Site-Directed Mutagenesis of the Carotenoid Isomerase Gene BnaCRTISO Alters the Color of Petals and Leaves in Brassica napus L.

Abstract

The diversity of petal and leaf color can improve the ornamental value of rapeseed and promote the development of agriculture and tourism. The two copies of carotenoid isomerase gene (BnaCRTISO) in Brassica napus (BnaA09.CRTISO and BnaC08.CRTISO) was edited using the CRISPR/Cas9 system in the present study. The mutation phenotype of creamy white petals and yellowish leaves could be recovered only in targeted mutants of both BnaCRTISO functional copies, indicating that the redundant roles of BnaA09.CRTISO and BnaC08.CRTISO are vital for the regulation of petal and leaf color. The carotenoid content in the petals and leaves of the BnaCRTISO double mutant was significantly reduced. The chalcone content, a vital substance that makes up the yellow color, also decreased significantly in petals. Whereas, the contents of some carotenes (lycopene, α-carotene, γ-carotene) were increased significantly in petals. Further, transcriptome analysis showed that the targeted mutation of BnaCRTISO resulted in the significant down-regulation of important genes BnaPSY and BnaC4H in the carotenoid and flavonoid synthesis pathways, respectively; however, the expression of other genes related to carotenes and xanthophylls synthesis, such as BnaPDS3, BnaZEP, BnaBCH1 and BCH2, was up-regulated. This indicates that the molecular mechanism regulating petal color variation in B. napus is more complicated than those reported in Arabidopsis and other Brassica species. These results provide insight into the molecular mechanisms underlying flower color variation in rapeseed and provides valuable resources for rapeseed breeding.

Clorf Encodes Carotenoid Isomerase and Regulates Orange Flesh Color in Watermelon (Citrullus lanatus L.)

“…To determine whether SNP 31,017,592 variation exists in the coding sequence region, the CDS of Clorf in W1-1 and W1-61 was then cloned. SNP 31,017,592 was still found in the last exon at the 5,077th bp position of Cla97C10G200950 in orange flesh W1-61 and caused a change from leucine to proline at the 560th amino acid which consistent with the resequencing data.…”

“…To investigate the allelic diversity of the Clorf gene in watermelon natural accessions, nucleotide variation of the Clorf locus in 18 resequenced accessions (Table S3) was examined (Figure 3c). SNP 31,017,592 (A → G, located in the last exon of ClCrtiso at the 5,077th bp position) was stable present and distinguished W1-61 from 17 other watermelon accessions. These findings suggest that ClCrtiso might be the most likely candidate gene for the Clorf locus.…”

Precise A∙T to G∙C base editing in the allotetraploid rapeseed (Brassica napus L.) genome

“…In addition, we revealed that the single mutants of BnaA09. CRTISO had a similar yellow flower phenotype as the wild type, which was in accordance with the previous study that two copies of the BnaCRTISO gene function redundantly in regulating the flower color (H. Li et al, 2022).…”

Genomic evidence supports the genetic convergence of a supergene controlling the distylous floral syndrome

“…In addition, we found that the candidate anther‐position gene contained a unique mutation, which caused a change in coding in the amino acid sequence. Mutations can affect gene expression and/or functions (Feng et al ., 2006; Li et al ., 2020, 2022; Perez‐Santangelo et al ., 2022) altering floral phenotype. Given the similar expression patterns of GeGA3OX in the S‐ and H‐morphs, it is possible that this mutation caused the failure of anther elevation through functional loss or neofunctionalization in the H‐morph.…”