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

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.

GtMYB3 with the SRDX motif, which is a dominant negative regulatory domain, confirmed that GtMYB3 is also a positive regulator of several downstream flavonoid biosynthetic genes in gentian flowers 12 .
The conversion of delphinidin to gentiodelphin involves four modification steps (Fig. 1). Namely, delphinidin 3-O-glucoside is formed by the glycosylation of delphinidin via Gt3GT activity. Thereafter, additional sugar and acyl modifications are catalyzed by three key enzymes, including two glycosyltransferases, Gt5GT and Gt3′GT, and one acyl transferase, Gt5/3′AT, to produce gentiodelphin (Fig. 1). Regarding the polyacylated gentiodelphin structure, in vitro biochemical analyses suggested that the glycosylation and subsequent acylation of the 3′-hydroxy group of the B-ring of delphinidin aglycone are important for the stabilization and intramolecular sandwich-type stacking of two gentiodelphin caffeic acids, ultimately leading to a more intense blue coloration 5,[13][14][15] . However, the functions of these modification enzymes in vivo have not been comprehensively characterized in gentian because of a lack of available lines in which these genes have been mutated. We previously generated mutant lines in which the expression levels of one (Gt5/3′AT) or two (Gt5/3′AT and GtF3′5′H) genes were knocked down by RNA interference (RNAi), and revealed that two transgenic lines exhibited abnormal flower coloration due to the decreased acylation of anthocyanins 16 . However, the effects were somewhat ambiguous because of the insufficient suppression of the target genes. The expression of the target genes was only downregulated rather than eliminated in the RNAi lines, and we were unable to analyze the effects of the complete inhibition of the expression of the anthocyanin modification genes. Furthermore, there are no available reports describing the suppression of Gt5GT and Gt3′GT expression. Genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system has recently been used to target specific genes in various higher plants, including ornamental plants, such as DFR in Japanese morning glory (Ipomoea nil) 17 , F3H in torenia 18 , and PDS in gentian 19 . Therefore, it is now possible to produce and analyze gene knockout mutant lines targeting genes of interest even in horticultural plant species.
In this study, we used the CRISPR/Cas9 system to target three anthocyanin modification genes (Gt5GT, Gt3′GT, and Gt5/3′AT) and analyze the effects of the knockout of these genes on the pigmentation of gentian flowers. We generated genome-edited lines in which the three targeted anthocyanin modification genes were knocked out and subsequently evaluated the effects on flower color. We confirmed the genome was accurately edited by next-generation sequencing (NGS) before examining flower colors with a spectrophotometer and by a compositional analysis of anthocyanin pigments via HPLC. Our results verified the utility of CRISPR/Cas9-based genome editing and the importance of anthocyanin modification genes for the blue coloration of gentian flowers.

