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

Salt-induced expressions of iron superoxide dismutases (FSD2 and FSD3) are regulated by MKK5 that interacts with MEKK1 via the MKK5-MPK6-coupled signalling cascade.


Introduction
Reactive oxygen species (ROS) have long been known to be harmful to many cellular processes, however, a transient increase in ROS can also act as a signal that mediates the regulation of various cellular activities. Examples include responses to biotic or abiotic stresses, cell death, stomatal movement or root hair development (Gill and Tuteja, 2010;Acharya et al., 2013;Suzuki et al., 2013). As a first line of defence against ROS, the enzyme superoxide dismutase (SOD) catalyses the initial step in the Asada-Halliwell pathway in chloroplasts and converts superoxide to hydrogen peroxide and molecular oxygen (Bowler et al., 1994;Allen, 1995). SOD enzymes are classified into three groups according to their metal co-factor: iron SOD (FeSOD), manganese SOD (MnSOD), and copper-zinc SOD (Cu-Zn SOD) and are located in different cellular compartments (McKersie et al., 2000).
There is growing evidence to suggest that SODs and other anti-oxidative enzymes are closely associated with plant biotic or abiotic stress tolerance, although the signalling processes involved in stress-induced gene expression of SODs are largely unknown (Miller et al., 2008, van Breuseqem et al., 2008Li et al., 2013;Xing et al., 2013). Overexpressing some SODs has been demonstrated to enhance diverse stress tolerances, such as to low temperature and high light (McKersie et al., 2000;Huang et al., 2012) and other abiotic stresses (Gill and Tuteja, 2010). A decrease in MnSOD leads to reduced root growth and affects tricarboxylic acid cycle flux and mitochondrial redox homeostasis (Morgan et al., 2008). SaFeSOD cloned from Sonneratia alba, a highly salt tolerant mangrove tree, is expressed in leaf, stem, flower, fruit, and root tissues with the highest expression in leaf tissues (Wang et al., 2013). Under nitrogen supplemented conditions, overexpression of either SOD protein, especially FeSOD, conferred significant tolerance against oxidative stress (Raghavan et al., 2011). Nevertheless, the signalling processes of stress-induced gene expression of SODs are still largely unknown (McKersie et al., 2000;Miller et al., 2008;van Breusegem et al., 2008).
Several lines of evidence suggest that the MAPK signalling cascade is, at several levels, involved in plant biotic or abiotic stress-induced signalling and indeed, most plant MAPKs investigated to date have been linked to stress responses (Xing et al., 2008(Xing et al., , 2013Ding et al., 2013;Li et al., 2013;Sheikh et al., 2013). In studies with alfalfa (Medicago sativa), stress-induced MAPKK (SIMKK) was isolated in a yeast two-hybrid interaction screen using the MAP kinase SIMK as a bait and the specific activation of SIMK by SIMKK was subsequently observed upon salt stress in protoplasts, supporting the function of SIMKK as an upstream activator of SIMK in this pathway (Kiegerl et al., 2000). A complete Arabidopsis MAPK signalling cascade pathway (MEKK1, MKK4/MKK5, and MPK3/6) has been identified and shown to be involved in the induction of expression of the WRKY22/WRKY29 transcription factors by the bacterial protein flagellin (Asai et al., 2002). Moreover, Arabidopsis MKK2 has been shown to be activated by MEKK1 and to increase freezing and salt tolerance by activating its direct targets MPK4 and MPK6, as well as the expression of other stress-induced marker genes (Teige et al., 2004). Previous reports have also shown that hydrogen peroxide-mediated stress signalling requires MAP kinase cascades (Kovtun et al., 2000;Kumar and Klessig, 2000;Samuel et al., 2000;Zhang et al., 2006;Xing et al., 2008), suggesting an important role for MAPK signalling in the generation of ROS and detoxification.
The involvement of Arabidopsis MKK5 in the MAP kinase cascade was suggested by its response to flagellin-induced signalling (Asai et al., 2002). Furthermore, Arabidopsis MKK5 has been shown to be involved in hydrogen peroxide-mediated cell death and oxidative stress (Ren et al., 2002;Xing et al., 2013). Taken together, these findings suggest that Arabidopsis MKK5 might be involved in both abiotic and biotic stress signalling. However, the exact roles and detailed mechanisms of MKK5-mediated signalling remain unknown.
To elucidate the potential role of MAPK signalling cascades in salt stress responses, an Arabidopsis protoplast transient expression system was used, in which transcription of FeSOD genes is induced by NaCl, allowing the roles of MAPK cascade components to be systematically evaluated. Using this system, MEKK1 was identified via the MKK5-MPK6-coupled signalling associated with salt-induced FeSOD expression. These data suggest that this signalling pathway functions in response to salt stress and could potentially be utilized to enhance salt tolerance in crops.

