Generation of semi-dwarf rice (Oryza sativa L.) lines by CRISPR/Cas9-directed mutagenesis of OsGA20ox2 and proteomic analysis of unveiled changes caused by mutations.

Plant height (PH) is one of the most important agronomic traits of rice, as it directly affects the yield potential and lodging resistance. Here, semi-dwarf mutant lines were developed through CRISPR/Cas9-based editing of OsGA20ox2 in an indica rice cultivar. Total 24 independent lines were obtained in T₀ generation with the mean mutation rate of 73.5% including biallelic (29.16%), homozygous (47.91%) and heterozygous (16.66%) mutations, and 16 T-DNA-free lines (50%) were obtained in T₁ generation without off-target effect in four most likely sites. Mutations resulted in a changed amino acid sequence of mutant plants and reduced gibberellins (GA) level and PH (22.2%), flag leaf length (FLL) and increased yield per plant (YPP) (6.0%), while there was no effect on other agronomic traits. Mutants restored their PH to normal by exogenous GA₃ treatment. The expression of the OsGA20ox2 gene was significantly suppressed in mutant plants, while the expression level was not affected for other GA biosynthesis (OsGA2ox3 and OsGA3ox2) and signaling (D1, GIDI and SLR1) genes. The mutant lines showed decreased cell length and width, abnormal cell elongation, while increased cell numbers in the second internode sections at mature stage. Total 30 protein spots were exercised, and 24 proteins were identified, and results showed that OsGA20ox2 editing altered protein expression. Five proteins including, glyceraldehyde-3-phosphate dehydrogenase, putative ATP synthase, fructose-bisphosphate aldolase 1, S-adenosyl methionine synthetase 1 and gibberellin 20 oxidase 2, were downregulated in dwarf mutant lines which may affect the plant growth. Collectively, our results provide the insights into the role of OsGA20ox2 in PH and confirmed that CRISPR-Cas9 is a powerful tool to understand the gene functions.