ABA regulates activation, transcription and translation of MPK3
Abiotic stresses or exogenous ABA application often activates MAP kinase cascade (Danquah et al. 2015). To further elucidate the crosstalk between MAP kinases and ABA signaling, we first analysed the activation of MAP kinase under increasing concentration of exogenous ABA application. The activation of two MAP kinases at 42 and 46 kDa molecular weight was observed (Fig. 1a). We then monitored the protein level of two MAP kinases corresponding to the 42 and 46kDa of AtMPK3 and AtMPK6,respectively (Fig. 1a). Interestingly, it was found that the protein level of AtMPK3 was upregulated by increasing concentrations of ABA as compared to AtMPK6.To confirm this observation, we performed the same experiment in mpk3 mutant and probed it with anti-AtMPK3 and anti-AtMPK6 antibodies (Fig. 1b). It was observed that AtMPK3 protein increased with time during exogenous ABA application. These results suggested that ABA signalling regulates the MAP kinase cascade through MAP kinase activation and also by increasing the protein content of AtMPK3. To check whether AtMPK3 is also regulated transcriptionally by ABA, we analysed the transcript abundance of AtMPK3 and AtMPK6 in plants under exogenous ABA application in time dependent manner. We found that the transcript of AtMPK3was significantly upregulated as compared to AtMPK6 by ABA (Fig. 1c). Taken together, these data indicate that MAP kinase signaling is regulated by ABA not only in the activation of MAP kinase protein activity but also at the transcriptional and translational levels.
We further explored the upstream MAP kinase kinase of AtMPK3 and AtMPK6 in the ABA pathway. For this purpose, two previously well-known interactors of AtMPK3 and AtMPK6, AtMKK4 and AtMKK5 were used. We found that activation of these two MAP kinases were compromised in the mkk4 and mkk5 mutants suggesting their role in the AtMPK3 and AtMPK6 activation (Fig. 1d). In contrast to lower activation of AtMPK3, the ABA significantly upregulated the protein content of AtMPK3 in mkk4 and mkk5 as compared to wild type (Fig. 1d). This result suggests the role of MKK4 and MKK5 in the activation of MAP kinases during ABA treatment but not in protein accumulation.
ABI5 interacts with MPK3 promoter and regulates its transcription
The specific higher upregulation of AtMPK3 transcript and not ofAtMPK6 suggested that AtMPK3 might be regulated by one of the ABA responsive transcription factor/s. Analysis of 1kb promoter sequence of AtMPK3 indicated the presence of six core ABA responsive DNA elements (ACGT) (Fig. 2a). However, no such elements were present in the promoter of AtMPK6 (SupplementaryFig.S1). The two closest core elements in AtMPK3 promoter were present at -269th and -287thposition while the other four at -377th, -382nd, -890th and -926th positions upstream to ATG. To find the putative ABA responsive TF that might regulate the AtMPK3 expression, we analyzed the transcript level of a well-characterised ABA responsive TF, ABI5. The expression of ABI5 was found to be upregulated by the ABA(Fig. 2b). On the basis of this observation, we hypothesized that ABI5 might bind to AtMPK3 promoter and regulate theAtMPK3 expression. To test the interaction of AtABI5 withAtMPK3 promoter, we performed the in-vitro DNA binding assay employing electrophoretic mobility shift assay (EMSA) (Fig. 2c). We used AtMPK3 promoter fragments (-256 to -402 bp) containing four ACGT elements and performed EMSA using HIS-ABI5 protein. We found that increasing ABI5 protein concentrations showed a gel shift and use of unlabelled probe diminished the complex formation, suggesting the specificity of DNA-protein interaction. This result suggests that ABI5 interacts with AtMPK3 promoter. Further, we checked the protein level of AtMPK3 in the wild type, mpk3 andabi5-8 mutants in the presence of ABA. We found that the level of AtMPK3 protein decreased in ABA treated samples in the abi5 mutant as compared to the wild type (Fig. 2d). Taken together, we can conclude that ABI5 regulates AtMPK3 expression in an ABA-dependent manner.
