Copper (Cu) is an essential micronutrient for plant growth. However, the molecular mechanisms underlying Cu trafficking and distribution to different organs in rice (Oryza sativa) are poorly understood. Here, we report the function and role of Antioxidant Protein1 (OsATX1), a Cu chaperone in rice. Knocking out OsATX1 resulted in increased Cu concentrations in roots, whereas OsATX1 overexpression reduced root Cu concentrations but increased Cu accumulation in the shoots. 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 increased significantly in OsATX1-overexpressing plants and decreased in osatx1 mutants compared with the wild type. The osatx1 mutants also showed a higher Cu concentration in older leaves. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that OsATX1 interacts with the rice heavy metal P_1B-ATPases HMA4, HMA5, HMA6, and HMA9. These results suggest that OsATX1 may function to deliver Cu to heavy metal P_1B-ATPases for Cu trafficking and distribution in order to maintain Cu homeostasis in different rice tissues. In addition, heterologous expression of OsATX1 in the yeast (Saccharomyces cerevisiae) cadmium-sensitive mutant Δycf1 increased the tolerance to Cu and cadmium by decreasing their respective concentrations in the transformed yeast cells. Taken together, our results indicate 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.

Summary: Salt stress negatively affects rice growth, development and yield. Metabolic adjustments contribute to the adaptation of rice under salt stress. Branched‐chain amino acids (BCAA) are three essential amino acids that cannot be synthesized by humans or animals. However, little is known about the role of BCAA in response to salt stress in plants. Here, we showed that BCAAs may function as scavengers of reactive oxygen species (ROS) to provide protection against damage caused by salinity. We determined that branched‐chain aminotransferase 2 (OsBCAT2), a protein responsible for the degradation of BCAA, positively regulates salt tolerance. Salt significantly induces the expression of OsBCAT2 rather than BCAA synthesis genes, which indicated that salt mainly promotes BCAA degradation and not de novo synthesis. Metabolomics analysis revealed that vitamin B5 (VB5) biosynthesis pathway intermediates were higher in the OsBCAT2‐overexpressing plants but lower in osbcat2 mutants under salt stress. The salt stress‐sensitive phenotypes of the osbcat2 mutants are rescued by exogenous VB5, indicating that OsBCAT2 affects rice salt tolerance by regulating VB5 synthesis. Our work provides new insights into the enzymes involved in BCAAs degradation and VB5 biosynthesis and sheds light on the molecular mechanism of BCAAs in response to salt stress.

Grain essential amino acid (EAA) levels contribute to rice nutritional quality. However, the molecular mechanisms underlying EAA accumulation and natural variation in rice grains remain unclear. Here we report the identification of a previously unrecognized auxin influx carrier subfamily gene, OsAUX5, which encodes an amino acid transporter that functions in uptake of multiple amino acids. We identified an elite haplotype of Pro::OsAUX5^Hap2 that enhances grain EAA accumulation without an apparent negative effect on agronomic traits. Natural variations of OsAUX5 occur in the cis elements of its promoter, which are differentially activated because of the different binding affinity between OsWRKY78 and the W-box, contributing to grain EAA variation among rice varieties. The two distinct haplotypes were shown to have originated from different Oryza rufipogon progenitors, which contributed to the divergence between japonica and indica. Introduction of the indica-type Pro::OsAUX5^Hap2 genotype into japonica could significantly increase EAA levels, indicating that indica-type Pro::OsAUX5^Hap2 can be utilized to increase grain EAAs of japonica varieties. Collectively, our study uncovers an WRKY78-OsAUX5-based regulatory mechanism controlling grain EAA accumulation and provides a potential target for breeding EAA-rich rice.