Calcium-dependent protein kinase 16 phosphorylates and activates the aquaporin PIP2;2 to regulate reversible flower opening in Gentiana scabra.

Flower opening is important for successful pollination in many plant species, and some species repeatedly open and close their flowers. This is thought to be due to turgor pressure changes caused by water influx/efflux, which depends on osmotic oscillations in the cells. In some ornamental plants, water-transporting aquaporins, also known as plasma membrane intrinsic proteins (PIPs), may play an important role in flower opening. However, the molecular mechanism(s) involved in corolla movement are largely unknown. Gentian (Gentiana spp.) flowers undergo reversible movement in response to temperature and light stimuli; using gentian as a model, we showed that the Gentiana scabra aquaporins GsPIP2;2 and GsPIP2;7 regulate repeated flower opening. In particular, phosphorylation of a C-terminal serine residue of GsPIP2;2 is important for its transport activity and relates closely to the flower re-opening rate. Furthermore, GsPIP2;2 is phosphorylated and activated by the calcium (Ca2+)-dependent protein kinase GsCPK16, which is activated by elevated cytosolic Ca2+ levels in response to temperature and light stimuli. We propose that GsCPK16-dependent phosphorylation and activation of GsPIP2;2 regulate gentian flower re-opening, with stimulus-induced Ca2+ signals acting as triggers.

Flower color modification in Torenia fournieri by genetic engineering of betacyanin pigments.

BACKGROUND: Betalains are reddish and yellow pigments that accumulate in a few plant species of the order Caryophyllales. These pigments have antioxidant and medicinal properties and can be used as functional foods. They also enhance resistance to stress or disease in crops. Several plant species belonging to other orders have been genetically engineered to express betalain pigments. Betalains can also be used for flower color modification in ornamental plants, as they confer vivid colors, like red and yellow. To date, betalain engineering to modify the color of Torenia fournieri-or wishbone flower-a popular ornamental plant, has not been attempted. RESULTS: We report the production of purple-reddish-flowered torenia plants from the purple torenia cultivar "Crown Violet."  Three betalain-biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were constitutively ectopically expressed under the cauliflower mosaic virus (CaMV) 35S promoter, and their expression was confirmed by quantitative real-time PCR (qRT-PCR) analysis. The color traits, measured by spectrophotometric colorimeter and spectral absorbance of fresh petal extracts, revealed a successful flower color modification from purple to reddish. Red pigmentation was also observed in whole plants. LC-DAD-MS and HPLC analyses confirmed that the additional accumulated pigments were betacyanins-mainly betanin (betanidin 5-O-glucoside) and, to a lesser extent, isobetanin (isobetanidin 5-O-glucoside). The five endogenous anthocyanins in torenia flower petals were also detected. CONCLUSIONS: This study demonstrates the possibility of foreign betacyanin accumulation in addition to native pigments in torenia, a popular garden bedding plant. To our knowledge, this is the first report presenting engineered expression of betalain pigments in the family Linderniaceae. Genetic engineering of betalains would be valuable in increasing the flower color variation in future breeding programs for torenia.

Identification of a novel viral factor inducing tumorous symptoms by disturbing vascular development in planta.

Plant viruses induce various disease symptoms that substantially impact agriculture, but the underlying mechanisms of viral disease in plants are poorly understood. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Here, we show that a gene fragment of gentian Kobu-sho-associated virus, which is designated as Kobu-sho-inducing factor (KOBU), induces gall formation accompanied by ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. Transgenic gentian expressing KOBU exhibited tumorous symptoms, confirming the gall-forming activity of KOBU. Surprisingly, KOBU expression can also induce differentiation of an additional leaf-like tissue on the abaxial side of veins in normal N. benthamiana and gentian leaves. Transcriptome analysis with Arabidopsis thaliana expressing KOBU revealed that KOBU activates signaling pathways that regulate xylem development. KOBU protein forms granules and plate-like structures and co-localizes with mRNA splicing factors within the nucleus. Our findings suggest that KOBU is a novel pleiotropic virulence factor that stimulates vascular and leaf development. IMPORTANCE While various mechanisms determine disease symptoms in plants depending on virus-host combinations, the details of how plant viruses induce symptoms remain largely unknown in most plant species. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Our findings demonstrate that a gene fragment of gentian Kobu-sho-associated virus (GKaV), which is designated as Kobu-sho-inducing factor, induces the gall formation accompanied by the ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. The molecular mechanism by which gentian Kobu-sho-associated virus induces the Kobu-sho symptoms will provide new insight into not only plant-virus interactions but also the regulatory mechanisms underlying vascular and leaf development.