ABI5 shows molecular interaction with MPK3 and MPK6
The activation of two MAP kinases, AtMPK3 and AtMPK6 by ABA treatment suggests that MAP kinase cascade might regulate the post-translational modification of ABA responsive transcription factors. To test this possibility whether ABI5 is an interacting partner of AtMPK3, we first performed the yeast-two hybrid assay using ABI5 variants, full length (ABI5 FL), N-terminal deleted (ABI5C) and C-terminal deleted (ABI5N) (Fig. 3a). We found that AtMPK3 strongly interacted with AtABI5 full length and ABI5Nbut not with the ABI5C (Fig. 3b). AtABI5-AtMPK3 protein-protein interaction was further validated by in-vitro pulldown assay (Supplementary Fig. S2a). The GST-MPK3 and HIS-AtABI5 were expressed in bacteria, purified and pulled down using GST sepharose beads. The proteins were detected using anti-HIS and anti-AtMPK3 antibodies. The data clearly shows that AtMPK3 and AtABI5 interacts in-vitro. To get further insight into the interaction of two proteins in-vivo, we performed BiFC assay in tobacco leaves. The interaction between AtABI5 and AtMPK3 was observed within the nucleus (Fig. 3c).No fluorescence signal was observed in the AtMPK3 and AtABI5 when used independently. We also analysed the interaction between another ABA activated kinase, MPK6 with ABI5 in-planta and we found these proteins to be interacted within nucleus (Supplementary Fig. S2b). Thus,the data clearly suggest that AtABI5 interacts with AtMPK3/6in-planta and the interaction takes place in the nucleus.
ABI5 is phosphorylated by MPK3 at serine-314
The in-vivo interaction between AtABI5 and AtMPK3 led us to investigate the in-vitro phosphorylation of protein using bacterially purified AtABI5 and AtMPK3 proteins. We found that AtABI5 is strongly phosphorylated in-vitro by AtMPK3 which also showed auto-phosphorylation (Fig. 3d). In addition to AtMPK3, AtMPK6 which was found to be activated during ABA treatment also phosphorylated AtABI5 in-vitro (Supplementary Fig. S3). The phosphorylation of substrates by MAP kinases are mediated at serine/threonine residues followed by a characteristic proline amino acid (Singh and Sinha, 2015). Analysis of AtABI5 protein sequence showed a putative serine residue at 314th position (Supplementary Fig. S4a). Multiple protein sequence alignment from other plants indicated its evolutionary conservation (Supplementary Fig. S4b). We replaced this serine-314 to alanine, a non-phosphorytable amino acid (AtABI5S314A) (now onward termed as phospho-null)and again performed the in-vitro phosphorylation assay using AtMPK3. The phosphorylation of AtABI5S314A was completely abolished (Fig. 3e) suggesting that MAP kinase phosphorylates AtABI5 at serine-314 position which is evolutionarily conserved.
Phosphorylation of AtABI5 by AtMPK3 regulates its subcellular localization and dimerization
To further investigate the role of AtABI5 phosphorylation by MAP kinase in-vivo, along with phospho-null variant AtABI5S314A, we generated a phospho-mimetic variant AtABI5S314D(Serine (S) at 314 was changed to Aspartic acid (D)) and performed localization of both the variants in tobacco leaves. The localization of phospho-mimetic version was solely found in the nucleus (Fig. 4a). However, the phospho-null version was localized to the cytosol in addition to the nucleus (Fig. 4b). In the cytosol, AtABI5S314A was localized to the peripheral plasma membrane as detected by plasmolysis. Thus, it can be suggested that phosphorylation of AtABI5 by MAP kinase might regulate its subcellular localization. Next, we analyzed the dimerization of AtABI5 by BiFC assay using AtABI5 mutant variants. The wildtype AtABI5 formed a dimer within the nucleus (Fig. 4c). However, interaction between two phospho-null protein molecules of AtABI5 was seen inboth the cytoplasm and the nucleus (Fig. 4d) similar to that of its subcellular localization. Surprisingly, the interaction was abolished between the two phospho-mimetic protein molecules of AtABI5 (Fig. 4e). Additionally, no interaction was observed between phospho-null and phospho-mimetic AtABI5 variants (Fig. 4f). No fluorescence was observed in the case of negative controls (Supplementary Fig. S5a). We also checked the protein expression in these samples from infiltrated N. benthamiana leaves. The expression of all the and no significant difference in the expression was observed (Supplementary Fig. S5b). Taken together, phosphorylation of AtABI5 not only regulates its subcellular localization but also inhibits its nuclear dimerization.