Plant materials and stress treatments
Arabidopsis thaliana (L.) plants were kept in a growth chamber at 22 ± 2 °C with 16 h light and 8 h dark and a relative humidity of 90%. RNAi gene-silenced plants of MKK5 and plants from MKK5-RNAi lines, MKK5-7, named mkk5 were generated and chosen for this study (Xing et al., 2013). For NaCl treatment of germination, wild-type and mutant seedlings growing on MS agar plates (MS complete medium with 30 g/l sucrose and 7 g/l agar) were transferred onto filter paper saturated with 150 mM NaCl and the plants were incubated under light for various times. For NaCl treatment of protoplasts, isolated protoplasts from Arabidopsis leaves were subjected to a final NaCl concentration of 150 mM for 10 min.
Total RNA extraction, semi-quantitative reverse transcription-PCR and northern blot analysis Seedlings (mutant and wild-type plants) were grown for 2 weeks on MS agar plates under continuous light and then treated with NaCl as described above. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and stored at −80 °C until further use. Total RNA was used as a template for first-strand cDNA synthesis using the SuperScript II First-Strand Synthesis System for reverse transcription (RT)-PCR (Invitrogen). The PCR volume was 25 µl, containing 100 ng of each primer, 2 mM each dNTP, 0.5 µl cDNA, and 0.75 units of Taq DNA polymerase (Invitrogen), and the reactions were run in a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, MA, USA). PCR products were separated on 1.5% agarose gels.
For northern blot analysis, 15 µg of total RNA was fractionated on a 1.0% formaldehyde-containing agarose gel with an RNA molecular weight marker (Promega Corp., Madison, WI, USA) and blotted onto a nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Aylesbury, UK) overnight at room temperature. Equal loading of RNA was confirmed using ethidium bromide staining of the gel prior to transfer. Probes were labelled with [α-32 P] dCTP using a random-primed DNA labelling kit (Megaprime, Amersham) and hybridization was performed according to the manufacturer's recommendations.

Protoplasts preparation and transformation
Arabidopsis protoplasts were isolated using a modified protocol from Abel and Theologis (1994). Leaves from 4-week old Arabidopsis plants were washed in distilled water and incubated with an enzyme solution containing 1% Cellulase R10, 0.25% Macerozyme R10 (both from Yakult, Tokyo, Japan), and 0.4 M mannitol for about 3-4 h at 23 °C with gentle shaking (50 rpm). The protoplast suspension was filtered through a net with a pore diameter of 150 µM. After centrifugation (60 g, 2 min), the supernatant was discarded and the cells were re-suspended in washing solution and washed twice in washing buffer containing Magma solution. The concentration of protoplasts was determined and the viability of the cells was verified by staining with FDA (fluoresce in diacetate) and subsequently by fluorescent microscopy (Zeiss Axioskop) (Nunberg and Thomas, 1993). Plasmid DNA solutions were adjusted with 0.5 M mannitol and mixed with 5 × 10 5 protoplasts. After addition of an equal volume of PEG 6000 solution, the suspension was incubated at room temperature for 10 min and washed with washing and incubation (WI) solution before being re-suspended in WI solution. The transformed cells were incubated in the dark (23 °C, 30 rpm, overnight) and the suspensions were divided into different tubes, treated with NaCl solution and incubated for the appropriate times.