Results
Production of genome-edited gentian plants. Adventitious shoots were selected on medium containing 0.5 or 0.75 mg l −1 bialaphos. A total of 35, 41, and 37 bialaphos-resistant putative transgenic lines were obtained for the transformation experiments involving the Gt5GT, Gt3′GT, and Gt5/3′AT constructs, respectively, of which 23, 36, and 31 lines, respectively, were confirmed to be transgenic by the PCR amplification of part of the pcoCas9 sequence using crude DNA samples. The genome-edited shoots were then screened by a direct Sanger sequence analysis of the PCR products amplified by the primer sets presented in Fig. 2b. Several clones of the PCR products from potential genome-edited shoots were subjected to further subcloning, and the sequences, including the target sites, were determined. Finally, we obtained two, two, and five lines that may have biallelic mutations at the Gt5GT, Gt3′GT, and Gt5/3′AT target sites, respectively. For each transformed construct, two genome-edited knockout lines were selected and grown in a closed greenhouse until flowering.
Next-generation sequencing analysis of genome-edited gentian plants. The results of the amplicon sequence analyses of all target sites in the transformants are summarized in Table 1. We excluded non-specific or low-copy fragments that were thought to be derived from PCR and/or sequence errors, and determined the number of major fragments of each allele. The number of the major Gt5GT, Gt3′GT, and Gt5/3′AT genomic DNA amplicons in the WT plants represented 68.8% (24,402/35,448), 72.7% (21,762/29,916), and 75.8% (33,661/44,435) of the total number of fragments, respectively. Analyses of SNP sequences revealed the gentian genome includes two alleles for Gt5GT and Gt5/3′AT, but four alleles for Gt3′GT. The counted read numbers among individual gene alleles of RNA were almost equal (less than 2.5-fold) in WT plants, except for Gt3′GT allele 4, which was counted approximately 5-to 10-fold higher than that of alleles 1-3. Amplicon sequence analyses also indicated that each transformant contained several characteristic mutations, such as deletions of 1-30 bases or more than 200 bases between the target 1 and target 2 sequences, depending on the transgenic lines and target genes ( Table 1). All Gt5GT and Gt5/3′AT transformants were confirmed to have heterozygous biallelic mutations. Interestingly, the inversion of a 202-base sequence and a 27-base deletion were also confirmed in one allele of Gt5/3′AT #50. Additionally, we were unable to distinguish between alleles 2, 3, and 4 in Gt3′GT #11 because of a lack of SNPs due to a 221-bp deletion. Regarding Gt3′GT #35, some alleles were not detected, probably because of one or more large deletions or base changes to primer sites. However, for both Gt3′GT lines, the remaining amplified alleles were affected by mutations that caused frameshifts. Although the ratio of fragment counts for each allele differed between the genomic DNA and transcripts, we detected a consistent mutation pattern between the genomic DNA and transcripts in all transgenic lines. Therefore, these transgenic lines were confirmed as gene knockout lines, and they were analyzed further.
Petal phenotypes of the genome-edited gentian plants. At approximately 4 months after plants were first acclimated, we detected changes to the flower color phenotypes in the transformants in a closed greenhouse. Figure 3a presents the abaxial and adaxial sides of flower petals of representative transformants. Specifically, the Gt5GT #106, Gt3′GT #11, and Gt5/3′AT #60 lines produced pale red violet, dull pink, and pale mauve flowers, respectively. The colorimetric values of the adaxial side of the flower limb area for the Gt5GT (#47 and #106), Gt3′GT (#11 and #35), and Gt5/3′AT (#50 and #60) transformants are summarized in Fig. 3b. The L*, a*, b*, and C* values for all transformants were significantly higher and/or lower than the corresponding values for the WT plants (Fig. 3b). The hue angles of Gt3′GT (#11 and #35) plants, which produced flowers with dull pink petals, exceeded 300°. The C* values and hue angles are presented in a scatter plot to visualize the color distribution (Fig. 3c). Although the two-dimensional coordinated positions of the Gt5GT and Gt5/3′AT lines overlapped (Fig. 3c), the L* values of the Gt5/3′AT lines were significantly higher than those of the Gt5GT lines (Fig. 3b). Moreover, the hue angles were significantly greater in the Gt3′GT lines than in the WT plants and in the other transformants. Furthermore, the C* value was significantly lower in all transformants than in the WT plants. Thus, the color distribution clearly indicated that all transformants differed from the WT plants. The absorption spectra of the transformants are provided in Fig. 3d. The absorbance values were high for the WT plants, with two peaks (at 580 and 620 nm). In contrast, all transformants had absorbance values less than 1.0, with a slight decrease at 600 nm. The absorbance of the Gt3′GT lines shifted more intensely at shorter wavelengths than the Gt5GT and Gt5/3′AT lines.

Analysis of anthocyanins in genome-edited gentian plants. The petal extracts of the transformants
and WT control were separated by HPLC (at 520 nm). The chromatograms and major peaks of Gt5GT #106, Gt3′GT #11, Gt5/3′AT #60, and WT plants (i.e., the same lines as in Fig. 3a) are presented in Fig. 4a,b. Eleven peaks were detected in the samples. Minor peaks (peak area <100,000) were excluded from analyses. We www.nature.com/scientificreports www.nature.com/scientificreports/ designated the peaks as An1 to An11 based on the retention times. A co-chromatography and analysis of absorbance spectra indicated peaks An1, An2, An5, An8, An9, and An10 corresponded to six standards. Anthocyanin compositions calculated based on the peak area at 520 nm are summarized in Table 2. In WT plants, one major www.nature.com/scientificreports www.nature.com/scientificreports/ peak (An10) corresponding to gentiodelphin accounted for 91% of the total peak area, whereas gentiodelphin was undetectable in all transformants. In the Gt5GT, Gt3′GT, and Gt5/3′AT lines, the major peaks were An5, An9, and An1, which corresponded to delphinidin 3-glucoside (61% or 74%), delphinidin 3-glucosyl-5-caffeoyl-glucoside (90% or 100%), and delphinidin 3,5,3′-triglucosides (57% or 75%), respectively. The presence of minor peaks implied a small amount of unidentified anthocyanins accumulated in the transformants ( Fig. 4a and Table 2). Similar results were obtained for other genome-edited lines ( Supplementary Fig. S1). The anthocyanin concentrations in flower petal extracts were calculated and expressed as delphinidin equivalents ( Supplementary Fig. S2). Gt5GT-edited lines showed significant increase in anthocyanin concentrations compared with WT and other genome-edited lines.

