In the Arabidopsis genome, approximately 80 MAP3Ks (mitogen-activated protein kinase kinase kinases) have been identified. However, only a few of them have been characterized, and the functions of most MAP3Ks are largely unknown. In this paper, we report the function of MAP3K16 and several other MAP3Ks, MAP3K14/15/17/18, whose expression is salt-inducible. We prepared
The plant hormone abscisic acid (ABA) plays important roles in plant growth and development (Finkelstein, 2013). Plants are exposed to various adverse environmental conditions such as drought, high salinity, and extreme temperatures during their life cycle. Under these conditions, ABA level increases, and ABA mediates protective responses to the abiotic stresses (Nambara and Marion-Poll, 2005). For instance, ABA minimizes water loss through transpiration by promoting stomatal closure and, at the same time, inhibiting stomatal opening under water deficit condintion (Kim et al., 2010). It also controls the synthesis of compatible osmolytes and coordinates other responses to protect cells from the damage caused by various stresses (Xiong et al., 2002). Additionally, ABA functions, in general, as a negative regulator of plant growth under the stress conditions (Finkelstein, 2013). It inhibits seed germinaton, seedling establishment and the onset of postgermination growth, and subsequent seedling growth. Cellular ABA level is high also in developing seeds, and it plays a key role in the synthesis of storage components and establishing seed dormancy during seed maturation (Holdsworth et al., 2008). Most of the cellular processes controlled by ABA entail changes in gene expression, and numerous genes are known to be regulated by ABA (Fujita et al., 2011).
The molecular components comprising the ABA response pathway have been identified by various genetic and biochemical studies, and the core signaling pathway leading to the ABA-dependent gene expression has been unraveled (Cutler et al., 2010; Yoshida et al., 2015). ABA is perceived by PYR/PYL/RCAR family receptor proteins (Ma et al., 2009; Park et al., 2009). The ABA-bound receptor proteins interact with the members of the clade A subfamily of PP2Cs (e.g, ABI1, ABI2, and HAB1). The PP2Cs are negative regulators of ABA signaling and, in the absence of ABA, inactivate a subset of SnRK2s (e.g., OST1/SnRK2.6/SnRK2E, and SnRK2.2/SnRK2D, and SnRK2.3/SnRK2I) (Fujii and Zhu, 2009; Fujita et al., 2009). The receptor binding to the PP2Cs inhibits the phosphatase activity of PP2Cs, resulting in the activation of the SnRK2s. The activated SnRK2s phosphorylate the ABF/AREB/ABI5 family of bZIP class transcription factors that regulate ABA-responsive gene expression. Thus, the core ABA signaling pathway that leads to the ABA-regulated gene expression consists of PYR/PYL/RCAR-PP2Cs-SnRK2s-ABFs/AREBs/ABI5 (Fujii et al., 2009).
Mitogen-activated protein kinase (MAPK) signaling cascades play diverse roles in eukaryotic cell signaling (Colcombet and Hirt, 2008; Rodriguez et al., 2010). A typical MAPK cascade consists of three MAP kinases: MAP kinase kinase kinase (MAPKKK/MAP3K), MAPkinase kinase (MAPKK/MAP2K), and MAP kinase (MAPK). The signaling module, which is universal among eukaryotic kingdom, is activated by various internal or external stimuli, such as mitogen, hormones, developmental signal, pathogen, and abiotic stresses. The kinases comprising the module is sequentially activated by a relay of phosphorylation. A MAP3K, which is activated by a specific stimulus or stimuli, phosphorylate MAP2K, which, in turn, phosphorylate MAPK. The activated MAPKs phosphorylate downstream targets to modify their activities.