Production of yellow-flowered gentian plants by genetic engineering of betaxanthin pigments.

Genetic engineering of flower color provides biotechnological products such as blue carnations or roses by accumulating delphinidin-based anthocyanins not naturally existing in these plant species. Betalains are another class of pigments that in plants are only synthesized in the order Caryophyllales. Although they have been engineered in several plant species, especially red-violet betacyanins, the yellow betaxanthins have yet to be engineered in ornamental plants. We attempted to produce yellow-flowered gentians by genetic engineering of betaxanthin pigments. First, white-flowered gentian lines were produced by knocking out the dihydroflavonol 4-reductase (DFR) gene using CRISPR/Cas9-mediated genome editing. Beta vulgaris BvCYP76AD6 and Mirabilis jalapa MjDOD, driven by gentian petal-specific promoters, flavonoid 3',5'-hydroxylase (F3'5'H) and anthocyanin 5,3'-aromatic acyltransferase (AT), respectively, were transformed into the above DFR-knockout white-flowered line; the resultant gentian plants had vivid yellow flowers. Expression analysis and pigment analysis revealed petal-specific expression and accumulation of seven known betaxanthins in their petals to c. 0.06-0.08 µmol g FW-1 . Genetic engineering of vivid yellow-flowered plants can be achieved by combining genome editing and a suitable expression of betaxanthin-biosynthetic genes in ornamental plants.

Gentian FLOWERING LOCUS T orthologs regulate phase transitions: floral induction and endodormancy release.

Perennial plants undergo a dormant period in addition to the growth and flowering phases that are commonly observed in annuals and perennials. Consequently, the regulation of these phase transitions in perennials is believed to be complicated. Previous studies have proposed that orthologs of FLOWERING LOCUS T (FT) regulate not only floral initiation but also dormancy. We, therefore, investigated the involvement of FT orthologs (GtFT1 and GtFT2) during the phase transitions of the herbaceous perennial gentian (Gentiana triflora). Analysis of seasonal fluctuations in the expression of these genes revealed that GtFT1 expression increased prior to budbreak and flowering, whereas GtFT2 expression was induced by chilling temperatures with the highest expression occurring when endodormancy was released. The expression of FT-related transcription factors, reportedly involved in flowering, also fluctuated during each phase transition. These results suggested the involvement of GtFT1 in budbreak and floral induction and GtFT2 in dormancy regulation, implying that the two gentian FT orthologs activated a different set of transcription factors. Gentian ft2 mutants generated by CRISPR/Cas9-mediated genome editing had a lower frequency of budbreak and budbreak delay in overwintering buds caused by an incomplete endodormancy release. Our results highlighted that the gentian orthologs of FRUITFULL (GtFUL) and SHORT VEGETATIVE PHASE-like 1 (GtSVP-L1) act downstream of GtFT2, probably to prevent untimely budbreak during ecodormancy. These results suggest that each gentian FT ortholog regulates a different phase transition by having variable responses to endogenous or environmental cues, leading to their ability to induce the expression of distinct downstream genes.

Metabolic engineering of betacyanin in vegetables for anti-inflammatory therapy.

Betalains, which consist of the subgroups betaxanthins and betacyanins, are hydrophilic pigments that have classically been used for food colorants. Owing to their strong antioxidant property, their usefulness for application for therapeutic use is also expected. In addition, as betalains are mainly naturally available from plants of the order Caryophyllales, including beet (Beta vulgaris), metabolic engineering for betalain production in crops such as vegetables, fruits and cereals may provide new food resources useful for healthcare. Here we conducted metabolic engineering of betacyanins in tomato fruits and potato tubers. The transgenic tomato fruits and potato tubers with coexpression of betacyanin biosynthesis genes, CYP76AD1 from B. vulgaris, DOD (DOPA 4,5-dioxygenase) and 5GT (cyclo-DOPA 5-O-glucosyltransferase) from Mirabilis jalapa, under control of suitable specific promoters, possessed dark red tissues with enriched accumulation of betacyanins (betanin and isobetanin). The anti-inflammatory activity of transgenic tomato fruit extract was superior to that of wild-type fruit extract on macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS), as a result of decreased LPS-stimulated transcript levels of proinflammatory genes. These findings were in accord with the observation that administration of the transgenic tomato fruits ameliorated dextran sulfate sodium (DSS)-induced colitis as well as body weight loss and disease activity index in mice, via suppression of DSS-stimulated transcript levels of pro-inflammatory genes, including Tnf (encoding TNF-alpha), Il6, and Ptgs2 (encoding cyclooxygenae 2). Intriguingly, given the fact that the transgenic potato tuber extract failed to enrich the anti-inflammatory activity of macrophage cells, it is likely that metabolic engineering of betacyanins will be a powerful way of increasing the anti-inflammatory property of ordinary foods such as tomato.

