MAPK (mitogen-activated protein kinase) pathways have been implicated in stress signalling in plants. In the present study, we performed yeast two-hybrid screening to identify partner MAPKs for OsMKK (Oryza sativa MAPK kinase) 6, a rice MAPK kinase, and revealed specific interactions of OsMKK6 with OsMPK3 and OsMPK6. OsMPK3 and OsMPK6 each co-immunoprecipitated OsMKK6, and both were directly phosphorylated by OsMKK6 in vitro. An MBP (myelin basic protein) kinase assay of the immunoprecipitation complex indicated that OsMPK3 and OsMPK6 were activated in response to a moderately low temperature (12°C), but not a severely low temperature (4°C) in rice seedlings. A constitutively active form of OsMKK6, OsMKK6DD, showed elevated phosphorylation activity against OsMPK3 and OsMPK6 in vitro. OsMPK3, but not OsMPK6, was constitutively activated in transgenic plants overexpressing OsMKK6DD, indicating that OsMPK3 is an in vivo target of OsMKK6. Enhanced chilling tolerance was observed in the transgenic plants overexpressing OsMKK6DD. Taken together, our data suggest that OsMKK6 and OsMPK3 constitute a moderately low-temperature signalling pathway and regulate cold stress tolerance in rice.

INTRODUCTION

Low temperature is one of the major environmental factors that limits the productivity and geographical distribution of a number of crops. Chilling (low but non-freezing) temperatures affect seed germination, seedling growth and seed fertility of rice (Oryza sativa L.) [1]. Owing to its origin in tropical and subtropical climates, rice is more sensitive to low temperature stress than are other cereal crops such as wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) [2,3]. Exposure of rice plants at the tetrad stage to a moderately low temperature (12°C) for 4 days resulted in male sterility in 80% of spikelets due to abnormal development of pollen grains [4,5]. Although agricultural loss associated with capricious chilling-induced reduced fertilization is quite significant in rice production in cooler temperate regions, the sensing and signal transduction mechanism for moderately low temperature is largely unknown.

MAPK (mitogen-activated protein kinase) cascades are conserved signalling pathways for transducing extracellular stimuli into cellular responses in eukaryotes [6]. MAPK cascades consist of three sequentially phosphorylating and activating components, a MEKK/MAPKKK (MAPK kinase kinase), a MKK/MAPKK (MAPK kinase) and a MAPK (MPK). MPKs phosphorylate a variety of substrates including transcription factors, protein kinases and cytoskeleton-associated proteins [7].

MAPK pathways have been implicated in a wide variety of plant biotic and abiotic stress responses. Overexpression of a constitutively active form of the Nicotiana MAPKKK NPK1 enhances freezing tolerance in maize [8,9]. Ectopic overexpression of a maize MAPKK, ZmMKK4 (Zea mays MKK4), in Arabidopsis confers cold- and salt-stress tolerance [10]. In the Arabidopsis genome, 20 MAPK, 10 MAPKK and more than 60 MAPKKK genes have been identified [11]. Whereas a complete cascade consisting of At (Arabidopsis) MEKK1–AtMKK4/AtMKK5–AtMPK3/AtMPK6 has been identified as participating in flagellin-mediated innate immune signalling [12], another cascade, AtMEKK1–AtMKK2–AtMPK4/AtMPK6, is involved in cold- and salt-stress tolerances. Overexpression of a constitutively activated form of AtMKK2, AtMKK2DD, where serine/threonine residues in the phosphorylation motif (S/TXXXXXS/T) are substituted by aspartic acid, enhances tolerance against cold and salt stresses [13,14]. In the rice genome, 15 MAPK, eight MAPKK and 75 MAPKKK genes have been annotated [11,15]. These protein kinases are considered to form specific complexes in response to multiple biotic and abiotic stresses in rice. The transcripts of many Os (Oryza sativa) MPKs are induced by abiotic and biotic stresses [16]. However, only OsMPK3 and OsMPK6 have been functionally characterized. OsMPK3 (also known as OsMAP1 or OsMAPK5) positively regulates drought, salt and cold stress tolerances, and negatively modulates broad-spectrum disease resistance [17]. OsMPK6 is activated by a fungal elicitor and regulates disease resistance in rice [1820]. OsMKK4, one of the eight OsMKKs, has been shown to activate OsMPK3 and OsMPK6 in vitro and in vivo, and the OsMKK4–OsMPK6 cascade mediates a fungal chitin elicitor-activated signal and regulates defence responses in rice [19]. However, an entire MAPK cascade for low-temperature signalling has not yet been determined.

We have previously reported that the transcripts of OsMKK6 and OsMPK3 are concomitantly induced in response to a moderately low temperature (12°C), but not a severely low temperature (4°C) in rice seedlings. Interaction of OsMKK6 with OsMPK3 was also detected in yeast two-hybrid assays [21]. In the present paper, we present several lines of evidence that OsMKK6 and OsMPK3 constitute a MAPK cascade for moderately low temperature signalling and regulate chilling tolerance in rice.

EXPERIMENTAL

Plant materials and stress treatments

Seeds of japonica rice (O. sativa L. cv. Yukihikari) were surface-sterilized, imbibed and germinated in the incubator. Seedlings were grown hydroponically at 25°C for 7 days under continuous illumination in a growth chamber. Plants were subjected to 12°C (moderately low temperature) or 4°C (severely low temperature) treatments for 0, 1, 3, 6, 12 and 24 h as described previously [21]. Shoot tissues were collected at the designated time points, immediately frozen in liquid nitrogen and stored at −80°C until use.

