A novel positive feedback mechanism of ABI5 phosphorylation by MPK3 regulates ABA signaling in Arabidopsis

doi:10.21203/rs.3.rs-1335024/v1

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A novel positive feedback mechanism of ABI5 phosphorylation by MPK3 regulates ABA signaling in Arabidopsis

https://doi.org/10.21203/rs.3.rs-1335024/v1

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Seed germination is a crucial physiological process regulated by both environmental and endogenous phytohormones. ABA negatively regulates seed germination, post-germination growth and floral transition, however, the cross talks between multiple regulatory pathways involved in seed germination are still unclear. Here, we show that ABA activates two MAP kinases, AtMPK3/AtMPK6 and selectively regulates the transcript of AtMPK3 through ABI5, a master regulator of ABA signaling in Arabidopsis thaliana. As a feedback loop, AtMPK3 interacts and phosphorylates ABI5 at the serine-314 position.ABI5 phosphorylation by MAP kinase positively regulates ABI5 nuclear localization and negatively regulates its dimerization. Further, subcellular localization of ABI5 phospho-null protein suggests the role of phosphorylation in regulation of its cytoplasmic stability and its nuclear dimerization. Overexpression of phospho-null ABI5 in abi5-8 mutant restored the ABA sensitivity during seed germination and delayed the floral transition as compared to phospho-mimeticABI5. Additionally, overexpression of constitutive phosphorylated form of ABI5 in abi5-8 mutant suggests that phosphorylation makes ABI5 partially inactive. Furthermore, phospho-null ABI5 plants showed drought sensitive phenotype whereas, mpk3, mkk4,mkk5, abi5-8 and phospho-mimetic ABI5 plants showed drought tolerant phenotype. Our findings present a new insight between MAP kinase cascade and ABA signaling which collectively regulates the ABA response through ABI5 phosphorylation.

ABA

AtABI5

MAP kinase

AtMPK3 and AtMPK6

This study shows the involvement of AtMPK3 in regulating the function of bZIP transcription factor, ABI5 in ABA signaling. ABI5 in turn regulates AtMPK3 in a positive feedback loop mechanism.

During various developmental phases and abiotic stress, plants employ a conserved abscisic acid (ABA) signaling which integrates multiple complex pathways that are regulated at both the transcriptional and post-translational levels(Nakashima et al. 2009). A well-studied ABA pathway in Arabidopsisinvolves an ABA receptor and a core ABA signaling kinase-phosphatase modules(Fujii et al. 2007). Activated kinase phosphorylates ABA-responsive transcription factors (TFs) such as ABF and a bZIP transcription factor, Abscisic acid-insensitive 5 (ABI5)(Nakamura et al. 2001; Nakashima et al. 2009; Dai et al. 2013). ABA signaling regulates the plant growth and development from seed germination to the floral transition and also integrates the stress responses via cross talk with multiple signaling and hormone pathways(Piskurewicz et al. 2008; Wang et al. 2013).

ABI5 is a master regulator of ABA signaling and knockout of ABI5 results in ABA-insensitive phenotype which was used to characterize a number of new factors in ABA metabolism and signaling(Lopez-Molina and Chua, 2000; Lopez-Molina et al. 2002). Presence of ABA strictly regulates the expression of ABI5 at transcriptional level and also the function of ABI5 at post-translational stages by covalent modifications(Miura et al. 2009; Nakashima et al. 2009; Liu and Stone, 2010, 2013). Transgenic plantsoverexpressing ABI5 are hypersensitive to ABA, however they grow normally in the absence of ABA(Lopez-Molina et al. 2001). Further, this report suggests that ABA induced post-translational modifications (PTMs) are crucial for ABA–induced regulations. Additionally, floral transition occurs early in the abi5 mutants and its overexpression delays flowering(Wang et al. 2013). Thus, ABA exerts the inhibitory effect on floral transition through ABI5 which upregulates the expression of FLOWERING LOCUS C (AtFLC) which in turn inhibits the flowering(Wang et al. 2013). However, floral transition is also regulated by other factors such as ABI4, independent of ABI5(Shu et al. 2018). Therefore, floral transition is a complex mechanism governed by ABA.