Identification and characterization of xanthone biosynthetic genes contributing to the vivid red coloration of red-flowered gentian.

Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin-based polyacylated anthocyanins. However, recent breeding programs developed several red-flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high-performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin-based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red-flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6-O-glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol-6-O-(6'-O-malonyl)-glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3-O-glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell-free protein expression system and assayed. We determined that a UDP-glucose-dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red-flowered gentians via copigmentation effects.

Genome Analysis Revives a Forgotten Hybrid Crop Edo-dokoro in the Genus Dioscorea.

A rhizomatous Dioscorea crop 'Edo-dokoro' was described in old records of Japan, but its botanical identity has not been characterized. We found that Edo-dokoro is still produced by four farmers in Tohoku-machi of the Aomori prefecture, Japan. The rhizomes of Edo-dokoro are a delicacy to the local people and are sold in the markets. Morphological characters of Edo-dokoro suggest its hybrid origin between the two species, Dioscorea tokoro and Dioscorea tenuipes. Genome analysis revealed that Edo-dokoro likely originated by hybridization of a male D. tokoro to a female D. tenuipes, followed by a backcross with a male plant of D. tokoro. Edo-dokoro is a typical minor crop possibly maintained for more than 300 years but now almost forgotten by the public. We hypothesize that there are many such uncharacterized genetic heritages passed over generations by small-scale farmers that await serious scientific investigation for future use and improvement by using modern genomics information.

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

The elongation of flower longevity increases the commercial value of ornamental plants, and various genes have been identified as influencing flower senescence. Recently, EPHEMERAL1 (EPH1), encoding a NAC-type transcription factor, was identified in Japanese morning glory as a gene that promotes flower senescence. Here we attempted to identify an EPH1 homolog gene from cultivated Japanese gentians and characterized the same with regard to its flower senescence. Two EPH1-LIKE genes (EPH1La and EPH1Lb), considered as alleles, were isolated from a gentian cultivar (Gentiana scabra × G. triflora). Phylogenetic analyses revealed that EPH1L belongs to the NAM subfamily. The transcript levels of EPH1L increased along with its senescence in the field-grown flowers. Under dark-induced senescence conditions, the gentian-detached flowers showed the peak transcription level of EPH1L earlier than that of SAG12, a senescence marker gene, suggesting the involvement of EPH1L in flower senescence. To reveal the EPH1L function, we produced eph1l-knockout mutant lines using the CRISPR/Cas9 system. When the flower longevity was evaluated using the detached flowers as described above, improved longevity was recorded in all genome-edited lines, with delayed induction of SAG12 transcription. The degradation analysis of genomic DNA matched the elongation of flower longevity, cumulatively indicating the involvement of EPH1L in the regulation of flower senescence in gentians.

De Novo Transcriptome Analysis Reveals Flowering-Related Genes That Potentially Contribute to Flowering-Time Control in the Japanese Cultivated Gentian Gentiana triflora.

Japanese cultivated gentians are perennial plants that flower in early summer to late autumn in Japan, depending on the cultivar. Several flowering-related genes, including GtFT1 and GtTFL1, are known to be involved in regulating flowering time, but many such genes remain unidentified. In this study, we obtained transcriptome profiling data using the Gentiana triflora cultivar 'Maciry', which typically flowers in late July. We conducted deep RNA sequencing analysis using gentian plants grown under natural field conditions for three months before flowering. To investigate diurnal changes, the plants were sampled at 4 h intervals over 24 h. Using these transcriptome data, we determined the expression profiles of leaves based on homology searches against the Flowering-Interactive Database of Arabidopsis. In particular, we focused on transcription factor genes, belonging to the BBX and MADS-box families, and analyzed their developmental and diurnal variation. The expression levels of representative BBX genes were also analyzed under long- and short-day conditions using in-vitro-grown seedlings, and the expression patterns of some BBX genes differed. Clustering analysis revealed that the transcription factor genes were coexpressed with GtFT1. Overall, these expression profiles will facilitate further analysis of the molecular mechanisms underlying the control of flowering time in gentians.