Assay for transient GUS activity
The suspensions of protoplasts were centrifuged (60 g, 2 min) and washed with 0.5 M mannitol. Pellets were dissolved in 105 µl extraction buffer and extracts were vortexed and kept at −80 °C for at least 1 h or in liquid nitrogen for several minutes. GUS activity was quantified according to Fütterer et al. (1990) and a modified protocol for suppressing endogenous GUS activity (Kosugi et al., 1990). The protein concentration was measured according to the manufacturer's instructions (Bio-Rad, Hercules, CA, USA) with BSA as a standard. GUS activity was measured in the supernatant after adding the substrate 4-methyl-umbelliferyl-β-d-glucuronide (MUG). MUG was hydrolysed by GUS protein into 4-methyl-umbelliferone or 7-hydroxy-4-methyl-cumarin (4-MU) and glucuronide, and GUS activity determined in a dark micro-titre plate (Nunc GmbH & Co. KG, Wiesbaden, Germany) using the HTS 7000 plus Bioassay Reader (emission, 460 nm; excitation, 365 nm). GUS activity was normalized according to the expression derived from pHBT95-GUS with no fusion protein (average 204 pmol of 4-MU produced per minute per microgram of protein obtained from six independent experiments).

Expression and purification of GST fusion proteins
Full-length Arabidopsis MKK5, MPK3, MPK4, and MPK6 cDNAs were obtained using RT-PCR, cloned into the pENTR-TOPO cloning vector (Invitrogen) and sequenced. The Escherichia coli Strain BL-21 codon plus (Stratagene, LA Jolla, CA, USA) was transformed with the expression constructs, which was prepared by subcloning the genes into pGEX-6P-1 vector (Amersham Pharmacia Biotech). Growth of bacteria and isolation of recombinant GST fusion protein was as described in Matsuoka et al. (2002).

Protein extraction, immunoprecipitation and kinase activity assay
The following steps were carried out at 4 °C unless otherwise stated. Plant tissues (3:1 buffer volume:fresh weight) were homogenized with a pestle and mortar in 100 mM Tris-HCl buffer (pH 8.0) containing 2 mM EDTA, 5 mM DTT, 10% glycerol, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 0.3 µM aprotinin. The homogenate was filtered through four layers of muslin cloth and centrifuged at 12,000 g for 40 min. The supernatant was desalted with a Sephadex G-25 column equilibrated with buffer suitable for the individual enzymes. The desalted supernatants were stored in aliquots at −80 °C. The protein concentration was determined using the protein assay kit (Bio-Rad) with BSA as a standard.
Protein extracts (0.5 mg) were incubated with 50 µl antibody at 4 °C overnight. Protein G-agarose beads (50 μl) were added and incubated for 2 h at 4 °C. The protein-antibody complex on the beads was collected and washed three times in ice-cold PBS and before re-suspension in protein sample buffer.
The coding regions of MPK3, MPK4, and MPK6 were cloned into the pGEX-6P-1 vector and expressed as GST fusion proteins in BL21 codon plus E. coli cells (see above). Kinase inactive GST-MPK fusion proteins were generated by exchanging a conserved lysine reside in the ATP binding domains to methionine and arginine using the Quick Change kit (Stratagene). The point mutations were performed as described by Teige et al. (2004). Inactive GST-MPK (2 μg) was incubated in 20 µl of kinase reaction buffer (50 mM Tris, pH 7.5, 10 mM MgCl 2 , 1 mM DTT, 0.1 mM ATP, and 8 µCi of 32 P-ATP) with immunoprecipitated GST fused to MKK5 from protoplasts. Kinase reactions were stopped after 30 min by adding 4 µl SDS loading buffer and heating for 5 min at 95 °C. Reaction products were analysed by SDS-PAGE, autoradiography, and Coomassie brilliant blue R250 staining.
In-gel kinase assays were performed essentially as described by Katou et al. (1999) with some modifications. Briefly, samples (20 µg) of total protein or immunoprecipitate from 400 µg of total protein were separated on a 10% SDS-polyacrylamide gel polymerized in the presence of 0.25 mg/ml bovine brain myelin basic protein (Sigma). After electrophoresis, SDS was removed by washing the gel in buffer (25 mM Tris-HCl pH 8.0, 0.1 mM Na 3 VO 4 , 5 mM NaF, 0.5 mM DTT, 0.5 mg/ml BSA, and 0.1% Triton X-100) three times (30 min each) at room temperature. After 1 h denaturation in a denaturing buffer containing guanidine, the kinases were allowed to renature overnight at 4 °C with five changes of renaturing buffer (25 mM Tris, pH 7.5, 1 mM DTT, 0.1 mM Na 3 VO 4 , and 5 mM NaF). The phosphorylation of myelin basic protein was performed in 30 ml reaction buffer (25 mM Tris, pH 7.5, 0.1 mM Na 3 VO 4 , 12 mM MgCl 2 , 2 mM EGTA, and 1 mM DTT) with a pre-reaction for 30 min, then 0.2 µM ATP and 50 µCi of 32 P-ATP in reaction buffer was added and the reactions incubated at room temperature for 60-90 min. The gel was then transferred into washing buffer at room temperature for at least 6 h with six changes of buffer. Finally, the gel was dried on filter paper and autoradiographed.

