Abstract
Calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of abscisic acid (ABA) and abiotic stress signaling in plants and is believed to act upstream of mitogen-activated protein kinase (MAPK) in ABA signaling. However, it is unclear how CCaMK activates MAPK in ABA signaling. Here, we show that OsDMI3, a rice (Oryza sativa) CCaMK, directly interacts with and phosphorylates OsMKK1, a MAPK kinase (MKK) in rice, in vitro and in vivo. OsDMI3 was found to directly phosphorylate Thr-25 in the N-terminus of OsMKK1, and this Thr-25 phosphorylation is OsDMI3-specific in ABA signaling. The activation of OsMKK1 and its downstream kinase OsMPK1 is dependent on Thr-25 phosphorylation of OsMKK1 in ABA signaling. Moreover, ABA treatment induces phosphorylation in the activation loop of OsMKK1, and the two phosphorylations, in the N-terminus and in the activation loop, are independent. Further analyses revealed that OsDMI3-mediated phosphorylation of OsMKK1 positively regulates ABA responses in seed germination, root growth, and tolerance to both water stress and oxidative stress. Our results indicate that OsMKK1 is a direct target of OsDMI3, and OsDMI3-mediated phosphorylation of OsMKK1 plays an important role in activating the MAPK cascade and ABA signaling.
In rice, the calcium/calmodulin-dependent protein kinase directly phosphorylates and activates a mitogen-activated protein kinase, which plays an important role in abscisic acid signaling.
Introduction
Abscisic acid (ABA) is a major plant hormone that regulates plant growth and development and plant responses to environmental stresses. Under drought and salt stress, plants accumulate ABA, which is perceived by the ABA receptors pyrabactin resistance 1 (PYR1)/PYR1-like/regulatory component of ABA receptor. ABA binds to the ABA receptors, which then interact with and inhibit the group A type 2C protein phosphatases (PP2Cs), resulting in the activation of the subclass III sucrose nonfermenting1-related protein kinase 2s (SnRK2s; Ma et al., 2009; Park et al., 2009). The activated SnRK2s phosphorylate downstream targets, such as transcription factors for stress-responsive gene regulation, NADPH oxidases for reactive oxygen species production, and ion channels for stomatal closure (Umezawa et al., 2013; Wang et al., 2013). In addition to SnRK2s, other protein kinases, such as the plasma-membrane receptor-like kinase GHR1 (Hua et al., 2012; Sierla et al., 2018), mitogen-activated protein kinases (MAPKs), calcium (Ca2+)-dependent protein kinases (CDPKs), and Ca2+/calmodulin (CaM)-dependent protein kinase (CCaMK), have also been shown to be involved in ABA signaling (Umezawa et al., 2014; Zhu, 2016; Qi et al., 2018; Chen et al., 2020).
The MAPK cascade is a major signaling pathway in all eukaryotes, linking the perception of stimuli to cellular responses. The MAPK cascade is highly conserved and a typical MAPK cascade consists of three protein kinases: a MAPK kinase kinase (MAPKKK or MKKK or MEKK), a MAPK kinase (MAPKK or MKK or MEK), and a MAPK (MPK), which are sequentially activated by phosphorylation. Once activated, MAPKs phosphorylate various downstream protein targets, such as transcription factors, phospholipases, protein kinases, metabolic enzymes, cytoskeletal, and microtubule-associated proteins, leading to the activation of cellular responses (Umezawa et al., 2014; de Zelicourt et al., 2016; Dóczi and Bögre, 2018; Zhang et al., 2018). There are 80 MAPKKKs, 10 MAPKKs, and 20 MAPKs encoded in the Arabidopsis (Arabidopsis thaliana) genome (MAPK Group, 2002; Colcombet and Hirt, 2008), and 75 MAPKKKs, 8 MAPKKs, and 17 MAPKs in rice (Hamel et al., 2006; Rao et al., 2010). Arabidopsis MPK3, MPK4, and MPK6, and their orthologs in other plant species have been widely demonstrated to play a crucial role in the response of plants to various biotic and abiotic stresses (Colcombet and Hirt, 2008; Rodriguez et al., 2010; Šamajová et al., 2013; de Zelicourt et al., 2016; Li et al., 2017a; Zhang et al., 2017, 2018; Zhao et al., 2017; Dóczi and Bögre, 2018).
Some complete MAPK cascades involved in biotic and abiotic stress signaling in plants have been identified. In Arabidopsis, diverse pattern recognition receptors activate two MAPK cascades: the first one is composed of MEKK1–MKK1/2–MPK4 (Suarez-Rodriguez et al., 2007; Gao et al., 2008), and the second of MAPKKK3/5–MKK4/5–MPK3/6 (Bi et al., 2018; Sun et al., 2018). Similarly, in rice, OsMAPKKK11/18–OsMKK4–OsMPK3/6 (Yamada et al., 2017) and OsMAPKKK24–OsMKK4–OsMPK3/6 (Wang et al., 2017) were also reported to function in chitin signaling. In the response of plants to abiotic stresses, a recent study revealed that the MEKK1–MKK2–MPK4 cascade positively regulates freezing tolerance, and the MKK4/5–MPK3/6 cascade negatively regulates freezing tolerance (Zhao et al., 2017). In ABA signaling, the ABA-activated MAPKKK17/18–MKK3–MPK1/2/7/14 cascade is involved in stress signaling (Danquah et al., 2015) and timing of senescence (Matsuoka et al., 2015). A recent study reported that the MAPKKK20 (AIK1)–MKK5–MPK6 cascade functions in the ABA-mediated regulation of primary root growth and stomatal response (Li et al., 2017b).
CCaMK, as a decoder of Ca2+ signals, is a crucial regulator of root nodule and arbuscular mycorrhizal symbioses (Singh and Parniske, 2012; Poovaiah et al., 2013). CCaMK is also involved in the responses of plants to abiotic stresses (Ma et al., 2012; Shi et al., 2012, 2014; Zhu et al., 2016; Ni et al., 2019) and biotic stresses (Wang et al., 2015). Previous studies have indicated that the rice CCaMK OsDMI3 is a positive regulator of ABA responses, including seed germination, root growth, antioxidant defense, and tolerance to both water stress and oxidative stress (Shi et al., 2012, 2014; Ni et al., 2019). A recent study revealed that in the absence of ABA, the PP2C OsPP45 directly interacts with OsDMI3 to inactivate OsDMI3 by dephosphorylation, and in the presence of ABA, ABA-induced H2O2 production inactivates OsPP45, resulting in OsDMI3 activation (Ni et al., 2019). OsDMI3 functions upstream of OsMPK1 (also called OsMPK6), a major ABA-activated MAPK, to regulate the antioxidant defense systems in ABA signaling (Shi et al., 2014). However, the molecular mechanism of OsDMI3-mediated activation of OsMPK1 in ABA signaling remains to be determined.
Here, we show that OsDMI3 directly interacts with and phosphorylates the upstream activator of OsMPK1, OsMKK1 in vitro and in vivo, and OsDMI3-mediated phosphorylation of OsMKK1 is required for the activation of the OsMKK1–OsMPK1 cascade in ABA signaling. Genetic evidence demonstrates that OsDMI3-mediated phosphorylation of OsMKK1 plays an important role in ABA signaling. These findings uncover an important noncanonical MKK activation mechanism, which directly connects CCaMK to the MAPK cascade in ABA signaling.
Results
OsDMI3 does not interact directly with OsMPK1, but interacts with its upstream activators OsMKK1 and OsMKK6
To determine whether a direct interaction between OsDMI3 and OsMPK1 exists in plant cells, yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC) assays were performed. Experimental results showed that OsDMI3 did not interact with OsMPK1 either in yeast (Saccharomyces cerevisiae) cells (Supplemental Figure S1A) or in onion (Allium cepa) epidermis cells (Supplemental Figure S1B). Moreover, an in vitro phosphorylation assay showed that OsDMI3 did not directly phosphorylate OsMPK1 (Supplemental Figure S1C). These results suggest that OsDMI3 does not directly activate OsMPK1 in plant cells.
In a canonical MAPK cascade, MAPK is activated by its upstream MKK. In rice, OsMPK1 was shown to interact with OsMKK1 (OsMEK2), OsMKK6 (OsMEK1), OsMKK3 (OsMEK8a), OsMKK4 (OsMEK6), OsMKK5 (OsMEK7b), and OsMKK10-2 (OsMEK3) (Singh et al., 2012). To determine whether OsDMI3 can interact with these MKKs, Y2H, and BiFC assays were conducted. OsDMI3 was shown to interact with the group A MKKs, OsMKK1, and OsMKK6, in both yeast cells (Figure 1A; Supplemental Figure S2, A and B) and onion epidermis cells (Figure 1C; Supplemental Figure S2, C and D). Further, glutathione S-transferase (GST) pull-down assays showed that OsDMI3 was able to directly interact with OsMKK1 and OsMKK6 in vitro (Figure 1B). The OsDMI3–OsMKK1 and OsDMI3–OsMKK6 interactions were also detected in co-immunoprecipitation (Co-IP) assays when OsDMI3-Myc was coexpressed with OsMKK1-Flag or OsMKK6-Flag in rice protoplasts. Immunoblot (IB) analyses using an anti-Flag antibody revealed that both OsMKK1-Flag and OsMKK6-Flag interacted with OsDMI3-Myc in rice protoplasts (Figure 1D).
Figure 1.
OsDMI3 interacts with OsMKK1/OsMKK6 both in vitro and in vivo. A, Y2H assay. SD-Trp-Leu-His-Ade/AbA/X-α-gal medium was used for testing the interaction between OsDMI3 and OsMKK1 (left) or OsMKK6 (right). BD-P53/AD-SV40 was used as a positive control, and BD-Lam/AD-SV40 was used as a negative control. B, GST pull-down assay. The equal amount of GST and GST-DMI3 were incubated with His-OsMKK1 (left) or His-OsMKK6 (right) in GST beads. GST-OsDMI3 was detected with anti-GST antibody, and both His-OsMKK1 and His-OsMKK6 were detected with anti-His antibody. C, BiFC analysis. The indicated constructs were transiently expressed in onion epidermis cells. Co-expression of SYFPN-OsDMI3 plus SYFPC or SYFPC-OsMKK1 plus SYFPN (left) or SYFPC-OsMKK6 plus SYFPN (right) were used as negative controls. Scale bars, 100 µm. D, Co-IP analysis. OsDMI3-Myc and OsMKK1-Flag (left) or OsMKK6-Flag (right) were co-transformed into rice protoplasts, and the protein extracts were immunoprecipitated using an anti-Myc antibody and were detected with anti-Flag (OsMKK1/OsMKK6) and anti-Myc (OsDMI3) antibodies. Protein input was shown by IB analysis of protein extracts before IP using antibodies against the respective tags. Molecular mass markers in kD are shown on the left. All experiments were repeated at least three times with similar results
To determine which regions of both OsDMI3 and OsMKK1 are required for the interaction, a series of deletion derivatives of the two proteins were generated and then tested for the interaction using Y2H and luciferase complementation imaging (LCI) assays. Y2H assays showed that OsMKK1 interacted with the EF (helix–loop–helix) hand domain (337–516 amino acids) of OsDMI3 (Supplemental Figure S3A). LCI assays confirmed the in vivo interaction between OsMKK1 and the EF-hand domain of OsDMI3 (Supplemental Figure S3B). Similarly, OsDMI3 was shown to interact with the N-terminus domain (1–65 amino acids) of OsMKK1 in Y2H (Supplemental Figure S4A) and LCI assays (Supplemental Figure S4B). Moreover, the interaction domains between OsDMI3 and OsMKK6 were also tested by Y2H assay and by LCI assay. Experimental results showed that the EF-hand domain (337–516 amino acids) in the C-terminus of OsDMI3 (Supplemental Figure S5, A and B) interacted with the N-terminus domain (1–71 amino acids) of OsMKK6 (Supplemental Figure S6, A and B). Taken together, these results indicate that the OsDMI3–OsMKK1/OsMKK6 interaction involves the EF-hand domain of OsDMI3 and the N-terminus domain of OsMKK1/OsMKK6.