DNA sequencing and phylogenetic analysis

DNA sequencing was carried out using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). The BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) was used for the sequencing reactions. Analysis of DNA sequences was performed using GENETYX software (Software Development). Multiple amino acid sequence alignments of kinase domains of MAPK families from rice and Arabidopsis were performed using the ClustalW alignment program available at DDBJ (http://www.ddbj.nig.ac.jp/index-e.html). The phylogenetic tree was constructed using the neighbour-joining method.

Plasmids

For the yeast two-hybrid assays, the yeast bait plasmid pHyblex/Zeo (Invitrogen) was used to express OsMKK6 (GenBank® accession number AK059461) fused in-frame to the LexA DNA-binding domain. Ten OsMPK genes, including OsMPK3 (GenBank® accession number AK104834), OsMPK4 (GenBank® accession number AK111579), OsMPK6 (GenBank® accession number AK111942), OsMPK14 (GenBank® accession number AK119650), OsMPK16 (GenBank® accession number AK068981), OsMPK17-1 (GenBank® accession number AK066531), OsMPK20-1 (GenBank® accession number AK065930), OsMPK20-3 (GenBank® accession number AK100081), OsMPK20-4 (GenBank® accession number AK072082) and OsMPK21-1 (GenBank® accession number AK070644), were each fused in-frame with the B42 activation domain of the prey plasmid pYESTrp2 (Invitrogen). All primers used for the cloning are shown in Supplementary Table S1 (at http://www.BiochemJ.org/bj/443/bj4430095add.htm).

For recombinant protein induction, the pQE31 vector (Novagen) was used to express an N-terminal His6-tagged OsMKK6. The pGEX-6P-3 vector (GE Healthcare) was used to express GST (glutathione transferase)-fused proteins of OsMPK3, OsMPK3m, OsMPK6, OsMPK6m, OsMKK6, OsMKK6D and OsMKK6DD. All primers used for this cloning are shown in Supplementary Tables S2 and S3 (at http://www.BiochemJ.org/bj/443/bj4430095add.htm).

Yeast two-hybrid assays

Saccharomyces cerevisiae strain L40 [MATa his3Δ200 trp1-901 leu2-3112 ade2 LYS2::(4lexAop-HIS3) URA3::(8lexAop-lacZ) GAL4)] was used as the host yeast strain. The hybrid Hunter™ (version D, Invitrogen) yeast two-hybrid system was utilized to evaluate the physical interaction between OsMKK6 and OsMPK proteins according to the manufacturer's instructions. L40 cells were co-transformed with bait (pHybLex/Zeo) and prey (pYES-Trp2) plasmids as described previously [21].

The ability to drive expression of the HIS3 reporter gene was assessed by growth of the yeast transformants containing the bait and prey plasmids on selective YC medium lacking tryptophan, leucine and histidine. LacZ expression was quantified by measuring β-galactosidase activity using ONPG (o-nitrophenyl β-D-galactopyranoside) as a substrate, as described previously [22].

Recombinant protein preparation

GST-fused proteins, including GST–OsMPK3, GST–OsMPK3m, GST–OsMPK6, GST–OsMPK6m, GST–OsMKK6, GST–OsMKK6D and GST–OsMKK6DD, were induced in Escherichia coli BL21 (DE3) cells, followed by sonication and affinity purification with glutathione–Sepharose 4B according to the manufacturer's instructions (GE Healthcare). Protein concentration was measured with the Bio-Rad Protein Assay kit using BSA as a standard. Where indicated, the GST tag was removed from the GST-fused proteins by digestion with PreScission protease (GE Healthcare) at 4°C overnight. His-tagged OsMKK6 protein was similarly produced in E. coli M15 cells, and purified according to the manufacturer's instructions (Novagen).

In vitro pull-down assay

Pull-down assays were performed as described previously [23]. His-tagged OsMKK6 was pulled down, subjected to SDS/PAGE (15% gel), and detected by Western blotting with an anti-His monoclonal antibody (Santa Cruz Biotechnology).

Site-directed mutagenesis

The GeneEditor™ in vitro site-directed mutagenesis system (Promega) was utilized to introduce kinase-negative mutations into pGEX-OsMPK3, pGEX-OsMPK6, pGEX-OsMPK3m and pGEX-OsMPK6m, as well as to generate pGEX-OsMKK6DD, the constitutively active form of pGEX-OsMKK6. All primers used for the mutagenesis are shown in Supplementary Tables S2 and S3.

In vitro autophosphorylation and kinase assays

In vitro autophosphorylation and one-step kinase assays were performed using purified recombinant proteins as described previously [24]. For autophosphorylation assays, 1 μg of recombinant protein (except for OsMKK6 where 0.5 μg was used) was incubated in 20 μl of kinase reaction buffer [50 mM Tris (pH 7.5), 10 mM MgCl2, 10 mM MnCl2, 1 mM EGTA and 1 mM DTT (dithiothreitol)] in the presence of 1 μCi of [γ-32P]ATP. The MPK3 phosphorylation activity of OsMKK6, OsMKK6D and OsMKK6DD was determined in the same kinase reaction buffer containing 1 μg of OsMPK3m as a substrate. After incubation at 30°C for 30 min, the reaction was stopped by the addition of 2× SDS/PAGE sample loading buffer (20 μl). An aliquot (5 μl) from this was separated by SDS/PAGE (15% gel), and the kinase activities were detected by autoradiography.

