Molecular characterization of an anthocyanin-related glutathione S-transferase gene in Japanese gentian with the CRISPR/Cas9 system

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 Phitype 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.

Anthocyanin Pigments: Beyond Aesthetics

“…Gene editing offers a faster method to develop these crops. New CRISPR-Cas9 (CRISPR for Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful genome editing tool that has recently been widely adopted in model organisms [ 87 , 88 ].…”

Isolation and Functional Analysis of EPHEMERAL1-LIKE (EPH1L) Genes Involved in Flower Senescence in Cultivated Japanese Gentians

“…Furthermore, we performed RT-PCR analysis and detected the same sequences corresponding to the edited genomic sequences in each of the three lines. These results clearly showed that the functional knockout of EPH1L was achieved as in the case of the genes involved in flower color [ 21 , 22 ] and overwintering [ 35 ] in gentians.…”

“…We used the Japanese cultivated gentian “Albireo” ( Gentiana scabra × Gentiana triflora ) as a WT in this study. The details of this cultivar are described in the manuscript by Tasaki et al [ 22 ]. In this study, WT plants were grown in the fields of the Iwate Agricultural Research Center under natural conditions or in a greenhouse at the Iwate Biotechnology Research Center (IBRC).…”

“…Studies on various traits of Japanese gentian flowers (e.g., color [ 11 , 12 ], flower shape [ 13 , 14 ], flowering time [ 15 ], and floral odor [ 16 ]) have been performed so far. Furthermore, in recent years, knowledge about the metabolic pathway of flower colors in Japanese gentian has accumulated through molecular biology techniques [ 17 , 18 , 19 , 20 , 21 , 22 ]. In addition, the development of DNA markers to distinguish the flower color and shape is progressing based on these findings [ 23 , 24 , 25 ].…”

“…Photosynthesis occurring in the corolla and the movement of the flower while opening and closing may affect flower longevity; however, detailed analyses regarding flower senescence have not been performed. In these experiments, biological tools, such as virus-induced gene silencing and genome editing, have been developed and found helpful in characterizing the gene functions in gentians [ 13 , 21 , 22 , 27 ]. Among studies conducted on the effect of ethylene treatment against senescence, it has been reported that G. scabra is highly sensitive [ 28 ], while other gentians ( G. dahurica , G. kochiana , G. sino-ornata [ 29 ], and G. triflora [ 30 ]) are less sensitive.…”