OsDMI3-dependent activation of OsMKK1 but not OsMKK6 in ABA signaling
To determine whether OsDMI3 mediates the activation of both OsMKK1 and OsMKK6 in ABA signaling, we first tested if ABA induces the activation of both OsMKK1 and OsMKK6 in planta. Two independent OsMKK1- or OsMKK6-overexpressing (OE) lines (OsMKK1-OE1, OsMKK1-OE2, OsMKK6-OE1, and OsMKK6-OE2) and two independent OsMKK1- or OsMKK6-knockout (KO) lines (osmkk1-KO1, osmkk1-KO2, osmkk6-KO1, and osmkk6-KO2) were generated, and then specific anti-OsMKK1 and anti-OsMKK6 antibodies were prepared. The activities of OsMKK1 and OsMKK6 in rice plants were determined by an IP kinase assay with myelin basic protein (MBP) as a substrate. In the osmkk1-KO lines (Supplemental Figure S7A) and the osmkk6-KO lines (Supplemental Figure S7D) generated by the CRISPR/Cas9 system, the activities of both OsMKK1 (Supplemental Figure S7, B and C) and OsMKK6 (Supplemental Figure S7, E and F) were undetectable. By contrast, the OsMKK1-OE lines and the OsMKK6-OE lines exhibited high activities of OsMKK1 and OsMKK6. ABA treatment induced a rapid increase in the activities of both OsMKK1 (Figure 2, A and B) and OsMKK6 (Figure 2, C and D) in rice leaves, with a significant increase at 30 min of ABA treatment and a maximal increase at 90 min of ABA treatment. The specificity of the anti-OsMKK1 and anti-OsMKK6 antibodies was proven by immunoblotting analysis using the proteins extracted from the leaves of wild-type (WT), OsMKK1-OE1, and osmkk1-KO1 plants (Supplemental Figure S8A) or OsMKK6-OE1 and osmkk6-KO1 plants (Supplemental Figure S8B).
Figure 2.
The activation of OsMKK1 but not OsMKK6 Is OsDMI3-dependent in ABA signaling. A and C, ABA-induced increases in the activities of OsMKK1 (A) and OsMKK6 (C) in rice leaves. The rice seedlings were treated with 100 µM ABA for various times as indicated. OsMKK1 and OsMKK6 were immunoprecipitated from rice leaves, and the activities of OsMKK1 and OsMKK6 were analyzed by an IP kinase assay using MBP as substrate. OsMKK1 and OsMKK6 input were analyzed by IB using anti-OsMKK1 antibody and anti-OsMKK6 antibody, respectively. β-actin was used as the total protein loading control. Molecular mass markers in kD are shown on the left. B and D, The relative activities of (B) OsMKK1 in (A) and (D) OsMKK6 in (C). Kinase activities were quantitated by ImageJ software, and the activities of OsMKK1 and OsMKK6 in the control leaves treated with ABA for 0 min were set to 1, respectively. E and G, The activities of OsMKK1 (E) and OsMKK6 (G) in OsDMI3-OE1, osdmi3-KO1, and WT. The rice seedlings were treated with 100 µM ABA for 30, 60, and 90 min, and the activities of OsMKK1 and OsMKK6 were analyzed by an IP kinase assay using MBP as substrate. OsMKK1 and OsMKK6 input were analyzed by IB using anti-OsMKK1 antibody and anti-OsMKK6 antibody, respectively. β-actin was used as the total protein loading control. Molecular mass markers in kD are shown on the left. F and H, The relative activities of (F) OsMKK1 in (E) and (H) OsMKK6 in (G). Kinase activities were quantitated using ImageJ software, and the activities of OsMKK1 and OsMKK6 in WT plants treated with ABA for 0 min were set to 1, respectively. All experiments were repeated at least three times with similar results. In (B), (D), (F), and (H), values are means ± sem of three independent experiments. Means denoted by the same letter did not significantly differ at P < 0.05 according to Duncan’s multiple range test
Then, we used the OsDMI3-KO mutant osdmi3-KO1 and the OsDMI3-OE line OsDMI3-OE1 (Ni et al., 2019) to investigate whether OsDMI3 is required for the ABA-induced activation of both OsMKK1 and OsMKK6. Under the nontreated conditions, the OsDMI3-OE1 plants showed increased activity of OsMKK1, but the osdmi3-KO1 plants showed no obvious change in OsMKK1 activity, compared with WT plants (Figure 2, E and F). After ABA treatment, the ABA-induced increase in OsMKK1 activity was slightly enhanced in OsDMI3-OE1 plants, but was substantially inhibited in osdmi3-KO1 plants, indicating that ABA-induced activation of OsMKK1 is OsDMI3-dependent (Figure 2, E and F). However, there was no difference among OsDMI3-OE1, osdmi3-KO1, and WT plants in the activity of OsMKK6 under the nontreated conditions, and ABA treatment induced the same increase in OsMKK6 activity in these plants, indicating that ABA-induced activation of OsMKK6 is OsDMI3-independent in rice plants (Figure 2, G and H).
Finally, we investigated whether ABA-induced activation of both OsMKK1 and OsMKK6 is Ca2+-dependent. Treatment with 2 mM CaCl2 induced the activation of OsMKK1 (Supplemental Figure S9, A and B) but not OsMKK6 (Supplemental Figure S10, A and B), and pretreatments with the Ca2+ chelator ethylene glycol tetraacetic acid (EGTA) and the Ca2+ channel blocker LaCl3 inhibited the ABA-induced increase in OsMKK1 activity (Supplemental Figure S9, C and D) but not in OsMKK6 activity (Supplemental Figure S10, C and D), indicating that ABA-induced activation of OsMKK1 is Ca2+-dependent, and ABA-induced activation of OsMKK6 is Ca2+-independent. Furthermore, the Ca2+-induced increase in OsMKK1 activity was greatly reduced in osdmi3-KO1 plants, indicating that OsDMI3 makes a major contribution to the Ca2+-induced activation of OsMKK1 in rice plants (Supplemental Figure S9, E and F).
We also investigated whether OsMKK1 mediates the activation of OsDMI3 in ABA signaling. Under the nontreated conditions, there was no difference among OsMKK1-OE1, osmkk1-KO1, and WT plants in the activity of OsDMI3, and ABA treatment induced the same increase in OsDMI3 activity in these plants, indicating that OsMKK1 is not involved in the activation of OsDMI3 in ABA signaling (Supplemental Figure S11, A and B).
OsDMI3 phosphorylates Thr-25 in the N-terminus of OsMKK1
To determine if OsMKK1 or OsMKK6 can be phosphorylated by OsDMI3, both in vitro and in vivo protein phosphorylation assays were conducted. In vitro kinase assays showed that OsDMI3 extracted from ABA-treated rice leaves phosphorylated both His-OsMKK1 (Supplemental Figure S12A) and His-OsMKK6 (Supplemental Figure S12B). To investigate whether this phosphorylation by OsDMI3 also occurs in plant cells, the osdmi3-KO1 and WT plants were treated with ABA, and the phosphorylated proteins of OsMKK1 and OsMKK6 were detected by immunoblotting using biotinylated Phos-tag. Under the nontreated conditions, there was no difference between WT and osdmi3-KO1 in the phosphorylation levels of OsMKK1 (Supplemental Figure S12, C and D) and OsMKK6 (Supplemental Figure S12, E and F). ABA treatment induced a significant increase in the phosphorylation levels of OsMKK1 (Supplemental Figure S12, C and D) and OsMKK6 (Supplemental Figure S12, E and F) in WT plants, and the time course of ABA-induced phosphorylation of OsMKK1 and OsMKK6 is consistent with that of ABA-induced activation of OsMKK1 and OsMKK6 (Figure 2, E–H). In the osdmi3-KO1 plants, the ABA-induced increase in the phosphorylation of OsMKK1 was substantially reduced (Supplemental Figure S12, C and D), but the ABA-induced increase in the phosphorylation of OsMKK6 was not affected (Supplemental Figure S12, E and F). These results indicate that ABA-induced OsMKK1 phosphorylation is OsDMI3-dependent and ABA-induced OsMKK6 phosphorylation is OsDMI3-independent.
We next sought to determine which amino acid residues in OsMKK1 are phosphorylated by OsDMI3. The recombinant OsMKK1 was pretreated with calf intestine alkaline phosphatase (CIAP) to remove the preexisting phosphorylation events and then incubated with OsDMI3, and the phosphorylation sites were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. Thr-25 of OsMKK1 was identified as the residue phosphorylated by OsDMI3 (Supplemental Table S1; Supplemental Figure S13). To further determine the phosphorylation site of OsMKK1 by OsDMI3, Thr-25 of OsMKK1 was mutated to Ala to create the nonphosphorylatable mutant OsMKK1T25A. In vitro kinase assays showed that the OsMKK1T25A mutant protein was not phosphorylated by OsDMI3 (Figure 3A), indicating that Thr-25 in OsMKK1 is the residue phosphorylated by OsDMI3 in vitro.
Figure 3.
