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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

Activation of the MAP kinases, MPK 3 and MPK6, by DEX-inducible expression of a constitutively active MAPK kinase, MKK5. (A) Six week old plants were sprayed with 20 μM DEX to induce expression of a constitutively active MAPK kinase 5, MKK5DD, in Col-0 ecotype background (abbreviated as Col-0 DD). Leaves were harvested at the indicated time points after DEX treatment and used for immunodetection of phosphorylated MAPKs (α-pTEpY). The expected positions of MPK3/6 are marked. Col-0 KR designates the corresponding control plants expressing a kinase-inactive version of MKK5. (B) Prolonged MPK3/6 activation, after DEX treatment, lead to death of the plants—visible as tissue collapse after 24 h. (Note that for some lines, new leaves emerged from the central meristem some days after the DEX treatment). Photos of the plants were taken 2 week after DEX treatment. The core experiment (within red box) compares the Col-0 KR (control) with Col-0 DD (active MKK5DD) lines. Additional transgenic lines expressing the MKK5DD in the indicated mutant background are also included (For detection of phosphorylated MAPKs for these lines, please see Figure S1).
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Figure 1: Activation of the MAP kinases, MPK 3 and MPK6, by DEX-inducible expression of a constitutively active MAPK kinase, MKK5. (A) Six week old plants were sprayed with 20 μM DEX to induce expression of a constitutively active MAPK kinase 5, MKK5DD, in Col-0 ecotype background (abbreviated as Col-0 DD). Leaves were harvested at the indicated time points after DEX treatment and used for immunodetection of phosphorylated MAPKs (α-pTEpY). The expected positions of MPK3/6 are marked. Col-0 KR designates the corresponding control plants expressing a kinase-inactive version of MKK5. (B) Prolonged MPK3/6 activation, after DEX treatment, lead to death of the plants—visible as tissue collapse after 24 h. (Note that for some lines, new leaves emerged from the central meristem some days after the DEX treatment). Photos of the plants were taken 2 week after DEX treatment. The core experiment (within red box) compares the Col-0 KR (control) with Col-0 DD (active MKK5DD) lines. Additional transgenic lines expressing the MKK5DD in the indicated mutant background are also included (For detection of phosphorylated MAPKs for these lines, please see Figure S1).

Mentions: Substitution of two serines by “phosphomimicking” aspartic acid (D) in the kinase activation loop of the parsley MAPK kinase 5 (MKK5) generates a constitutively active kinase (abbreviated as MKK5DD) that can phosphorylate and activate downstream MAPKs (Lee et al., 2004). Transgenic Arabidopsis thaliana (Col-0) plants were created with a dexamethasone (DEX)-inducible expression of this heterologous MKK5DD (hereafter named Col-0 DD) (see material and methods for generation and validation of the transgenic plants). Two immunoreactive bands representing phosphorylated MAPKs are detected in plant extracts of such transgenic plants after 4–6 h of DEX treatment (Figure 1A), which is not seen in corresponding control plants (Col-0 KR) that express the kinase-inactive version of MKK5 (MKK5KR). Previous experiments using MAPK-specific antibodies have defined these two bands to be MPK3 and MPK6 (Bethke et al., 2009) and this is now further confirmed by the loss of one of the MAPK bands in the respective mpk3 or mpk6 mutant background (see Figure S1).


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)

Activation of the MAP kinases, MPK 3 and MPK6, by DEX-inducible expression of a constitutively active MAPK kinase, MKK5. (A) Six week old plants were sprayed with 20 μM DEX to induce expression of a constitutively active MAPK kinase 5, MKK5DD, in Col-0 ecotype background (abbreviated as Col-0 DD). Leaves were harvested at the indicated time points after DEX treatment and used for immunodetection of phosphorylated MAPKs (α-pTEpY). The expected positions of MPK3/6 are marked. Col-0 KR designates the corresponding control plants expressing a kinase-inactive version of MKK5. (B) Prolonged MPK3/6 activation, after DEX treatment, lead to death of the plants—visible as tissue collapse after 24 h. (Note that for some lines, new leaves emerged from the central meristem some days after the DEX treatment). Photos of the plants were taken 2 week after DEX treatment. The core experiment (within red box) compares the Col-0 KR (control) with Col-0 DD (active MKK5DD) lines. Additional transgenic lines expressing the MKK5DD in the indicated mutant background are also included (For detection of phosphorylated MAPKs for these lines, please see Figure S1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Activation of the MAP kinases, MPK 3 and MPK6, by DEX-inducible expression of a constitutively active MAPK kinase, MKK5. (A) Six week old plants were sprayed with 20 μM DEX to induce expression of a constitutively active MAPK kinase 5, MKK5DD, in Col-0 ecotype background (abbreviated as Col-0 DD). Leaves were harvested at the indicated time points after DEX treatment and used for immunodetection of phosphorylated MAPKs (α-pTEpY). The expected positions of MPK3/6 are marked. Col-0 KR designates the corresponding control plants expressing a kinase-inactive version of MKK5. (B) Prolonged MPK3/6 activation, after DEX treatment, lead to death of the plants—visible as tissue collapse after 24 h. (Note that for some lines, new leaves emerged from the central meristem some days after the DEX treatment). Photos of the plants were taken 2 week after DEX treatment. The core experiment (within red box) compares the Col-0 KR (control) with Col-0 DD (active MKK5DD) lines. Additional transgenic lines expressing the MKK5DD in the indicated mutant background are also included (For detection of phosphorylated MAPKs for these lines, please see Figure S1).
Mentions: Substitution of two serines by “phosphomimicking” aspartic acid (D) in the kinase activation loop of the parsley MAPK kinase 5 (MKK5) generates a constitutively active kinase (abbreviated as MKK5DD) that can phosphorylate and activate downstream MAPKs (Lee et al., 2004). Transgenic Arabidopsis thaliana (Col-0) plants were created with a dexamethasone (DEX)-inducible expression of this heterologous MKK5DD (hereafter named Col-0 DD) (see material and methods for generation and validation of the transgenic plants). Two immunoreactive bands representing phosphorylated MAPKs are detected in plant extracts of such transgenic plants after 4–6 h of DEX treatment (Figure 1A), which is not seen in corresponding control plants (Col-0 KR) that express the kinase-inactive version of MKK5 (MKK5KR). Previous experiments using MAPK-specific antibodies have defined these two bands to be MPK3 and MPK6 (Bethke et al., 2009) and this is now further confirmed by the loss of one of the MAPK bands in the respective mpk3 or mpk6 mutant background (see Figure S1).

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