The Relationship Between TATA-box Insertion Located In Promoter Of Iron Transporter (IRT1) And The Tolerance Of Iron Deficiency In Apple Rootstocks

Iron absorption is an essential process for plant growth and development. Foliar chlorosis caused by low-iron stress has a large impact to the yield and quality for fruit trees. IRTl, a important Fe2+transporters in iron transport system, plays a key role in iron absorption of Malus. This study analyzed the gene relative expression of related pathways in iron efficient genotype Malus xiaojinensis and the inefficient genotype Malus baccata under the condition of the iron deficiency, and detected the sequence of MdIRT1promoter of Malus xiaojinensis, Malus baccata and their pregnant. Results showed that TATA-box insertion is common in Malus xiaojinensis, Malus baccata. This study analysised the TATA-box function for IRT1and its ecological evolution and try to clarified the key element of IRT1promoter TATA-box effect and molecular mechanism in Malus xiaojinensis and Malus baccata resistance to iron deficiency which will lay a foundation for further study about the molecular mechanisms of apple rootstocks resistance to iron deficiency.Iron efficient genotype Malus xiaojinensis and iron inefficient genotype Malus baccata response differently under the condition of iron deficiency. The gene relative expression analysis showed that iron absorption, ethylene, and abscisic acid metabolism related genes responsed earlier than other pathways. Compared to Malus baccata, the iron absorption gene (IRT1) and transport gene (YSL5and SAMS1) responsed earlier under the condition of iron deficiency. The gene expression of IRT1is an important gene for apple rootstocks which affects the ability of the apple plant resistance to iron deficiency.The sequencing result of IRT1in Malus xiaojinensis and Malus baccata showed a difference in the two rootstocks. There is a ATTATAA insertion in the upstream of IRT1promoter in Malus xiaojinensis. The ATTATAA was predicted as a TATA-box which is a core element for the transcription start in the upstream of promoter. This sequencing detection of IRT1in the pregnant of Malus xiaojinensis X Malus baccata showed that the ratio of TATA-box insertion and without TATA-box insertion is more than or equal to1in iron deficiency tolerant rootstocks while the ratio is less than1in iron deficiency sensitive rootstocks. These results pointed out that TATA-box insertion in the upstream of IRT1promoter has close relationship with tolerance of iron deficiency. To investigate the function of this TATA-box insertion, we determined the promoter activity using transiently expression in tobacco leaves. IRT1.1Pro::GUS promoter had a higher activity than IRT1.2Pro::GUS as indicated by the transient GUS gene expression. We also found the TATA-box insertion tightly linked to a base mutation in coding region+96bp (G/T), forming a missense mutation, resulting in the amino acid change from E to D. To assess the effects of the sense mutation of Md1RT1CDS on Fe uptake, we have used functional complementation of a yeast mutant DEY1453(fet3fet4). Both IRT1.1and IRT1.2can complement the mutant strain defective in high-affinity Fe uptake. Therefore, the linked base mutation does not affect the function of IRTl.To determine the variation origin of TATA-box, we detected the IRT1promoter in M. toringoides, progeny of M. toringoides×M. kansuensis, M. maerkangensis, which are the wild relatives of M. xiaojinensis. The result showed that there was no TATA-box insertion in IRT1promoter of these plants except for M. xiaojinensis. So we speculated that the TATA-box insertion was originated along with the evolution of M. xiaojinensis from other Malus species. Examination of14seedlings of M. baccata showed that the11seedlings were homozygous genotypes without TATA-box insertion (IRT1.2). However, three of the14seedlings came from Shanxi or Liaoning, both alkaline soil regions with lower soil available Fe content, were heterozygous with TATA-box. The results indicates that insertion of the promoter is functionally conserved in apple genotypes, and also the insertion evolved during the long period when apple genotypes were used as rootstocks due to the poor soil available Fe.