OsDMI3 directly phosphorylates OsMKK1 at Thr-25 to regulate OsMKK1 activity. A, In vitro phosphorylation of OsMKK1 and mutated OsMKK1 (T25A) by OsDMI3. Protein extracts from ABA-treated rice leaves were immunoprecipitated with anti-OsDMI3 antibody. His-OsMKK1 and mutated His-OsMKK1 proteins were used as substrates and subjected to an in-gel kinase assay. Thr-25 phosphorylation was analyzed by IB using an anti-pT25 OsMKK1 antibody. OsDMI3 protein was tested by IB with anti-OsDMI3 antibody, and His-OsMKK1 and mutated His-OsMKK1 proteins were determined by IB with anti-His antibody. B, Thr-25 phosphorylation is OsDMI3-dependent in ABA signaling. OsDMI3-OE1, osdmi3-KO1, and WT plants were treated with 100 µM ABA for 30, 60, and 90 min, and Thr-25 phosphorylation was tested by IB with anti-pT25 OsMKK1 antibody. OsMKK1 input was analyzed by IB with anti-OsMKK1 antibody. β-actin was used as total protein loading control. C, ABA-dependence of Thr-25 phosphorylation under water stress. OsABA2-OE1, osaba2-KO1, and WT plants were treated with 20% PEG 4000 for 30, 60, and 90 min, and Thr-25 phosphorylation was tested by IB with anti-pT25 OsMKK1 antibody. OsMKK1 input was analyzed by IB with anti-OsMKK1 antibody. β-actin was used as total protein loading control. D, The N-terminal Thr-25 residue of OsMKK1 is conserved in monocots and dicots. N-terminal sequences of MKKs from Z. mays (ZmMKK1), A. thaliana (AtMKK1), G. max (GmMKK1), M. truncatula (MtMKK1), and O. sativa (OsMKK1) were aligned. The number at the end of each line indicates the coordinate of the last residue. E, OsMKK1T25D enhances the activity of OsMKK1 in vitro. The upper gel shows substrate phosphorylation (MBP as substrate) and autophosphorylation activity of OsMKK1, OsMKK1T25A, and OsMKK1T25D, and the lower gel shows the corresponding Coomassie staining. F, The relative activities of substrate phosphorylation and autophosphorylation in (E). The activities of autophosphorylation and substrate phosphorylation of OsMKK1 were quantitated using ImageJ software, and the substrate phosphorylation activity of OsMKK1 was set to 1. G, Thr-25 phosphorylation is required for the activation of OsMKK1 in ABA signaling. OsMKK1-OE1, OsMKK1T25A-A1, OsMKK1T25D-D1, and WT plants were treated with 100 µM ABA for 90 min, and the activity of OsMKK1 was analyzed by an IP kinase assay using MBP as substrate. OsMKK1 input was analyzed by IB with anti-OsMKK1 antibody. β-actin was used as the total protein loading control. H, The relative activity of OsMKK1 in (G). Kinase activity was quantitated using ImageJ software. The activity of OsMKK1 in untreated WT was set to 1. All experiments were repeated at least three times with similar results. In (F) and (H), values are means ± sem of three independent experiments. Means denoted by the same letter did not significantly differ at P < 0.05 according to Duncan’s multiple range test. Molecular mass markers in kD are shown on the left
To determine if OsDMI3 is responsible for the phosphorylation of Thr-25 in OsMKK1 in rice plants, a specific anti-phospho-Thr-25 antibody was prepared. This antibody specifically recognized the phosphorylated Thr-25 of OsMKK1, but not the two mutants of OsMKK1 in which Thr-25 was converted to either Ala (OsMKK1T25A) or Asp (OsMKK1T25D, a phosphomimetic mutant) (Supplemental Figure S14). Under the nontreated conditions, OsMKK1 Thr-25 phosphorylation was not observed in WT and osdmi3-KO1 plants, but was strong in OsDMI3-OE1 plants (Figure 3B). ABA treatment induced a progressive increase in OsMKK1 Thr-25 phosphorylation in WT plants in a time-dependent manner during the 90-min treatment, and further enhanced OsMKK1 Thr-25 phosphorylation in OsDMI3-OE1 plants. However, this phosphorylation was completely blocked in osdmi3-KO1 plants, indicating that OsMKK1 Thr-25 phosphorylation is specifically dependent on OsDMI3 in ABA signaling (Figure 3B).
To further determine whether endogenous ABA also plays such a role under water stress, the OsABA2-OE line (OsABA2-OE1) and the OsABA2-KO line (osaba2-KO1) were generated (Supplemental Figure S15, A and B). ABA2 can catalyze the conversion of xanthoxin to abscisic aldehyde in ABA biosynthesis (Cheng et al., 2002; González-Guzmán et al., 2002; Ma et al., 2016). In osaba2-KO1 plants, the content of ABA was ∼20% of WT plants, but in OsABA2-OE1 plants, the content of ABA was ∼2.6 times that of WT plants (Supplemental Figure S15B). Water stress induced by polyethylene glycol (PEG) led to a progressive increase in OsMKK1 Thr-25 phosphorylation in WT plants in a time-dependent manner during the 90-min treatment, and the increase was further enhanced in OsABA2-OE1 plants (Figure 3C). In contrast, this phosphorylation induced by water stress was completely blocked in osaba2-KO1 plants, indicating that ABA is essential for water stress-induced Thr-25 phosphorylation of OsMKK1.
Notably, Thr-25 was highly conserved in the OsMKK1 orthologs in maize (Zea mays; ZmMKK1), Arabidopsis (A. thaliana; AtMKK1), soybean (Glycine max; GmMKK1), and Medicago truncatula (MtMKK1), suggesting that the phosphorylation sites of MKKs by CCaMKs are conserved in plants (Figure 3D).
Phosphorylation of OsMKK1 at Thr-25 enhances the activities of OsMKK1 and OsMPK1
To determine if Thr-25 phosphorylation plays a role in OsMKK1 activation, we mutated Thr-25 of OsMKK1 either to Ala to create a nonphosphorylatable mutant (OsMKK1T25A) or to Asp to create a phosphomimetic mutant (OsMKK1T25D). In vitro kinase assays showed that the activity of OsMKK1 in the mutant OsMKK1T25A did not change compared with that of OsMKK1, but the activity of OsMKK1 in the mutant OsMKK1T25D had a 2.6-fold increase (Figure 3, E and F). However, there was no difference in the autophosphorylation activity of OsMKK1 in OsMKK1, OsMKK1T25A, and OsMKK1T25D (Figure 3, E and F). Then, we further investigated whether Thr-25 phosphorylation regulates OsMKK1 activity in rice plants. OsMKK1T25A, OsMKK1T25D, and OsMKK1 were over-expressed in rice under the control of the 35S promoter. Two lines of each of OsMKK1T25A (A1 and A2), OsMKK1T25D (D1 and D2), and OsMKK1-OE (OE1 and OE2) were selected and used for further experiments, based on their increased levels of OsMKK1 transcript and OsMKK1 protein (Supplemental Figure S16, A and B). Under the nontreated conditions, the activity of OsMKK1 in OsMKK1-OE1 and OsMKK1T25A-A1 plants was 1.6- and 1.65-fold, respectively, higher than that in WT plants, while the activity of OsMKK1 in OsMKK1T25D-D1 plants was 2.4-fold higher than that in WT plants (Figure 3, G and H). After ABA treatment, OsMKK1 activity in WT, OsMKK1-OE1, and OsMKK1T25D-D1 plants increased to 2.2-, 2.5-, and 2.8-fold of that in WT plants without ABA treatment, respectively, while OsMKK1 activity in OsMKK1T25A-A1 plants increased only to 1.95-fold of that in WT plants without ABA treatment (Figure 3, G and H). These results clearly indicate that Thr-25 phosphorylation in OsMKK1 is required for the activation of OsMKK1 in ABA signaling.
OsMKK1 has been reported to interact with OsMPK1 (Singh et al., 2012). To determine whether Thr-25 phosphorylation of OsMKK1 by OsDMI3 directly regulates the activity of OsMPK1, we reconstituted these three components in vitro using recombinant OsMKK1, OsMKK1T25A, OsMKK1T25D, and OsMPK1 proteins, and OsDMI3 pulled down from extracts of ABA-treated plants. After incubation with these components, the phosphorylation level of OsMPK1 was monitored. Incubation of OsMPK1 with OsMKK1 or OsMKK1T25A exhibited a low phosphorylation level of OsMPK1, but incubation of OsMPK1 with OsMKK1T25D significantly increased the phosphorylation level of OsMPK1 (Figure 4, A and B). When OsDMI3 was incubated together with OsMKK1 and OsMPK1, the phosphorylation level of OsMPK1 was enhanced. However, this OsDMI3-mediated enhancement of OsMPK1 phosphorylation was blocked when OsDMI3 was incubated with OsMKK1T25A and OsMPK1 (Figure 4, A and B). These results indicate that Thr-25 phosphorylation of OsMKK1 by OsDMI3 directly regulates the phosphorylation level of OsMPK1.
Figure 4.
Thr-25 phosphorylation of OsMKK1 is required for the activation of OsMPK1 in ABA signaling. A, In vitro reconstitution of OsDMI3-OsMKK1-OsMPK1 pathway. Recombinant OsMKK1, OsMKK1T25A, and OsMKK1T25D were incubated with OsMPK1 in the presence or absence of OsDMI3 pulled down from extracts of ABA-treated plants, and the phosphorylation level of OsMPK1 was monitored with an in-gel kinase assay. The recombinant His-OsMKK1 was determined by IB with anti-His antibody, and the recombinant GST-OsMPK1 was determined by IB with anti-GST antibody. The amount of OsDMI3 protein was determined by IB with anti-OsDMI3 antibody. B, The relative phosphorylation level of OsMPK1 in (A). The phosphorylation level of OsMPK1 was quantitated by ImageJ software. The phosphorylation level of OsMPK1 incubated with OsMKK1 in the absence of OsDMI3 was set to 1. C, The activation of OsMPK1 is dependent on Thr-25 phosphorylation of OsMKK1 in ABA signaling. osmkk1-KO1, OsMKK1-OE1, OsMKK1T25A-A1, OsMKK1T25D-D1, and WT plants were treated with 100 µM ABA for 2 h, and the activity of OsMPK1 was analyzed by an IP kinase assay using MBP as substrate. OsMPK1 input was analyzed by IB with anti-OsMPK1 antibody. β-actin was used as total protein loading control. D, The relative activity of OsMPK1 in (C). Kinase activity was quantitated by ImageJ software. The activity of OsMPK1 in untreated WT was set to 1. All experiments were repeated at least three times with similar results. In (B) and (D), values are means ± sem of three independent experiments. Means denoted by the same letter did not significantly differ at P < 0.05 according to Duncan’s multiple range test. Molecular mass markers in kD are shown on the left
Then, we further investigated whether Thr-25 phosphorylation regulates OsMPK1 activity in rice plants. As shown in Figure 4, C and D, under the nontreated conditions, the activity of OsMPK1 in osmkk1-KO1, OsMKK1-OE1, and OsMKK1T25A-A1 plants was 0.70-, 1.33-, and 1.36-fold of that in WT plants, respectively, but the activity of OsMPK1 in OsMKK1T25D-D1 plants was 1.93-fold of that in WT plants. After ABA treatment, OsMPK1 activity in WT, osmkk1-KO1, OsMKK1-OE1, and OsMKK1T25D-D1 plants increased to 1.86-, 1.09-, 2.11-, and 2.35-fold of that in WT plants without ABA treatment, respectively, but OsMPK1 activity in OsMKK1T25A-A1 plants increased only to 1.52-fold of that in WT plants without ABA treatment (Figure 4, C and D). These results indicate that OsMKK1 is a key activator of OsMPK1 in ABA signaling and the ABA-mediated Thr-25 phosphorylation of OsMKK1 plays an important role in the activation of OsMPK1.
Moreover, we investigated whether Thr-25 phosphorylation of OsMKK1 by OsDMI3 could affect the interactions between OsDMI3 and OsMKK1 and between OsMKK1 and OsMPK1. Y2H assays and LCI assays showed that the interactions of OsDMI3–OsMKK1, OsDMI3–OsMKK1T25A, and OsDMI3–OsMKK1T25D were similar (Supplemental Figure S17, A and B) and the interactions of OsMKK1–OsMPK1, OsMKK1T25A–OsMPK1, and OsMKK1T25D–OsMPK1 were also similar (Supplemental Figure S17, C and D), indicating that Thr-25 phosphorylation of OsMKK1 does not affect the interactions between OsDMI3 and OsMKK1 and between OsMKK1 and OsMPK1.
