Identification Of Kinase Gene Which Phosphorylates The Boric Acid Channel Protein AtNIP5;1

Boron is an essential micronutrient element for plant growth and development.Under low boron stress,the model plant Arabidopsis primarily relies on the boric acid channel AtNIP5;1(nodulin 26-like intrinsic protein5;1)in the root to quickly absorb boron nutrition.AtNIP5;1 is located on the PM domain toward the soil-side of the outermost root cell layers,which is mediated by the phosphorylation of the conserved threonine residue(TPG motif)in the protein N-terminal region.Mitogen-activated protein kinase MAPK plays its role through phosphorylating the P X S/T P motif of the target protein.Thus,we speculate that MAPKs have the potential to phosphorylate AtNIP5;1.In this study,we conducted bioinformatics analysis of the AtMAPK family and then cloned them from Arabidopsis.The response of AtMAPKs to low boron stress and of AtNIP5;1 to the boron conditions was then investigated.Furthermore,we analyzed the interaction between AtMAPKs and AtNIP5;1 using a Bimolecular Fluorescence Complementation technology in tobacco and a protein interaction technology in yeast.These results provided a theoretical basis to reveal the molecular mechanism by which AtMAPKs phosphorylate AtNIP5;1.The main results are as follows:1.Bioinformatics analysis of AtMAPK family genesTwenty AtMAPK genes were identified from Arabidopsis,we found that there are large differences in the structure of 20 genes by visualizing the genomic structure of 20 genes through software.The result of phylogenetic tree analysis indicated that 20 genes can be divided into three sub-families,and there is a difference in their evolutionary rate.The result of protein sequence alignment revealed that there is a high homology and multiple conserved regions in the core functional regions of the protein sequence.We further cloned 16 genes of all.2.The response of AtMAPK family genes to low boron stressWe used Arabidopsis thaliana transcriptome data in response to low boron to construct a heat map,and the results showed that a total of 9 AtMAPK genes are expressed in high levels,but their expression is not primarily regulated by boron.We analyzed the expression of 20 genes at each time point when treated with boron deficiency for 0 h,3 h,12 h,and 72 h by fluorescence-quantitative PCR.Fluorescence quantitative results indicated that the expression level of AtMAPK10/11/12/13/14 is hardly detectable,and the expression level of AtMAPK4/6/8/17/18/20 is relatively high.Some of them showed low boron response.20 genes do not respond strongly to boron as a whole.3.The relationship between AtNIP5;1 protein and boronWe treated pNIP5;1:GFP-AtNIP5;1 Arabidopsis under low boron(15 ?M)and high boron(500 ?M)conditions for 24 h after being grown for 7 d with noamal boron,and observed the expression of GFP-AtNIP5;1 protein in the root through a laser confocal microscope.It was found that the expression level of GFP-AtNIP5;1 protein under high boron conditions is significantly lower than that under low boron conditions.We also treated the 5'-UTR(5' untranslated region)deletion mutant material pNIP5;1(?5'-UTR):GFP-NIP5;1 under high and low boron conditions for 24 h,then observed the expression and localization of GFP-AtNIP5;1 protein in roots under the same conditions.The results indicated that the expression and polar localization of GFP-AtNIP5;1 protein in the mutant do not change significantly under high and low boron conditions,These results revealed that the expression of AtNIP5;1 protein level is not regulated by boron.4.AtMAPK proteins interact with AtNIP5;1 proteinThe analysis of Bimolecular Fluorescence Complementation technology(BiFC)demonstrated that AtMAPK4/6/17/19,which have relatively high expression levels,can interact with AtNIP5;1 in tobacco.Besides,the yeast two-hybrid analysis revealed that AtMAPK3/4/6/7/14/17 could interact with AtNIP5;1.These results indicated that the AtMAPKs gene might be involved in the phosphorylation modification process of AtNIP5;1 protein.