Functional Analysis Of The Chloroplast Iron Transporter AtMTP7 In Arabidopsis

Iron(Fe)is an essential micronutrient for plant growth and development.Fe is cofactor for various enzymes and involved in metabolic activities such as photosynthesis,cell respiration,redox reactions.Fe deficiency will seriously affect the metabolism and development of plants.Plant metal tolerance proteins(MTP)are also known as a cation diffusion enhancer(CDF),which plays an important role in the absorption and maintenance of metal ions homeostasis in plants.As an important part of the MTP family,the function of the Fe/Zn-CDF subfamily is unclear.In this study,we identified and characterized the function of AtMTP7,a member of the Fe/Zn-CDF subfamily.It is mainly studied in the following aspects,including the target gene AtMTP7 expression pattern and histochemical staining,subcellular localization,yeast function complementation experiment,analysis of physiological phenotype of T-DNA insertion mutant plants and over-expressing plants.AtMTP7 plays an important role in maintaining ion homeostasis in chloroplasts under conditions of iron deficiency.The main findings are as follows:(1)AtMTP7 gene contains 13 exons and 12 introns and encodes a putative protein of 457 amino acids.The transmembrane topology of AtMTP7 was predicted by TMHMM.It predicted to have six transmembrane domains.DNAMAN was used to analyze multiple sequence alignments for Arabidopsis thaliana,rice,and Cucumis sativus of the Fe/Zn-CDF subfamily members.Homology alignment revealed that have a conserved sequence characteristic H ××× D(× indicates any amino acid)in the second transmembrane region.(2)In order to explore the expression pattern of AtMTP7 gene,the transcript level of AtMTP7 in different tissues was explored by qRT-PCR.The results showed that the expression level of AtMTP7 in the shoots was significantly higher than in the roots.ProAtMTP 7:G US vector was constructed to infect wild-type Arabidopsis thaliana.Histochemical staining results showed that AtMTP7 was expressed in roots,leaves and flowers,and the expression level in rosette leaves was higher than that in cauline leaves,but AtMTP7 not expressed in stem,root tip,petiole and peduncle.At the same time,the expression levels of AtMTP7 were analyzed under different concentrations of Fe2+,Zn2+,Mn2+,and Cu2+.The results showed that the expression of AtMTP7 was not induced by these ions.(3)The AtMTP7 gene was cloned.GFP was ligated to the C-terminus of AtMTP7,and a pXZP008-AtMTP7-GFP subcellular localization vector was constructed,which was transformed into Arabidopsis and rice protoplasts and expression in tobacco mesophyll cells.The results showed that AtMTP7 was localized in chloroplast.(4)The yeast expression vector pYES2-AtMTP7 was constructed,the recombinant plasmid pYES2-AtMTP7 and unloaded pYES2 were transformed into different yeast mutants using lithium acetate transformation method,and the unloaded pYES2 was transformed into wild-type yeast BY4741 as negative control.It was found that the expression of AtMTP7 led to iron-sensitive yeast mutant ?ccc1(ccc1 is an iron transporter protein located in the tonoplast and transport Fe from cytoplasm into the vacuole for detoxification)more sensitive under iron poisoning conditions.It revealed that AtMTP7 has the activity of transporting Fe2+,but has no activity of transporting other metal ions Mn2+,Zn2+,Cu2+,Cd2+,and Co2+.(5)We used homozygous mutants with T-DNA insertion and over-expressing plants to investigate the physiological function of AtMTP7.Observing phenotypic under iron deficiency or normal conditions by hydroponics and ICP-UES used to determine the metal content in different tissues.It was found that under iron deficiency conditions,compared with the wild type plants the shoot of the mutant mtp7 plant was worse and the leaves were more yellow.The dry weight of roots and shoots are lower than wild type.Further determination that Fe content was no significant difference between mutant and wild type plants in different tissue.Under iron deficiency conditions,plants over-expressing AtMTP7 grew better than wild type,but the Fe content did not change correspondingly.The above results indicate that AtMTP7 is an Fe transporter located in the chloroplast.AtMTP7 positively regulates Fe2+ accumulation in plants by transport Fe into the chloroplast and further distributed,which promoting the normal function of the chloroplast and maintaining the intracellular ion balance.