| Rice (Oryza sativa L.) is one of the most important food crops worldwide, and therefore, improving its quality and production is always the major goal of most agricultural scientific researchers. Nowadays, with the development of industry, the pollution of environment is increasingly serious, and in particular, the excess of heavy metals in lots of farmland has become a great threat to food security. In this case, to study the mechanism of rice plants absorbing and accumulating heavy metals and consequently helps cultivate green rice with high yield and good quality is of great significance. Manganese (Mn) is an essential micronutrient for plant growth, and too less or too much Mn accumulation will block the growth of plants. Cadmium (Cd) is a nonessential heavy metal, and its accumulation has a health risk for most organisms. In this study, we identified three natural resistance-associated macrophage protein (NRAMP) genes in rice, and characterized their exact roles in uptake or translocation of Mn and Cd. The main results are summarized as follows:Besides highly expressed in roots, OsNRAMPS was also found to be expressed in hulls and leaves, and moreover, the expression level in leaves decreased with leaf age. Based on experiments of GUS staining and in situ hybridization, high-magnification observations revealed that OsNRAMP5 was enriched in the vascular bundles of roots and shoots especially in the parenchyma cells surrounding the xylem. The osnramp5 mutant accumulated significantly less Mn in shoots than the wild-type plants even at high levels of Mn supply. Furthermore, a high supply of Mn could compensate for the loss in the root uptake ability in the mutant, but not in the root-to-shoot translocation of Mn, suggesting that the absence of OsNRAMP5 reduces the transport of Mn from roots to shoots. Additionally, roots of osnramp5 plants accumulated much higher Fe concentration than wild type when supplied with a low level of Mn. Further study showed that this can be explained by the induction of four essential Fe transporters, containing OsNRAMP1, OsIRT2, OsYSL2 and OsYSL15. A significant antagonism was observed between Mn and Cd in wild type rice plants but not in OsNRAMP5 knockout line, suggesting that this antagonism was mediated by OsNRAMP5. Furthermore, the analysis on sequence diversity of OsNRAMP5 showed that OsNRAMP5 was hyperconserved in evolutional process and strong selection pressure was acting on it.OsNRAMP3 encodes a plasma membrane-localized protein and was specifically expressed in vascular bundles, especially in phloem cells. Yeast complementation assay showed that OsNRAMP3 is a functional Mn-influx transporter. When OsNRAMP3 was absent, rice plants showed high sensitivity to Mn deficiency. Serious necrosis appeared on young leaves and root tips of the OsNRAMP3 knockout line cultivated under low Mn conditions, and high Mn supplies could rescue this phenotype. However, the necrotic young leaves of the knockout line possessed similar levels of Mn to the wild type, suggesting that the necrotic appearance was caused by disturbed distribution of Mn but not a general Mn shortage. Additionally, compared with wild type, leaf Mn content in osnramp3 plants was mostly in older leaves. We conclude that OsNRAMP3 is a vascular bundle-localized Mn-influx transporter involved in Mn distribution and contributes to remobilization of Mn from old to young leaves.The protein encoded by OsNRAMP6 was found to be localized in plasma membrane and was functional in transporting Cd2+, but didn’t possess Mn2+ transport activity. OsNRAMP6 was mainly expressed in rice shoots, especially in young leaves. When OsNRAMP6 was knocked out, rice plants showed significantly shorter height and root length than wild type, whereas no difference was found in Cd concentrations of roots, shoots and different leaves between the two lines. But up-regulating the expression of OsNRAMP6 caused significant increase of Cd concentration in old leaves. We inferred that OsNRAMP6 was involved in Cd accumulation in rice leaves. |
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