Phosphorylated Status Of AtNRT1.1 Is Functioned In Regulating Of Root Development And PTI Responses In Arabidopsis

Nitrate is the primary nitrogen source in most plants.The dual-affinity nitrate transceptor AtNRT1.1(Nitrate transporter 1.1)has two modes of transport and signaling governed by Thr-101(T101)phosphorylation.Here,using the Arabidopsis wild type plants(WT),chl1-5(an AtNRT1.1 null mutant),AtNRT1.1^T101A/chl1-5(anonphosphorylatable T101A mutant,hereafter abbreviated T101A),and AtNRT1.1^T101D/chl1-5(a phosphomimetic T101D mutant,hereafter abbreviated as T101D),we found that the phosphorylated forms of AtNRT1.1 were involved in regulating root development and pattern-triggered immunity(PTI)responses upon low-concentration nitrate(LN)in Arabidopsis by molecular,physiological methods and live-cell microscopy.First,we conducted RNA-seq experiments in roots of WT,chl1-5,T101A and T101D grown on medium containing 0.2 m M nitrate,and performed analysis of Venn diagrame,KEGG and GO function enrichment,hierarchical clustering.Cluster 3 and 8 gathered genes with upregulation in T101D compared with T101A,the functional categories of which were enriched to plant defense responses.Cluster 7 gathered genes with downregulation in T101D compared with T101A,the functional categories of which were enriched to auxin and other hormorne signaling.Secondly,through light sheet fluorescence microscopy,laser confocal microscope and stereoscope detection,we found that T101A plants grown in LN medium displayed strongly enhanced DR5::GFP expression in the primordia and young lateral roots(LRs)as well as higher LRs density compared to the T101D plants.These data indicate that nonphosphorylated AtNRT1.1 promotes LR growth in LN caused by the accumulation of auxin in the tips of LRs.Thirdly,upon flg22,chitin and Pep1 treatment,T101D grown in LN medium exhibited obviously enhanced PTI responses including calcium influx,membrane depolarization,callose deposition and production of reactive oxygen species as well as inhibition of primary root elongation compared to the T101A.These data suggest that phosphorylated AtNRT1.1 induces root PTI responses in LN,resulting the inhibition of primary root growth.In conclusion,the phosphorylated forms of AtNRT1.1 are involved in regulating root development and PTI responses upon LN in Arabidopsis.These results would deepen the understanding of AtNRT1.1-mediated regulation of root morphogenesis,and the effect of nitrate on disease resistance of plant root.