In the Arabidopsis genome, approximately 80 MAP3Ks, 10 MAP2Ks, and 20 MAPKs have been identified (Colcombet and Hirt, 2008). Numerous studies have been carried out to determine the functions of individual MAPKs, especially those of MAP2Ks and MAPKs. The studies show that they are involved in various cellular processes, such as plant development, hormone responses, disease resistance, and abiotic stress responses (Colcombet and Hirt 2008; Rodriguez et al., 2010). In several cases, the entire signaling cascades comprised of MAP3K-MAP2K-MAPK have been delineated. For instance, the MEKK1-MKK2-MPK4/6 modules are involved in salt and cold response (Teige et al., 2004), the MEKK1-MKK4/5-MPK3/6 modules function in innnate immunity (Asai et al., 2002), and the YODA-MKK4/5-MPK3/6 modules regulate the stomatal development (Lampard et al
Some MAPKs are involved in ABA response (Liu, 2012). The MKK1-MPK6 module mediates ABA response during seed germination by regulating
We are interested in the roles of MAP3Ks in ABA and/or abiotic stress signaling and set out to investigate the ABA-associated functions of several MAP3Ks. We chose MAP3K16 and other related MAP3Ks for our study. MAP3K16 is one of the several salt-inducible MAP3Ks, together with MAP3K14, MAP3K15, MAP3K17, and MAP3K18 (i.e., MAP3K14/15/17/18). MAP3K17 and MAP3K18 are also ABA-inducible. As a first step toward their functional analysis, we investigated whether MAP3K16 and the related MAP3Ks, MAP3K14/15/17/18, are involved in ABA response. In this paper, we show that MAP3K16 regulates a subset of ABA response and present the data suggesting its possible substrates. Our results further indicate that MAP3K14/15/17/18 play similar roles in ABA response.
RNA isolation, semi-quantitative RT-PCR, and real-time RT-PCR were performed described previously (Lee et al., 2015). Briefly, RNA was isolated employing the RNeasy plant mini kit (Qiagen) and treated with DNase I to remove contaminating DNA. For RT-PCR, the first strand cDNA was synthesized using Superscript III (Invitrogen), and qRT-PCR was carried out in a Bio-Rad CFX96 real-time PCR system using the primers shown in the Supplementary Table S2.
Transformation of Arabidopsis was performed according to Bechtold and Pelletier (Bechtold and Pelletier, 1998). For the promoter analysis, T3 homozygous lines were employed in histochemical GUS staining. For OX lines, more than 100 T1 transgenic lines were selected for each construct, and T3 generation homozygous lines were recovered from T2 generation lines segregating with 3:1 ratio of KanR and KanS seeds. Final phenotype analyses were carried out using T4 generation seeds of representative lines after preliminary analyses of T3 generation homozygous lines. The knockout (KO) mutant,
Phenotype analyses were performed using the seeds amplified from the confirmed lines. For the analyses of
Yeast two-hybrid assays were performed as described before (Choi et al., 2005; Lee et al., 2009). To prepare bait constructs, insert fragments were amplified using the primers shown in the Supplementary Table S2 and cloned into the pPC62LexA vector (Lee et al., 2009). The prey constructs were prepared by cloning the individual amplified fragments into pYESTrp2 (Invitrogen). Bait constructs were individually introduced into the reporter yeast L40 (
Pulldown assays were performed as described before (Choi et al., 2005) with minor modification. For ABR1, maltose–binding protein (MBP) was used as a negative control, and glutathione S-transferase (GST) was used as a negative control for MKKs (see below). Approximately 5 μg of fusion proteins, 30 μl of MBP (ABR1) or GST (MKKs) resin (50% slurry), and 20 μl of
MAP3K16 recombinant proteins were prepared employing the pMAL system (NEB) with the modification described previously (Lee et al., 2015). The entire coding region (amino acids 1-443) or the kinase domain (amino acids 1-263) were amplified and cloned into the Sal I/Eco RI sites of pMAL-c5X with a 6X His-tag. Protein induction and protein purification were carried out according to the supplier’s instruction, and the proteins were further purified employing the Ni-NTA resin (Qiagen). ABR1 recombinant protein was prepared in a similar way. The entire
Kinase assays were carried out as described (Choi et al., 2005; Lee et al., 2015). Briefly, approximately 0.5–1 μg of MAP3K16 recombinant proteins were incubated with similar amounts of substrates in a buffer (25 mM Tris–HCl, pH7.5, 10mM MgCl2, 10 μM ATP) containing 2 μCi of γ-32P for 30 min at 30°C. After the reaction, the reaction mixtures were separated by SDS-PAGE, and gels were stained with Coo-massie Brilliant Blue R, dried and autoradiographed.
