The Role Of MKP1, MPK3/6 And Ethylene Signaling Pathway In UV-B Regulation Of Nitrate Distribution And Its Molecular Mechanism

The most needed element of plant,besides carbon,is nitrogen,which is a necessary nutrient for plant growth and development.Therefore,agricultural production often promotes plant growth and development by applying nitrogen fertilizer.However,with the increasing application of nitrogen fertilizer,nitrogen pollution has become one of the major environmental problems facing mankind today.Therefore,it is important to study the mechanism of nitrogen absorption,transportation and distribution of plants.Nitrate is the main source of nitrogen for most plants.Plants absorb,transport and distribute nitrate through nitrate transporters.Previous studies have shown that various soil stresses,such as cadmium,salt and drought,regulate the expression of nitrate transporters by activating the plant hormone ethylene/jasmonic acid signaling pathway,thus affecting the distribution of nitrate and changing the growth,developnient and stress tolerance of plants.At the same time,a large number of studies also show that various bad factors can activate mitogen-activated protein kinase(MAPKs)pathway,but the role of MAPK signaling pathway in the regulation of nitrate distribution under adverse circumstances is not clear.With the destruction of the ozone layer,the enhancement of ultraviolet B(UV-B)radiation has become one of the major environmental problems facing humanity today.Previous studies have shown that enhanced UV-B radiation can inhibit plant growth and development,activate MPK3/6 and mitogen-activated protein kinase phosphatase MKP1,and also promote gene expression of ethylene biosynthesis rate-limiting enzyme ACC synthase(ACS)and ethylene biosynthesis,and MPK3/6 mediates UV-B radiation-induced ACS gene expression and ethylene production.However,the effect of UV-B radiation on nitrate distribution in plants and the role of MAPK and ethylene signaling pathways in UV-B regulation of nitrate distribution and their interrelations are unclear.Based on the above research status,the effects of 0.5 W.m-2 UV-B radiation on nitrate distribution and the effects and molecular mechanisms of MKP1,MPK3/6 and ethylene signaling pathways in UV-B radiation regulation of nitrate distribution in Arabidopsis thaliana were studied using wild-type Col-0 and Ws,as well as functional deletion mutants mpk3-1,mpk6-1,mkp1,mkplhnpk6 and ein3-l as experimental materials by means of gene expression analysis,nitrate content determination,protein kinase activity determination and protein interaction analysis.The main results and conclusions are as follows:1.0.5 W.m-2 UV-B radiation for 3 h could promote the expression of ERF transcription factors ORA59,ERF1B and ERF104 and nitrate transporter NRT1.8 in the aboveground part and roots,inhibit the expression of nitrate transporter NRT1.5 in the roots,and improve the ratio of leaves/shoots and roots/shoots distribution of nitrate in Col-0 and Ws.This indicates that UV-B radiation affects the distribution of nitrate in plants by regulating the expression of nitrate transporters.2.Mutant ein3-1 inhibited the promotion of UV-B radiation on the gene expression of ORA59,ERF IB,ERF104 and NRT1.8 in the aboveground part and roots,and also inhibited the promotion of UV-B radiation on the leaf/stem and root/stem partition ratio of nitrate,but did not affect the inhibition of UV-B radiation on the gene expression of NRT1.5 in the roots.This indicates that ethylene signal transduction element EIN3 participates in the signal transduction process in which UV-B radiation regulates nitrate distribution in plants.EIN3 promotes the expression of NRT1.8 gene by inducing the expression of ORA59,ERFIB and ERF104.However,EIN3 does not participate in the inhibition of NRT1.5 gene expression by UV-B radiation.3.UV-B radiation promoted the activation of MPK3 and MPK6 in the aboveground part,and its promotion was negatively regulated by MKP1,but UV-B radiation did not affect the activity of MPK3 and MPK6 in the roots;Mutants mpk3-1,mpk6-1 and mkpl/mpk6 inhibited the promotion of UV-B radiation on the gene expression of ORA59,ERF1B,ERF104 and NRT1.8 in the aboveground part and roots,and also inhibited the promotion of UV-B radiation on the leaf/stem and root/stem partition ratio of nitrate,but did not affect the inhibition of UV-B radiation on the gene expression of NRT1.5 in the roots.However,mutant mkpl enhanced the promotion of UV-B radiation on the expression of ORA59,ERF1B,ERF104,NRT1.8 genes in the aboveground part and roots,and also enhanced the promotion of UV-B radiation on the leaf/stem and root/stem partition ratio of nitrate,but did not affect the inhibition of UV-B radiation on the expression of NRT1.5 genes in the roots.This shows that MKP1 and its target protein MPK3/6 participate in the signal transduction process of UV-B radiation-induced ORA59,ERF1B and ERF104 gene expression and downstream NRT1.8 gene expression as negative and positive regulators respectively,and the role of MKP1 in this process depends on its regulation of MPK3/6 activity.However,MKP1 and its target protein MPK3/6 do not participate in the inhibition of NRT1.5 gene expression by UV-B radiation.4.Yeast two-hybrid experiments showed that MPK3 interacted directly with ORA59,ERF1B and ERF 104,while MPK6 interacted directly with ERF 104 only.It is suggested that MPK3 and MPK6 not only regulate the gene expression of ERF transcription factors from the transcription level,but also directly regulate the stability of ERF transcription factor proteins from the post-transcription level through direct interaction with transcription factors under UV-B radiation.Previous studies in our laboratory have shown that MPK3/6 mediates UV-B radiation-induced ACS gene expression and ethylene production.Based on the experimental results in this paper and the previous research results in our laboratory,we have concluded that the molecular mechanism of UV-B radiation regulating nitrate distribution is as follows:UV-B radiation activates MPK3 and MPK6(the process is negatively regulated by MKP1)in the aboveground part,activated MPK3/6 induces the biosynthesis of aboveground ethylene and transports it to the roots to promote the increase of ethylene level in the roots.Then ethylene relies on its signal transduction element EIN3 to induce the expression of ORA59,ERFIB and ERF104 genes in the aboveground part and roots.At the same time,the activated MPK3 and MPK6 may also directly regulate their protein stable types through interaction with ERF transcription factors.Afterwards,ORA59,ERF1B and ERF104 bind to the promoter of NRT1.8 gene to directly induce its expression,promoting NRT1.8 mediated nitrate unloading from xylem to root and leaf.In addition,UV-B radiation simultaneously inhibited the expression of NRT1.5 gene in the roots in a manner independent of MPK3/6 and ethylene signaling pathways,ultimately inhibiting NRT1.5 mediated loading of root nitrate into xylem,thus changing the distribution of nitrate in Arabidopsis thaliana organs.