The two modes of OsMKK1 phosphorylation in the N-terminus and in the activation loop are independent
In a canonical MAPK cascade, MKK is activated by MKKK through phosphorylation on two Ser/Thr residues in the conserved S/T-X5-S/T motif. In OsMKK1, the phosphorylation sites in the activation loop are Ser-215 and Thr-221 (Jagodzik et al., 2018). To test whether OsDMI3 affects the phosphorylation of OsMKK1 at Ser-215 and Thr-221 in ABA signaling, OsMKK1-Flag was introduced into the protoplasts of OsDMI3-OE1, osdmi3-KO1, and WT, and OsMKK1 proteins were immunoprecipitated from the transfected protoplasts with or without ABA treatment (Figure 5A). After tryptic digestion, phosphorylated peptides of Ser-215, Thr-221, and Thr-25 were identified by LC–MS/MS (Supplemental Figure S18, A–C) and quantified by parallel reaction monitoring (PRM) analysis. Experimental results showed that ABA treatment induced a significant increase in the phosphorylation of both Ser-215 and Thr-221 in the protoplasts of WT, and this ABA-induced increase was not affected in the protoplasts of both OsDMI3-OE1 and osdmi3-KO1 (Figure 5, C and D), indicating that the phosphorylation of both Ser-215 and Thr-221 in OsMKK1 is independent of OsDMI3 in ABA signaling. At the same time, PRM analysis showed that ABA-induced Thr-25 phosphorylation of OsMKK1 is specifically dependent on OsDMI3 (Figure 5B), which is consistent with the results from the anti-phospho-Thr-25 antibody (Figure 3B).
Figure 5.
The Two phosphorylation pathways of OsMKK1 in the N-terminus and in the activation loop are independent in ABA signaling. A, OsMKK1 protein immunoprecipitated from rice protoplasts. OsMKK1-Flag was introduced into rice protoplasts of OsDMI3-OE1, osdmi3-KO1, and WT. Transfected protoplasts were treated with 10 μM ABA for 10 min. Protein extracts were immunoprecipitated with anti-Flag antibody, followed by separation by SDS-PAGE and stained with Coomassie brilliant blue. β-actin was used as total protein loading control. B–D, ABA-induced phosphorylation of Thr-25 (B), Ser-215 (C), and Thr-221 (D) in OsMKK1 in rice protoplasts. OsMKK1 protein was cut out and digested by trypsin. After tryptic digestion, the phosphorylated peptides of Thr-25, Ser-215, and Thr-221 were quantified by PRM analysis. E, The phosphorylation of Thr-25 is independent of the phosphorylation of both Ser-215 and Thr-221 in vitro. Protein extracts from ABA-treated rice leaves were immunoprecipitated with anti-OsDMI3 antibody. His-OsMKK1 and mutated His-OsMKK1 (OsMKK1S215A/T221A and OsMKK1S215D/T221D) were incubated with OsDMI3 and ATP in vitro. The phosphorylation of OsMKK1 at Thr-25 was analyzed by IB with anti-pT25 OsMKK1 antibody. Equal loading of OsDMI3 protein was shown by IB with anti-OsDMI3 antibody. His-OsMKK1 and mutated His-OsMKK1 proteins were determined by IB with anti-His antibody. F, The phosphorylation of Thr-25 is not affected by the phosphorylation of both Ser-215 and Thr-221 in rice protoplasts. The indicated constructs were expressed in osmkk1-KO1 protoplasts, and the protoplasts were treated with 10 µM ABA for 10 min. OsMKK1 Thr-25 phosphorylation was tested by IB with anti-pT25 OsMKK1 antibody. OsMKK1 input was analyzed by IB using an anti-OsMKK1 antibody. β-actin was used as total protein loading control. All experiments were repeated at least three times with similar results. In (B–D), values are means ± sem of three independent experiments. Means denoted by the same letter did not significantly differ at P < 0.05 according to Duncan’s multiple range test. Molecular mass markers in kD are shown on the left
We also investigated whether the phosphorylation of both Ser-215 and Thr-221 affects the phosphorylation of Thr-25 in ABA signaling. Both Ser-215 and Thr-221 were mutated either to Ala (OsMKK1S215A/T221A) or to Asp (OsMKK1S215D/T221D), and OsMKK1 and the mutant forms of OsMKK1 were introduced into protoplasts isolated from osmkk1-KO1 mutant plants. The phosphorylation of OsMKK1 at Thr-25 was detected by IB with the anti-phospho-Thr-25 antibody. In vitro assays showed that there was no difference in the phosphorylation of Thr-25 in OsMKK1, OsMKK1S215A/T221A, and OsMKK1S215D/T221D in the presence of OsDMI3 and ATP (Figure 5E), indicating that the phosphorylation of both Ser-215 and Thr-221 does not affect the phosphorylation of Thr-25. Further, in vivo assays showed that under the nontreated conditions, OsMKK1 Thr-25 phosphorylation was not observed in WT protoplasts and in osmkk1-KO1 protoplasts transiently expressing OsMKK1 or OsMKK1S215A/T221A or OsMKK1S215D/T221D (Figure 5F). ABA treatment induced OsMKK1 Thr-25 phosphorylation in WT protoplasts and in osmkk1-KO1 protoplasts transiently expressing OsMKK1, and this phosphorylation was not affected in the osmkk1-KO1 protoplasts transiently expressing OsMKK1S215A/T221A or OsMKK1S215D/T221D (Figure 5F). These results indicate that the phosphorylation of Thr-25 is independent of the phosphorylation of both Ser-215 and Thr-221 in ABA signaling.
We next investigated whether the phosphorylation of both Ser-215 and Thr-221 in OsMKK1 also contributes to ABA-induced activation of OsMKK1. In vitro kinase assays showed that in the OsMKK1S215A/T221A mutant, the autophosphorylation and substrate phosphorylation of OsMKK1 were markedly lower than those of OsMKK1 WT, while in the OsMKK1S215D/T221D mutant, the autophosphorylation and substrate phosphorylation of OsMKK1 were significantly higher than those of OsMKK1 WT (Supplemental Figure S19, A and B). Further, in vivo assays showed that in the osmkk1-KO1 protoplasts transiently expressing OsMKK1S215A/T221A, the activity of OsMKK1 was significantly lower than that of the osmkk1-KO1 protoplasts transiently expressing OsMKK1 WT, but in the osmkk1-KO1 protoplasts transiently expressing OsMKK1S215D/T221D, the activity of OsMKK1 was much higher than that of the osmkk1-KO1 protoplasts transiently expressing OsMKK1 WT (Supplemental Figure S19, C and D). ABA treatment induced a significant increase in the activity of OsMKK1 in the protoplasts transiently expressing OsMKK1 WT, and the increase was further enhanced in the protoplasts transiently expressing OsMKK1S215D/T221D. However, this ABA-induced increase was significantly reduced in the protoplasts transiently expressing OsMKK1S215A/T221A (Supplemental Figure S19, C and D). These results indicate that the phosphorylation of both Ser-215 and Thr-221 in OsMKK1 is required for ABA-induced activation of OsMKK1. The only partial inhibition of the ABA-induced activation of OsMKK1 in the OsMKK1S215A/T221A protoplasts and the further enhancement of the ABA-induced activation of OsMKK1 in the OsMKK1S215D/T221D protoplasts also suggest that both OsMKKK and OsDMI3 are involved in the activation of OsMKK1 in ABA signaling.
OsMKK1 positively regulates ABA responses and tolerance to both water stress and oxidative stress
Previous studies have shown that OsDMI3 is a positive regulator of ABA responses, including seed germination, root growth, antioxidant defense, and tolerance of both water stress and oxidative stress (Shi et al., 2012, 2014; Ni et al., 2019). To determine whether OsMKK1 plays a similar role to OsDMI3 in these ABA responses, we first tested the role of OsMKK1 in the regulation of ABA responses in seed germination and root growth. Under the nontreated conditions, there were no obvious differences among OsMKK1-OE lines (OE1 and OE2), OsMKK1-KO lines (KO1 and KO2), and WT plants in seed germination (Figure 6, A and B) and primary root growth (Figure 6, C and D). ABA treatment markedly inhibited seed germination and primary root growth in WT. The ABA sensitivity of both seed germination and primary root growth was enhanced in OsMKK1-OE lines, and was reduced in osmkk1-KO lines. These results indicate that OsMKK1, like OsDMI3, positively regulates ABA responses in seed germination and root growth.
Figure 6.
The phosphorylation of Thr-25 enhances ABA sensitivity in seed germination and root growth. A, Seed germination in OsMKK1-OE (OE1 and OE2), OsMKK1T25A (A1 and A2), OsMKK1T25D (D1 and D2), osmkk1-KO (KO1 and KO2), and WT. The seeds of transgenic lines and WT were germinated and grown in 1/2 MS medium supplemented with different concentrations of ABA (0, 1, 5 μM) for 9 days after stratification. Scale bar, 3 cm. B, The seed germination rates of OsMKK1-OE (OE1 and OE2), OsMKK1T25A (A1 and A2), OsMKK1T25D (D1 and D2), and osmkk1-KO (KO1 and KO2) transgenic lines and WT under ABA treatments during 17 days after stratification. C, Seedling growth of OsMKK1-OE (OE1 and OE2), OsMKK1T25A (A1 and A2), OsMKK1T25D (D1 and D2), and osmkk1-KO (KO1 and KO2) transgenic lines and WT under ABA treatments for 8 days. Scale bar, 4 cm. D, Primary root lengths of OsMKK1-OE (OE1 and OE2), OsMKK1T25A (A1 and A2), OsMKK1T25D (D1 and D2), and osmkk1-KO (KO1 and KO2) transgenic lines and WT grown in different concentrations of ABA as indicated for 10 days. Approximately 48 seeds of each transgenic line were analyzed per replicate for each concentration of ABA in (A–D). In (A) and (C), experiments were repeated at least three times with similar results. In (B) and (D), values are means ± sem of three independent experiments
We next investigated whether OsMKK1 is involved in the tolerance of water stress and oxidative stress in rice plants. OsMKK1-OE, osmkk1-KO, and WT plants were treated with either PEG to simulate water stress or H2O2 to produce oxidative stress, and the phenotype of these plants under the stressed conditions was analyzed. Under the nonstressed conditions, there was no obvious morphological difference between the transgenic plants and the WT plants (Figure 7, A and B). When treated with 20% PEG (Figure 7A) or with 100 mM H2O2 (Figure 7B), the osmkk1-KO plants were more sensitive to water stress and oxidative stress compared with WT plants, and had lower survival rates after recovery by re-watering (Figure 7, C and D). In contrast, the OsMKK1-OE plants showed enhanced tolerance to water stress and oxidative stress after PEG and H2O2 treatments and had higher survival rates after recovery by re-watering. Consistent with the phenotype of these transgenic plants under the stressed conditions, the osmkk1-KO plants had more oxidative damage, indicated by the content of malondialdehyde (MDA) (Figure 7E) and the percentage of electrolyte leakage (Figure 7F), than WT plants under the stressed conditions, whereas the OsMKK1-OE plants had less oxidative damage than WT plants. These results indicate that OsMKK1 positively regulates the tolerance of rice plants to water stress and oxidative stress.
