| Chlorotic leaves are causing great losses on the yield and quality of fruit trees, tree potential and economic benefits, and therefore seek ways and means of correcting iron deficiency chlorosis is becoming increasingly important.In a previous study, we found that Malus xiaojinensis Cheng et Jiang is an iron-efficient species in the genus Malus. It can be used as a stock to aid normal growth and development in apple under iron-limited conditions. However, the molecular mechanism involved in the response to iron deprivation in this iron-efficient species is not clear. In this study, iron transport genes MxNRAMP1were cloned in M. xiaojinensis and the characteristics and functions were also analysed. To explore mode and role of MxNRAMP1gene involved in iron uptake and transport in xiaojinensis,trying to explain the mechanism of iron deficiency adaptive response from different angles and levels, provide a basis for further studies on molecular mechanism of resistance to iron deficiency of M. xiaojinensis. The main results were as follows:A1656bp NRAMP1cDNA was obtained from Malus xiaojinensis as expected.It encodes a predicted polypeptide of551amino acids, a molecular weight of about59.8kDa, Isoelectric point of8.05. Multiple sequence alignment showed that MxNrampl shared high homology with other species. The MxNRAMP1protein including10putative transmembrane domains (TMs) and the chloroplast transit peptide, it may be located in the chloroplast.There is no secretory pathway signal peptide in NRAMP1.The transcript abundance of MxNRAMP1was assessed in M. xiaojinensis supplemented with or without Fe. The expression of MxNRAMP1was higher in roots than in leaves, and was markedly induced by Fe deficiency in both roots and leaves. The transcript level of MxNRAMP1increased at least4-fold after Fe-deficiency treatment for3,6, and9d in roots. In comparison with roots, the induction of expression was delayed in young and old leaves. In leaves, MxNRAMP1showed remarkably higher expression under Fe limitation only at9d. Whether the up-regulation of MxNRAMP1after iron starvation are repressed by NPA treatment.To further verify the function of MxNRAMP1in plants, we transformed the MxNRAMP1gene into the model plant tobacco for over-expression. After the transgenic line, empty vector line and wild-type tobacco underwent iron-deficient hydroponics, the chlorophyll content in new leaves of the transgenic line was significantly higher than in the empty vector line and wild-type. Wild-type tobacco showed more yellowing in the leaves. New leaves and roots of the transgenic line had higher Fe and Mn concentrations than the empty vector line and the wild-type, while there was no clear difference in the Zn concentration. These results showed that MxNRAMP1over-expression facilitated iron uptake in transgenic tobacco roots and iron accumulation in roots and leaves, and therefore induced stronger resistance against iron stress. It had no effect on Zn transportation. Protein expression analysis of transgenic tobacco showed that MxNRAMP1protein expression is induced by iron deficiency in root.Onion subcellular localization revealed that the MxNRAMP1proteins were localized to the plasma membrane. Protoplast transient expression system and immune electron microscopy localization techniques were used to prove that MxNRAMP1mainly exists in the plasma membrane and vesicles. Interestingly, iron deficiency stress could induce the MxNRAMP protein to transport iron ions to specific organelles (lysosome and chloroplast) through vesicle endocytosis. These results showed that, in Malus xiaojinensis, MxNRAMP1not only plays an important role in iron absorption and transportation, it can also produce adaptive responses against iron deficiency through endocytosis. |
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