In vitro one- and two-step MBP (myelin basic protein) kinase activity assays of OsMKK6 were performed as described previously [25]. Briefly, for in vitro one-step MBP kinase assays, 0.5 μg of GST–OsMKK6 and 1 μg of GST–OsMPK3, GST–OsMPK6 or GST (control) were incubated in 20 μl of kinase reaction buffer as described above. For in vitro two-step MBP kinase assays, 1 μg of GST–OsMPK3, GST–OsMPK6 or GST (control) was incubated with 0.5 μg of GST–OsMKK6 in 15 μl of kinase reaction buffer with 50 μM unlabelled ATP for 15 min at 30°C. A 5 μl aliquot of kinase reaction buffer with MBP (5 μg) and 1 μCi of [γ-32P]ATP were then added to the kinase reaction buffer, and incubation was continued for 30 min at 30°C, before the reaction was stopped by the addition of SDS/PAGE sample loading buffer. The phosphorylated MBP was visualized by autoradiography after SDS/PAGE (15% gel).

RNA extraction and RT (reverse transcription)–PCR analysis

Total RNA was isolated from shoots using TRIzol® reagent (Invitrogen). First-strand cDNA was synthesized with the Gene Amp RNA PCR core kit (Applied Biosystems), and PCR amplification was performed with Ex-Taq DNA polymerase (TaKaRa) for 24 cycles of 30 s at 95°C, 30 s at 56°C and 30 s at 68°C, followed by 3 min at 68°C for the final extension, using transgene-specific primers for OsMKK6: 5′-GCAATTGTTGACCAGCCACC-3′ and 5′-GGAAATTCGAGCTCGGTACC-3′. The internal control ubiquitin (Ubi) gene (GenBank® accession number AK059011) was amplified with 5′-CCAGGACAAGATGATCTGCC-3′ and 5′-AAGAAGCTGAAGCATCCAGC-3′.

MBP kinase assay of immunoprecipitated OsMPK3 and OsMPK6

MBP kinase activity of immunoprecipitated OsMPK3 and OsMPK6 was determined as described previously [26] with a slight modification in the amounts of starting proteins. Total soluble protein (400 μg) from the shoot tissues of rice seedlings after 4°C and 12°C stress treatments was incubated with 50 μl of anti-OsMPK3 or anti-OsMPK6 antibodies.

Rice transformation

The full-length cDNA of OsMKK6 was amplified by PCR with the primers 5′-TCTAGAGGCTGATTACTCGAGCCG-3′ and 5′-GGTACCTCATTCCTCCCAATGAC-3′ (XbaI and KpnI sites respectively are underlined) and cloned into the pGEM-T Easy vector (Promega). To produce pGEM-MKK6DD, the DD mutation was introduced into pGEM-OsMK6 by replacing the corresponding region with that from pGEX-OsMKK6DD digested by NheI and NdeI. The resulting pGEM-OsMKK6DD plasmid was digested with XbaI and KpnI and inserted into the binary vector pActZH2 [27], forming pAct-MKK6DD, which was introduced into the rice cultivar Yukihikari by Agrobacterium-mediated transformation [28].

Chilling tolerance measurement

Transgenic plants (T2) were germinated and grown for 7 days on 0.5× Murashige and Skoog medium with 50 μg/l hygromycin under continuous light conditions. The seedlings were then transferred to a commercial soil matrix (Sankyo) and allowed to grow until the four-leaf stage. Chilling stress was imposed by transferring the plants to a growth chamber maintained at 4°C for 7 days under continuous light. The plants were subsequently grown at 25°C for 10 days under continuous light in a growth cabinet. Survival of the plants was scored by the emergence of new leaves.

RESULTS

OsMKK6 specifically interacts with OsMPK3 and OsMPK6 in vitro

In our previous study, interaction of OsMKK6 with OsMPK3 was detected using yeast two-hybrid assays [21]. To identify further possible targets of OsMKK6, a pair-wise yeast two-hybrid analysis was performed. Phylogenetic analysis identified four groups (A, B, C and D) within 15 rice OsMPKs [11]. Representative OsMPK genes from each group, including OsMPK3 and OsMPK6 (group A), OsMPK4 (group B), OsMPK14 (group C), and OsMPK17-1/OsBWMK1, OsMPK20-4/OsWJUMK, OsMPK16, OsMPK20-1, OsMPK20-3 and OsMPK21-1 (group D), were tested for interactions with OsMKK6. The interaction was examined first by assays for complementation of histidine auxotrophy. Of the ten combinations tested, only the OsMKK6–OsMPK3 and OsMKK6–OsMPK6 pairs showed apparent colony growth similar to the positive control on the selection medium (Figure 1A). Interaction was further analysed by measuring the β-galactosidase activity. Compared with the positive control pair, Fos–Jun, the activities of OsMKK6–OsMPK3 and OsMKK6–OsMPK6 pairs were 148.16% and 151.25% respectively. However, the other combinations, including the negative control, exhibited very low relative activities (Figure 1B). These data indicate that OsMKK6 interacts with OsMPK3 and OsMPK6 in yeast two-hybrid assays.