Mitogen-activated protein kinase (MAPK) pathway is an evolutionarily conserved signaling cascade which regulates several developmental and stress responses(Jalmi et al. 2018). During abiotic or drought stress, the ABA content is significantly enhanced and is known to activate several MAP kinases(Matsuoka et al. 2015; Li et al. 2017). Role ofMAPK cascades involved in plant growth and development as well as in stress responses have been elucidated (Xu and Zhang, 2015; Jalmi et al. 2018; Verma et al. 2019). The link between ABA and MAP kinase cascade is specifically well explored in primary root growth, stomatal responses and leaf senescence (Danquah et al. 2015; Khokon et al. 2015; Matsuoka et al. 2015; Li et al. 2017). In the guard cells, ROS activates MPK9 and MPK12 which regulate ABA response positively (Jammes et al. 2009). It has been reported that during ABA signaling, MAP kinase signaling transduces the signal to the transcriptional apparatus(Lu et al. 2002). Though, significant contributions have been made in understanding the role of MAP kinase cascade and ABA signaling during stomatal response which is usually a late growth response while the information in early developmental stages involving seed germination and post germination growth is still missing. The underlying mechanism for this crosstalk at molecular level needs further investigation.

The present work reports a novel regulation of AtABI5 by AtMPK3 and its role in seed germination, flowering and drought response. We identify the activation of two MAP kinases, AtMPK3 and AtMPK6 during exogenous application of ABA. Interestingly, ABA upregulates the expression of AtMPK3 transcriptionally through AtABI5 which binds to the ABA-responsive elements present selectively in AtMPK3promoter. Additionally, the ABA activated AtMPK3 interacts with AtABI5 in the nucleus and phosphorylates it at an evolutionarily conserved serine-314 residue. Phosphorylation of ABI5 regulates its subcellular localization and inhibits its dimerization. In addition, we further show that only phospho-null ABI5 could restore the ABA sensitivity when overexpressed in abi5-8 mutant background. However, both phospho-null and phospho-mimetic ABI5 transgenic plants exhibited delayed flowering but this phenotype was more pronounced in phospho-null transgenic plants. Drought stress analysis of MAP kinase mutants and ABI5 transgenic plants suggested that except phospho-null ABI5, all other mutants showed drought tolerance phenotype and better recovery.

Plant materials and growth conditions

The Arabidopsis thaliana ecotype Columbia (Col-0) and mutant seeds were grown as described in Verma et. al., 2019. The MS plates were supplemented with indicated concentrations of ABA (Sigma) for seed germination and ABA sensitivity assay. For production of overexpression transgenic plants, the coding sequences of ABI5 and its mutant variants (ABI5^S314A and ABI5^S314D) were cloned in pGWB5 binary vector using Gateway cloning method and transformed into Agrobacterium tumefaciens GV3101 strain. Positive constructs were introduced into abi5-8 mutant plants by floral-dip method. The subsequent seeds were screened on ½ MS plates supplemented with kanamycin and hygromycin. T3 generation seeds were used for further experiments.

Plant protein extraction and Immunoblotting

Arabidopsis seedlings were ground into fine powder using liquid nitrogen. Total proteins were extracted using extraction buffer (50 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10 mM Na₃VO₄, 10 mM NaF, 50 mM β-glycerolphosphate, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail, and 10% glycerol). Proteins were denatured by boiling with a 5X sodium dodecyl sulfate (SDS) loading buffer for 3 minutes. Immunoblotting was performed with phospho-ERK1/2 (pERK1/2) antibody (Cell signaling), anti-AtMPK3 (Sigma) and anti-AtMPK6 (Sigma) as described earlier (Singh and Sinha, 2016).

ABA sensitivity assay

WT and mutant seeds of mpk3 and abi5 were plated on ½ MS agar plate either without ABA (control) or with ABA (1µM, 2 µM, 3 µM and 5 µM). After vernalisation for 2 to 3 days, plates were transferred to constant light for germination. Germinated seeds were captured using a Nikon stereo microscope. More than 80 seeds of each genotype were taken and the assay was repeated at least three times and each time similar phenotype was observed.

