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Effects of knocking out three anthocyanin modification genes on the blue pigmentation of gentian flowers.

ABSTRACT: Genome editing by the CRISPR/Cas9 system has recently been used to produce gene knockout lines in many plant species. We applied this system to analyze Japanese gentian plants that produce blue flowers because of the accumulation of a polyacylated anthocyanin, gentiodelphin. Mutant lines in which anthocyanin modification genes were knocked out were examined to assess the contribution of each gene to the blue pigmentation of flowers. The targeted genes encoded anthocyanin 5-O-glycosyltransferase (Gt5GT), anthocyanin 3'-O-glycosyltransferase (Gt3'GT), and anthocyanin 5/3'-aromatic acyltransferase (Gt5/3'AT). The Gt5GT knockout lines accumulated delphinidin 3G, whereas the Gt3'GT knockout lines accumulated delphinidin 3G-5CafG as the major flower pigment. Knocking out Gt5/3'AT resulted in the accumulation of delphinidin 3G-5G-3'G and delphinidin 3G-5G as the primary and secondary pigments, respectively. These results indicated the existence of two pathways mediating the modification of delphinidin 3G-5G in flowers, with one involving a glycosylation by 3'GT and the other involving an acylation by 5/3'AT. The Gt5GT, Gt3'GT, and Gt5/3'AT transformants produced pale red violet, dull pink, and pale mauve flowers, respectively, unlike the vivid blue flowers of wild-type plants. Thus, the glycosylation and subsequent acylation of the 3'-hydroxy group of the B-ring in delphinidin aglycone is essential for the development of blue gentian flowers.

SUBMITTER: Tasaki K

PROVIDER: S-EPMC6825144 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Effects of knocking out three anthocyanin modification genes on the blue pigmentation of gentian flowers.

Tasaki Keisuke K Higuchi Atsumi A Watanabe Aiko A Sasaki Nobuhiro N Nishihara Masahiro M

Scientific reports 20191101 1

Genome editing by the CRISPR/Cas9 system has recently been used to produce gene knockout lines in many plant species. We applied this system to analyze Japanese gentian plants that produce blue flowers because of the accumulation of a polyacylated anthocyanin, gentiodelphin. Mutant lines in which anthocyanin modification genes were knocked out were examined to assess the contribution of each gene to the blue pigmentation of flowers. The targeted genes encoded anthocyanin 5-O-glycosyltransferase ...[more]

PMID: 31676875

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Project description:BACKGROUND:The cultivated potato Solanum tuberosum L. is the fourth most important crop worldwide. Anthocyanins synthesis and accumulation in potato tissues are considered as one of important traits related to stress resistance and nutritional value. It is considered that the major regulatory gene for anthocyanin biosynthesis is R2R3 MYB-encoding gene StAN1. However, the genetic control of pigmentation of different potato tissues is substantially under investigated. The development of genetic markers for breeding of potato with specific pigmentation pattern remains an actual task. RESULTS:We investigated 36 potato varieties and hybrids with different pigmentation of tubers and leaves. Sequence organization of regulatory R2R3 MYB (StAN1, StMYBA1, StMYB113), bHLH (StbHLH1, StJAF13) and WD40 (StWD40) genes potentially controlling anthocyanin biosynthesis has been evaluated. The results demonstrated a high variability in the StAN1 third exon and promoter region with the exception for 35?bp, containing elements for the transcription start and activation of gene expression in roots. The analysis of transcriptional activity of genes coding R2R3 MYBs, bHLHs and WD40 transcriptional factors in leaves of eight potato genotypes with different anthocyanin pigmentation was performed. The results showed a relation between the gene expression level and plant pigmentation only for the StAN1 and StWD40 genes, while other studied genes had either strong expression in all varieties and hybrids (StMYBA1, StbHLH1 and StJAF13) or they were not expressed at all (StMYB113). CONCLUSIONS:It was found that StAN1 is the major regulatory gene controlling potato anthocyanin synthesis. However, diagnostic markers developed for the functional StAN1 alleles (StAN1777 and StAN1816) can not be used efficiently for prediction of potato pigmentation patterns. It is likely that the sequence organization of StAN1 promoter is important for anthocyanin synthesis control and the development of additional diagnostic markers is necessary.

Project description:BackgroundFruit coloration of red-skinned grapevines is mainly due to anthocyanin pigments. We analysed a panel of nine cultivars that included extreme phenotypes for berry colour, ranging from green (absence of anthocyanins) to red, purple, violet and blue. Expression of six genes of the anthocyanin pathway coding for flavanone-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT), glutathione-S-transferase (GST), O-methyltransferase (OMT) and four transcription factors (MybA, MybB, MybC, MybD) was analysed by quantitative RT-PCR at four developmental stages from before the onset of ripening until full maturity and compared to anthocyanin metabolites.ResultsTotal anthocyanin content at full maturity correlated well with the cumulative expression of F3H, UFGT and GST throughout ripening. Transcripts of the last two genes were absent in the green-skinned cultivar 'Sauvignonasse', also known as 'Tocai friulano', and were at least 10-fold less abundant in pale red cultivars, such as 'Pinot gris' and 'Gewürztraminer', compared to fully coloured cultivars. Predominance of tri-hydroxylated anthocyanins (delphinidin, petunidin and malvidin) in cultivars bearing dark berries with violet and blue hue was associated with higher ratios of F3'5'H/F3'H transcription, compared to red-skinned cultivars. Higher levels of OMT transcripts were observed in berries of cultivars that accumulated methoxylated forms of anthocyanins more abundantly than non-methoxylated forms.ConclusionColour variation of the grape berry conforms to a peculiar pattern of genotype-specific expression of the whole set of anthocyanin genes in a direct transcript-metabolite-phenotype relationship. Cumulative mRNA levels of the structural genes and their relative abundance throughout ripening explained per se the final phenotype for anthocyanin content, anthocyanin composition, colour intensity and colour hue of grapes at berry maturity.

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Effects of knocking out three anthocyanin modification genes on the blue pigmentation of gentian flowers.

Publications

Effects of knocking out three anthocyanin modification genes on the blue pigmentation of gentian flowers.

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