Lycopene Is Enriched in Tomato Fruit by CRISPR/Cas9-Mediated Multiplex Genome Editing - PubMed (original) (raw)

Lycopene Is Enriched in Tomato Fruit by CRISPR/Cas9-Mediated Multiplex Genome Editing

Xindi Li et al. Front Plant Sci. 2018.

Abstract

Numerous studies have been focusing on breeding tomato plants with enhanced lycopene accumulation, considering its positive effects of fruits on the visual and functional properties. In this study, we used a bidirectional strategy: promoting the biosynthesis of lycopene, while inhibiting the conversion from lycopene to β- and α-carotene. The accumulation of lycopene was promoted by knocking down some genes associated with the carotenoid metabolic pathway. Finally, five genes were selected to be edited in genome by CRISPR/Cas9 system using _Agrobacterium tumefaciens_-mediated transformation. Our findings indicated that CRISPR/Cas9 is a site-specific genome editing technology that allows highly efficient target mutagenesis in multiple genes of interest. Surprisingly, the lycopene content in tomato fruit subjected to genome editing was successfully increased to about 5.1-fold. The homozygous mutations were stably transmitted to subsequent generations. Taken together, our results suggest that CRISPR/Cas9 system can be used for significantly improving lycopene content in tomato fruit with advantages such as high efficiency, rare off-target mutations, and stable heredity.

Keywords: CRISPR/Cas9 system; carotenoid metabolic pathway; genome editing; lycopene; tomato fruits.

PubMed Disclaimer

Figures

FIGURE 1

FIGURE 1

Selection of target genes and designing of CRISPR/Cas9 binary expression cassette. (A) A map of the target genes in the carotenoid metabolic pathway. The green boxes represent the key substances in the metabolic pathway. The red and orange boxes show the two substances, lycopene and β-carotene, respectively. A solid arrow indicates a direct effect, and a dashed arrow indicates an indirect effect. The selected target genes are represented by purple boxes, and the red asterisks represent the sites at which the target genes act on the pathway. G3P, glyceraldehyde 3-phosphate; DXS, 1-deoxy-

D

-xylulose 5-phosphate synthase; GGPPS, geranylgeranyl pyrophosphate synthase; PDS, phytoene desaturase; ZISO, z-carotene isomerase. (B) Five target genes were selected according to the synthesis and metabolism pathways of lycopene, and six target sites were designed. The target sequences are marked in red, and small rectangle frames indicate the PAM. Straight lines and boxes are the introns and exons of the target genes, respectively. (C) Structures of the pYLCRISPR/Cas9-Lycopene binary vectors. HPT(-H) encodes hygromycin B phosphotransferase. The six targets designed are represented by solid boxes in different colors, and the promoters used for each target are shown.

FIGURE 2

FIGURE 2

Editing of each target site in the pYLCRISPR/Cas9-lycopene expression cassette. (A) Total editing efficiency of the six targets in the pYLCRISPR/Cas9-lycopene expression cassette. The mutation rate is the ratio of the number of mutations detected to that of the total number of plants in which mutations are detected. (B) Specific types of each target among the six target sites in the pYLCRISPR/Cas9-lycopene expression cassette. Green, orange, purple, and blue represent homozygous, biallelic, heterozygous, and chimeric mutations, respectively. (C) Large fragment deletion on Blc. Target sequence is labeled with yellow. Small rectangle frames indicate the protospacer adjacent motifs. The transcriptional direction of the Blc is shown. (D) Special DNA inversion on SGR1. The red and blue parts represent the targets T1 and T2, respectively. Gradient change of color between yellow and white indicates the transcriptional direction of SGR1.

FIGURE 3

FIGURE 3

Determination of lycopene content in tomato fruit at Br+7 from different mutant groups. (A) List of different groups of lycopene mutants according to the different combinations of mutant genes. (B) HPLC results of crude extracts from tomato fruit samples in different groups. a, b, lycopene and β-carotenoids, respectively. (C,D) Contents of lycopene and β-carotenoids of tomato fruit in the five different groups and WT, respectively. Error bars represent standard deviation. Different lowercase letters show statistically significant difference according to ANOVA followed by Duncan’s test (p < 0.05).

FIGURE 4

FIGURE 4

TEM images of epidermal cells in tomato fruit of WT and lycopene mutants at Br+7. c, carotenoid containing structures; e, plastid envelope; l, crystal line. Bar, 1 μm.

FIGURE 5

FIGURE 5

Phenotype of tomato fruit at different ripening stages. Three groups of transgenic tomato fruits were photographed at different times after the breaker stage of ripening and compared with that in WT. Sections were obtained at Br+7. Each group photograph was obtained from the same tomato fruit. e, exocarp; i, endocarp and f, flesh of the tomato fruit.

Similar articles

Cited by

References

    1. Barry C. S., McQuinn R. P., Chung M. Y., Besuden A., Giovannoni J. J. (2008). Amino acid substitutions in homologs of the STAY-GREEN protein are responsible for the green-flesh and chlorophyll retainer mutations of tomato and pepper. Plant Physiol. 147 179–187. 10.1104/pp.108.118430 - DOI - PMC - PubMed
    1. Brooks C., Nekrasov V., Lippman Z. B., Van Eck J. (2014). Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol. 166 1292–1297. 10.1104/pp.114.247577 - DOI - PMC - PubMed
    1. Čermák T., Baltes N. J., Čegan R., Zhang Y., Voytas D. F. (2015). High-frequency, precise modification of the tomato genome. Genome Biol. 16:232. 10.1186/s13059-015-0796-9 - DOI - PMC - PubMed
    1. Chappell J., Wolf F., Proulx J., Cuellar R., Saunders C. (1995). Is the reaction catalyzed by 3-hydroxy-3-methylglutaryl coenzyme a reductase a rate-limiting step for isoprenoid biosynthesis in plants? Plant Physiol. 109 1337–1343. 10.1104/pp.109.4.1337 - DOI - PMC - PubMed
    1. Chen W., He S., Liu D., Patil G. B., Zhai H., Wang F., et al. (2015). A sweetpotato geranylgeranyl pyrophosphate synthase gene, IbGGPS, increases carotenoid content and enhances osmotic stress tolerance in Arabidopsis thaliana. PLoS One 10:e0137623. 10.1371/journal.pone.0137623 - DOI - PMC - PubMed

LinkOut - more resources