Molecular Mechanism Of Cold Stress Responses Regulated By Protein Kinases MPK3/MPK6 In Arabidopsis

Low temperature is a major abiotic stress that adversely affects plant growth and development,productivity and ecological distribution.Plants have evolved complicated and precise mechanisms involving altered physiological and biochemical processes to adapt to cold stress.Extensive studies have shown that cold stress rapidly induces the expression of CBFs,which in turn activates downstream cold-responsive(COR)genes,thus resulting in acquired freezing tolerance in plants.The expression of CBFs is regulated by several transcription factors,including ICE1.ICE1 is a bHLH transcription factior that has been shown a master regulator of CBF gene expression and freezing tolerance.The stability and transcriptional activity of ICE1 are precisely modulated by several regulators.Previous studies indicated that ubiquitination of ICE1 mediated by E3 ligase HOS1 promotes the degradation of ICE1,whereas sumolation of ICE1 mediated by SUMO E3 ligase SIZ1 stabilizes ICE1.JAZ1 and JAZ4,the repressors of jasmonate signaling,physically interact with ICE1 and attenuate the transcriptional activity of ICE1.Meanwhile,the protein kinase OST1 phosphorylates ICE1,which in turn inhibits the degradation ICE1 mediated by HOS1 and enhances the transcriptional activity of ICE1.Therefore,the posttranslational modification of ICE 1 is critical for its function.MPK3 and MPK6 were isolated by a yeast two-hybrid screen as an interaction protein of ICE1.MPK3 and MPK6 belong to MAPK protein kinase family which has twenty members in Arabidopsis.We further found that MPK3/MPK6 interacted with ICE1 in vitro using pull-down assays and in vivo using coimmunoprecipitation(Co-IP)assays.We performed a freezing tolerance assay using the knockout mutants of MPK3 and MPK6.Under non-acclimated(NA)and cold-acclimated(CA)conditions,the mpk3 and mpk6 mutants displayed substantially increased freezing tolerance compared to the wild type.The cold-induction of CBFs and CBF target genes was dramatically elevated in mpk3-2 and mpk6-3 mutants compared to the wild type after cold treatment.MPK3 and MPK6 are homologous proteins,which has similar function in the processes of plant growth and development and response to environmental stresses.Therefore,we examined the freezing tolerance of the conditional loss-of-function mpk3 mpk6 double mutant,which was also called inhibitor-sensitized MPK3 variant-rescued(MPK3SR)plants.The NA-PP1-treated MPK3SR plants showed dramatically increased freezing tolerance and expression of CBFs and CBF target genes compared to the untreated ones.To explore the genetic interaction of MPK3/MPK6 and ICE1,we generated mpk3-2 icel-2 and mpk6-3 icel-2 double mutants.The mpk3-2 icel-2 and mpk6-3 icel-2 double mutants displayed similar freezing sensitivity to the icel-2 mutant.Furthermore,the cold-induction of CBFs and their target genes in mpk3-2 icel-2,and mpk6-3 icel-2 double mutants was decreased as the icel-2 mutant was.These results suggest that MPK3 and MPK6 negatively regulate the expression of CBF genes and plant freezing tolerance.Further studies found that MPK3/MPK6 phosphorylated ICE1 in vitro.We further analyzed the ICE1 protein sequence and found six potential MAPK phosphorylation sites(Ser94,Ser203,Thr366,Thr382,Thr384,and Ser403).When all six Ser/Thr residues were mutated to Ala(ICE16A),the phospho-inactive mutant form of ICE1 could be no longer phosphorylated by MPK3/MPK6.These results suggest that the phosphorylation of ICE1 occurs on the six MAPK phosphorylation sites under cold stress.The icel-2 mutant plants expressing pICE1:ICE16A-GFP showed enhanced freezing tolerance and cold-induced CBFs expression comparable to the wild type.Meanwhile,phosphorylation of ICE1 by MPK3/MPK6 compromises ICE1 function in stomatal development.The icel-2 mutant plants expressing pICE1:ICE16A-GFP had more stomatal cells than the wild type.These results suggest that phosphorylation of ICE1 mediated by MPK3/MPK6 is required for its function.The stability of ICE 1 was significantly increased in mpk3 and mpk6 plants under cold treatment.The ubiquination of ICE16A was reduced and the stability of ICE16A was increased under cold stress.However,phosphorylation of ICE1 by MPK3/MPK6 did not affect the HOS1-ICE1 interaction.Meanwhile,MPK3/MPK6 attenuated the transcriptional activity of ICE 1 in transient assays.Moreover,the transcriptional activity of ICE16A was significantly increased.These results reveal that phosphorylation of ICE1 by MPK3/MPK6 affects the stability and transcriptional activity of ICE1 under cold stress.In summary,we uncover MPK3 and MPK6 as novel regulators in CBF-dependent cold signaling.MPK3 and MPK6 are activated and then phosphorylate ICE1,which consequently inhibit the stability and transcription activity of ICE1 under cold treatment.Therefore,MPK3/MPK6 contribute to the enhancement of ICE1 degradation to inhibit the expression of CBFs and plant freezing tolerance.This study provides the molecular mechamism of MAPK cascades regulating cold signaling and is helpful to understand how plants coordinate environmental stress and growth process.