Insights on the enhancement of chilling tolerance in Rice through over-expression and knock-out studies of OsRBCS3.

Chilling stress is an important environmental factor that affects rice ( L.) growth and yield, and the booting stage is the most sensitive stage of rice to chilling stress. In this study, we focused on , a rice gene related to chilling tolerance at the booting stage, which encodes the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit in photosynthesis. The aim of this study was to elucidate the role and mechanism of in rice chilling tolerance at the booting stage. The expression levels of under chilling stress were compared in two rice cultivars with different chilling tolerances: Kongyu131 (KY131) and Longjing11 (LJ11). A positive correlation was found between expression and chilling tolerance. Over-expression (OE) and knock-out (KO) lines of were constructed using over-expression and CRISPR/Cas9 technology, respectively, and their chilling tolerance was evaluated at the seedling and booting stages. The results showed that OE lines exhibited higher chilling tolerance than wild-type (WT) lines at both seedling and booting stages, while KO lines showed lower chilling tolerance than WT lines. Furthermore, the antioxidant enzyme activities, malondialdehyde (MDA) content and Rubisco activity of four rice lines under chilling stress were measured, and it was found that OE lines had stronger antioxidant and photosynthetic capacities, while KO lines had the opposite effects. This study validated that plays an important role in rice chilling tolerance at the booting stage, providing new molecular tools and a theoretical basis for rice chilling tolerance breeding.