Color-related chlorophyll and carotenoid concentrations of Chinese kale can be altered through CRISPR/Cas9 targeted editing of the carotenoid isomerase gene BoaCRTISO

Abstract

The carotenoid isomerase gene (BoaCRTISO) of Chinese kale was targeted and edited using the CRISPR/Cas9 system in the present study. The results showed a high mutation rate (81.25%), and 13 crtiso mutants were obtained. Only two types of mutations, insertions and replacements, were found. Both the total and individual carotenoid and chlorophyll concentrations of the biallelic and homozygous mutants were reduced, and the total levels declined by 11.89-36.33%. The color of the biallelic and homozygous mutants changed from green to yellow, likely reflecting a reduction in the color-masking effect of chlorophyll on carotenoids. The expression levels of most carotenoid and chlorophyll biosynthesis-related genes, including CRTISO, were notably lower in the mutants than in the WT plants. In addition, the functional differences between members of this gene family were discussed. In summary, these findings indicate that CRISPR/Cas9 is a promising technique for the quality improvement of Chinese kale and other Brassica vegetables.

Keywords: Molecular engineering in plants; Secondary metabolism; Transgenic plants.

Fig. 1. Vector map and BoaCRTISO mutations.

a CRISPR/Cas9-induced mutations in Chinese kale. The target sequence is indicated in blue, the PAM sequence (NGG) is underlined in red, the mutated bases are indicated in red font, and the asterisks indicate spacing between bases. WT wild-type plant, M # number of mutants, i # number of base insertions, r # number of base replacements. M1 is a biallelic mutant with two kinds of sequences. M3, M6, and M16 are homozygous mutants with one kind of sequence. b Number of different mutant types. c Frequency of different mutant types

Fig. 2. Phenotype of the crtiso mutants.

a Phenotype of the crtiso mutants and wild-type plants at 6 weeks after transplanting. WT wild-type plant, M1 biallelic plant, M3, M6, M16 homozygous plants. b Color parameters of the crtiso mutants and wild-type plants at 6 weeks after transplanting. The data are expressed as the means of three replicates. The same letter in the same column indicates no significant differences among values (p < 0.05) according to the LSD test

Fig. 3. Pigment composition and concentrations in mutant and wild-type (WT) plants.

a Concentrations of carotenoids and chlorophyll in the leaves of the mutants and WT plants at 6 weeks after transplanting. b Concentrations of carotenoids and chlorophyll in the bolting stems of the mutants and WT plants at 6 weeks after transplanting. WT wild-type plant, M1 biallelic mutant, M3, M6, M16 homozygous mutants. The data are expressed as the means ± SDs. The same letter in the same histogram indicates that there is no significant difference between the values tested, according to the LSD (p < 0.05)

Fig. 4. Expression levels of genes related to carotenoid biosynthesis and degradation.

The leaves and bolting stems of mutants and WT plants were sampled 6 weeks after transplanting. The main axis represents the amount of gene expression in the leaves, and the secondary axis represents the amount of gene expression in the bolting stems. WT wild-type plant, M1 biallelic mutant, M3, M6, M16 homozygous mutants. GGPP geranylgeranyl diphosphate, PSY phytoene synthase, PDS phytoene desaturase, ZDS ζ-carotene desaturase, Z-ISO ζ-carotene isomerase, CRTISO carotenoid isomerase, LCYe lycopene ε-cyclase, LCYb lycopene β-cyclase, ε-OHase ε-carotene hydroxylase, β-OHase β-carotene hydroxylase, VDE violaxanthin de-epoxidase, ZEP zeaxanthin epoxidase, NXS neoxanthin synthase, CCD carotenoid cleavage dioxygenase

Fig. 5. Expression levels of genes related to chlorophyll biosynthesis and degradation.

The leaves and bolting stems of mutants and WT plants were sampled 6 weeks after transplanting. The main axis represents the amount of gene expression in the leaves, and the secondary axis represents the amount of gene expression in the bolting stems. WT wild-type plant, M1 biallelic mutant, M3 M6, M16 homozygous mutants. ALAD 5-aminolevulinic acid dehydratase, HemE1 glutamyl tRNA reductase, ChlI magnesium-chelatase I, ChlD magnesium-chelatase D, ChlH magnesium-chelatase H, CS chlorophyll synthase, CLH chlorophyllase, PaO pheide a oxygenase, PPH pheophytinase, RCCR red Chl catabolite reductase, NYC nonyellow coloring

Fig. 6. Schematic diagram of the results of this study.

The solid frames indicate the presence of the substance in Chinese kale, and the dashed frames indicate the absence of the substance in Chinese kale. The blue genes were downregulated in the mutants, the red genes were upregulated in the mutants, and the black genes did not change significantly in terms of their expression. The down arrow next to a pigment indicates a decrease in its content. ⊥ indicates suppression