Figure 7.
The phosphorylation of Thr-25 positively regulates the tolerance of rice plants to water stress and oxidative stress. A and B, Phenotypes of OsMKK1-OE (OE1 and OE2), OsMKK1T25A (A1 and A2), OsMKK1T25D (D1 and D2), osmkk1-KO (KO1 and KO2), and WT plants exposed to water stress (A) or oxidative stress (B). Ten-day-old rice seedlings were treated with 20% PEG 4000 (A) or 100 mM H2O2 (B) for 18 days, and then recovered by re-watering for 7 days. Approximately 40 rice seedlings of each transgenic line were used per replicate. Scale bars, 4.5 cm. C and D, The survival rate (%) of the rice plants exposed to water stress (C) or oxidative stress (D) after recovery by re-watering for 7 days shown in (A) and (B). E and F, The content of MDA (E) and the percent leakage of electrolyte (F) in the leaves of OsMKK1-OE (OE1 and OE2), OsMKK1T25A (A1 and A2), OsMKK1T25D (D1 and D2), osmkk1-KO (KO1 and KO2), and WT plants exposed to water stress or oxidative stress. Ten-day-old seedlings were treated with 20% PEG 4000 or 100 mM H2O2 for 2 days, and then leaves were sampled for the determination of MDA content and electrolyte leakage (%). Approximately 40 seedlings of each transgenic line were used per replicate. In (A) and (B), experiments were repeated at least three times with similar results. In (C–F), values are means ± sem of three independent experiments. Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test
Phosphorylation of OsMKK1 by OsDMI3 enhances ABA sensitivity and tolerance to both water stress and oxidative stress
To test whether Thr-25 phosphorylation of OsMKK1 contributes to the function of OsMKK1 in ABA signaling, OsMKK1-OE (OE1 and OE2), OsMKK1T25A (A1 and A2), OsMKK1T25D (D1 and D2), and WT plants were treated with ABA, PEG, and H2O2, respectively, and the phenotype of these plants under the stressed conditions was analyzed. Under the nontreated conditions, there were no obvious differences between the transgenic lines and the WT in seed germination (Figure 6, A and B), primary root growth (Figure 6, C and D), and the growth of seedlings (Figure 7, A and B). After ABA treatment, there were significant differences among OsMKK1-OE, OsMKK1T25A, and OsMKK1T25D lines in seed germination (Figure 6, A and B) and primary root growth (Figure 6, C and D), in which OsMKK1T25D lines exhibited a significantly enhanced sensitivity to ABA compared with OsMKK1-OE lines, but OsMKK1T25A lines displayed a markedly reduced sensitivity to ABA, indicating that Thr-25 phosphorylation makes an important contribution to the function of OsMKK1 in these ABA responses. Further, OsMKK1T25D plants exhibited enhanced tolerance to water stress (Figure 7A) and oxidative stress (Figure 7B) compared with OsMKK1-OE plants, with a higher survival rate after recovery by re-watering (Figure 7, C and D) and a lower oxidative damage (Figure 7, E and F). By contrast, OsMKK1T25A plants showed reduced tolerance to water stress and oxidative stress, with a lower survival rate after recovery by re-watering and a higher level of oxidative damage. These results indicate that Thr-25 phosphorylation plays an important role in OsMKK1-regulated tolerance of rice plants to water stress and oxidative stress.
To further confirm that it is indeed the OsDMI3–OsMKK1 pathway that regulates the ABA response and tolerance to water stress and oxidative stress in rice plants, the mutants osdmi3-KO/OsMKK1-OE, osdmi3-KO/OsMKK1T25A, osdmi3-KO/OsMKK1T25D (Supplemental Figure S16, C and D), osmkk1-KO/OsDMI3-OE (Supplemental Figure S16, E and F), and osmkk1-KO/osdmi3-KO (Supplemental Figure S16G) were generated, and the phenotypes of these transgenic lines under ABA treatment and stress conditions were analyzed. Compared with the OsDMI3-OE line, the osmkk1-KO/OsDMI3-OE line exhibited a dramatic reduction in the sensitivity of seed germination to ABA (Figure 8, A and B) and in the tolerance to water stress (Figure 8C) and oxidative stress (Figure 8D), with a lower survival rate (Figure 8, E and F), higher levels of oxidative damage (Supplemental Figure S20, A and B), and faster water loss (Supplemental Figure S20C). The sensitivity of the osmkk1-KO/OsDMI3-OE line to ABA and the tolerance to water stress and oxidative stress were close to, but still significantly higher than those of the osmkk1 line. These results suggest that OsDMI3 mediates these responses partly through the action of OsMKK1. On the other hand, the sensitivity of the osdmi3-KO/OsMKK1-OE line to ABA and the tolerance to water stress and oxidative stress are much lower than those of the OsMKK1-OE line but significantly higher than those of the osdmi3-KO line, suggesting that the role of OsMKK1 in these responses is partly dependent on OsDMI3. Further, the osdmi3-KO/OsMKK1T25A line displayed a similar phenotype to the osdmi3-KO/OsMKK1-OE line in its sensitivity to ABA and tolerance to water and oxidative stress, but the osdmi3-KO/OsMKK1T25D line exhibited a greatly enhanced sensitivity to ABA and a greatly enhanced tolerance to water stress and oxidative stress, indicating that OsDMI3-mediated Thr-25 phosphorylation of OsMKK1 plays an important role in ABA responses. Moreover, compared with the osdmi3-KO line and the osmkk1-KO line, the osmkk1-KO/osdmi3-KO double mutant was less sensitive to ABA treatment (Figure 8, A and B) and displayed lower tolerance to water stress and oxidative stress (Figures 8, C–F), suggesting that, in addition to the OsDMI3-OsMKK1 pathway, there are other targets of OsDMI3 and another activation pathway of OsMKK1 involved in the ABA responses. Taken together, these results support the notion that the OsDMI3-OsMKK1 pathway is involved in the regulation of ABA responses.
Figure 8.
The OsDMI3-OsMKK1 pathway regulates the ABA response and tolerance to water stress and oxidative stress. A, Seed germination in osdmi3-KO1 (dmi3), OsDMI3-OE1 (3OE1), osmkk1-KO1 (mkk1), OsMKK1-OE1 (1OE1), osmkk1-KO/osdmi3-KO (mkk1/dmi3), osmkk1-KO/OsDMI3-OE (mkk1/3OE), osdmi3-KO/OsMKK1-OE (dmi3/1OE), osdmi3-KO/OsMKK1T25A (dmi3/T25A), osdmi3-KO/OsMKK1T25D (dmi3/T25D), and WT. The seeds of transgenic lines and WT were germinated and grown in 1/2 MS medium supplemented with different concentrations of ABA (0, 1, 5 μM) for 12 days after stratification. Approximately 40 rice seedlings of each transgenic line were used per replicate for each concentration of ABA. Scale bar, 3 cm. B, The seed germination rates of the transgenic lines shown in (A) and WT under ABA treatments during 17 days after stratification. C and D, Phenotypes of the transgenic plants shown in (A) and WT plants exposed to water stress (C) or oxidative stress (D). Ten-day-old rice seedlings were treated with 20% PEG 4000 (C) or 100 mM H2O2 (D) for 12 days, and then recovered by re-watering for 7 days. Approximately 40 rice seedlings of each transgenic line were used per replicate. Scale bars, 4.5 cm. E and F, The survival rate (%) of the rice plants exposed to water stress (E) or oxidative stress (F) after recovery by re-watering for 7 days shown in (C) and (D). In (A), (C), and (D), experiments were repeated at least three times with similar results. In (B), (E), and (F), values are means ± SEM of three independent experiments. In (E) and (F), means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test
Discussion
CCaMK (DMI3) has been shown to be an important positive regulator of ABA and abiotic stress signaling in plants (Shi et al., 2012, 2014; Zhu et al., 2016; Ni et al., 2019). A double-negative regulatory pathway for the activation of the rice CCaMK OsDMI3 in ABA signaling has been established, whereby the type 2C protein phosphatase OsPP45 inactivates OsDMI3 by direct dephosphorylation in the absence of ABA, but in the presence of ABA, ABA-induced H2O2 inhibits OsPP45 activity, resulting in OsDMI3 activation (Ni et al., 2019). Once activated, CCaMK can phosphorylate its protein targets. In maize, the NAC (NAM, ATAF1/2, and CUC2) transcription factor ZmNAC84 has been identified as a direct target of ZmCCaMK (Zhu et al., 2016). ZmNAC84 interacted with and was phosphorylated by ZmCCaMK, and the phosphorylation at Ser-113 of ZmNAC84 by ZmCCaMK is essential for ABA-induced antioxidant defense. Moreover, it was also shown that OsDMI3 and OsMPK1 are in the same pathway in ABA signaling, in which OsDMI3 functions upstream of OsMPK1 to regulate the activities of antioxidant enzymes and the production of H2O2 (Shi et al., 2014). However, it is unknown whether OsMPK1 is a direct target of OsDMI3 in ABA signaling and, if not, how OsDMI3 induces the activation of OsMPK1 in ABA signaling.
In this study, we established that OsDMI3 does not interact directly with OsMPK1 (Supplemental Figure S1), but interacts with the OsMPK1 activators OsMKK1 and OsMKK6 (Figure 1; Supplemental Figure S2). We found that OsDMI3 is required for ABA-induced phosphorylation and activation of OsMKK1 but not OsMKK6 (Figure 2; Supplemental Figure S12). We further identified that OsDMI3 directly phosphorylates OsMKK1 at Thr-25 (Figure 3A; Supplemental Figure S13), and this Thr-25 phosphorylation is OsDMI3-specific in ABA signaling (Figure 3B) and ABA-specific under water stress (Figure 3C). Our genetic evidence indicated that OsMKK1 is a positive regulator of ABA responses, including seed germination, root growth, and tolerance of both water stress and oxidative stress, and the phosphorylation of OsMKK1 by OsDMI3 makes an important contribution to the function of OsMKK1 in the ABA responses (Figures 6–8).
In MAPK signaling, the interactions between MAPKs and their activators, substrates, and inactivators are commonly achieved through specific docking interactions (Bardwell, 2006; Bigeard and Hirt, 2018; Krysan and Colcombet, 2018). The docking interactions mediated by a common docking domain (CD domain) in the C-terminal region of MAPKs and a docking domain (D-domain) in the N-terminal tail of MKKs have been extensively characterized in animals and plants. D-domains can also be found in MAPK regulatory proteins and substrates. However, the protein domains involved in MKKK–MKK interactions have been less well characterized. In mammalian cells, it was found that the C termini of MEK1, MKK3, MKK4, MKK6, and MKK7 contain a conserved docking site, termed domain for versatile docking, which binds to the kinase domain of their specific upstream MKKKs, and such docking interactions are required for the activation of these MKKs (Takekawa et al., 2005). In Arabidopsis, it was shown that the C-terminal region of MKKK20 is required for the interaction with MKK3 (Bai and Matton, 2018). Surprisingly, only the full-length MKK3 interacted with MKKK20, the N-terminus, kinase domain, and the C-terminus of MKK3 did not interact with MKKK20. In this study, we found that among the six rice MKKs we tested (OsMKK1, OsMKK6, OsMKK3, OsMKK4, OsMKK5, and OsMKK10-2), only the group A MKKs, OsMKK1, and OsMKK6, interact with OsDMI3 (Figure 1; Supplemental Figure S2), and the interactions are mediated by the EF-hand domain in the C-terminus of OsDMI3 and the N-terminus domain of OsMKK1 or OsMKK6 (Supplemental Figures S3–S6). The interaction domains between OsDMI3 and OsMKKs are distinct from those between MKKKs and MKKs in animal systems (Takekawa et al., 2005), suggesting that the docking domain in MKKs for MKKKs is different from that in MKKs for their regulatory proteins.