Interaction analyses of OsMPKs with OsMKK6

Figure 1
Interaction analyses of OsMPKs with OsMKK6

(A) His auxotroph assay of yeast L40 cells containing pHyblex-OsMKK6 with different pYESTrp-OsMPK plasmids. YC plus histidine medium (left-hand panel) and YC His-deficient medium (right-hand) were used. (B) Quantitative β-galactosidase assay of yeast extract from each strain. The numbers on the x-axis indicate combinations of the bait plasmid and different prey plasmids, namely: pHyblex+pYESTrp (1, negative control); Fos2+Jun (2, positive control); pHyblex-OsMKK6+OsMPK3 (3); OsMPK6 (4); OsMPK4 (5); OsMPK14 (6); OsMPK17-1 (7); OsMPK20-4 (8); OsMPK16 (9); OsMPK20-1 (10); OsMPK20-3 (11); or OsMPK21-1 (12). Results are means±S.D. from three independent experiments. (C) In vitro protein pull-down assay of OsMKK6 with OsMPK3 and OsMPK6. Purified recombinant His–OsMKK6 protein was incubated with GST–OsMPK3, GST–OsMPK6 or GST (control), and pulled down with glutathione–Sepharose 4B beads. After washing and elution, the bound protein was subjected to Western blot analysis with an anti-His monoclonal antibody.

Figure 1
Interaction analyses of OsMPKs with OsMKK6

(A) His auxotroph assay of yeast L40 cells containing pHyblex-OsMKK6 with different pYESTrp-OsMPK plasmids. YC plus histidine medium (left-hand panel) and YC His-deficient medium (right-hand) were used. (B) Quantitative β-galactosidase assay of yeast extract from each strain. The numbers on the x-axis indicate combinations of the bait plasmid and different prey plasmids, namely: pHyblex+pYESTrp (1, negative control); Fos2+Jun (2, positive control); pHyblex-OsMKK6+OsMPK3 (3); OsMPK6 (4); OsMPK4 (5); OsMPK14 (6); OsMPK17-1 (7); OsMPK20-4 (8); OsMPK16 (9); OsMPK20-1 (10); OsMPK20-3 (11); or OsMPK21-1 (12). Results are means±S.D. from three independent experiments. (C) In vitro protein pull-down assay of OsMKK6 with OsMPK3 and OsMPK6. Purified recombinant His–OsMKK6 protein was incubated with GST–OsMPK3, GST–OsMPK6 or GST (control), and pulled down with glutathione–Sepharose 4B beads. After washing and elution, the bound protein was subjected to Western blot analysis with an anti-His monoclonal antibody.

To test further the interaction of OsMKK6 with OsMPK3 and OsMPK6 in the above yeast two-hybrid system, an in vitro pull-down assay was performed. His–OsMKK6 was pulled down by GST–OsMPK3 and by GST–OsMPK6, but not by GST, as indicated by an obvious band with a molecular mass corresponding to His–OsMKK6 (41 kDa) (Figure 1C). This demonstrates that OsMKK6 specifically interacts with OsMPK3 and OsMPK6 in vitro.

OsMKK6 directly phosphorylates OsMPK3 and OsMPK6 in vitro

To determine whether OsMKK6 directly phosphorylates OsMPK3 and OsMPK6 in vitro, kinase-deficient mutants of OsMPK3 and OsMPK6, OsMPK3m (D180A) and OsMPK6m (D211A) respectively, were created by site-directed mutagenesis. The mutations were introduced into the conserved DFG motif DFGXXXXXXXXXXXTEY (mutated amino acid underlined) within subdomain VII [24]. The recombinant mutant proteins were tested for autophosphorylation and kinase activities. As expected, OsMPK3m and OsMPK6m exhibited no autophosphorylation activity under conditions that supported wild-type MPK activity (Figure 2A). OsMPK3m and OsMPK6m were subsequently used as the substrates for one-step kinase assays to determine if OsMKK6 directly phosphorylates OsMPK3 and OsMPK6 in vitro. As shown in Figure 2(B), phosphorylation of OsMPK3m and OsMPK6m was manifested in the presence of OsMKK6, suggesting that OsMPK3 and OsMPK6 are targets of phosphorylation by OsMKK6 in vitro.

In vitro autophosphorylation and one-step kinase activity assays of OsMKK6, OsMPK3 and OsMPK6

Figure 2
In vitro autophosphorylation and one-step kinase activity assays of OsMKK6, OsMPK3 and OsMPK6

(A) Autophosphorylation assays for OsMKK6, OsMPK3 and OsMPK6. Purified recombinant GST–OsMPK3, GST–OsMPK3m, GST–OsMPK6, GST–MPK6m, OsMKK6 or GST was incubated at 30°C for 30 min, and autophosphorylation was detected by autoradiography after SDS/PAGE (15% gel). (B) In vitro one-step kinase activity assay of OsMKK6 against OsMPK3 and OsMPK6. Purified recombinant GST–OsMPK3m or GST–OsMPK6m was incubated with or without OsMKK6, and phosphorylation was detected by autoradiography after SDS/PAGE (15% gel).

Figure 2
In vitro autophosphorylation and one-step kinase activity assays of OsMKK6, OsMPK3 and OsMPK6

(A) Autophosphorylation assays for OsMKK6, OsMPK3 and OsMPK6. Purified recombinant GST–OsMPK3, GST–OsMPK3m, GST–OsMPK6, GST–MPK6m, OsMKK6 or GST was incubated at 30°C for 30 min, and autophosphorylation was detected by autoradiography after SDS/PAGE (15% gel). (B) In vitro one-step kinase activity assay of OsMKK6 against OsMPK3 and OsMPK6. Purified recombinant GST–OsMPK3m or GST–OsMPK6m was incubated with or without OsMKK6, and phosphorylation was detected by autoradiography after SDS/PAGE (15% gel).