Discussion
In this study, we used the CRISPR/Cas9 system to edit three anthocyanin modification genes (Gt5GT, Gt3′GT, and Gt5/3′AT) in the Japanese gentian genome. In preliminary experiments, we obtained genome-edited gentian plants by applying one sgRNA targeting one site. However, because the frequency of the genome editing greatly varied depending on the genes and target sites, we decided to use two sgRNAs to target two sites on a single gene (Fig. 2b). The use of multiple sgRNAs reportedly increases the efficiency of Cas9-mediated genome editing by increasing the mutation rate of the target genes [20][21][22] . It is also expected to introduce large fragment deletions between the two target sites. In the current study, we obtained one line (Gt3′GT #11) with a large sequence deletion (Table 1). Additionally, the biallelic mutation efficiencies based on a Sanger sequence analysis were 5.7% (2/35 lines), 4.9% (2/41 lines), and 8.6% (5/58 lines) for the Gt5GT, Gt3′GT, and Gt5/3′AT transformants, respectively (data not shown). These efficiencies are much lower than the reported efficiencies for other plants, such as torenia (80%) 18 , morning glory (75%) 17 , and rice (67.6%) 23 . The reason for this difference remains unclear, but one possibility is that the CaMV 35 S promoter-mediated expression of SpCas9 is considerably silenced in gentian 24 . Another possibility is that the high heterogeneity of the large gentian genome (approximately 5 Gb, 2n = 2 × = 26) contributes to the observed inefficiencies. Our NGS analysis of DNA and RNA revealed that the number of major allele fragments represented 66.9%-95.4% of the total number of fragments (Supplementary Table S2). The minor fragments contained SNPs and/or In/dels around the target sequence, but these variations were likely derived from PCR or NGS errors. Alleles 3 and 4 in Gt3′GT-edited line #35 were not detected in our NGS analysis, suggesting the mutations were located outside the sequenced region amplified with primers #426 and #427. An analysis of RNA amplicons indicated that the number of non-mutated WT allele fragments accounted for less than 0.05% of the total number of fragments. This result may have been due to technical errors during the NGS analysis or because of the extremely low chimerism of the remaining unedited cells. However, the expression of a  www.nature.com/scientificreports www.nature.com/scientificreports/ few non-edited sequences in gentian flowers is unlikely to affect the absorbance of the petal surface evaluated with a spectrophotometer or the peak area values determined by HPLC. Thus, we concluded that Gt5GT-, Gt3′GT-, and Gt5/3′AT-edited lines can be used to evaluate flower coloration during an in vivo analysis. To increase the efficiency of gentian genome editing, we are now assessing the effects of several modifications to the CRISPR/ Cas9 system, including to the promoters, the sgRNA scaffold structure, and the selection conditions during the A. tumefaciens-mediated transformation step.
The absorption spectrum of the surface of the fresh petals of WT plants revealed the highest absorbance occurred at 580 nm, with an additional peak at 620 nm (Fig. 3d). The characteristic peaks detected for the WT gentian petals were also observed for the petals of other plant species with blue flowers, including Commelina communis (metal complex pigment), Platycodon grandiflorus (polyacylated anthocyanin), and Felicia amelloides (intermolecular co-pigmentation with flavone C-glycosides) (Supplementary Fig. S3). However, the absorption spectra of the Gt5GT-, Gt3′GT-, and Gt5/3′AT-edited knockout lines were lower than that of the WT control, with a slight decrease at 600 nm. The a*, b*, L*, and C* values of the genome-edited knockout lines accumulating intermediate delphinidin derivatives varied from the corresponding values of the WT plants (Fig. 3b). Specifically, the b* value for blue-yellow color components (blue in the negative direction) considerably increased in all transformants.
Flower color is determined by not only the anthocyanin kinds but also the anthocyanin amounts in petals. Our result indicated that Gt5GT-edited lines showed about 2-fold increase in anthocyanin concentrations compared with WT and other lines (Supplementary Fig. S2). This may also contribute to the observed change of flower color in Gt5GT lines. Although the reason remains unknown, anthocyanin transport activity and/or anthocyanin stability in vacuole may be different among anthocyanin molecules. Further analysis is needed to elucidate the effects of anthocyanin modifications on flower color pigmentation in gentian. However, our results clearly confirmed the actual gentiodelphin biosynthetic pathway and its contribution to blue color development previously only reported from in vitro chemical analysis.
In summary, we confirmed the importance of gentiodelphin for the blue coloration of gentian flowers by analyzing genome-edited lines in which specific anthocyanin modification genes were knocked out by the CRISPR/ Cas9 system. Our data provide novel insights regarding the gentiodelphin biosynthetic pathway and verify the co-pigmentation bluing effects on flower petals. We are now developing new CRISPR/Cas9 vectors and optimizing the method for selecting genome-edited knockout lines. We are also generating various genome-edited knockout lines suitable for horticultural, physiological, and pathological studies. We believe the CRISPR/Cas9 system will accelerate future gentian studies.

