Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress

Abstract

Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5-MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases.

Keywords: Arabidopsis; FSD; iron superoxide dismutase; mitogen-activated protein kinase 6; mitogen-activated protein kinase kinase 5; salt stress..

Fig. 1.

FSD1, FSD2, and FSD3 expression following NaCl treatment. (A) Northern blot analysis of FSD1, FSD2, and FSD3 expression. Arabidopsis tissues from different points of the time course were used for RNA gel blot analysis. Total RNA was extracted from wild-type plants without stress treatment (control) or subjected to 150mM NaCl, and incubated for different times. The actin gene ACT2 was used as a loading control. (B) Relative GUS activity, driven by the FSD1, FSD2, or FSD3 promoters, showed the response to NaCl. The FSD1, FSD2, and FSD3 promoters were fused to GUS and tested for their response to NaCl in a protoplast transient expression assay. All experiments were repeated at least three times with similar results.

Fig. 2.

Involvement of MKK5 in salt stress responses. RNA gel blot analysis of FSD2 and FSD3 transcript levels in response to salt stress in wild-type Arabidopsis plants, or the mkk2, mkk4, and mkk5 mutants. Total RNA was extracted from wild-type (WT) or mutant plants without stress treatment (control, C) or subjected to NaCl (150mM) for 4h (S). The actin gene ACT2 was used as a loading control. (B) Relative GUS activity driven by FSD2 or FSD3 promoter, respectively, showed the response of FSD2 and FSD3 to NaCl. The promoters of the FSD2 and FSD3 genes were fused to the GUS reporter gene and tested for their response to NaCl in transiently transformed protoplasts from wild-type plants or the mkk5 mutants. The MKK5 genes were cloned and co-transformed into protoplasts of the mkk5 mutant. Transformed protoplasts were pre-incubated 1h with 5 M PD98059 before treatment with 150mM NaCl. Protoplasts were isolated from wild-type (M) and the mkk5 mutant (m) leaves. (C) Relative GUS activity driven by FSD2 or FSD3 promoter, respectively, showed the response of FSD2 and FSD3 to NaCl. The promoters of the FSD2 and FSD3 genes were fused to the GUS reporter gene and tested for their response to NaCl in transiently transformed protoplasts from wild-type plants or the mkk2 and mkk4 mutants. The MKK2 and MKK4 genes were cloned and co-transformed into protoplasts of the mkk2 or mkk4 mutant, respectively. Protoplasts were isolated from wild-type (M) and mutant (m) leaves. All experiments were repeated at least three times with similar results.

Fig. 3.

NaCl activates MPK3 and MPK6 through MKK5. (A) MAPK activation with or without NaCl 10min and expression of each MAPK. (B) Salt-triggered activation of MPK3, MPK4, and MPK6 in Arabidopsis. Kinetics of MPK3, MPK4, and MPK6 activation were measured in wild-type and mkk5 mutant plants in response to salt stress. MPK3, MPK4, and MPK6 were immunoprecipitated from leaf cell extracts of salt-induced plants. MPK activity was measured in immunocomplex kinase assays using myelin basic protein (MBP) as a substrate and the levels of MPK3, MPK4, and MPK6 proteins were measured by western blot analysis. (C) Relative GUS activity driven by the MPK3, MPK4, or MPK6 promoter, respectively, showed MPK3, MPK4, and MPK6 response to NaCl in transiently transformed protoplasts of wild-type (M) and mkk5 mutant (m). The MKK5 genes were cloned and co-transformed into protoplasts of the mkk5 mutant. (D) In vitro phosphorylation of MPK3, MPK4, and MPK6 by active MKK5. GST-tagged MKK5 was immunoprecipitated from Arabidopsis protoplasts before and 10min after salt stress treatment. Immunoprecipitated MKK5 was subsequently used for phosphorylation of recombinant kinase inactive GST-MPK3, GST-MPK4, and GST-MPK6, respectively. Phosphorylation of MPKs was analysed by autoradiography after SDS-PAGE. MKK5 protein was detected using a GST antibody. (E) Relative GUS activity driven by the FSD2 and FSD3 promoters showed the FSD2 and FSD3 response to NaCl in transient expression assays using protoplasts from wild-type (M) or mpk3 and mpk6 mutants (m). The MKK5 genes were cloned and co-transformed into protoplasts of the mkk5 mutant. All experiments were repeated at least three times with similar results.

Fig. 4.

Activation of MKK5 by salt stress. (A) MKK5 activity was determined after transient expression in plant cells upon salt stress. GST epitope-tagged MKK5 was immunoprecipitated from Arabidopsis protoplasts following NaCl (150mM) treatments for 10min. MKK5 kinase activity was determined by in vitro kinase assays using kinase inactive GST-MPK6 as a substrate. (B) Relative GUS activity driven by the MKK5 promoter in wild-type, mkk5, and MKK5-overexpressing plants induced by NaCl. The MKK5 promoter was fused to GUS and tested for its response to NaCl in transient expression assays using protoplasts. All experiments were repeated at least three times with similar results.

Fig. 5.

Phenotypic analysis of and overexpressing plants. (A) The salt-sensitive phenotype of MKK5-RNAi plants, mkk5, was investigated by germination assays of wild-type [Col-0 (WT)], mkk5, and MKK5 overexpressing plants on agar plates, with 0mM or 150mM NaCl. Seeds were sterilized, stratified, and plated. Germination was visually determined after10 d. (B) Salt-sensitive phenotype of mkk5 plants and salt tolerance of MKK5 overexpressing lines. (C) The production of superoxide in seedlings response to salt stress (300mM NaCl) for 10 d. (D) SOD activities in seedlings of wild-type, mkk5, and MKK5-OE response to salt stress (300mM NaCl) for 10 d. All experiments were repeated at least three times with similar results.

Fig. 6.

MKK5 specifically interacts with MEKK1. (A) MKK5 strongly interacted with MEKK1 in the yeast two-hybrid system. Yeast strains containing pEXP^TM32-MKK5 as bait and pEXP^TM22-MEKK1 as prey were grown on the following medium to screen for transformants: 1, SC medium lacking Leu and Trp for 48h; 2, SC medium lacking Leu, Trp, and Ura for an additional 48h to select the transformants; 3, SC medium lacking Leu and Trp and adding 0.2% of 5-fluoroorotic acid (5-FOA) for an additional 48h to select the cells containing interacting proteins; 4, SC medium lacking Leu, Trp, and His and adding 100mM 3-Amino-1,2,4-Triazole (3AT) for an additional 48h to confirm the interaction; 5, YPAD medium 48h, then an X-Gal assay was performed on the membrane to confirm the results. The pEXP^TM22 empty prey vector was used as negative control. (B) Quantitative analysis of -galactosidase activity of the yeast strains in liquid culture showing the interaction between MKK5 and MEKK1. Values are means of data from at least three independent experiments. (C) Relative GUS activity driven by MKK5, FSD2, or FSD3 promoters, respectively, showed the response to NaCl in transiently transfected protoplasts of wild-type (M) or mekk1 (m) plants. The MEKK1 genes were cloned and co-transformed into protoplasts of the mekk1 mutant. All experiments were repeated at least three times with similar results.