Interestingly, although both OsMKK1 and OsMKK6 can be phosphorylated by OsDMI3 in vitro, only ABA-induced phosphorylation and activation of OsMKK1 are OsDMI3-dependent, while ABA-induced phosphorylation and activation of OsMKK6 are OsDMI3-independent (Figure 2; Supplemental Figure S12). These results indicate that OsMKK1 is, but OsMKK6 is not, a physiological substrate of OsDMI3. This opens up the interesting question of why OsDMI3 cannot phosphorylate OsMKK6 in ABA signaling. In yeast and animal systems, multiple mechanisms have been shown to be involved in the specificity of protein phosphorylation, including (1) the structures of the kinase and the substrate, (2) local and distal interactions between the kinase and the substrate, (3) the formation of complexes with scaffolding and adaptor proteins that spatially regulate the kinase, and (4) systems-level effects such as competition, multisite phosphorylation, and kinetic proofreading (Ubersax and Ferrell, 2007; Miller and Turk, 2018). Plants may employ similar mechanisms to maintain the specificity of protein phosphorylation (Pitzschke, 2015; Bigeard and Hirt, 2018; Dóczi and Bögre, 2018; Krysan and Colcombet, 2018). Because no crystal structures of OsDMI3, OsMKK1, and OsMKK6 have yet been elucidated, we do not know whether there exists a structural constraint between OsDMI3 and OsMKK6, which prevents OsDMI3 from phosphorylating OsMKK6 in ABA signaling. In addition, there might exist a systems-level competition between OsMKK1 and OsMKK6, which may explain why OsDMI3 fails to phosphorylate OsMKK6 in ABA signaling.
A canonical MAPK cascade is composed of three sequentially activating kinases: an MKKK phosphorylates and activates an MKK on two Ser and/or Thr residues in a conserved (S/T)-X5-(S/T) motif in plants and (S/T)-X3-(S/T) in yeast and animals (Ren et al., 2002; Hamel et al., 2012), which then phosphorylates a MAPK by dual phosphorylation of the conserved TXY motif located in its activation loop (Hamel et al., 2012; Jagodzik et al., 2018). This three-component MAPK cascade is highly conserved in eukaryotes (Rodriguez et al., 2010; Hamel et al., 2012; Bigeard and Hirt, 2018; Jagodzik et al., 2018). However, some noncanonical MAPK pathways have also been reported in both animals and plants. The mammalian MAPK p38α has been shown to be activated by direct interaction with the protein TAB1 (transforming growth factor-β-activated protein kinase 1-binding protein 1) (Ge et al., 2002) or through Tyr-323 phosphorylation by T cell receptor-proximal tyrosine kinases (Salvador et al., 2005), and both pathways induce the cis-autophosphorylation of its activation loop. In plants, several different regulatory mechanisms for the MKK-independent activation of MAPKs have been described. In a salt stress response, Arabidopsis AtMPK6 can be activated by the binding of phosphatidic acid (Yu et al., 2010). Under mechanical wounding, the full activation of AtMPK8 requires direct binding of CaMs in a Ca2+-dependent manner in addition to MKK3 phosphorylation (Takahashi et al., 2011). In an immune response, the rice CDPK OsCPK18 is able to activate OsMPK5 by phosphorylating two conserved Thr residues (Thr-14 and Thr-32) in the N-terminal region of OsMPK5 without affecting the phosphorylation of its TXY motif (Xie et al., 2014). Moreover, a recent study showed that the transmembrane kinases (TMKs) TMK1 and TMK4 directly and specifically interact with and phosphorylate MKK4/5, which is required for auxin to activate MKK4/5-MPK3/6 signaling (Huang et al., 2019). However, the phosphorylation sites of MKK4/5 by these TMKs were not identified. Therefore, it is not clear whether or not these TMKs are MKKK-like kinases. In this study, we show an OsDMI3-dependent activation pathway of OsMKK1 in rice, which is distinct from these reported examples of unconventional MAPK activation in plants and animals. We found that OsDMI3 directly phosphorylates Thr-25 in the N-terminus of OsMKK1, but not the two Ser/Thr residues (Ser-215/Thr-221) in the conserved S/T-X5-S/T motif (Figure 3, A–C), and this phosphorylation does not affect the autophosphorylation of OsMKK1 (Figure 3, E and F). The activation of OsMKK1 (Figure 3) and OsMPK1 (Figure 4) is dependent on Thr-25 phosphorylation of OsMKK1 by OsDMI3 in ABA signaling. Our data indicate that OsDMI3-mediated phosphorylation of OsMKK1 is an important regulatory mechanism for OsMPK1 activation in ABA signaling (Figure 9).
Figure 9.
A working model for OsDMI3-mediated activation of MAPK cascade in ABA signaling. ABA induces the activation of OsDMI3, and the activated OsDMI3 directly phosphorylates OsMKK1 at Thr-25 to enhance its activity, thus resulting in the activation of OsMPK1. On the other hand, ABA also induces the phosphorylation of both Ser-215 and Thr-221 in OsMKK1, which are the phosphorylation sites in the activation loop of OsMKK1 by OsMKKK, showing that an OsMKKK-dependent pathway is involved in the regulation of OsMKK1 activity in ABA signaling. The two modes of OsMKK1 phosphorylation are independent and the OsDMI3-mediated phosphorylation of OsMKK1 makes a major contribution to the activation of OsMKK1 and the function of OsMKK1 in ABA signaling. However, the OsMKKK for the ABA-induced activation of the OsMKK1-OsMPK1 cascade is unknown. Moreover, it is also unclear whether a Ca2+/CaM-dependent pathway functions upstream of the canonical MAPK cascade in ABA signaling
Interestingly, the N-terminal Thr-25 residue of OsMKK1 is conserved in monocots and dicots, such as maize, Arabidopsis, soybean, and M. truncatula (Figure 3D). In legumes, CCaMKs have been shown to play key roles in mediating symbiotic relationships with rhizobial and arbuscular mycorrhizae (Singh and Parniske, 2012; Poovaiah et al., 2013). A recent study by quantitative phosphoproteomic analyses suggested that MAPK-mediated phosphorylation signaling may be involved in the rhizobia–legume symbiosis (Zhang et al., 2019). It is possible that the CCaMK-MKK-MAPK pathway might also be involved in the establishment of symbiotic relationships.
However, in this study, our results also showed that ABA-induced phosphorylation and activation of OsMKK1 were not fully inhibited in the osdmi3 mutant (Figure 2, E and F; Supplemental Figure s12, C and D). Our genetic results showed that the role of OsMKK1 in ABA responses is only partly dependent on OsDMI3 (Figure 8). These results indicate that an OsDMI3-independent pathway is also involved in the activation of OsMKK1 in ABA signaling. Further, we found that ABA treatment not only induces an increase in the phosphorylation of OsMKK1 Thr-25, but also induces an increase in the phosphorylation of both Ser-215 and Thr-221 (Figure 5, B–D), which are the phosphorylation sites in the activation loop of OsMKK1 by OsMKKK (Jagodzik et al., 2018). The phosphorylation of both Ser-215 and Thr-221 in OsMKK1 is also essential for the activation of OsMKK1 in ABA signaling (Supplemental Figure S19), suggesting that OsMKKK is also involved in the activation of OsMKK1 in ABA signaling. However, this ABA-induced increase in the phosphorylation of both Ser-215 and Thr-221 is not affected by OsDMI3. On the other hand, the phosphorylation of Thr-25 is also independent of the phosphorylation of both Ser-215 and Thr-221 in ABA signaling (Figure 5, E and F). Taken together, our results suggest that the phosphorylation of OsMKK1 by both OsDMI3 and OsMKKK is independent, and the full activation of OsMKK1 requires both OsDMI3 and OsMKKK in ABA signaling (Figure 9).
In Arabidopsis, several complete MAPK cascades, such as MAPKKK17/18–MKK3–MPK1/2/7/14 (Danquah et al., 2015; Matsuoka et al., 2015) and MAPKKK20–MKK5–MPK6 (Li et al., 2017b), have been shown to be involved in ABA signaling. However, the exact OsMKKK for the ABA-induced activation of the OsMKK1–OsMPK1 cascade is unknown. Moreover, in this study, Ca2+ was shown to be required for ABA-induced activation of OsMKK1, but the Ca2+-induced increase in OsMKK1 activity was not completely blocked in osdmi3-KO1 plants (Supplemental Figure S9), indicating that the other Ca2+-dependent pathway is also involved in ABA-induced activation of OsMKK1. Accumulating data have demonstrated that the receptor-like protein kinases/receptor-like proteins are key regulators of MAPK cascades (Xu and Zhang, 2015; Zhang et al., 2018). CRLK1, a Ca2+/CaM-regulated receptor-like kinase, has been reported to play a critical role in plant responses to cold stress (Yang et al., 2010a; Zhao et al., 2017). CRLK1 physically interacts with and phosphorylates MEKK1 (Yang et al., 2010b; Furuya et al., 2013). CRLK1 and CRLK2 positively regulate the cold response, possibly by activating the MEKK1–MKK2–MPK4 pathway and by suppressing the cold activation of the MKK4/5–MPK3/6 pathway (Zhao et al., 2017). However, whether such a Ca2+/CaM-regulated receptor-like kinase is involved in the ABA-activated OsMKKK–OsMKK1–OsMPK1 cascade remains to be determined (Figure 9).
Materials and methods
Plant materials and constructs
Rice (Oryza sativa) plants used in this study include Nipponbare (WT), osdmi3, and OsDMI3 (Ni et al., 2019). Rice seedlings were grown under previously described conditions (Zhang et al., 2014). When the second leaves were fully expanded, they were collected and used for various investigations.
All transgenic lines, including the OE lines of OsMKK1, OsMKK6, and OsABA2, the KO lines of osmkk1, osmkk6, and osaba2, the osdmi3-KO mutant plants OE OsMKK1 (osdmi3/OsMKK1), OsMKK1T25A (osdmi3/OsMKK1T25A), and OsMKK1T25D (osdmi3/OsMKK1T25D), the osmkk1-KO mutant plants OE OsDMI3 (osmkk1/OsDMI3), and the double mutant osmkk1 osdmi3, were generated by Biogel Company (Hangzhou, China). To generate OsMKK1, OsMKK6, OsMKK1T25A, OsMKK1T25D, OsABA2, and OsDMI3 constructs, OsMKK1, OsMKK6, and the mutated OsMKK1 coding sequences were inserted into the pCAMBIA 1304 vector by NcoI and SpeI sites, the coding sequence of OsDMI3 was inserted into the pCAMBIA 1304 vector by KpnI and SpeI sites, and the coding sequence of OsABA2 was inserted into the pCAMBIA 1304 vector by NcoI and KpnI sites. The constructs were introduced into Agrobacterium strain EHA105 and transformed into rice (O. sativa sub. japonica cv Nipponbare) under the control of the CaMV 35S promoter. The transgenic plants were selected on 50 μg mL−1 hygromycin. The homozygous T3 seeds of transgenic plants were used for further analysis.