RNA isolation and quantitative real time PCR (qRT-PCR) analysis

Total RNA was extracted from control and treated samples using Trizol reagent sigma (#T9424). The cDNA was prepared using RevertAid H Minus First Strand cDNA synthesis kit (#K1632, Thermo Scientific) and qRT-PCR were performed as described previously (Singh and Sinha, 2016). The Actin gene was used as internal reference to normalise the expression of target gene. The primer sets used in the analysis are given in the Supplementary Table S1.

Yeast-two hybrid assay

The interaction between MPK3 and ABI5 was checked using the Matchmaker yeast two-hybrid system (BD Biosciences) as described previously with slight modifications (Sheikh et al. 2013; Raghuram et al. 2015). Briefly, the N-/C-terminal deletion construct of ABI5 along with its full length CDS and full length MPK3 were cloned in the pGADT7 and pGBKT7 vectors. The yeast transformation was performed according to the Fast yeast transformation kit (Gbioscience: GZ‐1).

In-vitro GST-pulldown assay

For in-vitro protein-protein interaction study, bacterially purified GST-AtMPK3 and His-AtABI5 were incubated in the binding buffer (50 mM Tris-HCl, pH 7.5, 150 mMNaCl, 0.2% (v/v) glycerol, 0.1% (v/v) Triton X-100, 1 mM EDTA, pH 8.0, 1 mM PMSF, 0.1% (v/v) Nonidet P-40) over night at 4°C. After washing with 1x PBS for three-four times, the bound proteins were separated on 10% SDS-PAGE. Interacting proteins were detected by immunoblot with anti-his and anti-AtMPK3 antibodies.

Bimolecular fluorescence complementation (BiFC) assay and sub-cellular localization

For BiFC assay, MPK3 and ABI5 (along with mutant ABI5^S314A and ABI5^S314D) were cloned in frame with binary vectors pSPYCE M and pSPYNE 173 which contains C- terminal half (cYFP) and n-terminal half (nYFP) of yellow fluorescence protein (YFP), respectively. For localization studies, the ABI5 WT and mutant variants (ABI5^S314Aand ABI5^S314D) were cloned in pGWB5 which has green fluorescence protein (GFP) at its amino- terminal. Nicotiana benthamiana leaves were infiltrated and fluorescence were monitored as described previously (Raghuram et al. 2015).

Bacterial protein expression and in-vitro phosphorylation assay

MPK3 and ABI5 CDS were cloned in frame with GST (pGEX4t2) and 6xHis (pET28a) tags, respectively. The proteins were induced in BL21 strain of E. coli by addition of 1mM IPTG at 28°C. The recombinant proteins tagged with GST and 6xHis were purified by affinity chromatography using GST-sepharose beads and Ni-NTA beads, respectively. The total proteins were quantified by Bradford reagent (Sigma) and purity was analysed by SDS-PAGE. The in-vitro phosphorylation of ABI5 was performed as described previously (Raghuram et al. 2015).

Multiple protein sequence alignment and Site - directed mutagenesis (SDM) analysis

The multiple protein sequences were aligned from different plant species with Arabidopsis thaliana ABI5 and the conserved amino acids were analysed using the Uniprot database (https://www.uniprot.org/). The putative and invariant amino acids were marked and indicated. The conserved invariant and single putative MAP kinase site, S-314 was used for SDM analysis. The list of primers used is mentioned in Supplementary Table S1, primers list. The ABI5-pET28a and ABI5- pENTR vectors were used as templates for PCR based SDM. The PCR reaction was digested with DpnI restriction enzyme overnight and the resultant reaction was transformed in E. coli DH5α competent cells. The mutation was confirmed by DNA sequencing.

Electrophoretic mobility shift assay (EMSA)

The DNA-protein interaction between AtMPK3 promoter and ABI5 transcription factor was performed by EMSA. The DNA sequence containing the ACGT core element (-402 to -256 base pair) were PCR amplified, radiolabelled and used for the binding experiment with bacterially purified ABI5-His proteins as described previously (Singh and Sinha, 2016). Briefly, radiolabelled probes were either incubated alone or with increasing protein concentration of His-ABI5 in the reaction buffer (20mM HEPES, pH 7.4; 10mM KCl; 0.5mM EDTA; 0.5mM DTT; 1mM MgCl₂; 3% Glycerol and 1 μg of poly(dI-dC) at room temperature for 30 minutes. The DNA-protein complex was separated on 4% PAGE using 0.5x TBE running buffer and auto-radiographed using typhoon.

