NAT1 And NAT2 Are Required For Copper And Iron Homeostasis In Arabidopsis Thaliana

Plant metabolism requires light,water and specific elements to allow plants growth and development.Copper(Cu)and iron(Fe)are considered to be essential micronutrients and play a crucial role in many physiological processes in plants.A variety of metal transporter families have been found to be involved in the absorption and translocation of iron and copper in plants,including members of heavy metal ATPase(HMA)proteins and yellow stripe like(YSL)transporters.Copper and iron can be chelated by specific metal ligands or directly transported by transporters.Common metal ligands include nicotianamine(NA),phytochelatin(PC),2 '-deoxymugineic acid(DMA),glutathione(GSH)and other substances.The mechanisms of copper and iron absorption,translocation and distribution in plants are been studied in depth,but there are already many details remain to be explained about the mechanisms of copper and iron long distance transport.In this research,two genes that may be involved in ion transport in Arabidopsis thaliana were screened and named NAT1 and NAT2 respectively.NAT1 and NAT2 are both members of the nitrate transporter 1/peptide transporter(NPF)family.They are closely related homologues,which share 74.1% amino acid similarity by sequence alignment.We obtained knockout mutants nat1,nat1,nat1nat2 mutants by using CRISPR-Cas9 technology.In the normal artificial soil,we found that nat1 and nat2 have similar phenotypes with wild-type Col-0,but the nat1nat2 double mutants have interveinal chlorosis of young leaves,lower plant height and silique length,fewer seeds during the growth period,indicating that the two genes may be have similar function.We measured mineral concentrations of Col-0,nat1,nat2,nat1nat2 mutants in leaves and seeds by ICP-MS technology.There is no significant change in Col-0 and nat1,nat2 single mutants,but nat1nat2 double mutants have largely decrease in the content of Cu,Cobalt(Co)and Fe while compared with the wild type.We observed concentration of Cu and Co were decreased by 70% and 50% in leaves.For seeds,concentration of Cu and Fe were also dropped significantly by 40% and 60%.These ionome change phenotype in nat1nat2 leaves and seeds suggests that NAT1 and NAT2 may function in ion uptake or long-distance transport in root and shoot.The ion content was also measured of wild type and nat1nat2 samples taken from different tissues.The results showed that,the Cu and Co contents in the roots of nat1nat2 mutants significantly increased comparing with Col-0,but the contents in the aboveground tissues,including stem,old and young leaves,flower,silique and mature seeds,significantly decreased.However,the content of Fe in nat1nat2 mutants only decreased in young leaves,flower,silique and mature seeds.Thus,these results suggest that NAT1 and NAT2 function in Cu and Co other than Fe root to shoot translocation.X-ray Fluorescence Spectrometer technology was used to detect the seedling roots and mature seeds of Col-0,nat1,nat2 and nat1nat2 mutants respectively,it is consistent with the ICP-MS results.The results showed that only seeds of nat1nat2 mutants were significantly different from those of Col-0.We also constructed NAT1 and NAT2 gene overexpression vectors to transform Col-0 plants,then these transgenic positive seedlings were grown on the medium containing excessive Cu and Co.It was observed that compared with the wild type,the leaves of NAT1 and NAT2 overexpressed plants significantly turned yellow and showed higher sensitivity.According to the above experimental results,it is speculated that NAT1 and NAT2 participate in the transport process of Cu and Co f root-shoot translocation and whereas Fe efficiently remobilization from leaves during the reproductive phase to flowers and seeds.The distribution of NAT1 and NAT2 proteins in cells was observed in a dot?like pattern in subcellular localization analysis.In addition to the above ion phenotypes,the nat1nat2 double mutants has multiple defects in reproduction.The seed germination rate of nat1nat2 double mutants also decreased,with only about 3%.It is found that the color of seeds in immature silique and mature seeds of double mutant nat1nat2 was more pale than Col-0.The Col-0,nat1,nat2 and nat1nat2 mutants in the same mature stage were immersed in seed Transparent buffer,it was found that the nat1nat2 double mutants could not successfully developed.In addition,exogenous Cu and Fe supply in soil can partially restore sterile phenotype of nat1nat2.The arrested embryo phenotype of nat1nat2 seeds is the result of Fe and Cu deficiency.Therefore,it is speculated that NAT1 and NAT2 can affect the normal growth and reproductive development of Arabidopsis thaliana by regulating the transport of copper and iron.Our results illustrate a new idea for the long distance transport of Cu and Fe in plants and a new direction for the regulation of the reproductive growth and development of Arabidopsis thaliana.