To determine if OsMKK6 activates OsMPK3 and OsMPK6 in vitro, one-step and two-step kinase assays were performed using MBP as an artificial substrate [29]. As shown in Figure 3, OsMPK3, OsMPK6 and OsMKK6 alone each had low phosphorylation activity against MBP in the in vitro one-step kinase assays. However, in the two-step assays, MBP kinase activities were strongly enhanced when OsMPK3 or OsMPK6 was incubated with OsMKK6, indicating that OsMKK6 activates the MPK activities of OsMPK3 and OsMPK6. Taken together, these in vitro data suggest that OsMPK3 and OsMPK6 are the putative MPK signalling components for OsMKK6.

In vitro two-step MBP kinase assays of OsMKK6–OsMPK3 and OsMKK6–OsMPK6

Figure 3
In vitro two-step MBP kinase assays of OsMKK6–OsMPK3 and OsMKK6–OsMPK6

Purified recombinant GST–OsMPK3, GST–OsMPK6 or GST was first incubated individually or together with GST–OsMKK6 in the presence of unlabelled ATP at 30°C for 15 min. MBP and [γ-32P]ATP were then added to the reaction, and it was further incubated for 30 min at 30°C. Phosphorylation was detected by autoradiography after SDS/PAGE (15% gel).

Figure 3
In vitro two-step MBP kinase assays of OsMKK6–OsMPK3 and OsMKK6–OsMPK6

Purified recombinant GST–OsMPK3, GST–OsMPK6 or GST was first incubated individually or together with GST–OsMKK6 in the presence of unlabelled ATP at 30°C for 15 min. MBP and [γ-32P]ATP were then added to the reaction, and it was further incubated for 30 min at 30°C. Phosphorylation was detected by autoradiography after SDS/PAGE (15% gel).

OsMPK3 and OsMPK6 are activated by 12°C, but not 4°C in rice seedlings

In our previous report, OsMKK6 mRNA was found to accumulate under moderately low temperature (12°C) stress [21]. To confirm the function of OsMKK6 in moderately low temperature signalling in rice, activation of OsMPK3 and OsMPK6 in response to chilling stress was determined. Immunoprecipitation of OsMPK3 and OsMPK6 was performed from 12°C- or 4°C-treated rice seedlings using anti-OsMPK3 and anti-OsMPK6 antibodies respectively. MBP kinase activities of immunoprecipitated OsMPK3 and OsMPK6 showed similar changes in response to moderately low temperature stress. OsMPK3 and OsMPK6 MBP kinase activities were initially induced at 1 h, remained high at 3 h and then declined after 6 h to the basal level after 12 h of 12°C stress. By contrast, MBP kinase activities of immunoprecipitated OsMPK3 and OsMPK6 were not induced, but rather repressed, in response to the 4°C stress. These results indicate that OsMPK3 and OsMPK6 are specifically activated by moderately low temperature stress in rice seedlings (Figure 4).

Changes in MBP kinase activities of immunoprecipitated OsMPK3 and OsMPK6 after chilling stress in rice

Figure 4
Changes in MBP kinase activities of immunoprecipitated OsMPK3 and OsMPK6 after chilling stress in rice

Total soluble proteins were extracted from the shoots of 7-day-old rice seedlings exposed to 4°C or 12°C stress for the indicated periods and subsequently immunoprecipitated with anti-OsMPK3 or anti-OsMPK6 antibodies. The immunoprecipitated complexes were subjected to in vitro one-step kinase assays in the presence of MBP and [γ-32P]ATP.

Figure 4
Changes in MBP kinase activities of immunoprecipitated OsMPK3 and OsMPK6 after chilling stress in rice

Total soluble proteins were extracted from the shoots of 7-day-old rice seedlings exposed to 4°C or 12°C stress for the indicated periods and subsequently immunoprecipitated with anti-OsMPK3 or anti-OsMPK6 antibodies. The immunoprecipitated complexes were subjected to in vitro one-step kinase assays in the presence of MBP and [γ-32P]ATP.

A double-substitution mutant of OsMKK6, OsMKK6DD, constitutively activates the kinase activities of OsMPK3 and OsMPK6 in vitro

To obtain constitutively active forms of OsMKK6, OsMKK6D and OsMKK6DD were produced by introducing a substitution of Ser221 and an additional substitution of Thr227 by aspartate respectively. These substitutions were introduced within the phosphorylation motif (SXXXXXT), and may mimic phosphorylated serine/threonine residues [30]. Kinase activities of the wild-type and mutant OsMKK6 proteins were determined by in vitro one-step kinase assays using OsMPK3m as a substrate. OsMKK6DD showed a substantially higher kinase activity against MPK3m than did the wild-type OsMKK6. By contrast, OsMKK6D showed weaker kinase activity in vitro (Figure 5A).

In vitro MBP kinase activities of OsMKK6–OsMPK3 and OsMKK6–OsMPK6

Figure 5
In vitro MBP kinase activities of OsMKK6–OsMPK3 and OsMKK6–OsMPK6

(A) In vitro autophosphorylation and OsMPK3 kinase activity of different forms of OsMKK6. Purified recombinant GST–OsMKK6, GST–OsMKK6D, GST–OsMKK6DD or GST was incubated with OsMPK3m in kinase reaction buffer. (B) In vitro one-step and two-step MBP kinase assays of OsMKK6DD, OsMPK3 and OsMPK6. Purified recombinant GST–OsMPK3, GST–OsMPK6 and GST–OsMKK6DD were incubated individually or in combination in kinase reaction buffer including MBP and [γ-32P]ATP as described above.