Immunocomplex kinase assays
The method for kinase assays has been described by Xing et al. (2008Xing et al. ( , 2013. Kinase inactive MPK6-GST protein (1 μg) immunoprecipitated from Arabidopsis protoplast using anti-GST was incubated with MKK5 immunoprecipitated from Arabidopsis seedlings using anti-MKK5 in the kinase reaction mixture [20 µl of kinase reaction buffer containing 50 mM Tris (pH 7.5), 1 mM DTT, 10 mM MgCl 2 , 0.1 mM ATP, and 9.7 × 10 -3 Bq of 32 P-ATP] for 30 min at room temperature. The reaction was stopped after 30 min by adding 4 µl of SDS loading buffer and heating for 5 min at 95 °C. Reaction products were analysed by autoradiography after SDS-PAGE. MPK3-GST, MPK4-GST, and MPK6-GST fusion proteins were generated by exchanging a conserved lysine residue in the ATP binding domains to methionine and arginine using the Quick Change kit (Stratagene). The point mutations for MPK6 were K92M and K93R (Teige et al., 2004).

Generation of MKK5 overexpressing plants
Full-length Arabidopsis MKK5 cDNA was obtained by RT-PCR, cloned into the pENTR-TOPO cloning vector (Invitrogen) and sequenced. After the LR reaction, MKK5 cDNA was inserted into the pGWB5-DHA vector, and the vector named pGWB5-DHA-MKK5. Transgenic Arabidopsis plants expressing the DHA-tagged MKK5 under control of the CaMV 35S promoter were generated using the floral dip method (Clough and Bent, 1998) and either Col-0 wild-type plants or mkk5 plants. Transformed plants were selected for growth on kanamycin containing media. Second generation plants were used for experiments.

Determination of O 2
− production and SOD activity in Arabidopsis leaves O 2 − production in Arabidopsis leaves of treated and control plants was determined according to the method of Able et al. (1998) by monitoring the reduction of 3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulphonic acid hydrate (XTT) in the presence of O 2 − . Leaves (1 g) were frozen in N 2 , ground, and then homogenized with 5 ml of 50 mM TRIS-HCl buffer (pH 7.5) and centrifuged at 5000 g for 10 min. The reaction mixture of 1 ml contained 50 µg of supernatant proteins, 50 mM TRIS-HCl buffer (pH 7.5), and 0.5 mM XTT. The reduction of XTT was determined at 470 nm for 5 min. Corrections were made for the background absorbance in the presence of 50 units SOD. O 2 − production rate was calculated using an extinction coefficient of 2.16 × 10 4 M −1 cm −1 . SOD (EC 1.12.1.11) was measured in the leaves as previously described (Xing et al. 2013).

Yeast two-hybrid interactions
Yeast two-hybrid interactions were performed using the ProQuest TM Two-Hybrid System (Invitrogen) according to the manufacturer's instructions.

