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. 2002 Dec 2;21(23):6483-93.
doi: 10.1093/emboj/cdf646.

Distinct regulation of salinity and genotoxic stress responses by Arabidopsis MAP kinase phosphatase 1

Roman Ulm  1 , Kazuya Ichimura, Tsuyoshi Mizoguchi, Scott C Peck, Tong Zhu, Xun Wang, Kazuo Shinozaki, Jerzy Paszkowski
Affiliations

Distinct regulation of salinity and genotoxic stress responses by Arabidopsis MAP kinase phosphatase 1

Roman Ulm et al. EMBO J. .

Abstract

The Arabidopsis genome contains 20 genes encoding mitogen-activated protein kinases (MAPKs), which drastically outnumbers genes for their negative regulators, MAP kinase phosphatases (MKPs) (five at most). This contrasts sharply with genomes of other eukaryotes where the number of MAPKs and MKPs is approximately equal. MKPs may therefore play an important role in signal integration in plants, through concerted regulation of several MAPKs. Our previous studies identified Arabidopsis MKP1 and showed that its deficiency in the mkp1 mutant results in plant hypersensitivity to genotoxic stress. Here, we identify a set of MAPKs that interact with MKP1, and show that the activity level of one of these, MPK6, is regulated by MKP1 in vivo. Moreover, using expression profiling, we identified a specific group of genes that probably represent targets of MKP1 regulation. Surprisingly, the identity of these genes and interacting MAPKs suggested involvement of MKP1 in salt stress responses. Indeed, mkp1 plants have increased resistance to salinity. Thus MKP1 apparently plays a pivotal role in the integration and fine-tuning of plant responses to various environmental challenges.

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Figures

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Fig. 1. MKP1 function in genotoxic stress relief depends on its catalytic cysteine. Five-day-old seedlings were transferred to liquid medium supplemented with MMS at the concentration indicated. The picture of representative seedlings was taken after 2 weeks of further growth. Ws, Wassilewskija wild type; mkp1, mkp1 mutant; line 6, mkp1 complemented by ectopic expression of MKP1 (homozygous progeny); mkp1 MKP1(C235S), representative line of mkp1 with ectopic expression of MKP1(C235S) (heterozygous progeny); mkp1 MKP1, representative line of mkp1 complemented by ectopic expression of wild-type MKP1 (heterozygous progeny).
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Fig. 2. MKP1 interacts with MPKs 3, 4 and 6. (A) MKP1 was expressed in yeast cells with the indicated MAPKs (MPK1–9) or with the empty vector pVP16. Each of the MKP1-interacting MAPKs was also expressed with the empty vector pBTM116 used to express MKP1. Qualitative interaction assays for His3 and lacZ were scored from no interaction (–) to strong interaction (++++). LacZ activity is shown as standard units × 1000. Values reported are the average of the assays of four independent transformants. (B) Co-pull-down of baculovirus-produced Py-MKP1 from insect cell lysate with GST–MPK6. The upper panel depicts immunoblot analysis of GST pull-downs with anti-Glu–Glu antibodies (detecting the Py epitope) and the lower panel a parallel Coomassie-stained SDS–polyacrylamide gel.
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Fig. 3. MPK6 is activated by genotoxic stress. (A) Nine-day-old seedlings of Columbia (Col) wild type or transgenic for GFP or MPK6–GFP were subjected to the indicated genotoxic stress treatment. Soluble protein extracts were used for MBP in-gel kinase assays or immunoblot analysis with anti-MPK6 (Ab6NT1) antibody. (B) The protein kinase activity of immunoprecipitated MPK4 (with Ab4CT1) and MPK6 (Ab6NT1) was assayed by MBP in-gel kinase assay. Nine-day-old seedlings were irradiated with 0, 1 or 2 kJ/m2 UV-C as indicated and samples taken 10 min after treatment. (C) MPK6 protein kinase activity level in response to UV-C determined by MKP1 level. Ab6NT1-immunoprecipitated MPK6 from Ws, mkp1 mutant and MKP1-overexpressing line 6 after UV-C irradiation, as indicated. An example of a control IP without antibody is shown (identical results were obtained for all other samples).
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Fig. 4. MPK6 is constitutively transcribed in response to genotoxic stress, while MPK3 is induced. RNA gel blot analysis of 10 µg of total RNA isolated from UV-C-treated (0.5 kJ/m2) 14-day-old seedlings. Blots were hybridized sequentially with specific probes for MKP1, MPK3, MPK4 and MPK6. Radiant Red-stained rRNA is shown as loading control.
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Fig. 5. Oligonucleotide array comparing Ws and mkp1. (A) The 21 genes overexpressed (≥5-fold) in mkp1 are complemented in line 6. Black bars depict genes that are induced by UV-C (0.5 kJ/m2) during 3 h post-irradiation (≥3-fold). (B) Genes with an altered basal expression level in mkp1. Bold font indicates genes that are induced by UV-C during the 3 h time course (≥3-fold). The asterisk indicates that the expression level in wild type is very low and the signals are near background. Therefore, the fold changes of these genes are only approximate. (C) MKP1-dependent regulation of the 21 genes in mkp1 overexpressed in response to UV-C treatment. Time points (x-axis; 0, 0.5, 1 and 3 h after irradiation) and expression levels (y-axis) are as indicated. Black bar, wild-type Ws; dark-gray bar, mkp1; light-gray bar, line 6.
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Fig. 5. Oligonucleotide array comparing Ws and mkp1. (A) The 21 genes overexpressed (≥5-fold) in mkp1 are complemented in line 6. Black bars depict genes that are induced by UV-C (0.5 kJ/m2) during 3 h post-irradiation (≥3-fold). (B) Genes with an altered basal expression level in mkp1. Bold font indicates genes that are induced by UV-C during the 3 h time course (≥3-fold). The asterisk indicates that the expression level in wild type is very low and the signals are near background. Therefore, the fold changes of these genes are only approximate. (C) MKP1-dependent regulation of the 21 genes in mkp1 overexpressed in response to UV-C treatment. Time points (x-axis; 0, 0.5, 1 and 3 h after irradiation) and expression levels (y-axis) are as indicated. Black bar, wild-type Ws; dark-gray bar, mkp1; light-gray bar, line 6.
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Fig. 6. Elevated resistance of mkp1 to salt stress. Two-week-old seedlings were transferred to plates containing the indicated concentration of NaCl (0–225 mM). Ws, Wassilewskija wild-type; mkp1, mkp1 mutant; line 6, mkp1 complemented by ectopic expression of MKP1; C235S-1/C235S-2, two independent lines of mkp1 with ectopic expression of MKP1(C235S). Pictures were taken after 8 days of further growth, and representative plates from five replicates are shown.
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