To examine the stress-induction pattern of
We investigated the expression pattern of
To investigate the
ABA plays a pivotal role in mediating water-deficit response. Thus, the reduced ABA sensitivity during early seedling growth suggested that the
To further investigate the function of MAP3K16, we acquired its knockout (KO) line,
As mentioned earlier,
The plants overexpressing each of
MAP3K16 and MAP3K14/15/17/18 belong to the MAP3K family proteins (Ichimura et al
It is possible that MAP3K16 may interact with proteins other than MKKs. To address the possibility, we performed yeast two-hybrid screening employing the full-length MAP3K16 as bait. We screened approximately 4.5 million yeast transformants obtained with a cDNA expression library prepared from salt- and ABA-treated seedling RNAs (Choi et al., 2000). Several putative positive clones, including APUM10 (At1g35750), CAD7 (At4g37980), ABR1 (At5g64750), and GDA1 (At4g19180), were isolated from the screen. Among the isolates, we chose ABR1 for further analysis, because it is known to be a negative regulator of ABA response (Pandey et al., 2005). As shown in Fig. 7A, ABR1 interacted with the full-length MAP3K16, and it also interacted with the kinase domain alone albeit with lower intensity (Fig. 7A). The interaction between the full-length MAP3K16 and ABR1 was confirmed by pulldown assays (Fig. 7B), in which
Once confirmed the interaction between the full-length MAP3K16 and ABR1, we performed a deletion study to map the interaction domain of ABR1. Various ABR1 deletion constructs shown in Figs. 7D and 7E were prepared, and their interactions with the full-length MAP3K16 were investigated by two-hybrid assay (Fig. 7F). Deletion of the N-terminal portion (amino acids 1-180) did not abolish the interaction, indicating that the remaining portion, i.e., fragment AP2C consisted of the AP2 domain (amino acids 181-260) and the C-terminal portion (amino acids 261-391) is sufficient for the interaction. On the other hand, the C-terminal deletion (amino acids 261-391) abolished the interaction, suggesting that it is essential for the interaction and that the remaining portion (i.e., fragment NAP2, amino acids 1-260) is not sufficient for the interaction. Although necessary, C-terminal portion alone could not interact with MAP3K16. In summary, the deletion analysis showed that both AP2 domain and the C-terminal portion of ABR1 are necessary for the interaction.
To investigate the enzymatic activity of MAP3K16, we carried out
Next, we investigated whether MAP3K16 could phosphorylate ABR1. In the above assay to examine MyBP phosphorylation, recombinant ABR1 was also phosphorylated by the constitutive active form (i.e., the kinase domain-containing fragment) of MAP3K16 (Fig. 8A, lane 4), when it was added to the reaction mixture with MyBP. In a separate assay employing ABR1 as an only substrate, ABR1 was phosphorylated by MAP3K16 (Fig. 8B, lanes 3, 4). Thus, our results indicate that the constitutive active form of MAP3K16 could phosphorylate ABR1
The primary goal of our current study is to find out MAP3Ks that are involved in ABA and stress responses. Toward the end, we searched database and chose MAP3K16 and several other ABA- or salt-inducible MAP3Ks for functional analyses.