Figure 5
In vitro MBP kinase activities of OsMKK6–OsMPK3 and OsMKK6–OsMPK6

(A) In vitro autophosphorylation and OsMPK3 kinase activity of different forms of OsMKK6. Purified recombinant GST–OsMKK6, GST–OsMKK6D, GST–OsMKK6DD or GST was incubated with OsMPK3m in kinase reaction buffer. (B) In vitro one-step and two-step MBP kinase assays of OsMKK6DD, OsMPK3 and OsMPK6. Purified recombinant GST–OsMPK3, GST–OsMPK6 and GST–OsMKK6DD were incubated individually or in combination in kinase reaction buffer including MBP and [γ-32P]ATP as described above.

To test if OsMKK6DD can hyperactivate the downstream targets OsMPK3 and OsMPK6, a two-step kinase assay was carried out. Compared with the MBP-phosphorylating activity of OsMPK3, OsMPK6 or OsMKK6DD alone, MBP was hyperphosphorylated by the combination of OsMKK6DD with OsMPK3 or OsMPK6 (Figure 5B). Similarly, a two-step in-gel kinase assay also revealed that only a single band, corresponding to the molecular mass of OsMPK3 (approximately 43 kDa) or OsMPK6 (approximately 45 kDa) respectively, phosphorylated MBP (results not shown). These results indicate that OsMKK6DD is constitutively active and readily phosphorylates the downstream MAPKs in vitro.

Overexpression of constitutively active OsMKK6 activates OsMPK3 and enhances chilling tolerance in rice

To characterize the possible functional interaction of OsMKK6 with OsMPK3 and OsMPK6 in vivo, transgenic plants overexpressing OsMKK6DD were created. In three independent transgenic lines, overexpression of OsMKK6DD was determined by RT–PCR. The three lines showed high levels of OsMKK6DD expression as compared with the wild-type and empty vector transgenic plants (Figure 6A).

Expression of OsMKK6 and activities of OsMPK3 and OsMPK6 in transgenic plants

Figure 6
Expression of OsMKK6 and activities of OsMPK3 and OsMPK6 in transgenic plants

(A) RT–PCR analysis of transgene expression in rice plants transformed with OsMKK6DD (three lines are shown: DD2, DD7 and DD8). Total RNA was extracted from 2-week-old rice seedlings and RT–PCR was carried out with specific primers. (B) MBP kinase assays of immunoprecipitated OsMPK3 and OsMPK6. Total soluble protein was extracted from 7-day-old rice seedlings of wild-type and OsMKK6DD-overexpressing transgenic plants. OsMPK3 and OsMPK6 were immunoprecipitated with anti-OsMPK3 and anti-OsMPK6 antibodies respectively. MBP kinase assays of the immunoprecipitated OsMPK3 and OsMPK6 were performed as described above. Vec, vector; Wt, wild-type.

Figure 6
Expression of OsMKK6 and activities of OsMPK3 and OsMPK6 in transgenic plants

(A) RT–PCR analysis of transgene expression in rice plants transformed with OsMKK6DD (three lines are shown: DD2, DD7 and DD8). Total RNA was extracted from 2-week-old rice seedlings and RT–PCR was carried out with specific primers. (B) MBP kinase assays of immunoprecipitated OsMPK3 and OsMPK6. Total soluble protein was extracted from 7-day-old rice seedlings of wild-type and OsMKK6DD-overexpressing transgenic plants. OsMPK3 and OsMPK6 were immunoprecipitated with anti-OsMPK3 and anti-OsMPK6 antibodies respectively. MBP kinase assays of the immunoprecipitated OsMPK3 and OsMPK6 were performed as described above. Vec, vector; Wt, wild-type.

The activities of OsMPK3 and OsMPK6 were determined in shoot tissues of transgenic plants using immunoprecipitation kinase assays with MBP as a substrate. In the three transgenic lines, constitutive hyperactivation of OsMPK3 was observed, indicating that OsMPK3 is an in vivo target of OsMKK6. By contrast, the activity of OsMPK6 in the transgenic lines was comparable with that of wild-type or the empty vector control line (Figure 6B).

The three transgenic lines overexpressing OsMKK6DD were then tested for cold-stress tolerance. These transgenic lines did not show significant differences in growth and development except that a slightly dwarf phenotype was observed in adult plants of one line (DD2 line; results not shown). As shown in Figure 7(A), the three transgenic lines overexpressing OsMKK6DD showed improved chilling tolerance compared with the wild-type plants. The transgenic plants developed new leaves often under these conditions, demonstrating increased vigour, whereas the wild-type plants displayed reduced vigour with many dead leaves. As a result, survival rates of the transgenic plants were higher than that of wild-type plants (Figure 7B).

Cold tolerance of transgenic plants overexpressing OsMKK6DD

Figure 7
Cold tolerance of transgenic plants overexpressing OsMKK6DD

Rice seedlings at the four-leaf stage were exposed to 4°C for 7 days under continuous illumination and subsequently allowed to recover at 25°C for 10 days. The survival rates of wild-type (WT) and transgenic seedlings were counted and compared. (A) Photograph of representative pots for each line. (B) Surviving plants were counted in four pots per line. Results are means±S.D. of the four pots.