Salt-Induced FeSODs in Arabidopsis
Salt-induced expression of FSD genes in Arabidopsis was investigated by northern blot analysis using RNA extracted from leaves. Treatment of plants with 150 mM NaCl led to a significant increase in the expression of FSD2 and FSD3, with FSD2 responding earlier than FSD3. The FSD2 gene transcript increased within 1 h of treatment, peaked at 4 h, and remained high until 6 h, while FSD3 transcription was induced within 2 h and remained high until 6 h. In contrast, FSD1 transcript level was not altered by NaCl treatment (Fig.1A).
To further characterize FeSOD gene activation, the promoters of FSD1, FSD2, and FSD3 (1.2 kb before the ATG) were fused to the β-glucuronidase reporter gene (GUS) and tested for response to NaCl in transiently transfected protoplasts. Consistent with the northern blot data obtained with the endogenous genes, the FSD2 and FSD3 promoters but not those of FSD1 were activated by NaCl (Fig. 1B). Although the FSD1 gene was expressed, it was not induced by NaCl. An earlier report indicated that FSD1 transcription is under the control of a circadian clock (Kliebenstein et al., 1998); however, these results did not show that FSD2 or FSD3 are controlled by a circadian clock mechanism (data not shown).

Salt-induced FSD signalling operates through MKK5
In a previous study all the 10 known mutants of the MAPKK family in Arabidopsis were screened for downstream signalling components (Xing et al., 2007(Xing et al., , 2008(Xing et al., , 2013. These MAPKK mutants were tested for FSD gene expression under salt stress in this study. Interestingly, the salt-activated gene expression of FSD2 and FSD3 that is apparent in wild-type plants was absent in the MKK5-RNAi plants (mkk5) but still present in MKK4-RNAi lines (mkk4). MKK4 is classified in the same subfamily as MKK5 and has the highest degree of DNA sequence homology to MKK5. Although MKK2 has also been shown to respond to NaCl-induced salt stress (Teige et al., 2004), the NaCl-induced increases of FSD2 and FSD3 transcripts were not blocked in MKK2 mutant, mkk2 plants ( Fig. 2A). These results suggest that MKK5 is a strong candidate for mediating the salt-induced FSD expression.
To determine whether the FSD2 and FSD3 promoters were activated by salt through MKK5 signalling, protoplasts isolated from mkk5 leaves were transiently transfected with a pFSD2-GUS and a pFSD3-GUS reporter construct. In the 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 150 mM 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. mkk5 protoplasts, neither of the two promoters was activated by NaCl (Fig. 2B), although transient expression of wild-type MKK5 restored NaCl-induced activation of the promoters in the mkk5 mutant protoplasts (Fig. 2B). Consistent with these results, PD98059, a broad-spectrum MAPK kinase inhibitor, partially impaired the ability of NaCl to activate the FSD2 and FSD3 promoters-most notably that of FSD2 (Fig. 2B).
It was observed that the FSD2 and FSD3 promoters were activated by salt in a protoplast assay using protoplasts isolated from leaves of the mkk2 and mkk4 RNAi lines. Co-transformation of MKK2 or MKK4 with FSD2 or FSD3 promoters into protoplasts from mkk2 or mkk4 mutant leaves, respectively, showed no difference in activation patterns compared with wild-type (Fig. 2C). Together these results suggest that neither salt-activated MKK2 nor MKK4, which is homologous to MKK5, is involved in salt-induced FSD signalling.