In our transgenic analyses, overexpression of
ABA affects several aspects of plant growth and development (Finkelstein, 2013), such as seed germination, seedling establishment (i.e., cotyledon greening/expansion and onset of vegetative growth) and subsequent postgermination growth, stress tolerance, and seed dormancy. Although ABA generally plays an inhibitory role during germination and postgermination growth, it inhibits postgermination seedling growth more efficiently than the germination process (Lopez-Molina et al., 2001). The ABA insensitivity of the
Our transgenic analyses of other salt-inducible MAP3Ks (summarized in the Supplementary Table S1), MAP3K14/15/17/18, indicate that they are likely to play similar roles to MAP3K16 in ABA and stress responses. The OX lines of the genes were ABA-insensitive during seedling establishment (i.e., cotyledon greening/expansion) stage and susceptible to water deficit (Fig. 5 and Supplementary Fig. S3). Like
It is noteworthy that the results of our transgenic analyses suggest the negative regulatory role of MAP3K14/15/16/17/18 in ABA response. The conclusion is based on our observation of OX phenotypes and the phenotypes of single KO lines without complementation analyses. Thus, the results must be interpreted with caution. Nonetheless, most distinct OX phenotypes (i.e., ABA insensitivity during seedling establishment stage and drought susceptibility) were observed with all OX lines (Supplementary Table S1), and opposite and complementary phenotypes were observed with their respective KO lines. Collectively, the results strongly support the conclusion that MAP3K14/15/16/17/18 play mainly negative regulatory roles in ABA response.
Recombinant MAP3K16 protein possesses kinase activity (Fig. 8), and, in an effort to identify putative MKK substrates, we found out that it interacts with MKK3 (Fig. 6, Supplementary Fig. S4). Consistent with this observation, recombinant MKK3 was phosphorylated by MAP3K16
Our results suggest that ABR1 may also be a substrate of MAP3K16. ABR1 was isolated as one of the positive clones in the two-hybrid screen to isolate MAP3K16-interacting proteins. Additionally, recombinant ABR1 was phosphorylated by MAP3K16
While our work is in progress, the function of MAP3K17 and MAP3K18 has been reported. According to Danquah et al. (2015), MAP3K17/18 activate MKK3, which in turn activates MPK1/2/7/14. The MAPK cascade is activated by the core ABA signaling module, and the
In summary, our study demonstrated that MAP3K16 functions mainly as a negative regulator of ABA response during early seedling growth and in water-deficit response, although it plays a positive role in the regulation of root growth. MAP3K16 possesses a kinase activity and could phosphorylate ABR1, a negative regulator of ABA response. MAP3K16 also interacts with MKK3 and phosphorylate it
ABA- and stress-induced expression of
|MAP3Ks||Gene ID||ABA||Salt (NaCl)||Mannitol||Cold||Untreated|
|MAP3K14||At2g30040||1.28 ± 0.21||6.04 ± 0.31||4.54 ± 0.40||0.66 ± 0.11||29.25/189.91|
|MAP3K15||At5g55090||1.35 ± 0.05||11.71 ± 2.69||1.88 ± 0.18||0.38 ± 0.05||1.25/7.8|
|MAP3K16||At4g26890||2.89 ± 0.25||11.72 ± 0.98||2.98 ± 0.26||0.17 ± 0.02||8.21/29.4|
|MAP3K17||At2g32510||11.47 ± 1.58||62.36 ± 0.25||32.60 ± 0.70||0.66 ± 0.30||19.83/33.43|
|MAP3K18||At1g05100||68.76 ± 6.95||333.72 ± 54.23||413.35 ± 27.02||1.43 ± 2.03||2.06/3.36|
Seedlings were treated with 1/4 MS or 1/4MS containing 100 μM ABA, 250 mM NaCl, or 600 mM mannitol for 4 h before RNA isolation. Experiments were done in triplicates, and the numbers indicate relative expression levels compared with those of ¼ MS.
Relative expression level compared with the expression level in untreated plants. Seedlings were placed at 4°C for 24 h before RNA isolation.
Expression values are from e-FP Browser (