Search

Abstract Background The blue pigmentation of Japanese gentian flowers is due to a polyacylated anthocyanin, gentiodelphin, and all associated biosynthesis genes and several regulatory genes have been cloned and characterized. However, the final step involving the accumulation of anthocyanins in petal vacuoles remains unclear. We cloned and analyzed the glutathione S-transferases (GSTs) in Japanese gentian that are known to be involved in anthocyanin transport in other plant species. Results We cloned GST1, which is expressed in gentian flower petals. Additionally, this gene belongs to the Phi-type GST clade related to anthocyanin biosynthesis. We used the CRISPR/Cas9-mediated genome editing system to generate loss-of-function GST1 alleles. The edited alleles were confirmed by Sanger and next-generation sequencing analyses. The GST1 genome-edited lines exhibited two types of mutant flower phenotypes, severe (almost white) and mild (pale blue). The phenotypes were associated with decreased anthocyanin accumulation in flower petals. In the GST1 genome-edited lines, sugar-induced stress conditions inhibited the accumulation of anthocyanins in stems and leaves, suggestvhing that GST1 is necessary for stress-related anthocyanin accumulation in organs other than flowers. These observations clearly demonstrate that GST1 is the gene responsible for anthocyanin transport in Japanese gentian, and is necessary for the accumulation of gentiodelphin in flowers. Conclusions In this study, an anthocyanin-related GST gene in Japanese gentian was functionally characterized. Unlike other biosynthesis genes, the functions of GST genes are difficult to examine in in vitro studies. Thus, the genome-editing strategy described herein may be useful for in vivo investigations of the roles of transport-related genes in gentian plants.

Abstract Background CRISPR/Cas9 technology is one of the most powerful and useful tools for genome editing in various living organisms. In higher plants, the system has been widely exploited not only for basic research, such as gene functional analysis, but also for applied research such as crop breeding. Although the CRISPR/Cas9 system has been used to induce mutations in genes involved in various plant developmental processes, few studies have been performed to modify the color of ornamental flowers. We therefore attempted to use this system to modify flower color in the model plant torenia (Torenia fournieri L.). Results We attempted to induce mutations in the torenia flavanone 3-hydroxylase (F3H) gene, which encodes a key enzyme involved in flavonoid biosynthesis. Application of the CRISPR/Cas9 system successfully generated pale blue (almost white) flowers at a high frequency (ca. 80% of regenerated lines) in transgenic torenia T0 plants. Sequence analysis of PCR amplicons by Sanger and next-generation sequencing revealed the occurrence of mutations such as base substitutions and insertions/deletions in the F3H target sequence, thus indicating that the obtained phenotype was induced by the targeted mutagenesis of the endogenous F3H gene. Conclusions These results clearly demonstrate that flower color modification by genome editing with the CRISPR/Cas9 system is easily and efficiently achievable. Our findings further indicate that this system may be useful for future research on flower pigmentation and/or functional analyses of additional genes in torenia.