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Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

Lassowskat I, Böttcher C, Eschen-Lippold L, Scheel D, Lee J - Front Plant Sci (2014)

Bottom Line: Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6.An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation.Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the "PEN" pathway required for penetration resistance to filamentous pathogens).

View Article: PubMed Central - PubMed

Affiliation: Department of Stress and Developmental Biology, Leibniz Institute of Plant Biochemistry Halle/Saale, Germany.

ABSTRACT
Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the "PEN" pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org).

No MeSH data available.


Related in: MedlinePlus

Regulation of global proteome after DEX treatment to induce MPK3/6 activation. (A) The relative abundance was normalized to the maximum value for each protein and a heat map generated by hierarchical clustering according to the MeV (Multiexperiment Viewer) algorithm. This resulted in a cluster of 480 up-regulated proteins, another with 145 down-regulated proteins and a third cluster of 72 proteins showing fluctuating changes. (B) Comparison of the total number of proteins with altered abundance between genotypes. To exclude any bias when comparing genotypes, total numbers of proteins detected within each genotype (right y-axis), which is relatively constant (~2000 throughout all genotypes), are also shown.
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Figure 4: Regulation of global proteome after DEX treatment to induce MPK3/6 activation. (A) The relative abundance was normalized to the maximum value for each protein and a heat map generated by hierarchical clustering according to the MeV (Multiexperiment Viewer) algorithm. This resulted in a cluster of 480 up-regulated proteins, another with 145 down-regulated proteins and a third cluster of 72 proteins showing fluctuating changes. (B) Comparison of the total number of proteins with altered abundance between genotypes. To exclude any bias when comparing genotypes, total numbers of proteins detected within each genotype (right y-axis), which is relatively constant (~2000 throughout all genotypes), are also shown.

Mentions: To elucidate the defense metabolism reprogramming by MPK3/6 activation, we performed a global proteome analysis of leaf material collected at 0, 2, 4, 6, 8, 10, 12, and 24 h after DEX spraying of the transgenic MKK5DD plants compared to the MKK5KR control plants. The time points were selected to cover the period where the rise in activated MAPKs (~4 h, Figure S1) and the rise in metabolites levels (typically starting at 6–12 h after DEX, c.f. Figure 2 and Figure S2) were detectable. Due to low protein yield, the 36 h timepoint was excluded. Triplicate samples (with each sample consisting of three plants pooled together) were collected for each genotype and time point. Two micrograms of extracted protein were digested with trypsin and separated with a 150-min LC gradient for LC-MS measurements. Quantitation of protein changes was performed with the Progenesis LC-MS software package and compared within each genotype. The heat map in Figure 4A illustrates the ~700 proteins with altered abundance (>2-fold, ANOVA, p < 0.05, for at least one of the measured time points) for the Col-0 genotype. Abundances for 480 and 145 proteins were gradually up- or down-regulated, respectively, during the 24 h period, while another group of 72 proteins showed fluctuating up- and down-regulation in an irregular manner (Figure 4A, Table S4). By contrast, the Col-0 KR plants had considerably less number of proteins with altered abundance (Table S3). With exception of the mpk3 mutant with its list of 789 proteins, all other tested mutants had fewer proteins with altered abundance (i.e., 267–501, Figure 4B, Tables S5–S9). Since the total number of detected proteins is comparable between genotypes (i.e., ~2000 in all cases, Figure 4B), the reduced number of proteins showing altered abundance in some mutants is not due to overall lower protein detection in a particular sample. Rather, it suggests that the changes in protein abundance are dependent on ethylene and ROS signaling. For mpk6, the reduced number of protein changes suggests an important contribution of MPK6 but it may also be in part through diminished ethylene biosynthesis since MPK6 is a major MAPK for stabilizing ACC synthase levels through phosphorylation (Liu and Zhang, 2004; Joo et al., 2008).


Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

Lassowskat I, Böttcher C, Eschen-Lippold L, Scheel D, Lee J - Front Plant Sci (2014)

Regulation of global proteome after DEX treatment to induce MPK3/6 activation. (A) The relative abundance was normalized to the maximum value for each protein and a heat map generated by hierarchical clustering according to the MeV (Multiexperiment Viewer) algorithm. This resulted in a cluster of 480 up-regulated proteins, another with 145 down-regulated proteins and a third cluster of 72 proteins showing fluctuating changes. (B) Comparison of the total number of proteins with altered abundance between genotypes. To exclude any bias when comparing genotypes, total numbers of proteins detected within each genotype (right y-axis), which is relatively constant (~2000 throughout all genotypes), are also shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202796&req=5

Figure 4: Regulation of global proteome after DEX treatment to induce MPK3/6 activation. (A) The relative abundance was normalized to the maximum value for each protein and a heat map generated by hierarchical clustering according to the MeV (Multiexperiment Viewer) algorithm. This resulted in a cluster of 480 up-regulated proteins, another with 145 down-regulated proteins and a third cluster of 72 proteins showing fluctuating changes. (B) Comparison of the total number of proteins with altered abundance between genotypes. To exclude any bias when comparing genotypes, total numbers of proteins detected within each genotype (right y-axis), which is relatively constant (~2000 throughout all genotypes), are also shown.
Mentions: To elucidate the defense metabolism reprogramming by MPK3/6 activation, we performed a global proteome analysis of leaf material collected at 0, 2, 4, 6, 8, 10, 12, and 24 h after DEX spraying of the transgenic MKK5DD plants compared to the MKK5KR control plants. The time points were selected to cover the period where the rise in activated MAPKs (~4 h, Figure S1) and the rise in metabolites levels (typically starting at 6–12 h after DEX, c.f. Figure 2 and Figure S2) were detectable. Due to low protein yield, the 36 h timepoint was excluded. Triplicate samples (with each sample consisting of three plants pooled together) were collected for each genotype and time point. Two micrograms of extracted protein were digested with trypsin and separated with a 150-min LC gradient for LC-MS measurements. Quantitation of protein changes was performed with the Progenesis LC-MS software package and compared within each genotype. The heat map in Figure 4A illustrates the ~700 proteins with altered abundance (>2-fold, ANOVA, p < 0.05, for at least one of the measured time points) for the Col-0 genotype. Abundances for 480 and 145 proteins were gradually up- or down-regulated, respectively, during the 24 h period, while another group of 72 proteins showed fluctuating up- and down-regulation in an irregular manner (Figure 4A, Table S4). By contrast, the Col-0 KR plants had considerably less number of proteins with altered abundance (Table S3). With exception of the mpk3 mutant with its list of 789 proteins, all other tested mutants had fewer proteins with altered abundance (i.e., 267–501, Figure 4B, Tables S5–S9). Since the total number of detected proteins is comparable between genotypes (i.e., ~2000 in all cases, Figure 4B), the reduced number of proteins showing altered abundance in some mutants is not due to overall lower protein detection in a particular sample. Rather, it suggests that the changes in protein abundance are dependent on ethylene and ROS signaling. For mpk6, the reduced number of protein changes suggests an important contribution of MPK6 but it may also be in part through diminished ethylene biosynthesis since MPK6 is a major MAPK for stabilizing ACC synthase levels through phosphorylation (Liu and Zhang, 2004; Joo et al., 2008).

Bottom Line: Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6.An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation.Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the "PEN" pathway required for penetration resistance to filamentous pathogens).

View Article: PubMed Central - PubMed

Affiliation: Department of Stress and Developmental Biology, Leibniz Institute of Plant Biochemistry Halle/Saale, Germany.

ABSTRACT
Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the "PEN" pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org).

No MeSH data available.


Related in: MedlinePlus