Figure 7
Cold tolerance of transgenic plants overexpressing OsMKK6DD

Rice seedlings at the four-leaf stage were exposed to 4°C for 7 days under continuous illumination and subsequently allowed to recover at 25°C for 10 days. The survival rates of wild-type (WT) and transgenic seedlings were counted and compared. (A) Photograph of representative pots for each line. (B) Surviving plants were counted in four pots per line. Results are means±S.D. of the four pots.

DISCUSSION

OsMPK3 is a genuine target of OsMKK6

On the basis of previous work, we hypothesized the possible involvement of OsMKK6 in moderate chilling stress signalling. A pair-wise interaction search revealed that OsMKK6 interacts with OsMPK3 and OsMPK6, both of which belong to MAPK group A. Previously, a comprehensive interactor screen of 204 rice protein kinases supported the physical interaction of OsMKK6 with OsMPK6, although no data on the possible interaction with OsMPK3 was included [31]. Although an interaction between MKK6 and MPK4 was not detected in our system, the interaction between OsMKK6 and OsMPK3 was confirmed in vitro and in vivo. It is likely that additional interactions in this signalling pathway exist, which can be expected to be revealed by further detailed in vivo analyses in combination with in vitro studies.

Comparison with the group A MKK pathway in Arabidopsis

In rice, MAPKK group A contains two genes, OsMKK1 and OsMKK6 [11]. OsMKK1 transcription is characterized as being inducible by abiotic stresses such as salt, cold, heat and drought. [15]. Transcription of OsMKK6 is also induced by abiotic stress in rice [21]. However, functional characterization of group A MAPKKs in rice has not been explored. By contrast, MAPKK group A contains three genes in Arabidopsis, AtMKK1, AtMKK2 and AtMKK6. The Arabidopsis group A MAPKKs have been well characterized in combination with their MAPK counterparts. The transcripts of AtMKK1 and AtMKK2 are induced by salt and cold stresses [32]. The AtMKK1–AtMPK6 module is involved in an ABA (abscisic acid)-dependent signalling cascade leading to stress responses, including H2O2 production [33]. AtMKK2 phosphorylates and activates AtMPK4 and AtMPK6, and thus forms the AtMKK2–AtMPK4/AtMPK6 cascade that regulates the freezing tolerance [14]. It will be interesting to determine if OsMKK6 and AtMKK6 have an evolutionally conserved function in stress signalling. A phylogenetic study has shown that most groups of plant MKK and MPK have evolved before monocot and dicot separation and most closely related proteins within the groups are considered orthologous [11]. AtMKK6 is the most closely related Arabidopsis MKK to OsMKK6 within the group A MKKs. It has been demonstrated that AtMKK6 phosphorylates AtMPK3 and AtMPK6 in vitro [34]. However, in contrast with OsMKK6, AtMKK6 expression is not known to be induced by cold, salt or drought stresses according to the eFP Browser microarray database (http://bar.utoronto.ca/welcome.htm). Further in vivo detailed characterization of AtMKK6 activity is required in order to clarify whether OsMKK6 and AtMKK6 have evolved distinct functions.

Function of plant MKKDD in stress tolerance

Substitutions of serine/threonine in the activation loop with aspartic acid/glutamic acid in AtMKK1 (a MAPKK) have been suggested to mimic phosphorylation of serine/threonine [30]. Transgenic tobacco plants overexpressing NtMEK2DD display greatly enhanced disease resistance [34,35], and similar functions have been identified in other plant species; for example AtMKK2EE in cold and salt stresses in Arabidopsis [36] and StMEK1DD in blight disease resistance in potato [37]. We constructed a constitutively active form of OsMKK6, OsMKK6DD, by mutating the serine/threonine in the activation loop (S-XXXXX-T) to aspartic acid and found enhanced tolerance to cold stress in the resulting plants (Figures 5, 6 and 7). These data suggest that OsMKK6 is involved in chilling stress tolerance in rice. Although in some cases an inducible promoter is required to express activated MKKs due to negative or lethal effects of ectopic transgene expression, it was confirmed in the present study that transgenic rice plants constitutively expressing OsMKK6DD did not show such a negative growth phenotype.

OsMPK3 and OsMPK6 are involved in chilling stress signalling

In our previous study, OsMPK3 was first identified as a moderate chilling (12°C)-inducible gene [21]. The results of the present study further reveal that the kinase activity of OsMPK3 is inducible by moderately low temperature (12°C) stress. Induction of kinase activities upon cold stress has been reported for a few OsMPK3-related proteins, such as AtMPK3 in Arabidopsis [32] and SAMK (SAM domain-containing protein kinase) in alfalfa [38]. OsMPK3 and OsMPK6 have also been established as elicitor-activated MAPKs and are involved in disease resistance in rice [1820]. Although it is not fully known how various stress signals are distinguished within the plant cell, it is possible that different combinations of MAPKKs and MAPKs provide specificity to achieve appropriate responses. The present study suggests that the OsMKK6–OsMPK3 module is a component of the moderate chilling stress signalling that leads to chilling tolerance in rice.