Flowering phenotype analysis

The flowering phenotype was analysed according to the previous study(Wang et al. 2013; Shu et al. 2018). Seeds were directly sown on the potted soil and flowering phenotypes were monitored till bolting. Images were taken every day. For phenotyping, at least three independent replicates were performed with at least 12 pots of each genotype.

Drought stress analysis

The drought stress response was conducted using mpk3, mkk4, mkk5, abi5-8, ABI5^S314A::abi5-8 and ABI5^S314D::abi5-8 transgenic plants according to previously described (Harb et al. 2010; Li et al. 2017). The soil grown 25 to 30 days old plants of each genotype in at least 12 pots, were subjected to drought stress conditions without watering for 14 days until the plants showed drying and leaf chlorosis phenotype. To examine the recovery response, plants were re-watered for one week and successively recovery was monitored every day. The plants which were unable to recover after 7 days, considered as sensitive and those recovered with green leaves, termed as tolerant. The experiment was repeated three times and similar phenotype was reported.

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 -269^th and -287^thposition while the other four at -377^th, -382^nd, -890^th and -926^th 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 314^th 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 (AtABI5^S314A) (now onward termed as phospho-null)and again performed the in-vitro phosphorylation assay using AtMPK3. The phosphorylation of AtABI5^S314A 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 AtABI5^S314A,we generated a phospho-mimetic variant AtABI5^S314D(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, AtABI5^S314A 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 (AtABI5^S314A) and phospho-mimetic(AtABI5^S314D) 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 (AtABI5^S314A) 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 (AtABI5^S314A) and phospho-mimetic(AtABI5^S314D) forms of AtABI5 during drought stress. We found that abi5-8 mutant and phospho-mimetic AtABI5^S314D complemented plants showed insensitive phenotype (Fig. 6c). Interestingly, when the mutant is complemented with phospho-null AtABI5^S314A, 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 AtABI5^S314A and AtABI5^S314D variants were grown up till flowering (Fig. 6d). We found that overexpression of phospho-null AtABI5^S314A delayed the flowering as compared to abi5-8 mutant (Supplementary Fig. S7b). However, interestingly it was observed that phospho-mimetic variant AtABI5^S314Dshowed 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.

ABI5, a bZIP type transcription factor is one of the master regulators which has been well characterized with respect to ABA signaling and stress responses. It has been shown that ABA signaling not only regulates the seed germination but also participates in the floral transition and drought stress where ABI5 is one of the ABA responsive transcription factors (Wang et al. 2013). Phosphorylation of ABI5 by SnRK2 kinases positively regulates the ABA signalling (Nakashima et al. 2009). However, involvement of other unknown kinase in the regulation of this bZIP transcription factor was not well characterized so far. In this study, we report the role of ABA activated MAP kinases, AtMPK3/AtMPK6 in the phospho-regulation of ABI5 during plant development and stress response.

MAP kinase pathways are one of the evolutionary conserved signaling cascades which regulate several developmental and stress responses. MAP kinase cascades are reported to be involved in several ABA signaling including stomatal regulation and stress responses (Wang et al. 2007; Danquah et al. 2014; de Zelicourt et al. 2016). However, there are a very few reports where a direct target of ABA activated MAP kinase has been identified and functionally characterized (Lu et al. 2002). It has been previously shown that an AP2/ERF class transcription factor, ABI4 is phosphorylated by AtMPK3/AtMPK6 and regulates the retrograde signaling in Arabidopsis and is the only well characterized ABA-insensitive transcription factor till now (Guo et al. 2016). In this study, we have identified that ABI5 is an interactor and phosphorylation target of AtMPK3/AtMPK6 by several biochemical and molecular approaches. We found that ABA not only upregulates the AtMPK3 and AtMPK6 kinase activity but selectively induces the transcription and translation of AtMPK3 via AtABI5 transcription factor. In turn, both ABA activated MAP kinases interact and phosphorylateABI5.