The involvement of specific MAPKs in NaCl-induced FSD signalling
To determine which MAPKs are involved in salt-induced FSD signalling, six MAPKs were targeted that collectively represent four of the five MAPK subfamilies, and which may exhibit distinct functions based on sequence homology analysis (Mizoguchi et al., 2000, Tena et al., 2000Hamel et al., 2006). The coding sequences of these MAPKs were each introduced into the pHBT95 vector in frame with the coding sequence of a GST, transiently expressed in protoplasts, immunoprecipitated with an anti-GST antibody, and tested for in vitro MAP kinase activity. MPK3, MPK4, and MPK6 all showed strong activation following NaCl treatment (Fig. 3A), while MPK7 showed a small activation but an overall weak expression. MPK2 and MPK9 did not show an altered expression (Fig. 3A).
To investigate the molecular mechanism of MKK5 action in salt-induced FSD signalling, MPK3, MPK4, and MPK6 activation was analysed in wild-type and mkk5 plants. NaCl activation of MPK3 and MPK6, which belong to the same subfamily, but not MPK4, in wild-type, decreased in the mkk5 mutant protoplasts unless functional MKK5 was coexpressed in the mkk5 mutant (Fig. 3B). The activation of MPK6 was absent in mkk5 mutant protoplasts, whereas the activation of MPK3 was decreased, suggesting that MPK3 is not regulated to the same extent by MKK5 in salt-induced FSD signalling. To investigate whether the MPK3, MPK4, 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 (150 mM) for 4 h (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 1 h with 5 µM PD98059 before treatment with 150 mM 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 wildtype (M) and mutant (m) leaves. All experiments were repeated at least three times with similar results. and MPK6 genes were activated transcriptionally in wild-type and the mkk5 mutant, the promoters of MPK3, MPK4, and MPK6 genes were fused to GUS and tested for their response to NaCl in transiently transfected protoplasts of wild-type and the mkk5 mutant. Consistent with the MAPK activity results, NaCl activation of MPK3 and MPK6 promoters, but not the MPK4 promoter, was blocked in mkk5 and restored when co-expressed with MKK5 (Fig. 3C). Together, these results suggest that MPK3 and MPK6, but not MPK4, are involved in MKK5-mediated salt-induced FSD signalling and that another signal is required to regulate NaCl-induced MPK4 signalling.
To investigate the phosphorylation targets of MKK5 in vitro, recombinant kinase inactive GST fusion proteins of MPK3, MPK4, and MPK6 were expressed and purified. MKK5 was expressed under the control of the 35S CaMV promoter, immunoprecipitated from transiently transformed protoplasts and tested for its ability to phosphorylate MPK3, MPK4, and MPK6 in vitro after activation by salt stress for 10 min. Consistent with the above results, both MPK3 and MPK6, but not MPK4, were phosphorylated by MKK5 (Fig. 3D).
To directly test the role of MPK3 and MPK6 in saltinduced FSD signalling, protoplasts isolated from mpk3 or mpk6 mutant leaves were transiently transformed with the FSD2-GUS and FSD3-GUS reporter constructs. In the mpk6 mutant protoplasts, neither of the two promoters was activated by NaCl and the relative GUS activity of the two promoters was restored when co-expressing MPK6 in mpk6 mutant protoplasts. In contrast, the two promoters maintained the same activated level in the mpk3 mutant protoplasts as the wild-type (Fig. 3E). Thus, it appears that while the salt-induced expression of FSD2 and FSD3 requires MPK6, it does not require MPK3, even though both of them can be activated by MKK5.

NaCl activation of MKK5
Salt activation of the FSD2 and FSD3 promoters was not observed in mkk5 protoplasts, so it was examined whether GST-tagged MKK5 was immunoprecipitated from Arabidopsis protoplasts before and 10 min 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.
NaCl could activate MKK5 in Arabidopsis leaves. MKK5 activity was present from 0.5 to 6 h after treatment and then dramatically decreased to basal levels (Fig. 4A). To confirm these results, MKK5-overexpressing plants (MKK5-OE) were constructed, and, consistent with the results using protoplasts, NaCl activation of the MKK5 promoter was absent in mkk5 but increased in MKK5-OE plants (Fig. 4B). These studies also demonstrated that the Arabidopsis protoplast transient expression system can offer a rapid and reliable tool for studying NaCl-induced FSD signalling based on early gene transcription.

MKK5-overexpressing plants exhibit salt tolerance phenotypes
The MKK5-OE plants showed no obvious phenotype under normal conditions, but large differences when they were stressed with salt treatments (Fig. 5B). To analyse salt tolerance of the MKK5-OE and mkk5 plants, their germination efficiency on salt-containing media was evaluated. The mkk5 plants germinated at a much lower rate than either wild-type or MKK5-OE, while the latter showed a slightly improved ability to germinate on salt-containing media (Fig. 5A). A quantification of the differences in germination of the different lines on salt-containing media confirmed the qualitative analysis: the germination rate of the mkk5 plants was only 50% of that of wild-type. As shown in Fig. 5B, MKK5-OE plants exhibited increased salt tolerance compared with wild-type and the mkk5 plants, so while wild-type and MKK5-overexpressing plants survived the salt stress conditions, the mkk5 plants were sensitive to salt stress. Notably, the increased superoxide production was relatively much lower in MKK5-overexpressing plants than in the mkk5 and wild-type plants, and SOD activity was also higher in MKK5-overexpressing plants (Fig. 5C, D). Similar results were obtained with 10 independent MKK5-OE lines in at least three independent assays. Taken together, these results confirm the importance of MKK5 in conferring salt stress tolerance.

