Transcriptomic and physiological analysis ofOsCAO1knockout lines using the CRISPR/Cas9 system in rice

Key message The altered rice leaf color based on the knockout of CAO1 gene generated using CRISPR/Cas9 technology plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced senescence in rice. Rice chlorophyllide a oxygenase (OsCAO1), identified as the chlorophyll b synthesis under light condition, plays a critical role in regulating rice plant photosynthesis. In this study, the development of edited lines with pale green leaves by knockout ofOsCAO1gene known as a chlorophyll synthesis process is reported. Eighty-one genetically edited lines out of 181 T(0)plants were generated through CRISPR/Cas9 system. The edited lines have short narrow flag leaves and pale green leaves compared with wild-type 'Dongjin' plants (WT). Additionally, edited lines have lower chlorophyll b and carotenoid contents both at seedling and mature stages. A transcriptome analysis identified 580 up-regulated and 206 downregulated genes in the edited lines. The differentially expressed genes (DEGs) involved in chlorophyll biosynthesis, magnesium chelatase subunit (CHLH), and glutamate-1-semialdehyde2, 1-aminomutase (GSA) metabolism decreased significantly. Meanwhile, the gel consistency (GC) levels of rice grains, chalkiness ratios and chalkiness degrees (CD) decreased in the edited lines. Thus, knockout ofOsCAO1influenced growth period, leaf development and grain quality characters of rice. Overall, the result suggests thatOsCAO1also plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced rice senescence.

Automated summary

CRISPR/Cas9-mediated knockout of the OsCAO1 gene in rice leads to changes in leaf color, morphology, and chlorophyll content, affecting growth, leaf development, and grain quality characteristics. Knockout of OsCAO1 also influences the gene expression involved in chlorophyll biosynthesis and metabolism, as well as rice grain quality traits such as gel consistency and chalkiness.