MPK3- and MPK6-Mediated ICE1 Phosphorylation Negatively Regulates ICE1 Stability and Freezing Tolerance in Arabidopsis

•Cold activates mitogen-activated protein kinases MPK3 and MPK6•MPK3/MPK6 phosphorylate and destabilize the ICE1 protein•MPK3/MPK6 activation attenuates plant freezing tolerance Low temperatures affect plant growth, development, productivity, and ecological distribution. Expression of the C-repeat-binding factor (CBF) transcription factors is induced by cold stress, which in turn activates downstream cold-responsive (COR) genes that are required for the acquisition of freezing tolerance. Inducer of CBF expression 1 (ICE1) is a master regulator of CBFs, and ICE1 stability is crucial for its function. However, the regulation of ICE1 is not well understood. Here, we report that mitogen-activated protein kinase 3 (MPK3) and MPK6 interact with and phosphorylate ICE1, which reduces its stability and transcriptional activity. Consistently, the mpk3 and mpk6 single mutants and the mpk3 mpk6 double mutants show enhanced freezing tolerance, whereas MPK3/MPK6 activation attenuates freezing tolerance. Phosphor-inactive mutations of ICE1 complement freezing sensitivity in the ice1-2 mutant. These combined results indicate that MPK3/MPK6 phosphorylate and destabilize ICE1, which negatively regulates CBF expression and freezing tolerance in plants. Low temperatures affect plant growth, development, productivity, and ecological distribution. Expression of the C-repeat-binding factor (CBF) transcription factors is induced by cold stress, which in turn activates downstream cold-responsive (COR) genes that are required for the acquisition of freezing tolerance. Inducer of CBF expression 1 (ICE1) is a master regulator of CBFs, and ICE1 stability is crucial for its function. However, the regulation of ICE1 is not well understood. Here, we report that mitogen-activated protein kinase 3 (MPK3) and MPK6 interact with and phosphorylate ICE1, which reduces its stability and transcriptional activity. Consistently, the mpk3 and mpk6 single mutants and the mpk3 mpk6 double mutants show enhanced freezing tolerance, whereas MPK3/MPK6 activation attenuates freezing tolerance. Phosphor-inactive mutations of ICE1 complement freezing sensitivity in the ice1-2 mutant. These combined results indicate that MPK3/MPK6 phosphorylate and destabilize ICE1, which negatively regulates CBF expression and freezing tolerance in plants. Low temperatures substantially attenuate plant growth, development, and geographical distribution, and adversely affect crop quality and productivity. Temperate plants can acquire cold tolerance after exposure to low but nonfreezing temperatures (called cold acclimation), which enhances their survival under freezing stress. 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ICE1 encodes a MYC-like basic-helix-loop-helix transcription factor that binds to canonical MYC cis-elements (CANNTG) in CBF promoters and activates their expression (Chinnusamy et al., 2003Chinnusamy V. Ohta M. Kanrar S. Lee B.H. Hong X. Agarwal M. Zhu J.K. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.Genes Dev. 2003; 17: 1043-1054Crossref PubMed Scopus (1206) Google Scholar, Ding et al., 2015Ding Y. Li H. Zhang X. Xie Q. Gong Z. Yang S. OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis.Dev. Cell. 2015; 32: 278-289Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Emerging evidence indicates that ICE1 is regulated posttranslationally by several factors, including high expression of osmotically responsive gene 1 (HOS1), SAP and Miz (SIZ1), and open stomata 1 (OST1) (Ding et al., 2015Ding Y. Li H. Zhang X. Xie Q. Gong Z. Yang S. OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis.Dev. Cell. 2015; 32: 278-289Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, Dong et al., 2006Dong C.H. Agarwal M. Zhang Y. Xie Q. Zhu J.K. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1.Proc. Natl. Acad. Sci. USA. 2006; 103: 8281-8286Crossref PubMed Scopus (511) Google Scholar, Miura et al., 2007Miura K. Jin J.B. Lee J. Yoo C.Y. Stirm V. Miura T. Ashworth E.N. Bressan R.A. Yun D.J. Hasegawa P.M. SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis.Plant Cell. 2007; 19: 1403-1414Crossref PubMed Scopus (577) Google Scholar). HOS1 acts as a RING-type ubiquitin E3 ligase that ubiquitinates and mediates the cold-induced degradation of ICE1, which in turn negatively regulates CBF expression at low temperatures (Dong et al., 2006Dong C.H. Agarwal M. Zhang Y. Xie Q. Zhu J.K. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1.Proc. Natl. Acad. Sci. USA. 2006; 103: 8281-8286Crossref PubMed Scopus (511) Google Scholar). SIZ1-mediated ICE1 sumoylation stabilizes ICE1 and controls CBF expression (Miura et al., 2007Miura K. Jin J.B. Lee J. Yoo C.Y. Stirm V. Miura T. Ashworth E.N. Bressan R.A. Yun D.J. Hasegawa P.M. SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis.Plant Cell. 2007; 19: 1403-1414Crossref PubMed Scopus (577) Google Scholar). Our recent study showed that OST1/SnRK2.6, a Ser/Thr protein kinase, originally identified in ABA signaling, is activated by cold stress, subsequently interacts with and phosphorylates ICE1, which suppresses HOS1-mediated ICE1 degradation and positively regulates CBF expression and freezing tolerance (Ding et al., 2015Ding Y. Li H. Zhang X. Xie Q. Gong Z. Yang S. OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis.Dev. Cell. 2015; 32: 278-289Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Previous studies showed that MYB15 and jasmonate ZIM-domain 1/4 also physically interact with ICE1 to repress CBF expression and negatively regulate freezing tolerance (Agarwal et al., 2006Agarwal M. Hao Y. Kapoor A. Dong C.H. Fujii H. Zheng X. Zhu J.K. A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance.J. Biol. Chem. 2006; 281: 37636-37645Crossref PubMed Scopus (623) Google Scholar, Hu et al., 2013Hu Y. Jiang L. Wang F. Yu D. Jasmonate regulates the inducer of CBF expression-C-repeat binding factor/DRE binding factor1 cascade and freezing tolerance in Arabidopsis.Plant Cell. 2013; 25: 2907-2924Crossref PubMed Scopus (465) Google Scholar). 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Plant Biol. 2010; 61: 621-649Crossref PubMed Scopus (780) Google Scholar). In plants, MAP3Ks are activated by stimulated plasma membrane receptors. Activated MAP3Ks phosphorylate and activate MAP2Ks, which in turn phosphorylate and activate MAPKs (Rodriguez et al., 2010Rodriguez M.C. Petersen M. Mundy J. Mitogen-activated protein kinase signaling in plants.Annu. Rev. Plant Biol. 2010; 61: 621-649Crossref PubMed Scopus (780) Google Scholar). Several lines of evidence suggest that the MAPK pathway mediated by MEKK1-MKK2-MPK4/6 has a positive role in plant responses to cold stress (Teige et al., 2004Teige M. Scheikl E. Eulgem T. Doczi R. Ichimura K. Shinozaki K. Dangl J.L. Hirt H. The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis.Mol. Cell. 2004; 15: 141-152Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar, Yang et al., 2010bYang T. Shad Ali G. Yang L. Du L. Reddy A.S. Poovaiah B.W. Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants.Plant Signal. Behav. 2010; 5: 991-994Crossref PubMed Scopus (88) Google Scholar). MPK4 and MPK6 are activated by cold stress in plants (Ichimura et al., 2000Ichimura K. Mizoguchi T. Yoshida R. Yuasa T. Shinozaki K. Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6.Plant J. 2000; 24: 655-665Crossref PubMed Google Scholar, Teige et al., 2004Teige M. Scheikl E. Eulgem T. Doczi R. Ichimura K. Shinozaki K. Dangl J.L. Hirt H. The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis.Mol. Cell. 2004; 15: 141-152Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). MKK2 is also activated by cold stress in plants, and consequently activates MPK4 and MPK6 (Teige et al., 2004Teige M. Scheikl E. Eulgem T. Doczi R. Ichimura K. Shinozaki K. Dangl J.L. Hirt H. The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis.Mol. Cell. 2004; 15: 141-152Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). CRLK1 interacts with MEKK1, leading to MAPK activation and freezing tolerance (Yang et al., 2010bYang T. Shad Ali G. Yang L. Du L. Reddy A.S. Poovaiah B.W. Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants.Plant Signal. Behav. 2010; 5: 991-994Crossref PubMed Scopus (88) Google Scholar). These results suggest that the MAPK cascade participates in plant cold-stress responses. However, the underlying mechanisms of MAPK activity in cold signaling have not been elucidated. Here, we show that MPK3 and MPK6 interact with and phosphorylate ICE1 in Arabidopsis thaliana. MPK3/MPK6 phosphorylation of ICE1 inhibits its transcriptional activity and facilitates ubiquitination-mediated ICE1 degradation under cold stress, thereby negatively regulating plant freezing tolerance and CBF expression. Our results identify a mechanism in which MPK3/MPK6 negatively regulate plant freezing tolerance by phosphorylating and destabilizing ICE1. ICE1 is regulated by multiple posttranslational modifications, which are crucial for its stability and transcriptional activity (Ding et al., 2015Ding Y. Li H. Zhang X. Xie Q. Gong Z. Yang S. OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis.Dev. Cell. 2015; 32: 278-289Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, Dong et al., 2006Dong C.H. Agarwal M. Zhang Y. Xie Q. Zhu J.K. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1.Proc. Natl. Acad. Sci. USA. 2006; 103: 8281-8286Crossref PubMed Scopus (511) Google Scholar, Miura et al., 2007Miura K. Jin J.B. Lee J. Yoo C.Y. Stirm V. Miura T. Ashworth E.N. Bressan R.A. Yun D.J. Hasegawa P.M. SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis.Plant Cell. 2007; 19: 1403-1414Crossref PubMed Scopus (577) Google Scholar). To further explore ICE1 regulation, we performed a yeast two-hybrid assay to identify interacting proteins. The full-length ICE1 protein had strong self-activation activity, whereas the C-terminal region of ICE1 (from 358 to 494 amino acids, required for protein-protein interaction) abolished its activity (Agarwal et al., 2006Agarwal M. Hao Y. Kapoor A. Dong C.H. Fujii H. Zheng X. Zhu J.K. A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance.J. Biol. Chem. 2006; 281: 37636-37645Crossref PubMed Scopus (623) Google Scholar). So we chose this C-terminal region cloned into the pGBKT7 vector as the bait in our assay (Figure S1A). This experiment identified MPK3 and MPK6 as interacting proteins, and they were selected for further study. We performed yeast two-hybrid to confirm the interaction of full-length MPK3/MPK6 with full-length ICE1 (Ding et al., 2015Ding Y. Li H. Zhang X. Xie Q. Gong Z. Yang S. OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis.Dev. Cell. 2015; 32: 278-289Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar) (Figure 1A). Then, we examined the ICE1 functional domains required for interaction with MPK3/MPK6 in yeast. ICE1 contains a serine-rich region at the N terminus, and a MYC-like basic-helix-loop-helix domain and possible zipper region at the C terminus (Figure 1B). The ICE1 C-terminal region strongly interacted with MPK3 and MPK6, whereas deletion of the C-terminal region reduced these interactions, indicating that the ICE1 C-terminal region is required for interaction with MPK3/MPK6 in yeast (Figures 1B and S1B). Next, we performed a glutathione S-transferase (GST) pull-down assay to confirm the interactions between MPK3/MPK6 and ICE1 in vitro. We tagged ICE1 with 6×His and incubated the tagged protein with GST-MPK3, GST-MPK6, or GST alone. The results showed that His-ICE1 was pulled down by GST-MPK3 and GST-MPK6, but not by GST alone (Figure 1C), indicating that ICE1 directly binds to MPK3/MPK6 in vitro. We performed a coimmunoprecipitation (coIP) assay using Nicotiana benthamiana leaves transiently co-expressing 35S:HA-Flag-MPK3/MPK6 (35S:HF-MPK3/MPK6) and 35S:Myc-ICE1 constructs. Protein extracts were immunoprecipitated with anti-hemagglutinin (HA) agarose, and the precipitated proteins were analyzed by immunoblotting with anti-Myc antibody. A band with the expected mobility of Myc-ICE1 was successfully detected in the anti-HA immunoprecipitates of leaves