MEKK1 interacts with MKK5
The MEKK1 gene has previously been shown to be upregulated by both cold and salt stresses (Mizoguchi et al., 1997;Teige et al., 2004) and it has also been shown that the MEKK1-MKK4/5-MPK3/6 module acts downstream of the flagellin receptor FLS2 and upstream of the WRKY22 and WRKY29 transcription factors (Asai et al., 2002). Having established that MKK5 is involved in salt-induced FSD signalling, it was hypothesized that MKK5 might interact with MEKK1 under salt stress conditions. It was examined whether MKK5 and MEKK1 interact physically using the yeast two-hybrid system. Yeast strains containing pEXP-MKK5 were used as a bait, and MEKK1, ANP1, and ANP2 were chosen as candidates for upstream MKK5 action, since they have been shown to be activated by oxidative stress (Kovtun et al., 2000;Krysan et al., 2002). As can be seen in Fig. 6A, MEKK1 interacted strongly with MKK5 but not with ANP1 or ANP2. The coloured product produced in the assay appeared and this was confirmed by a quantitative β-galactosidase assay (Fig. 6B). Finally, it was tested whether MKK5, the transcript levels of FSD2, and FSD3 were affected in wild-type and the mekk1 mutant, by fusing their promoters to GUS and testing their response to NaCl in transiently transfected protoplasts of wild-type and mekk1 mutant. Consistent with the results from the yeast two-hybrid assay, the activation of MKK5, FSD2, and FSD3 promoters were all arrested in the mekk1 mutant (Fig. 6C), suggesting that MEKK1 acts upstream of MKK5 and is involved in NaCl-induced FSD signalling.

