions. J. Proteome Res. 2013;12:4846–4861. PubMed

Hossain Z, Khatoon A, Komatsu S. Soybean proteomics for unraveling abiotic stress response mechanism. J. Proteome Res. 2013;12:4670–4684. PubMed

Singh R, Jwa NS. Understanding the responses of rice to environmental stress using proteomics. J. Proteome Res. 2013;12:4652–4669. PubMed

Amme S, Matros A, Schlesier B, Mock HP. Proteome analysis of cold stress response in Arabidopsis thaliana using DIGE-technology. J. Exp. Bot. 2006;57:1537–1546. PubMed

Wang H, Wang S, Lu Y, Alvarez S, Hicks LM, Ge X, Xia Y. Proteomic analysis of early-responsive redox-sensitive proteins in Arabidopsis . J. Proteome Res. 2012;11:412–424. PubMed PMC

Du H, Liang Y, Pei K, Ma K. UV radiation-responsive proteins in rice leaves: a proteomic analysis. Plant Cell Physiol. 2011;52:306–316. PubMed

Jiang Y, Yang B, Harris NS, Deyholos MK. Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots. J. Exp. Bot. 2007;58:3591–3607. PubMed

Foyer GH, Noctor G. Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications. Antioxidants Redox Signal. 2009;11:861–905. PubMed

Suzuki N, Koussevitzky S, Mttler R, Miller G. ROS and Redox Signalling in the Response of Plants to Abiotic Stress. Plant Cell Environ. 2012;35:259–270. PubMed

Apel K, Hirt H. REACTIVE OXYGEN SPECIES: Metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 2004;55:373–399. PubMed

Farmer EE, Mueller MJ. ROS-mediated lipid peroxidation and RES-activated signaling. Annu. Rev. Plant Biol. 2013;64:429–450. PubMed

Jacques S, Ghesquière B, Van Breusegem F, Gevaert K. Plant proteins under oxidative attack. Proteomics. 2013;13:932–940. PubMed

Wang Y, Lin A, Loake GJ, Chu G. H2O2-induced leaf cell death and the crosstalk of reactive nitric/oxygen species. J. Integr. Plant Biol. 2013;55:202–208. PubMed

Hideg E, Jansen MA, Strid A. UV-B exposure, ROS, and stress: inseparable companions or loosely linked associates? Trends Plant Sci. 2013;18:107–115. PubMed

Jaspers P, Kangasjärvi J. Reactive oxygen species in abiotic stress signaling. Physiol. Plant. 2010;138:405–413. PubMed

Hossain Z, Komatsu S. Contribution of proteomic studies towards understanding plant heavy metal stress response. Front. Plant Sci. 2013;3:310. PubMed PMC

González Besteiro MA, Bartels S, Albert A, Ulm R. Arabidopsis MAP kinase phosphatase 1 and its target MAP kinases 3 and 6 antagonistically determine UV-B stress tolerance, independent of the UVR8 photoreceptor pathway. Plant J. 2011;68:727–737. PubMed

Lee JS, Ellis BE. Arabidopsis MAPK phosphatase 2 (MKP2) positively regulates oxidative stress tolerance and inactivates the MPK3 and MPK6 MAPKs. J. Biol. Chem. 2007;282:25020–25029. PubMed

Xing Y, Jia W, Zhang J. AtMKK1 mediates ABA-induced CAT1 expression and H2O2 production via AtMPK6-coupled signaling in Arabidopsis . Plant J. 2008;54:440–451. PubMed

Kliebenstein DJ, Monde RA, Last RL. Superoxide dismutase in Arabidopsis: an eclectic enzyme family with disparate regulation and protein localization. Plant Physiol. 1998;118:637–650. PubMed PMC

Van Breusegem F, Slooten L, Stassart JM, Moens T, Botterman J, Van Montagu M, Inzé D. Overproduction of Arabidopsis thaliana FeSOD confers oxidative stress tolerance to transgenic maize. Plant Cell Physiol. 1999;40:515–523. PubMed

Morgan MJ, Lehmann M, Schwarzlander M, Baxter CJ, Sienkiewicz-Porzucek A, Williams TCR, Schauer N, Fernie AR, Fricker MD, Ratcliffe RG, et al. Decrease in manganese superoxide dismutase leads to reduced root growth and affects tricarboxylic acid cycle flux and mitochondrial redox homeostasis. Plant Physiol. 2008;147:101–114. PubMed PMC

Hodgson RA, Raison JK. Superoxide production by thylakoids during chilling and its implication in the susceptibility of plants to chilling-induced photoinhibition. Planta. 1991;183:222–228. PubMed

Torres MA, Dangl JL, Jones JDG. Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc. Natl. Acad. Sci. U. S. A. 2002;99:517–522. PubMed PMC

Jiang M, Zhang J. Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings. Planta. 2002;215:1022–1030. PubMed

Hao F, Wang X, Chen J. Involvement of plasma-membrane NADPH oxidase in nickel-induced oxidative stress in roots of wheat seedlings. Plant Sci. 2006;170:151–158.

