The Role Of MKP1 And MPK6 In The Regulation Of Stomatal Movement And Gene Expression In Arabidopsis Thaliana By UV-B Radiation

The signal transduction pathways of plants in response to ultraviolet B(UV-B)radiation can be divided into UV-B receptor UVR8-dependent UV-B-specific signaling pathways and UVR8-independent UV-B-nonspecific signaling pathways.The former can be activated at very low doses of UV-B radiation,and mainly mediates photomorphogenic responses and gene expression related to UV-B acclimation and tolerance,while the latter can be only activated by higher doses of UV-B radiation,and mainly mediate gene expression related to stress responses.At present,there are many studies on the composition,mechanism and physiological processes of the UV-B-specific signaling pathways,but it is less known about these processes of the UV-B-nonspecific signaling pathways.Mitogen-activated protein kinases(MPKs)and their corresponding mitogen-activated protein kinase phosphatases(MKPs)are conserved signaling molecules in eukaryotic cells.Under various biotic and abiotic stresses,plants often transduce the signals by activating some specific MPKs and/or MKPs.Recently,studies have shown that UV-B radiation mainly activates MPK3,MPK6 and MKP1 in plants,and that MKP1 affects plant resistance to UV-B stress via negatively regulating activities of MPK3 and MPK6.These results suggest that MKP1 and its targets MPK3 and MPK6 play important roles in the signal transduction of UV-B radiation in plants,but up to now,it is still unclear by which signaling pathways UV-B induces the activation of MPK3,MPK6 and MKP1,which physiological processes are regulated by the UV-B-activated MKP1 and its targets MPK3 and MPK6,and how MKP1,MPK3 or MPK6 regulate these physiological processes.Based on the above research status,this paper used Arabidopsis thaliana as the material,combined with the research methods and techniques of genetics,biochemistry,molecular biology and cell biology,mainly studied the following three aspects:(1)The roles of UVR8,COP1,HY5/HYH,heterotrimeric G protein,hydrogen peroxide(H2O2)and nitric oxide(NO)in the UV-B-induced activation of MPK3 and MPK6;(2)The roles of MKP1 and its targets MPK6 and MPK3 as well as their relationships with H2O2 and NO in UV-B-induced stomatal closure;(3)The roles of MKP1 and MPK6 in UV-B-regulated gene expression.The main results and conclusions are as follows:(1)High doses of UV-B radiation(equal to or above 0.3 Wm-2)could induce the activation of MPK3 and MPK6 in Arabidopsis leaves,and the suitable irradiation time was 3 h.Compared to that under visible light,0.5 Wm-2 UV-B radiation induced activation of MPK3 and MPK6 in leaves of wild-type,UVR8 signaling pathway component mutants uvr8,cop1 and hy5/hyh,G protein a subunit gpal mutants and nitrate reductases(NR)nial and nia2 mutants,but could not completely activate MPK6 and MPK3 in leaves of G protein ? subunit agbl and ? subunit aggl and agg2 mutants as well as NADPH oxidase AtrbohD and AtrbohF mutants.The results show that UV-B-induced activations of MPK3 and MPK6 in Arabidopsis leaves are independent on UVR8 signaling pathway,G protein a subunit and NR-sourced NO,but dependent on the G protein(3 and y subunits as well as H2O2 production by NADPH oxidase ATRBOHD and ATRBOHF.(2)Under different doses of UV-B irradiation for 3 h or under 0.5 Wm-2 UV-B radiation for different times,the level of endogenous NO in guard cells and the degree of stomatal closure in mkpl mutant were higher or greater than those in wild type and line6(a representative line of mkpl mutant complemented by the expression of wild-type MKP1),while the level of endogenous H2O2 in guard cells was same as that in wild type and line6.The results suggest that MKP1 acting as a negative regulator participates in UV-B guard cell signaling via inhibiting NO production in guard cells.(3)0.5 Wm-2 UV-B radiation induced production of H2O2 and NO in guard cells and the subsequent stomatal closure in wild-type and mpk3 mutants,also induced H2O2 generation in guard cells of mpk6 single mutants and mkp1/mpk6 double mutant,But the UV-B-induced NO production in guard cells and subsequent stomatal closure were impaired in mpk6 single mutants and mkp1/mpk6 double mutant;Exogenous H2O2 induced NO generation and stomatal closure in wild-type and mpk3 mutants under light,but these effects of exogenous H2O2 were impaired in mpk6 single mutants and mkp1/mpk6 double mutant under either light or UV-B radiation;Exogenous NO not only induced stomatal closure of wild-type and mutants mpk3,mpk6 and mkp1/mpk6 under light,also rescued the defect of UV-B-induced stomatal closure in mpk6 single mutants and mkp1/mpk6 double mutant.The results show that MPK6 acting as a positive regulator is involved in the signal transduction of UV-B-induced stomatal closure and functions downstream of H2O2 and upstream of NO,but MPK3 does not participate in the UV-B guard cell signaling.Moreover,the results also indicate that MKP1 negatively regulate UV-B-induced NO production in guard cells and subsequent stomatal closure via regulating MPK6 activity.(4)The transcriptome profiling analysis were performed by RNA sequencing technology(RNA-Seq)in wild-type and mutants mkp1 and mpk6 under either light or 0.5 Wm-2 UV-B radiation.Based on the differentially expressed genes(DEGs)with more than or equal to two fold changes in expression and false discovery rate(FDR)?0.05,we recognized 4988 UV-B response genes,among which 231 UV-B response genes were regulated by MKP1 and/or MPK6.Compared with the previously recognized genes regulated by the UVR8-dependent UV-B-specific pathways and the UVR8-independent UV-B-nonspecific pathways,respectively,it is found that both of MKP1 and MPK6 are important signal transduction molecules in the UVR8-independent UV-B-nonspecific pathways,which mediate the expression of some of the genes regulated by the UV-B-nonspecific pathways.A further analysis of 231 UV-B response genes regulated by MKP1 and/or MPK6,it is found that there are four relationships between MKP1 and MPK6 during regulation of the expression of UV-B response genes:(1)115 UV-B response genes are regulated only by MKP1 but not by MPK6;(2)97 UV-B response genes are regulated only by MPK6 but not by MKP1;(3)10 UV-B response genes are antagonistically regulated by MKP1 and MPK6;(4)9 UV-B response genes are identically regulated by both MKP1 and MPK6.(5)Bioinformatics analysis of 231 UV-B response genes,which are regulated by MPK6 and/or MKP1,showed that these genes are mainly related to phenylpropanoid biosynthesis,phenylalanine metabolism,phytohormone signal transduction,the metabolism of keratin,cork and wax,glutathione metabolism,flavonoids metabolism,nitrogen metabolism,amino acid synthesis,pyruvate metabolism,photosynthetic carbon fixation,starch and sucrose metabolism,biological rhythm and plant immune response,which suggest that MKP1 and MPK6 signaling pathways may regulate the UV-B responses in plants by affecting the above metabolic processes or signaling pathways.