MAP kinase mutants are hyposensitive to exogenous ABA during seed germination
ABA signaling regulates the seed germination and post-germination growth through ABI5. Mutation in the AtABI5 leads to ABA insensitive phenotype during seed germination (Lopez-Molina et al. 2001). To examine the role of AtMPK3 and ABA crosstalk during seed germination, we performed ABA sensitivity assay during seed germination in abi5-8 and mpk3 mutants along with the wild type Col-0 (Fig. 5). As expected, we found that wild type seeds were sensitive while abi5-8seeds were insensitive to exogenous ABA application (Supplementary Fig. S6). Interestingly, mpk3 mutants exhibited hyposensitive phenotype to ABA during seed germination as compared to wild type (Fig. 5a, 5b and 5c). At higher concentrations, the post-germination growth of mpk3 was arrested as compared to insensitive abi5-8 mutant. These results suggest that AtMPK3 participates in the ABA mediated inhibition of seed germination, however, hyposensitive phenotype of mpk3 mutant might be due to the other redundant MAP kinase such as AtMPK6 which is also activated by ABA and phosphorylates AtABI5.
Phosphorylation at serine-314 negatively regulates ABA response during seed germination
To further explore the role of AtABI5 phosphorylation in-vivo, we complemented the abi5-8 mutant by constitutive expression of phospho-null (AtABI5S314A) and phospho-mimetic(AtABI5S314D) forms of AtABI5. Semiquantitative gene expression analysis of ABI5was performed in these transgenic lines to ensure the equal expression of different mutant versions of ABI5 (Supplementary Fig. S7a). ABA sensitivity assay was performed using these lines (Fig.6). Interestingly, the phospho-null (AtABI5S314A) variant could complement the abi5-8 mutant as it showed sensitivity towards exogenous ABA application (Fig. 6a). However, the phospho-mimetic AtABI5 transgenic plants showed insensitive phenotype and was unable to complement the abi5-8 mutants. These results suggest that phosphorylation of AtABI5 makes it inactive and thus the phospho-mimetic AtABI5 transgenic plants phenotypically mimics the abi5-8 mutant during seed germination.
Overexpression of phospho-null ABI5 confers hypersensitivity to drought stress
ABA is known to play an important role during water deficient conditions or during drought stress (Lu et al. 2002). We therefore, were interested to study the effect of ABI5 phosphorylation by MPK3 during drought stress. To carry out this study, we first analyzed the role of MAP kinase pathway mutants during drought stress. We exposed WT, mpk3, mkk4 and mkk5 plants to water stress condition by withdrawing water for 14 days, then re-watered the plants for one week and assessed the recovery from drought stress. We found that during drought conditions all plants showed chlorosis and leaf damage (Fig. 6b). However, MAP kinase mutants, mpk3, mkk4 and mkk5 showed better tolerance to drought. Even the recovery was faster in MAPK mutants. These results indicate that MAP kinase signaling plays a crucial role during drought stress.
Similarly, we further extended our investigation using the abi5-8 transgenic plants constitutively overexpressing the phospho-null (AtABI5S314A) and phospho-mimetic(AtABI5S314D) forms of AtABI5 during drought stress. We found that abi5-8 mutant and phospho-mimetic AtABI5S314D complemented plants showed insensitive phenotype (Fig. 6c). Interestingly, when the mutant is complemented with phospho-null AtABI5S314A, the hypersensitive phenotype to drought was unable to recover during recovery. Taken together, these results clearly suggest that MKK4/MKK5-MPK3-ABI5 module regulates the drought stress response in Arabidopsis.
The reversible phosphorylation of ABI5 is crucial for flowering transition
AtABI5 is one of the master regulators of floral transition and reproductive growth. The abi5 mutants exhibit early flowering phenotype (Wang et al. 2013). To further characterize the role of ABI5 phosphorylation in the flowering response, the WT, abi5-8, and abi5-8 mutant lines complemented with AtABI5S314A and AtABI5S314D variants were grown up till flowering (Fig. 6d). We found that overexpression of phospho-null AtABI5S314A delayed the flowering as compared to abi5-8 mutant (Supplementary Fig. S7b). However, interestingly it was observed that phospho-mimetic variant AtABI5S314Dshowed intermediate flowering phenotype to that of abi5-8 and wild type but earlier than that of phospho-null (Fig. 6d). The observation indicates that the phosphorylation of AtABI5 by AtMPK3 has a role in regulating complex trait like flowering in A. thaliana.