Desikan R, Cheung M-K, Bright J, Henson D, Hancock JT, Neill SJ. ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells. J. Exp. Bot. 2004;55:205–212. PubMed

Foreman J, Demidchik V, Bothwell JHF, Mylona P, Miedema H, Torres MA, Linstead P, Costa S, Brownlee G, Jones JDG, et al. Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature. 2003;422:442–446. PubMed

Kim S-H, Woo D-H, Kim J-M, Lee S-Y, Chung WS, Moon Y-H. Arabidopsis MKK4 mediates osmotic-stress response via its regulation of MPK3 activity. Biochem. Biophys. Res. Commun. 2011;412:150–154. PubMed

Colcombet J, Hirt H. Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes. Biochem. J. 2008;413:217–226. PubMed

Takahashi F, Mizoguchi T, Yoshida R, Ichimura K, Shinozaki K. Calmodulin-Dependent Activation of MAP Kinase for ROS Homeostasis in Arabidopsis . Mol. Cell. 2011;41:649–660. PubMed

Yoshida S, Tamaoki M, Shikano T, Nakajima N, Ogawa D, Ioki M, Aono M, Kubo A, Kamada H, Inoue Y, Saji H. Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis thaliana . Plant Cell Physiol. 2006;47:304–308. PubMed

Chen Z, Young TE, Ling J, Chang S-G, Gallie DR. Increasing vitamin C content of plants through enhanced ascorbate recycling. Proc. Natl. Acad. Sci. U. S. A. 2003;100:3525–3530. PubMed PMC

Chen Z, Gallie D. R Dehydroascorbate Reductase Affects Leaf Growth, Development, and Function. Plant Physiol. 2006;142:775–787. PubMed PMC

Foyer GH, Noctor G. Ascorbate and Glutathione: The Heart of the Redox Hub. Plant Physiol. 2011;155:2–18. PubMed PMC

Conklin PL, Norris SR, Wheeler GL, Williams EH, Smirnoff N, Last RL. Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 1999;96:4198–4203. PubMed PMC

Van Breusegem F, Vranova E, Dat JF, Inzé D. The role of active oxygen species in plant signal transduction. Plant Sci. 2001;161:405–414.

Yao N, Tada Y, Park P, Nakayashiki H, Tosa Y, Mayama S. Novel evidence for apoptotic cell response and differential signals in chromatin condensation and DNA cleavage in victorin-treated oats. Plant J. 2001;28:13–26. PubMed

Gupta AS, Webb RP, Holaday AS, Allen RD. Overexpression of Superoxide Dismutase Protects Plants from Oxidative Stress (Induction of Ascorbate Peroxidase in Superoxide Dismutase-Overexpressing Plants) Plant Physiol. 1993;103:1067–1073. PubMed PMC

Kwon SY, Jeong YJ, Lee HS, Kim JS, Cho KY, Allen RD, Kwak SS. Enhanced tolerances of transgenic tobacco plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl viologen-mediated oxidative stress. Plant, Cell Environ. 2002;25:873–882. PubMed

Camp WV, Capiau K, Montagu MV, Inze D, Slooten L. Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Plants Overproducing Fe-Superoxide Dismutase in Chloroplasts. Plant Physiol. 1996;112:1703–1714. PubMed PMC

Eng J, McCormack A, Yates J. R An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass. Spectrom. 1994;5:976–989. PubMed

Knockout of MITOGEN-ACTIVATED PROTEIN KINASE 3 causes barley root resistance against Fusarium graminearum

Arabidopsis Iron Superoxide Dismutase FSD1 Protects Against Methyl Viologen-Induced Oxidative Stress in a Copper-Dependent Manner

TALEN-Based HvMPK3 Knock-Out Attenuates Proteome and Root Hair Phenotypic Responses to flg22 in Barley

Single Amino Acid Exchange in ACTIN2 Confers Increased Tolerance to Oxidative Stress in Arabidopsis der1-3 Mutant

Signaling Toward Reactive Oxygen Species-Scavenging Enzymes in Plants

Comparative proteomic study of Arabidopsis mutants mpk4 and mpk6

Advantages and limitations of shot-gun proteomic analyses on Arabidopsis plants with altered MAPK signaling