| Copper（Cu）is an essential micronutrient for growth and development of all living organisms,but can also be toxic in excess.It is vital to control Cu homeostasis for improving crop yields and nutritional quality,as well as human health.Antioxidant Protein1（ATX1）,the Cu chaperone is thought to be an important factor in maintaining Cu intracellular homeostasis.In this study,we functionally characterized an uncharacterized member of ATX1,OsATX1 in rice（Oryza sativa）,which is involved in the transport and distribution of Cu.RT-qPCR showed that OsATX1 expression was induced by a high concentration of Cu and cadmium（Cd）,but not affected by the excess of other metals,including iron（Fe）,manganese（Mn）,and zinc（Zn）.Analysis of the transgenic rice carrying the OsATX1promoter fused with GUS（β-Glucuronidase）and GFP（green fluorescent protein）revealed that it was localized in the exodermis and the xylem cells of roots,and in the leaf vascular bundles.Using the transient expression system to express OsATX1-GFP in rice mesophyll protoplasts and Nicotiana benthamiana leaf epidermal cells,we observed that OsATX1-GFP is localized to the cytoplasm,nucleus,and plasma membrane and also may adhere to endomembranes.Knocking out OsATX1 resulted in increased Cu concentrations in roots and reduced the Cu translocation from roots to shoots,whereas OsATX1 overexpression reduced root Cu concentrations but increased Cu accumulation in the shoots and the xylem sap,and the Cu translocation from roots to shoots.Under Cu deficiency,the growth of OsATX1overexpression plants was similar to that of the wild type.However,we found that overexpression of OsATX1 significantly reduced Cu tolerance in excess-Cu conditions.At the reproductive stage,the concentrations of Cu in developing tissues,including panicles,upper nodes and internodes,younger leaf blades,and leaf sheaths of the main tiller,were significantly increased in OsATX1-overexpressing plants and decreased in osatx1 mutants compared to that in wild type.The osatx1 mutants also showed a higher Cu concentration in older leaves.OsATX1 contains an MXCXXC Cu+-binding motif,which can bind to Cu+ion and form a homodimer.Yeast two-hybrid and bimolecular fluorescence complementation（BiFC）assays showed that OsATX1 interacts with the rice heavy metal P1B-ATPases OsHMA4,OsHMA5,OsHMA6,and OsHMA9.These results suggest that OsATX1 may function to deliver Cu to heavy metal P1B-ATPases for Cu trafficking and distribution in order to maintain Cu homeostasis in different rice tissues.Overexpression of OsATX1 increased Cd accumulation in the shoots and the xylem sap,whereas osatx1 mutants increased Cd accumulation in roots.Futhermore,overexpression of OsATX1 increased the Cd concentrations in all aboveground tissues at the flowering stage,suggesting that OsATX1 is also involved in Cd transport.In addition,heterologous expression of OsATX1 in the yeast（Saccharomyces cerevisiae）Cd-sensitive mutantΔycf1 increased the tolerance to Cu and Cd by decreasing their respective concentrations in the transformed yeast cells.Taken together,our results indicated that OsATX1 plays an important role in facilitating root-to-shoot Cu translocation and the redistribution of Cu from old leaves to developing tissues and seeds in rice. |