Discussion
Salt stress can modulate the activity of the antioxidant system (Khan et al., 2012), and several studies have demonstrated that antioxidant enzyme activities and levels of antioxidant compounds are increased in certain plants in response to salt stress (Meneguzzo et al., 1999;Shalata et al., 2001). As the first line of defence against ROS in plant cells, SODs react with the superoxide radical to produce H 2 O 2 (Scandalios, 1997). SODs have been extensively studied for many years and growing evidence suggests that they respond to many abiotic stresses and that SOD overexpressing plants have an enhanced tolerance of abiotic stresses (Miller et al., 2008, van Breusegem et al., 2008Li et al., 2013;Xing et al., 2013). This study was focused on FeSODs and analysing their response to salt stress. Of the three FeSODs from Arabidopsis, FSD2 GST epitope-tagged MKK5 was immunoprecipitated from Arabidopsis protoplasts following NaCl (150 mM) treatments for 10 min. 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. and FSD3, but not FSD1, were strongly regulated by salt stress. Possibly as a consequence of circadian regulation, the mRNA abundance of FSD1 was not very stable but was normally higher than that of FSD2 or FSD3 (Fig. 1A). Different FeSODs respond to stresses in various ways, suggesting complex mechanisms for regulating the expression of the SOD gene family, involving substantial signalling pathways crosstalk.
The specific ROS sensors that process and translate associated stresses have yet to be identified (Andreasson and Ellis, 2010). However, the crosstalk among ROS signalling, ROS scavenging enzymes, and MAPK cascades is complex and elaborate. There is evidence from several systems for the interaction of MAPKs with ROS (Samuel and Ellis, 2002;Pitzschke and Hirt, 2006;Xing et al., 2008Xing et al., , 2013. The MAPK signalling pathway, as one of the major signalling cascades, plays a crucial role in diverse cellular functions, and particularly the redox-regulated processes involved in cellular metabolism (Davletova et al., 2004;Joo et al., 2005;Pitzschke and Hirt, 2006;Xing et al., 2008Xing et al., , 2013. In some studies, H 2 O 2 is required for the activation of ZmMPK5 in maize leaves Lin et al., 2009). ZmCPK11 induces the activation of ZmMPK5 in ABA signalling by increasing the production of H 2 O 2 (Ding et al., 2013). Cai et al. (2014) found overexpression of ZmMKK1 alleviated ROS accumulation by maintaining high activities of ROS scavenging enzymes such as SOD, POD, CAT, and APX. Similarly, ZmMPK17 and ZmMKK4 transgenic tobacco plants have enhanced osmotic stress tolerance through high ROS scavenging ability (Kong et al., 2011;).
Although both MAPK signalling cascades and antioxidant systems are involved in biotic and abiotic stress, it is not clear how or if they interact. In a systematic screen of the 10 members of the Arabidopsis MAPKK family, it was found that transcription of the NaCl-activated FSD2 and FSD3 was significantly blocked in MKK5-RNAi plants-mkk5but not in mkk4-the MKK4-RNAi plants, which belongs to the same subfamily as MKK5. In the flagellin-induced pathway of the MEKK1-MKK4/MKK5-MPK3/MPK6 module, MKK4 and MKK5 are involved in the same process of regulating the downstream WRKY22 and WRKY29 transcription factors (Asai et al., 2002). MKK4 and MKK5 are paralogous MKKs acting upstream of the MPK3/MPK6, and play a key role in mediating many different stress signals and in plant development (Andreasson and Ellis, 2010). It is very interesting that MKK5 and MKK4 show different responses to salt stress, while MKK4 was not involved in the regulation of FSD (Fig. 2A). The present study reveals that MKK4 and MKK5 may operate in different pathways and that only MKK5 is involved in NaCl-induced salt stress. The regulation of MKK5 on FSD2/3 seems to be specific. The function of MKK4 in salt stress, in contrast to MKK5,  Miles et al. (2009) also showed that partial suppression of MKK5 expression in Arabidopsis is sufficient to induce ozone hypersensitivity, which would indicate that MKK4 and MKK5 are not fully redundant. However, it is still possible that the roles of MKK4 and MKK5 within the salt stress signalling network are redundant because there is no experimental evidence of a double mutant at present.
The NaCl-induced MAPK cascade leading to the activation of Arabidopsis MPK3, MPK4, and MPK6 is reminiscent of the activation of MPK4 and MPK6 by SIMK, under salt stress (Kiegerl et al., 2000). Teige et al. (2004) identified a salt-induced Arabidopsis MAPKK, MKK2, which is capable of activating MPK4 and MPK6. However, as shown in Fig 3A, these data further revealed an unexpected activation of MPK3. It was also shown that although MPK3, MPK4, and MPK6 are involved in salt stress responses, only MPK3 and MPK6 are regulated by MKK5 (Fig. 3C). In contrast to the activation of MPK4 and MPK6 by MKK2 following salt stress, the regulation of MPK3 and MPK6 by MKK5 is more complex, involving different MAPK signalling pathways. The MKK5-MPK3/MPK6 pathway is similar to the flagellin-induced MAPK signalling pathway, MKK4/MKK5-MPK3/MPK6 (Asai et al., 2002). The existence of a crosstalk between the different stress signals is suggested, and that MKK5, as an important component of MAPKs signalling cascades, plays a key role in plant responses to biotic and abiotic stresses. The MAPK family involves comprehensive protein interactions and different MAPKs may function to different stresses (Andreasson and Ellis, 2010). Together these data demonstrate that MKK5 is a key signal transducer in MAPKs signalling cascades involved in regulating different processes in response to multiple divergent stresses.

Supplementary data
Supplementary material is available at JXB online.   6. MKK5 specifically interacts with MEKK1. (A) MKK5 strongly interacted with MEKK1 in the yeast two-hybrid system. Yeast strains containing pEXP TM 32-MKK5 as bait and pEXP TM 22-MEKK1 as prey were grown on the following medium to screen for transformants: 1, SC medium lacking Leu and Trp for 48 h; 2, SC medium lacking Leu, Trp, and Ura for an additional 48 h to select the transformants; 3, SC medium lacking Leu and Trp and adding 0.2% of 5-fluoroorotic acid (5-FOA) for an additional 48 h to select the cells containing interacting proteins; 4, SC medium lacking Leu, Trp, and His and adding 100 mM 3-Amino-1,2,4-Triazole (3AT) for an additional 48 h to confirm the interaction; 5, YPAD medium 48 h, then an X-Gal assay was performed on the membrane to confirm the results. The pEXP TM 22 empty prey vector was used as negative control. (B) Quantitative analysis of β-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.