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

Tasaki, Keisuke; Higuchi, Atsumi; Watanabe, Aiko; Sasaki, Nobuhiro; Nishihara, Masahiro

doi:10.1038/s41598-019-51808-3

Cited by 41 publications

(35 citation statements)

References 27 publications

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“…The biallelic GST1 mutation efficiency was calculated as 13.0% (7/54 lines) based on the Sanger sequencing and NGS amplicon sequencing data (Additional file 1: Tables S1 and S2). This efficiency was slightly better than those of previous studies regarding the gentian anthocyanin modification genes Gt5GT (5.7%), Gt3′GT (4.9%), and Gt5/3′AT (8.6%) [22]. The diversity in the efficiencies was considered to be dependent on the target sequences.…”

Section: Discussioncontrasting

confidence: 63%

“…On the basis of this contig sequence, GST1 was isolated from the 'Albireo' cultivar (Gentiana triflora × Gentiana scabra) (i.e., WT) and functionally analyzed. Specifically, we targeted GST1 with the CRISPR/ Cas9 genome editing system, which has been used to functionally characterize gentian genes [22]. The biallelic GST1 mutation efficiency was calculated as 13.0% (7/54 lines) based on the Sanger sequencing and NGS amplicon sequencing data (Additional file 1: Tables S1 and S2).…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, we were the first to establish a CRISPR/Cas9 system targeting phytoene dehydrogenase (PDS) to examine gentians [10]. Moreover, we completed an in vivo functional analysis of anthocyanin modification genes, including 5GT, 3'GT, and 5/3'AT [22]. Thus, the CRISPR/Cas9 system can now be used to accelerate functional analyses of genes in gentians, which are non-model plants.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Discussioncontrasting

confidence: 63%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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“…It is very important to regulate anthocyanin synthesis in L. japonica breeding [22]. With respect to molecular breeding, the identi cation of the key genes involved in anthocyanin synthesis will enable the overexpression or knockout of these genes to facilitate color modi cation [23]. Therefore, this study began with the aim of identifying the essential genes that regulated the alteration of ower color and subsequently exploring the potential molecular mechanisms behind the development of ower color.…”

Section: Discussionmentioning

confidence: 99%

Time-Series Transcriptome Sheds Light on Anthocyanin Metabolism and Species Diversification Between two Lonicera Japonica Thunb Cultivars

Liu¹,

Ye²,

JingXiao³

et al. 2020

Preprint

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Background：‘Yujin 2’ is new variety of Lonicera japonica Thunb and its flower color can change from red to yellow; hence, it is a good model for investigating flower color development mechanisms. Results：High throughput transcriptome sequencing of seven flower development stages of Yujin No.2 was carried out, and 133,487 unigenes were annotated, among which 73,088 were differentially expressed. Then the real-time PCR analysis was carried out. Further, the number of up-regulated DEGs was higher than those that were down-regulated. Of these annotated DEGs, plant hormone signal transduction, phenylpropanoid biosynthesis, and flavonoid biosynthesis were active throughout the flowering process during each stage, whereas carotenoid biosynthesis was inactive in the S1-6 stages. Furthermore, phenylalanine synthesis was enhanced in the S1 phase; however, anthocyanin synthesis was weakened in the S5 and S6 phases, which may be consistent with the changes in petal color of ‘Yujin 2’ from red (S1) to white (S5) and gold (S6). The results showed that 114 unigenes were associated with anthocyanin metabolism, and 72 were significantly upregulated or downregulated. According to the analysis of TFs in anthocyanin metabolism, we obtained 47 transcription factors, which belonged to 18 families. The LjDFR, LjABCB1, LjMYC6, LjDDB2, and LjANS genes rapidly increased during the first three stages. However, only LjF3'5'H expression was significantly down-regulated at S5, which was consistent with anthocyanin accumulation. Conclusions：This study developed a transcriptome profile of flower color generation for L. japonica as well as annotated unigene sets of seven anthesis phases, thereby providing possibilities for improving the germplasm of L. japonica with genetic engineering technologies and cultivating new varieties of different colors.

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“…Using this information, molecular DNA markers to distinguish individuals with pink or white corollas from genotypes with blue-pigmented corollas have been developed [17,18]. Recently, novel technologies, such as radiation with heavy ion beams and genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system, have been applied to induce novel flower colors in gentian [19,20].…”

Section: Introductionmentioning

confidence: 99%

Morphological and cytological observations of corolla green spots reveal the presence of functional chloroplasts in Japanese gentian

et al. 2020

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Gentian is an important ornamental flower in Japan. The corolla of the majority of cultivated Japanese gentians have green spots, which are rarely encountered in flowers of other angiosperms. Little information is available on the functional traits of the green spots. In this study, we characterized the green spots in the Japanese gentian corolla using a number of microscopic techniques. Opto-digital microscopy revealed that a single visible green spot is composed of approximately 100 epidermal cells. The epidermal cells of a green spot formed a dome-like structure and the cell lumen contained many green structures that were granular and approximately 5 μm in diameter. The green structures emitted red autofluorescence when irradiated with 488 nm excitation light. Transmission electron microscopy revealed that the green structures contained typical thylakoids and grana, thus indicating they are chloroplasts. No grana were observed and the thylakoids had collapsed in the plastids of epidermal cells surrounding green spots. To estimate the rate of photosynthetic electron transfer of the green spots, we measured chlorophyll fluorescence using the MICROSCOPY version of an Imaging-PAM (pulse-amplitude-modulated) fluorometer. Under actinic light of 449 μmol m −2 s −1 , substantial electron flow through photosystem II was observed. Observation of green spot formation during corolla development revealed that immature green spots formed at an early bud stage and developed to maturity associated with chloroplast degradation in the surrounding epidermal cells. These results confirmed that the Japanese gentian corolla contains functional chloroplasts in restricted areas of epidermal cells and indicated that a sophisticated program for differential regulation of chloroplast formation and degradation is operative in the epidermis.

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

Cited by 41 publications

References 27 publications

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

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

Time-Series Transcriptome Sheds Light on Anthocyanin Metabolism and Species Diversification Between two Lonicera Japonica Thunb Cultivars

Morphological and cytological observations of corolla green spots reveal the presence of functional chloroplasts in Japanese gentian

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