The promoter analysis of both ABA activated MAP kinases, AtMPK3 and AtMPK6 suggested that only AtMPK3 has the core ABA responsive element (ACGT) while it is absent in AtMPK6 promoter. These observations were further corroborated with the transcript analysis of both kinases where only AtMPK3 showed higher transcript level under ABA treatment. As already reported, we also found that under ABA treatment, the expression of AtABI5 was upregulated (Liu and Stone, 2010). To find the role of AtABI5 in the transcriptional regulation of AtMPK3, we performed the EMSA and AtABI5 was found to interact with AtMPK3 promoter. Further, immunoblot analysis of AtMPK3 protein in abi5-8 mutant clearly indicated lower accumulation of AtMPK3protein under ABA treatment. However, the involvement of other ABA-responsive TFs along with AtABI5 where AtMPK3 protein was reduced as compared to wild type but not completely abolished under ABA treatment, cannot be ruled out. The feedback regulation where the TF regulating the expression of MAPKs were also the targets of respective MAPKs, have been reported in several signaling pathways (Sethi et al. 2014; Singh and Sinha, 2016). In the similar line, we found thatAtABI5 along with transcriptionally upregulating AtMPK3 also shows protein-protein interaction with it. The in-vivo interaction was observed in the nucleus where AtABI5 is known to regulate the transcripts of ABA-responsive genes(Liu and Stone, 2013). The AtABI5 protein has been shown to undergo multiple post-translational modifications including phosphorylation by SnRKs and BIN2 including ubiquitination/SUMOylation (SupplementaryFig.S8) (Miura et al. 2009; Nakashima et al. 2009; Liu and Stone, 2010, 2014). The in-vitro phosphorylation assay and site-directed mutagenesis conferred that AtABI5 is phosphorylated by AtMPK3 also and that this phosphorylation occurs at the evolutionarily conserved serine residue present at 314th position. Additionally, we found that other ABA-activated AtMPK6 which shows functional redundancy with AtMPK3, can phosphorylate AtABI5. Several reports suggest that protein activity and subcellular localization of most proteins and AtABI5 are regulated by post translational modifications (Liu and Stone, 2010, 2013; Guo et al. 2016). In our study, subcellular localization analysis using phospho-null and phospho-mimetic variant, AtABI5^S314A and AtABI5^S314D respectively showed a contrasting localization pattern. The phospho-mimetic AtABI5^S314D was exclusively localized to the nucleus, as the wild type AtABI5 reported earlier (Liu and Stone, 2013). However, the phospho-null AtABI5 was found to be localized in the cytoplasm also along with the nucleus. Thus phosphorylation of AtABI5 by MAP kinase at Serine-314 may regulate its subcellular localization and possibly its cytoplasmic stability. In an earlier study, mutation in the putative ubiquitination site in AtABI5 or use of MG132 inhibitor of proteasome significantly enhanced the level of the wild type protein in the cytoplasm (Liu and Stone, 2010, 2013), which is in corroboration with our observation of ABI5^S314A localization. Next, we also found that AtABI5 phosphorylation inhibits the homodimer formation in the nucleus (Nakamura et al. 2001). Therefore, it seems like both phosphorylation and dephosphorylation of AtABI5 is essential for its subcellular activity, which is also been reported in an earlier study by Dai et al. (2013).

ABI5 is a bZIP transcription factor which homodimerizes with its leucine zipper in bZIP domain and we found that Serine-314 residue is nearby bZIP domain and therefore reversible phosphorylation at this site may interfere with dimerization as observed in Fig. 4E and 4F. Furthermore, the serine-314 is also in the proximity of lysine-344 which is ubiquitinated and regulates its turnover in the cytoplasm (Liu and Stone, 2013). Their study shows that mutation at these sites significantly enhances the ABI5 protein localization in the cytoplasm, similarly as our phospho-null ABI5 variant protein. Therefore, it can be possible that reversible phosphorylation is crucial for ABI5 turnover by the interplay between phosphorylation and ubiquitination which further regulates the dimerization of ABI5 and downstream signaling.

