Mapping and functional analysis of high-copper accumulation mutant oshc1 in rice

Copper (Cu) is an essential but potentially toxic element in rice. Little is known about the mechanism of rice grain Cu accumulation. In this study, we identified a high copper accumulation in grain 1 (oshc1) mutant from the wild type indica rice cultivar 9311 (WT) mutant bank. Compared with those in WT, more Cu was shown to accumulate in the shoots of seedlings and the above-ground tissues except nodes although less total Cu content in oshc1. Further analysis showed that the mutant had an accelerated Cu transport ratio from roots to shoots and higher Cu concentration in xylem sap than WT. This phenomenon in oshc1 was controlled by a single recessive gene, which was identified as BGIOSGA007732, and named OsHMA4. The eight base frame-shift from 1021 to 1028 bp in the coding sequence of OsHMA4 led to a modification after the 341st amino acid and resulted in premature translation termination of OsHMA4 at the 377th amino acid. This may change the function of OsHMA4. Furthermore, the up-regulated OsCOPT7 and OsATX1 and down-regulated OsHMA4 probably decrease Cu compartmentalization in roots of oshc1. In summary, the frame-shift in OsHMA4 changes the function of OsHMA4 and the expression of genes relative to Cu transport in the mutant, which leads to more Cu transport upward and higher Cu accumulation in the rice grains. Moreover, oshc1 was more tolerance to Cu-shortage than WT, while more sensitive to Cu excess exposure than WT. However, RNA-Seq analysis shown that changes in transcription levels of genes in oshc1 involving in molecular function of ions binding and biological processes of cell wall organization and defense response to bio-stress. Which indicates that oshc1 is advantage to Cu limited condition than WT. This work reveals the mechanism of high Cu accumulation in the grains of oshc1 and provides a material to breed new cultivars with optimum levels of Cu in brown rice by crossing with other dominant varieties, which can be planted in different soils to ensure the yield and quality of rice.