Moderately low temperature activates distinct signals

Low temperature signalling mechanisms have been studied mostly in plants that show cold acclimation, such as Arabidopsis, wheat and alfalfa. These plants recognize severe chilling temperatures near 0°C and develop freezing tolerance for overwintering. By contrast, tropical and subtropical plants, such as maize and rice, are generally sensitive to chilling and do not develop freezing tolerance. Despite the lethal effects of severe chilling stress on rice, moderate chilling does not affect viability of the plants. However, moderate chilling has a great impact on pollen development and male sterility in rice [5]. The hypothesis that severe and moderately low temperatures elicit distinct molecular responses in rice is supported by the observation that several genes and proteins are differentially induced in response to these temperature ranges [21,39]. In the present study, we showed that activation of OsMPK3 and OsMPK6 occurs only when plants are treated with moderate chilling and not when they are subjected to severe chilling. Rice plants pre-treated with a moderately low temperature (12°C) for 1 day display enhanced tolerance against subsequent severe chilling stress (4°C) (H. Kato and R. Imai, unpublished work). These physiological studies suggest that moderate chilling temperatures can be perceived and cause the transmission of a distinctive stress signal in rice. Intriguingly, previous reports have revealed that a small temperature downshift (from 28 to 22°C) activates the transcription of several genes including COR15a (cold-regulated protein 15a) and ICE1 in Arabidopsis [40,41].

Possible model for moderate chilling stress signalling in rice

Our data suggests that the OsMKK6–OsMPK3 pathway is involved in moderate chilling stress signalling. The mechanism by which chilling temperatures activate this pathway is currently unknown. In maize, moderate chilling stress (14°C) leads to an immediate increase in H2O2 levels, which subsequently drop after 6 h [5]. We also observed a transient increase in H2O2 levels within 5 h in response to 12°C treatment in rice seedlings (H. Kato and R. Imai, unpublished work). Several plant MPKs, including AtMPK3 and AtMPK6, are activated by ROS (reactive oxygen species) [42,43]. Although direct evidence remains to be provided, it is possible that ROS may act as the first chemical signal that activates the OsMKK6–OsMPK3 pathway in response to moderate chilling (Figure 8). Our previous research showed that OsMPK3 and OsMPK6 are negatively regulated by OsTrx23, a thioredoxin h [44]. ROS may also regulate the OsMKK6–OsMPK3 pathway through redox control of OsTrx23. Whereas in vitro studies demonstrated that OsMKK6 activates both OsMPK3 and OsMPK6, activation of only OsMPK3 was observed in OsMKK6DD transgenic plants (Figure 6). One possible interpretation of this result is that there is some mechanism by which phosphorylation of OsMPK6 by OsMKK6 is inhibited in the transgenic plants. In this case, OsMPK6 should be activated by a MKK other than OsMKK6 in response to moderate chilling stress (Figure 8).

A model for moderate chilling stress signalling in rice

Figure 8
A model for moderate chilling stress signalling in rice

The schematic diagram summarizes our experimental results. Moderate chilling stress activates OsMKK6, which subsequently activates OsMPK3. OsMPK3 then phosphorylates (P) target proteins that are involved in severe chilling tolerance. OsMKK6 phosphorylates OsMPK6 in vitro, but this activity is blocked in vivo. OsMPK6 may be phosphorylated by an MKK other than OsMKK6 in response to moderate chilling stress. Moderate chilling stress also induces ROS accumulation, which causes reduced OsTrx23 (Trx23red), a negative regulator of OsMPK3 and OsMPK6, to become oxidized (Trx23ox). Trx, thioredoxin.

Figure 8
A model for moderate chilling stress signalling in rice

The schematic diagram summarizes our experimental results. Moderate chilling stress activates OsMKK6, which subsequently activates OsMPK3. OsMPK3 then phosphorylates (P) target proteins that are involved in severe chilling tolerance. OsMKK6 phosphorylates OsMPK6 in vitro, but this activity is blocked in vivo. OsMPK6 may be phosphorylated by an MKK other than OsMKK6 in response to moderate chilling stress. Moderate chilling stress also induces ROS accumulation, which causes reduced OsTrx23 (Trx23red), a negative regulator of OsMPK3 and OsMPK6, to become oxidized (Trx23ox). Trx, thioredoxin.

Abbreviations

     
  • At

    Arabidopsis

  •  
  • GST

    glutathione transferase

  •  
  • HIS

    histidine

  •  
  • LacZ

    β-galactosidase

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MAPKK

    MAPK kinase

  •  
  • MAPKKK

    MAPKK kinase

  •  
  • MBP

    myelin basic protein

  •  
  • Os

    Oryza sativa

  •  
  • ROS

    reactive oxygen species

  •  
  • RT

    reverse transcription

AUTHOR CONTRIBUTION

Guosheng Xie performed most of the experiments and wrote the paper. Hideki Kato participated in the kinase assay and the rice cold-tolerance testing. Ryozo Imai supervised the project and wrote the paper.

We thank Dr Shigemi Seo and Dr Yuko Ohashi of the National Institute of Agrobiological Sciences, Tsukuba, Japan for kindly providing the antibodies against OsMPK3 and OsMPK6. We would also like to thank Dr Derek Goto of Hokkaido University for critical reading of the paper prior to submission.

FUNDING

This work was supported, in part, by the JSPS (Japan Society of the Promotion of Science) [grant number 05F05689] and National Natural Science foundation of China [grant number 30871463]. G.X. was supported by a JSPS postdoctoral fellowship.

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Author notes

1

This paper is dedicated to the memory of Professor Ikuzo Uritani whose support and encouragement made this study possible.

Supplementary data