Next we functionally characterized the role of AtABI5 phosphorylation by MAP kinases in-vivo. We observed that overexpression of phospho-null AtABI5^S314A in abi5-8 mutant background restored the ABA sensitivity during seed germination under ABA treatment. Meanwhile, overexpression of phospho-mimetic AtABI5^S314D failed to rescue the phenotype and exhibited the phenotype of abi5-8 mutant. After functionally characterising the role of phosphorylation during seed germination, we investigated the developmental role of AtABI5 during floral transition. Previously, it has been shown that ABA delays the floral transition through AtABI5 which binds to the promoter of FLOWERING LOCUS C (FLC) (Wang et al. 2013). This study reported that abi5 mutants showed early flowering and overexpression of ABI5significantly delayed the same. Our finding suggested that both mutant versions of ABI5 delayed the flowering but phospho-null AtABI5 showed severe delay as compared to phospho-mimetic form which showed intermediate flowering phenotype. Thus, we concluded that reversible phosphorylation of AtABI5 by AtMPK3 is crucial for floral transition.

ABA signaling is also activated during water deficient condition or drought stress and MAP kinase cascade is known to regulate the stress response. Thus we further investigated the role of MAP kinase-ABI5 module during drought stress and recovery. Our data suggests that mpk3, mkk4 and mkk5 showed drought stress tolerance. We further found that abi5-8 mutant and phospho-mimetic AtABI5^S314D transgenic plants also showed drought insensitive phenotype similar to that of MAP kinase pathway mutants. However, phospho-null AtABI5^S314Atransgenic plants were found to be hypersensitive to drought. This data suggests that an interplay of MAP kinase and AtABI5 regulates the drought stress response in an ABA-dependent manner.

In summary, we can conclude that ABA regulates the activity of AtMPK3/AtMPK6 downstream of AtMKK4/AtMKK5. Moreover, AtMPK3 expression is also subjected to transcriptional regulation downstream to AtABI5. The activated AtMPK3 and AtMPK6, in-turn phosphorylate AtABI5 at serine-314 position which regulates its subcellular localization and dimerization. AtABI5 in turn interacts with AtMPK3 promoter in a positive feedback mechanism. This reversible phosphorylation functionally regulates the AtABI5 during seed germination, floral transition and drought stress in Arabidopsis. A simple working model of the involvement of MAPK in ABI5 function is depicted in Fig. 7. It is likely that MAP kinase cascade and ABA signaling crosstalk at multiple developmental pathways and during stress responses. This study provides an extensive effort towards understanding this molecular mechanism.

Acknowledgements

This work was supported by core grants from the National Institute of Plant Genome Research from the Department of Biotechnology (DBT), Government of India. PKB and DV are recipients of fellowship from the DBT and University Grant Commission (UGC) Government of India, respectively. NV is recipient of fellowship from DBT-BioCare Women Scientist scheme. AKS thanks TATA Innovation fellowship from Department of Biotechnology and Sir J.C. Bose fellowship from Science and Engineering Research Board, Government of India. Authors thank the Radioisotope facility, Confocal Microscopy Facility and the Central Instrumentation Facility of NIPGR, New Delhi, India. The authors are thankful to DBT-eLibrary Consortium (DeLCON) for providing access to e-resources.

Author contributions

PKB and AKS designed the experiments and overall study. PKB performed all the extensive experimental work with the help of DV and NV. PKB wrote the first draft of the manuscript. All participated in the data preparation and AKS participated in the discussion and progress of work. Final draft was approved by AKS.

Conflict of interest

The authors declare no conflict of interest.

Accession Numbers

The nucleotide and protein sequences used in this article can be found in the TAIR database under following accession numbers: AT3G45640 (MPK3), AT2G43790 (MPK6), AT2G36270 (ABI5), AT1G51660 (MKK4), AT3G21220 (MKK5), AT3G18780 (ACTIN2). The mpk3 (SALK_151594), mpk6 (Salk_073907) was used from our previous study and the following abi5-8 line (SALK_013163), mkk4 (SALK_058307), mkk5-1 (SALK_047797) were obtained from the Arabidopsis Biological Resource Center.

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A novel positive feedback mechanism of ABI5 phosphorylation by MPK3 regulates ABA signaling in Arabidopsis

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