To generate the osmkk1, osmkk6, osaba2, and osmkk1 osdmi3 mutant plants, the CRISPR/Cas9 system was used. The sgRNAs of OsMKK1, OsMKK6, OsABA2, and OsDMI3 are shown in Supplemental Figures S7, S15, and S16G. The single sgRNA was created in the BGK03 vector containing Cas9. The constructs were introduced into rice (O. sativa sub. japonica cv Nipponbare) by Agrobacterium-mediated transformation. Positive transgenic individuals were identified by sequencing analyses (for primers, Supplemental Table S3).
Y2H assay
Y2H analysis was performed using the Matchmaker Gold Y2H System (Clontech, Mountain View, CA, USA ) according to the manufacturer’s protocol. Full-length OsMKK1/OsMKK6 were separately cloned into pGADT7 vector at the SmaI-BamHI sites and the EcoRI-SmaI sites, and the constructs were transformed into the yeast strain Y187. The transformed Y187 yeast strain was mated with the Y2HGold yeast strain containing pGBKT7-OsDMI3 (Ni et al., 2019). The mating yeast cultures were spread on plates (SD/-Trp/-Leu/-His/-Ade) containing X-α-gal (40 μg mL−1) and AbA (0.125 μg mL−1). To determine the interaction intensity, dilutions of yeast cultures (10−0, 10−1, and 10−2) were spotted onto selection medium for blue color development. Photographs were taken after 5–7 days incubation at 30°C.
GST pull-down assay
OsDMI3-GST fusion protein was produced in Escherichia coli Rosetta (DE3; Novagen, Madison, WI, USA), and tested for interaction as described previously (Ni et al., 2019). Full-length coding sequences of OsMKK1/OsMKK6 were cloned into the pET30a vector (Novagen) at the KpnI-BamHI sites to generate His tag fusion proteins. Expression of His-OsMKK1/OsMKK6 in E. coli Rosetta (DE3) cells was induced with 0.5 mM isopropyl-β-D-thiogalactoside at 24°C for 6 h. Fusion proteins were purified using MagnetHis protein purification system (Promega, Madison, WI, USA) according to the manufacturer’s protocol.
For pull-down assay, GST or GST-OsDMI3 immobilized on the Magnet GST Particles (Promega) were incubated with His-OsMKK1/OsMKK6 in GST binding buffer (2 mM KH2PO4, 4.2 mM Na2HPO4, 10 mM KCl, 140 mM NaCl, 10% bovine serum albumin, pH 7.5) at 4°C for 1 h. The particles were washed three times with GST wash buffer (similar to GST binding buffer but without bovine serum albumin), separated on 12% SDS-PAGE gel, and detected by immunoblotting with anti-His antibody (Abmart, Shanghai, China; lot: 283874, 1:1,000, v/v) or anti-GST antibody (Abmart; lot:264160; 1:1,000, v/v).
BiFC assay
Full-length coding regions of OsMKK1/OsMKK6 were separately amplified and cloned into the BamHI-KpnI sites and the ClaI-KpnI sites of the pSPYNE vector to generate the pSPYNE-OsMKK1/OsMKK6 constructs. The pSPYCE-OsDMI3 construct was generated as described previously (Ni et al., 2019). The corresponding constructs were transiently cotransformed in onion epidermis cells using the particle bombardment (Bio-Rad) method as described previously (Lee et al., 2008). The YFP fluorescent signals were observed at 16 h after transformation using a confocal scanning microscope (Zeiss, Oberkochen, Germany).
Co-IP assay
For Co-IP assay, the construct OsDMI3-Myc was generated as described previously (Ni et al., 2019). OsMKK1/OsMKK6 were fused with Flag and separately cloned into the pXZP008 vector at BamHI-KpnI sites and SaII-KpnI sites to generate OsMKK1/OsMKK6-Flag constructs. The constructs were transiently expressed in rice protoplasts via PEG-mediated transfection (Ni et al., 2019). After 16 h incubation, the proteins of transfected protoplasts were extracted with Co-IP buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM Na3VO4, 10 mM NaF, 10% glycerol, 5 μg mL−1 leupeptin, 5 μg mL−1 aprotinin, 0.5% (v/v) Triton X-100, 0.5% (v/v) Nonidet P-40). The proteins were incubated with anti-Myc antibody bound to protein A beads for 2–3 h. The beads were collected and washed 3 times with wash buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM Na3VO4, 10 mM NaF, 10% glycerol, 0.5% [v/v] Triton X-100, 0.5% [v/v] Nonidet P-40), and then were boiled in 1× SDS loading buffer (50 mM Tris–HCl, 2% SDS, 0.1% bromophenol blue, 10% glycerol, 10 mM DTT). The immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotting with anti-Flag antibody (Abmart; lot:293674; 1:1,000, v/v).
LCI assay
For LCI assay, the full-length OsMKK1 and OsDMI3 were ligated into the BamHI/KpnI sites of the pC1300-nLUC vector and the KpnI/PstI sites of the pC1300-cLUC vector, respectively. Both the nLUC- and cLUC-fused constructs were transformed into Nicotiana benthamiana leaves using the Agrobacterium-mediated infiltration method (Ni et al., 2019). After 3 days of infiltration, luciferase activity was analyzed using chemiluminescence imaging (Tanon 5200 Multi, Tanon Biomart).
Immunocomplex kinase activity assay
Protein was extracted from rice leaves with buffer (100 mM Hepes, 10 mM Na3VO4, 10 mM NaF, 10% glycerol, 5 μg mL−1 leupeptin, 5 μg mL−1 aprotinin, 1 mM PMSF, 10 mM DTT). Protein content was determined by the method of Bradford (1976). For the determination of OsMKK1 and OsMKK6 activity, the total proteins (200 μg) were bound with anti-OsMKK1 antibody or anti-OsMKK6 antibody (2 μg, Abmart) in IP buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM Na3VO4, 10 mM NaF, 10% glycerol, 0.5% [v/v] Triton X-100, 0.5% [v/v] Nonidet P-40) overnight, and then incubated with protein A-sepharose beads for another 3 h. The immunoprecipitated proteins were incubated with 1 μg MBP (Sigma-Aldrich, St. Louis, MO, USA) in kinase buffer (25 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 0.1 mM Na3VO4, and 1 mM DTT) containing 10 μCi [γ32P]-ATP (3,000 Ci mM−1) at 30°C for 30 min. The reaction products were separated by 12% SDS-PAGE and analyzed by autoradiography using X-ray film or a phosphostorage screen (Typhoon TRIO; Amersham Biosciences, Little Chalfont, UK). To determine OsDMI3 and OsMPK1 activity, the immunocomplex kinase activity assay was performed as described previously (Ni et al., 2019). The relative levels of OsDMI3, OsMKK1, OsMKK6, and OsMPK1 activity were quantified by ImageJ, and were presented as values relative to those of the corresponding controls.
SDS-PAGE and IB analysis
Protein extracts (20 μg) were separated by 12% SDS-PAGE. After electrophoresis, the gel was transferred to a polyvinylidene difluoride membrane, and then incubated in phosphate-buffered saline/Tween (PBST) buffer (140 mM NaCl, 10 mM KCl, 2 mM KH2PO4, 8 mM Na2HPO4, 0.1% Tween-20 [v/w], pH 7.5) containing 5% (w/v) nonfat dry milk for 2 h at room temperature. The membrane was then washed 3 times with PBST buffer for 5 min. The blots were probed with anti-OsDMI3 antibody (ABclonal, Woburn, MA, USA; lot A17593), anti-OsMKK1 antibody (Abmart; lot 514727), anti-OsMKK6 antibody (Abmart; lot 514726), anti-OsMPK1 antibody (Beijing Protein Innovation, Beijing, China; lot AbP80140-A-SE), anti-pT25 OsMKK1 antibody (GenScript, Piscataway, NY, USA; lot C1450DA160), anti-ACT1 antibody (Beijing Protein Innovation; lot AbP80243-A-SE), anti-Myc antibody (Abmart; lot 294166), anti-Flag antibody (Abmart; lot 293674), anti-His antibody (Abmart; lot 283874), anti-GST antibody (Abmart; lot 264160), anti-GAL AD antibody (Abmart; lot 273164), and anti-HA antibody (Abmart; lot 294066). The anti-OsMKK1 antibody was raised against peptide (KDGLRIVSQSEE) of OsMKK1, the anti-OsMKK6 antibody was raised against peptide (IKKFEDKDLDLR) of OsMKK6, and the anti-pT25 OsMKK1 antibody was raised against peptide (LTQSGTFKDGDLLVN) of OsMKK1. The information on the other antibodies was described previously (Ni et al., 2019). First antibody and secondary antibody were used at 1:1,000 and 1:5,000 dilution, respectively. Chemiluminescence was detected with the enhanced chemiluminescence immunoblotting detection system (GE Healthcare, Chicago, IL, USA) and a camera (Tanon 5200 Multi, Tanon).
In vitro kinase assay
Total protein of rice leaves was extracted as described above, and the protein extract (200 μg) was incubated with anti-OsDMI3 antibody (2 μg; Abmart) and IP buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM Na3VO4, 10 mM NaF, 10% glycerol, 0.5% [v/v] Triton X-100, 0.5% [v/v] Nonidet P-40) to obtain the kinase OsDMI3. OsDMI3 was then incubated with 20 μg substrates (His-OsMPK1, His-OsMKK6, His-OsMKK1, and His-OsMKK1T25A) and 10 μCi [γ32P]-ATP in reaction buffer (25 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 0.5 mM CaCl2, 2 μM CaM [Sigma-Aldrich], 1 mM DTT) at 30°C for 30 min to perform the in vitro kinase assay. The reaction was stopped by mixing 5×SDS loading buffer, and the reaction mixtures were separated by 12% SDS-PAGE. The phosphorylated substrates were visualized by autoradiography.
For the in vitro kinase assay of OsMKK1 activity, 10 μg of His-OsMKK1, His-OsMKK1T25A, His-OsMKK1T25D, His-OsMKK1S215A/T221A, and His-OsMKK1S215D/T221D were respectively incubated with 1 μg MBP in kinase buffer (25 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 0.1 mM Na3VO4 and 1 mM DTT) and 10 µCi [γ-32P]-ATP at 30°C for 30 min. SDS sample buffer was then added to stop the reaction. After separation by 12% SDS-PAGE, the phosphorylated MBP was visualized by autoradiography.
