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

ABSTRACT

Chilling stress is an important environmental factor that affects rice (Oryza sativa L.) growth and yield, and the booting stage is the most sensitive stage of rice to chilling stress. In this study, we focused on OsRBCS3, 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 OsRBCS3 in rice chilling tolerance at the booting stage. The expression levels of OsRBCS3 under chilling stress were compared in two japonica rice cultivars with different chilling tolerances: Kongyu131 (KY131) and Longjing11 (LJ11). A positive correlation was found between OsRBCS3 expression and chilling tolerance. Over-expression (OE) and knock-out (KO) lines of OsRBCS3 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 OsRBCS3 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.

KEYWORDS:

• Rice
• OsRBCS3
• chilling tolerance
• Over-expression
• Knock-out

Disclosure statement

No potential conflict of interest was reported by the author(s).

Authors’ contributions

YH and RL designed the experiments. SS and YH performed the experiments and wrote the manuscript. CT, YH and SS analyzed the data. YH, CT, SS and RL revised the manuscript. All the authors have read and approved the manuscript.

Ethical approval

The authors have not performed any experiments involving human participants or animals for this study.

Availability of data and materials

All data supporting the findings of this study are available within the paper and its Supplementary Information.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592324.2024.2318514.

Additional information

Funding

This work was supported by the Research fees of Heilongjiang provincial research institutes (CZKYF2022-1-C012, CZKYF2023-1-C021, CZKYF2023-1-A004); Heilongjiang Province Agricultural Science and Technology Innovation Leap Forward Project Youth Science and Technology Innovation Fund Project (CX22YQ24); Heilongjiang Province Agricultural Science and Technology Innovation Leapfrog Project (CX23ZD01).