expressing HF-MPK3 and HF-MPK6 (Figure 1D). By contrast, no Myc-ICE1 was detected in the anti-HA immunoprecipitates of leaves expressing HF-tagged empty vector. These combined results indicate that MPK3 and MPK6 interact with ICE1 in vitro and in vivo. We hypothesized that the ICE1 interacting proteins MPK3 and MPK6 might be involved in plant cold-stress responses. Therefore, we performed freezing tolerance assays using the following Arabidopsis knockout mutants: mpk3-1 (Zhao et al., 2014Zhao C. Nie H. Shen Q. Zhang S. Lukowitz W. Tang D. EDR1 physically interacts with MKK4/MKK5 and negatively regulates a MAP kinase cascade to modulate plant innate immunity.PLoS Genet. 2014; 10: e1004389Crossref PubMed Scopus (101) Google Scholar), mpk3-2, mpk6-3, and mpk6-4 (Xu et al., 2008Xu J. Li Y. Wang Y. Liu H. Lei L. Yang H. Liu G. Ren D. Activation of MAPK kinase 9 induces ethylene and camalexin biosynthesis and enhances sensitivity to salt stress in Arabidopsis.J. Biol. Chem. 2008; 283: 26996-27006Crossref PubMed Scopus (258) Google Scholar). The mpk3 and mpk6 mutants displayed substantially increased freezing tolerance (reflected by survival rate) under non-acclimated (NA) and cold-acclimated (CA) conditions compared with the wild-type Columbia (Col) (Figures 2A−2D ). Next, we examined ion leakage, which is an indicator of stress-induced plasma membrane damage, in the mpk3 and mpk6 mutants. Ion leakage in the mpk3 and mpk6 mutants under NA and CA conditions was consistently lower than that in the wild type (Figures 2E and 2F). These results suggest that MPK3 and MPK6 are negative regulators of basal and acquired freezing tolerance in Arabidopsis. To further study the role of MPK3 and MPK6 in the freezing stress response, we examined freezing tolerance in the conditional loss-of-function mpk3 mpk6 double mutant, named MPK3SR (mpk3 mpk6 pMPK3:MPK3TG). The mpk3 mpk6 loss-of-function mutant is lethal, but can be rescued with MPK3TG, which is a 4-amino-1-tert-butyl-3-(1′-naphthyl)pyrazolo[3, 4-d]pyrimidine (NA-PP1)-sensitized version of MPK3. MPK3TG loses its function after the addition of NA-PP1 (Su et al., 2017Su J. Zhang M. Zhang L. Sun T. Liu Y. Lukowitz W. Xu J. Zhang S. Regulation of stomatal immunity by interdependent functions of a pathogen-responsive MPK3/MPK6 cascade and abscisic acid.Plant Cell. 2017; 29: 526-542Crossref PubMed Scopus (90) Google Scholar, Xu et al., 2014Xu J. Xie J. Yan C. Zou X. Ren D. Zhang S. A chemical genetic approach demonstrates that MPK3/MPK6 activation and NADPH oxidase-mediated oxidative burst are two independent signaling events in plant immunity.Plant J. 2014; 77: 222-234Crossref PubMed Scopus (137) Google Scholar). The NA-PP1-treated MPK3SR plants (line nos. 28 and 64) displayed increased freezing tolerance and reduced ion leakage compared with the wild-type, with or without cold acclimation (Figures 2G−2I and S2A−S2C). Similar freezing tolerance and ion-leakage responses were observed in another conditional mpk3 mpk6 double mutant, named MPK6SR (mpk3 mpk6 pMPK6:MPK6YG) (Su et al., 2017Su J. Zhang M. Zhang L. Sun T. Liu Y. Lukowitz W. Xu J. Zhang S. Regulation of stomatal immunity by interdependent functions of a pathogen-responsive MPK3/MPK6 cascade and abscisic acid.Plant Cell. 2017; 29: 526-542Crossref PubMed Scopus (90) Google Scholar, Xu et al., 2014Xu J. Xie J. Yan C. Zou X. Ren D. Zhang S. A chemical genetic approach demonstrates that MPK3/MPK6 activation and NADPH oxidase-mediated oxidative burst are two independent signaling events in plant immunity.Plant J. 2014; 77: 222-234Crossref PubMed Scopus (137) Google Scholar) after NA-PP1 treatment (Figures S2D−S2F). NA-PP1-treated MPK3SR plants (line no. 64) also displayed greater freezing tolerance than mpk6-3 mutants (Figures 2G−2I). These results further demonstrate that MPK3 and MPK6 negatively regulate plant freezing tolerance. MAPK activation requires dual phosphorylation of threonine (Thr) and tyrosine (Tyr) residues in the TXY motif by activated MAPKKs (Cobb and Goldsmith, 1995Cobb M.H. Goldsmith E.J. How MAP kinases are regulated.J. Biol. Chem. 1995; 270: 14843-14846Crossref PubMed Scopus (1662) Google Scholar). Previous studies reported that MPK3 and MPK6 can be activated by the expression of MKK5DD (T215D/S221D), a constitutively active form of MKK5 (Liu and Zhang, 2004Liu Y. Zhang S. Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis.Plant Cell. 2004; 16: 3386-3399Crossref PubMed Scopus (637) Google Scholar, Ren et al., 2002Ren D. Yang H. Zhang S. Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis.J. Biol. Chem. 2002; 277: 559-565Crossref PubMed Scopus (351) Google Scholar). We examined steroid-inducible gain-of-function MKK5DD transgenic Arabidopsis plants, and found that MPK3/MPK6 activation in dexamethasone-treated MKK5DD reduced freezing tolerance and increased ion leakage compared with that of control plants with or without cold acclimation (Figures S2G−S2I). These combined results suggest that the constitutively activated MKK5DD negatively regulates plant freezing tolerance. Having shown that MPK3 and MPK6 are involved in cold-stress responses and interact with the CBF master regulator ICE1, we next evaluated whether MPK3/MPK6 mediate plant freezing tolerance via the CBF-dependent pathway by analyzing the expression of CBFs and CBF target genes in mpk3-2 and mpk6-3. Cold-induced expression of CBF genes and targets, such as COR15A, KIN1, and RD29A, was significantly higher in mpk3-2 and mpk6-3 mutants than in wild-type plants (Figures 3A−3D ). Consistently, the expression of CBFs and CBF target genes in NA-PP1-treated MPK3SR plants was also higher than that in the wild-type plants under cold treatment (Figures S3A and S3B). By contrast, MPK3/MPK6 activation in MKK5DD dramatically reduced cold induction of CBFs and CBF target gene expression compared with that in wild-type plants (Figures S3C and S3D). These results suggest that MPK3 and MPK6 negatively regulate CBF gene expression. To explore the genetic interactions between MPK3/MPK6 and ICE1, we generated mpk3-2 ice1-2 and mpk6-3 ice1-2 double mutants. Consistent with our previous study (Ding et al., 2015Ding Y. Li H. Zhang X. Xie Q. Gong Z. Yang S. OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis.Dev. Cell. 2015; 32: 278-289Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar), the ice1-2 mutant was hypersensitive to freezing stress. The mpk3-2 ice1-2 and mpk6-3 ice1-2 double mutants resembled the ice1-2 mutant in terms of freezing sensitivity, ion leakage, and cold-induced expression of CBFs and their target genes (Figure 3). These results suggest that MPK3 and MPK6 act upstream of ICE1 to negatively regulate CBF gene expression. Previous studies reported that MPK3 and MPK6 physically interact with and phosphorylate their substrates to modulate plant physiological and biochemical processes (Mao et al., 2011Mao G. Meng X. Liu Y. Zheng Z. Chen Z. Zhang S. Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis.Plant Cell. 2011; 23: 1639-1653Crossref PubMed Scopus (517) Google Scholar, Meng et al., 2013Meng X. Xu J. He Y. Yang K.Y. Mordorski B. Liu Y. Zhang S. Phosphorylation of an ERF transcription factor by Arabidopsis MPK3/MPK6 regulates plant defense gene induction and fungal resistance.Plant Cell. 2013; 25: 1126-1142Crossref PubMed Scopus (273) Google Scholar, Zhang et al., 2015Zhang Y. Wang P. Shao W. Zhu J.K. Dong J. The BASL polarity protein controls a MAPK signaling feedback loop in asymmetric cell division.Dev. Cell. 2015; 33: 136-149Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). As MPK3 and MPK6 interact with ICE1, we hypothesized that ICE1 might be a substrate of MPK3 and MPK6. Therefore, we purified recombinant His-tagged ICE1 and performed in vitro phosphorylation assays. Recombinant His-MPK3 and His-MPK6 strongly phosphorylated ICE1 after activation by constitutively active MKK5DD (Figure 4A). By contrast, neither MPK3 nor MPK6 phosphorylated ICE1 in the absence of constitutively active MKK5DD (Figure 4A). We analyzed the ICE1 amino acid sequence and found six potential MAPK phosphorylation sites (Ser94, Ser203, Thr366, Thr382, Thr384, and Ser