Abstract
Rice (Oryza sativa L.) is one of the major crops in the world and significant increase in grain yield is constant demand for breeders to meet the needs of a rapidly growing population. The size of grains is one of major components determining rice yield and a vital trait for domestication and breeding. To increase the grain size in rice, OsSPL16/qGW8 was mutagenized through CRISPR/Cas9, and proteomic analysis was performed to reveal variations triggered by mutations. More specifically, mutants were generated with two separate guide RNAs targeting recognition sites on opposite strands and genomic insertions and deletions were characterized. Mutations followed Mendelian inheritance and homozygous and heterozygous mutants lacking any T-DNA and off-target effects were screened. The mutant lines showed a significant increase in grain yield without any change in other agronomic traits in T0, T1, and T2 generations. Proteomic screening found a total of 44 differentially expressed proteins (DEPs), out of which 33 and 11 were up and downregulated, respectively. Most of the DEPs related to pyruvate kinase, pyruvate dehydrogenase, and cell division and proliferation were upregulated in the mutant plants. Pathway analysis revealed that DEPs were enriched in the biosynthesis of secondary metabolites, pyruvate metabolism, glycolysis/gluconeogenesis, carbon metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and citrate cycle. Gene Ontology (GO) analysis presented that most of the DEPs were involved in the pyruvate metabolic process and pyruvate dehydrogenase complex. Proteins related to pyruvate dehydrogenase E1 component subunit alpha-1 displayed higher interaction in the protein-protein interaction (PPI) network. Thus, the overall results revealed that CRISPR/Cas9-guided OsSPL16 mutations have the potential to boost the grain yield of rice. Additionally, global proteome analysis has broad applications for discovering molecular components and dynamic regulation underlying the targeted gene mutations.
Highlights
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, State Key Laboratory for Conservation and Utilization of Subtropical Agricultural Bioresources, South China Agricultural University, Guangzhou 510642, China
The results showed that the two target sequences assembled in the intermediate vector through the Bsa I site were consistent with the designed single guided RNA (sgRNA) sequences, so the constructed vector is suitable for the step of Agrobacterium Mediated genetic transformation of rice (Figure 1)
After screening the differentially expressed proteins (DEPs), we found that proteins related to pyruvate kinase and dehydrogenase, cell division, and differentiation were up-regulated in mutant plants
Summary
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, State Key Laboratory for Conservation and Utilization of Subtropical Agricultural Bioresources, South China Agricultural University, Guangzhou 510642, China. The size of grains is one of major components determining rice yield and a vital trait for domestication and breeding. To increase the grain size in rice, OsSPL16/qGW8 was mutagenized through CRISPR/Cas, and proteomic analysis was performed to reveal variations triggered by mutations. The mutant lines showed a significant increase in grain yield without any change in other agronomic traits in T0 , T1 , and T2 generations. The overall results revealed that CRISPR/Cas9-guided OsSPL16 mutations have the potential to boost the grain yield of rice. Global proteome analysis has broad applications for discovering molecular components and dynamic regulation underlying the targeted gene mutations. The genetic regulation pathways of rice grain size are complex and diverse, which may involve ubiquitinated protein degradation, plant hormones, and the interaction between different signaling pathways
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