Generation of High Yielding and Fragrant Rice (Oryza sativa L.) Lines by CRISPR/Cas9 Targeted Mutagenesis of Three Homoeologs of Cytochrome P450 Gene Family and OsBADH2 and Transcriptome and Proteome Profiling of Revealed Changes Triggered by Mutations

Abstract

The significant increase in grain yield and quality are often antagonistic but a constant demand for breeders and consumers. Some genes related to cytochrome P450 family are known for rice organ growth but their role in controlling grain yield is still unknown. Here, we generated new rice mutants with high yield and improved aroma by simultaneously editing three cytochrome P450 homoeologs (Os03g0603100, Os03g0568400, and GL3.2) and OsBADH2 with the CRISPR/Cas9 system, and RNA-sequencing and proteomic analysis were performed to unveil the subsequent changes. High mutation efficiency was achieved in both target sites of each gene and the mutations were predominantly only deletions, while insertions were rare, and no mutations were detected in the five most likely off-target sites against each sgRNA. Mutants exhibited increased grain size, 2-acetyl-1-pyrroline (2AP) content, and grain cell numbers while there was no change in other agronomic traits. Transgene-DNA-free mutant lines appeared with a frequency of 44.44% and homozygous mutations were stably transmitted, and bi-allelic and heterozygous mutations followed Mendelian inheritance, while the inheritance of chimeric mutations was unpredictable. Deep RNA sequencing and proteomic results revealed the regulation of genes and proteins related to cytochrome P450 family, grain size and development, and cell cycle. The KEGG and hub-gene and protein network analysis showed that the gene and proteins related to ribosomal and photosynthesis pathways were mainly enriched, respectively. Our findings provide a broad and detailed basis to understand the role of CRISPR/Cas9 in rice yield and quality improvement.

Keywords: CRISPR/Cas9; cytochrome P450; mutations; proteome; rice; transcriptome; yield.

Figure 1

Illustration of target sites and assembly of single guided RNAs (sgRNAs) in pYLCRISPR/Cas9PubiH. (A) The illustration of eight sgRNAs in pYLCRISPR/Cas9. (B) Product length of all sgRNAs in the second round of PCR, M; molecular marker (2000 bp), T1-T8 represents target1-target8 (C) Sequencing confirmation of eight sgRNAs in pYLCRISPR/Cas9PubiH, PAM, protospacer adjacent motif.

Figure 2

The CRISPR/Cas9 system induced mutation frequency detection in T₀ generation and sequence alignment of heritable homozygous rice mutants. (A) The mutation efficiency of each sgRNA; (B) types of mutations with the number of mutation events; (C) PCR identification and sequence alignment of wild-type (WT) and four homozygous T₀ mutant lines (i.e., GXU7, GXU19, GXU24, and GXU28). T1–T8 represents Target1-Target8. Red letters are the sgRNA sites; letter in violet color represents inserted nucleotide sequence; the blue letters are protospacer adjacent motif (PAM) sequence; “d” and “i” indicates the deletions and insertion, respectively.

Figure 3

Grain phenotype and the 2-acetyl-1-pyrroline (2AP) levels of homozygous mutants and wild-type (WT) grains. (A) Grain phenotype of WT and homozygous mutant lines (GXU7-1, GXU19-1, GXU24-1, and GXU27-1). (B) The total ion chromatograms (TIC) of 2AP and TMP (as internal standard) in the T₁ homozygous mutant lines and WT. (C) 2AP levels of the mutants and WT. Values are means ± SD, n = 5, p ≤ 0.01, student’s t-test. ** indicates significant difference.

Figure 4

Anatomical observation and comparison of wild-type (WT) and mutant line (GXU7-1) grain cells. Transverse section of whole grain and paraffin section of the developed lemmas of WT (A) and GXU7-1 (B), respectively. (C) Cell number of the lemma. (D) Cell length of the outer epidermal lemma. Data are given as means ± SD, n = 5, p ≤ 0.01. * and ns represents significant and non-significant difference, respectively.

Figure 5

RT-qPCR-based assessment of target genes expression analysis and validation of transcriptome and proteomic experiments. (A) Expression level of Os03g0603100, Os03g0568400, GL3.2, and OsBADH2 in wild-type (WT) and mutant plants (B) Expression analysis of ten selected DEGs and (C) genes associated with DEPs. * Denotes a significant difference, student’s t-test, p ≤ 0.01, n = 3.