In vitro reconstitution assay of OsMPK1 phosphorylation
Ten micrograms of His-OsMKK1 or His-OsMKK1T25A or His-OsMKK1T25D was incubated with 20 μg GST-OsMPK1 in reaction buffer (25 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 2.5 mM MnCl2, 0.5 mM CaCl2 and 1 mM DTT) containing 10 µCi [γ-32P]-ATP at 30°C for 30 min. The reaction mixtures were then incubated with or without OsDMI3 pulled down from extracts of ABA-treated leaves at 30°C for another 30 min. The phosphorylated OsMPK1 was analyzed as described above (in vitro kinase assay).
In vivo phosphorylation assay
The total proteins were extracted from rice leaves with buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM Na3VO4, 10 mM NaF, 10% glycerol, 5 μg mL−1 leupeptin, 5 μg mL−1 aprotinin, 0.5% [v/v] Triton X-100, 0.5% [v/v] Nonidet P-40, 1% [v/v] phosphatase inhibitor cocktail 3 [Sigma-Aldrich]). OsMKK1 and OsMKK6 proteins were immunoprecipitated by anti-OsMKK1 antibody (Abmart; lot 514727; 1:1000, v/v) or anti-OsMKK6 antibody (Abmart; lot 514726; 1:1,000, v/v) bound to protein A beads, and separated by 12% SDS-PAGE. Phosphorylated proteins were detected by immunoblotting using Biotinylated Phos-tag as described previously (Kinoshita-Kikuta et al., 2007).
Mass spectrometry analysis
OsMKK1 was incubated with 1 μg CIAP in 100 μL of phosphatase buffer (50 mM KAc, 20 mM Tris–HAc, pH 7.9, 10 mM Mg(Ac)2, 100 μg mL−1 BSA) at 37°C for 10 min. The phosphatase was deactivated by heating at 80°C for 2 min. The dephosphorylated OsMKK1 was then incubated with OsDMI3 and ATP (200 nM) in kinase reaction solution (25 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 0.5 mM CaCl2, 2 μM CaM, 1 mM DTT) at 30°C for 30 min. The phosphorylated OsMKK1 was digested by trypsin and analyzed by MS as described previously (Gampala et al., 2007). The phosphopeptide sequence of OsMKK1 identified by LC–MS/MS analysis is listed in Supplemental Table S1.
PRM analysis
OsMKK1-Flag was introduced into rice protoplasts of OsDMI3-OE1, osdmi3-KO1, and WT respectively, and the transfected protoplasts were treated with 10 μM ABA for 10 min after 16 h incubation. Protein was extracted from protoplasts with buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM Na3VO4, 10 mM NaF, 10% glycerol, 5 μg mL−1 leupeptin, 5 μg mL−1 aprotinin, 0.5% [v/v] Triton X-100, 0.5% [v/v] Nonidet P-40, 1% [v/v] phosphatase inhibitor cocktail 3 [Sigma-Aldrich]), and the protein extracts were immunoprecipitated using anti-Flag antibody and separated by 12% SDS-PAGE. A pretest was performed to verify if the target phosphopeptides were detected by MS after the OsMKK1 protein was digested by trypsin. LC-PRM MS analysis (Peterson et al., 2012) was applied to quantify the target phosphopeptides. Briefly, the label-free protocol was used for phosphopeptide preparation. Each sample was spiked with the stable isotope-containing AQUA peptide as a standard internal reference, and tryptic peptides were loaded on stage tips of C18 for desalting prior to reversed-phase chromatography on one of the nLC-1200 easy systems (Thermo Scientific, Waltham, MA, USA). Then, 1 h LC gradients were performed with 5–35% acetonitrile for 45 min. Then, Q Exactive Plus MS was applied for PRM analysis. The raw data were analyzed via Skyline (MacCoss Lab, University of Washington; MacLean et al., 2010), wherein the intensity of signal produced by a certain phosphopeptide sequence could be quantified with respect to each sample and referenced to standards via normalization for each protein.
Site-directed mutagenesis
The mutated variants were achieved by site-directed mutagenesis using the Multi-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. The sequences of DNA oligonucleotides used in mutagenesis are listed in Supplemental Table S2.
Quantitative RT-PCR analyses
Total RNA was extracted with the RNAiso Plus kit (TaKaRa, Kyoto, Japan). Approximately 2 μg of total RNA was reverse-transcribed using a PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). Real-time quantitative RT-PCR was performed with TB Green Premix Ex Taq (TakaRa) using a 7500 real-time PCR system (Applied Biosystems Inc., Foster City, CA, USA). The expression level was normalized against that of the rice glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. The primers used are listed at Supplemental Table S3.
Phenotype analysis
For seed germination assay, the seeds of WT, OsMKK1, osmkk1, OsMKK1T25A, OsMKK1T25D, OsDMI3, osdmi3, osmkk1 osdmi3, osmkk1 OsDMI3, osdmi3 OsMKK1, osdmi3 OsMKK1T25A, and osdmi3 OsMKK1T25D were surface-sterilized and germinated on half-strengthMurashige and Skoog (MS) medium (0.8% [w/v] agar) with different concentrations of ABA (0, 1, and 5 μM). The seeds were incubated at 4°C for 2 days and then transferred to a growth chamber (16 h light/8 h dark; bulb-type fluorescent light; 200 μmol m−2 s−1 light intensity; 25°C) to germinate. The germination rate of seeds was scored at the indicated times.
For the detection of root growth, 4-day-old rice seedlings grown in the normal conditions were transferred to a nutrient solution containing different concentrations of ABA (0, 0.5, 1, 2.5, 5, and 10 μM) for another 8 days, and then the length of primary roots was measured.
For the analysis of survival rate, 10-day-old rice seedings were treated with 20% PEG 4000, 100 mM H2O2 for 12 or 18 days, and the survival rates were measured after recovery by re-watering for 7 days.
For the determination of oxidative damage to lipids and plasma membranes, the rice seedings were treated with 20% PEG 4000 or 100 mM H2O2 for 2 days, and the content of MDA and the percentage of electrolyte leakage were determined by the methods described previously (Jiang and Zhang, 2001).
For the determination of water loss, the fully expanded leaves of 10-day-old rice seedlings were detached, weighed, and placed on the laboratory bench. Weight loss of the detached leaves was monitored at the indicated time intervals as shown in Supplemental Figure S20C. Water loss was expressed as the percentage of initial fresh weight.
Determination of ABA content
Fresh leaves (0.5 g) of 10-day-old rice seedlings were collected, frozen in liquid nitrogen and ground in a mortar, and extracted in 7 mL extraction solution (methanol:water:acetic acid, 80:19:1) overnight at 4°C. After centrifugation at 8,000g for 20 min at 4°C, the supernatant was collected and eluted through a Sep-Pak C18 cartridge (Waters, Milford, MA, USA), which was preconditioned with methanol. The total extract solution was dried under nitrogen gas and then dissolved in 500 μL mobile phase (methanol:1% acetic acid, 45:55). The extract was filtered through 0.45 μm membrane filters before injection into the high-performance LC system. Detection was done with an absorbance detector at a wavelength of 254 nm. Quantification was obtained by comparing the peak areas with those of known amounts of ABA (Sigma, Shanghai, China).
Alignment analysis
Sequences of plant MKK1 proteins from different species were retrieved from NCBI and aligned using Alignx. The GenBank accession numbers are as follows: ZmMKK1, BT065734; AtMKK1, AT4g26070; GmMKK1, AY070230; and MtMKK1, AC144503.
Statistical analysis
Statistical analysis was conducted using the program SPSS Statistics version 22.0 (IBM, Armonk, NY, USA). Data were analyzed by ANOVA followed by Duncan’s multiple range test (P < 0.05 as the level of significance). The results of statistical analyses are shown in Supplemental Data Set S1.
Accession numbers
Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: OsDMI3, LOC_Os05g41090; OsMPK1, LOC_Os06g06090; OsACT1, LOC_Os03g50885; OsMKK1, LOC_Os06g05520; OsMKK6, LOC_Os01g32660; OsMKK3, LOC_Os06g27890; OsMKK4, LOC_Os02g54600; OsMKK5, LOC_Os06g09180; OsMKK10-2, LOC_Os03g12390; OsABA2, LOC_Os03g59610; OsGAPDH, LOC_Os02g38920.
Supplemental data
The following supplemental materials are available in the online version of this article.
Supplemental Figure S1. OsDMI3 does not interact with OsMPK1 and does not phosphorylate OsMPK1.
Supplemental Figure S2. OsDMI3 interacts with the group A MKKs, OsMKK1, and OsMKK6.
Supplemental Figure S3. OsMKK1 interacts with the EF-hand domain of OsDMI3.
Supplemental Figure S4. OsDMI3 interacts with the N-terminus domain of OsMKK1.
Supplemental Figure S5. OsMKK6 interacts with the EF-hand domain of OsDMI3.
Supplemental Figure S6. OsDMI3 interacts with the N-terminus domain of OsMKK6.
Supplemental Figure S7. Identification of osmkk1-KO and osmkk6-KO mutants.
Supplemental Figure S8. The specificity of the anti-OsMKK1 antibody and the anti-OsMKK6 antibody.
Supplemental Figure S9. Ca2+ is required for ABA-induced activation of OsMKK1.
Supplemental Figure S10. ABA-induced activation of OsMKK6 is Ca2+-independent.
Supplemental Figure S11. OsMKK1 does not affect the activation of OsDMI3 in ABA signaling.
Supplemental Figure S12. OsDMI3 phosphorylates OsMKK1 but not OsMKK6 in ABA signaling.
Supplemental Figure S13. Identification of the phosphorylation site of OsMKK1 by OsDMI3 in vitro.
Supplemental Figure S14. The specificity of the anti-pT25 OsMKK1 antibody.
Supplemental Figure S15. Identification of osaba2-KO mutant.
Supplemental Figure S16. The expression of OsMKK1 and OsDMI3 and the levels of OsMKK1 and OsDMI3 proteins in the transgenic lines.
Supplemental Figure S17. Phosphorylation of OsMKK1 at Thr-25 does not affect the interaction between OsDMI3 with OsMKK1 and between OsMKK1 with OsMPK1.
Supplemental Figure S18. Identification of the phosphorylated peptides of OsMKK1 in rice protoplasts.
Supplemental Figure S19. The phosphorylation of both Ser-215 and Thr-221 in OsMKK1 is required for the activation of OsMKK1 in ABA signaling.
Supplemental Figure S20. The OsDMI3-OsMKK1 pathway regulates the physiological response to water stress and oxidative stress.
Supplemental Table S1. Phosphopeptide sequence of OsMKK1 identified by LC–MS/MS analysis.
Supplemental Table S2. DNA oligonucleotides used for site-directed mutagenesis.
Supplemental Table S3. PCR primers used in this study.
Supplemental Data Set S1. Statistical analysis of ANOVA results for the data shown in the figures.
Supplementary Material
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant nos. 31671606 and 31971824) and the National Basic Research Program of China (Grant no. 2012CB114306).
Conflict of interest statement: None declared.
M. J. conceived the project. M.J. and M.C. designed the experiments. M.C. performed most of the experiments. J.C., M.S., C.Q., and G.Z. performed some of the experiments. M.J., L.N., and A.Z. analyzed data. M.J., M.C., and L.N. wrote the manuscript.
The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell) is: Mingyi Jiang (myjiang@njau.edu.cn).
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