Materials and Methods
Plant materials and transformation. The blue-flowered gentian cv. Albireo (Gentiana triflora × Gentiana scabra) plants that were transformed in this study were maintained in an in vitro culture. Half-strength Murashige and Skoog (MS) medium containing 3% (w/v) sucrose and 0.2% (w/v) gellan gum was used as the basal culture medium. The growth conditions in an incubation room were as follows: 22 °C under a 16-h light/8-h dark photoperiod, with light supplied by cool white fluorescent lamps. Plants were transformed according to a modified version of a previously described Agrobacterium tumefaciens-mediated procedure 24 . Briefly, leaf sections were excised and co-cultivated with A. tumefaciens cells harboring a CRISPR/Cas9 vector (Fig. 2a) on half-strength MS medium supplemented with 100 μM acetosyringone. After a 5-day co-cultivation, the leaf sections were transferred to selection medium containing 0.5 or 0.75 mg l −1 bialaphos (Meiji-Seika Co. Ltd., Tokyo, Japan), 500 mg l −1 cefotaxime (Claforan; Aventis Pharma, France), 10 mg l −1 thidiazuron [N-phenyl-N′-(1,2,3-thiadiazol-5-yl) urea], and 1 mg l −1 1-naphthaleneacetic acid. Adventitious shoots were induced from calli dedifferentiated from the edge of leaves on selection medium and transferred to half-strength MS medium solidified with 0.25% (w/v) gellan gum and supplemented with 3% (w/v) sucrose, 500 mg l −1 cefotaxime, and 0.75 mg l −1 bialaphos for rooting. The production of transgenic material was first confirmed by PCR, which was completed with crude extracts from shoots, MightyAmp DNA polymerase (TaKaRa, Shiga, Japan), and primers #61_Cas9U1898 and #62_Cas9L2260 specific for pcoCas9 (Supplementary Table S1). The regenerated plants were acclimated as previously described 25 .

Measurement of gentian petal colors.
To evaluate the flower color phenotypes of the genome-edited knockout lines and the untransformed wild-type (WT) plants, the adaxial surface of freshly collected petals was analyzed spectrophotometrically. For each line, the colorimetric values (L*, a*, and b*) and the absorption spectra of the adaxial surface of the limb area of fresh petals were measured with the CM-3600A spectrophotometer (Konica Minolta, Tokyo, Japan). The chroma C* value and the hue angle were also calculated. Tukey's HSD test was performed to test differences among sample means for significance.
Anthocyanin analysis. The anthocyanin compositions of petal extracts were analyzed by HPLC. Fresh petals were collected from greenhouse-grown plants and stored at −80 °C until used. The petals were soaked in 80% methanol containing 0.1% (v/v) trifluoroacetic acid to extract the anthocyanins, which were then separated with an HPLC-photodiode array detector system [PU-4180 PUMP, MD-4015 photodiode array detector, and ChromNAV (version 2.02) software; JASCO, Tokyo, Japan] equipped with a COSMOSIL PBr column (4.6 mm internal diameter × 250 mm; Nacalai Tesque, Kyoto, Japan). We used a linear gradient elution (2 ml min −1 ) of 10%-80% acetonitrile in 1% aqueous phosphoric acid over 50 min.  Supplementary Table S1. The PCR products were purified and sequenced with the BigDye Terminator (version 1.1) Cycle Sequencing Kit and the ABI PRISM 3130xl or 3500 Genetic Analyzers (Applied Biosystems, Foster City, CA, USA).
Next-generation sequencing analysis. The genomic DNA and transcripts in the petals of the genome-edited lines and the WT control underwent an Illumina NGS analysis. Genomic DNA was isolated from the leaves of transgenic and WT gentian plants with the GeneElute Genome DNA Isolation system (Sigma-Aldrich, St Louis, MO, USA). Total RNA was isolated from the petals of transgenic gentian plants with the RNeasy Plant Mini kit (Qiagen, GmbH, Hilden, Germany) and treated with DNase I (TaKaRa) according to the manufacturer's instructions. The extracted RNA samples were reverse transcribed with PrimeScript II (TaKaRa). The subsequent library preparation, PCR amplification, amplification check, and library quantification check were performed as previously described 18 . The primer positions and target sites are indicated in Fig. 2b. Additional details regarding the primers (e.g., index) are listed in Supplementary Table S1. A bioinformatics analysis was performed as previously described 18 . Briefly, the resulting raw sequence reads were pre-processed with the FASTX toolkit, and the read pairs were merged into a single contiguous sequence (fragment) with a fastq-join script 28 . The unique fragments were then counted.