Post-transcriptional gene silencing of the chalcone synthase gene CHS causes corolla lobe-specific whiting of Japanese gentian.

MAIN CONCLUSION: Post-transcriptional gene silencing of the chalcone synthase gene CHS specifically suppresses anthocyanin biosynthesis in corolla lobes and is responsible for the formation of a stripe type bicolor in Japanese gentian. The flower of Japanese gentian is a bell-shaped corolla composed of lobes and plicae, which is painted uniformly blue. However, the gentian cultivar 'Hakuju' shows bicolor phenotype (blue-white stripe corolla), in which anthocyanin accumulation is suppressed only in corolla lobes. Expression analysis indicated that steady-state levels of chalcone synthase (CHS) transcripts were remarkably reduced in corolla lobes compared with plicae during petal pigmentation initiation. However, no significant difference in expression levels of other flavonoid biosynthetic structural and regulatory genes was detected in its lobes and plicae. On feeding naringenin in white lobes, anthocyanin accumulation was recovered. Northern blotting probed with CHS confirmed the abundant accumulation of small RNAs in corolla lobes. Likewise, small RNA-seq analysis indicated that short reads from its lobes were predominantly mapped onto the 2nd exon region of the CHS gene, whereas those from the plicae were scarcely mapped. Subsequent infection with the gentian ovary ringspot virus (GORV), which had an RNA-silencing activity, showed the recovery of partial pigmentation in lobes. Hence, these results strongly suggested that suppressing anthocyanin accumulation in the lobes of bicolored 'Hakuju' was attributed to the specific degradation of CHS mRNA in corolla lobes, which was through post-transcriptional gene silencing (PTGS). Herein, we revealed the molecular mechanism of strip bicolor formation in Japanese gentian, and showed that PTGS of CHS was also responsible for flower color pattern in a floricultural plant other than petunia and dahlia.

Acute diffuse thyroid swelling after fine-needle aspiration: A case report and review of the literature.

Fine-needle aspiration (FNA) is the first-line and a cost-effective examination method of nonfunctional thyroid nodules. Acute transient thyroid swelling after an FNA is a rare complication, and to date, only 14 cases have been reported in the English literature. Herein, we report a case of a 26-year-old woman with acute transient thyroid swelling, which occurred after an ultrasound-guided FNA of a thyroid nodule. Although the patient had undergone an FNA without complication 2 years previously, the second FNA caused acute thyroid swelling. The present case emphasizes the potential risk of acute thyroid swelling associated with every FNA procedure.

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

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.

The Host Stomatal Density Determines Resistance to Septoria gentianae in Japanese Gentian.

Plant stomata represent the main battlefield for host plants and the pathogens that enter plant tissues via stomata. Septoria spp., a group of ascomycete fungi, use host plant stomata for invasion and cause serious damage to agricultural plants. There is no evidence, however, showing the involvement of stomata in defense systems against Septoria infection. In this study, we isolated Septoria gentianae 20-35 (Sg20-35) from Gentiana triflora showing gentian leaf blight disease symptoms in the field. Establishment of an infection system using gentian plants cultured in vitro enabled us to observe the Sg20-35 infection process and estimate its virulence in several gentian cultivars or lines. Sg20-35 also entered gentian tissues via stomata and showed increased virulence in G. triflora compared with G. scabra and their interspecific hybrid. Notably, the susceptibility of gentian cultivars to Sg20-35 was associated with their stomatal density on the adaxial but not abaxial leaf surface. Treatment of EPIDERMAL PATTERNING FACTOR-LIKE 9 (EPFL9/STOMAGEN) peptides, a small secreted peptide controlling stomatal density in Arabidopsis thaliana, increased stomatal density on the adaxial side of gentian leaves as well. Consequently, treated plants showed enhanced susceptibility to Sg20-35. These results indicate that stomatal density on the adaxial leaf surface is one of the major factors determining the susceptibility of gentian cultivars to S. gentianae and suggest that stomatal density control may represent an effective strategy to confer Septoria resistance.

Inhibition of post-transcriptional gene silencing of chalcone synthase genes in petunia picotee petals by fluacrypyrim.

In petals of picotee petunia (Petunia hybrida) cultivars, margin-specific post-transcriptional gene silencing (PTGS) of chalcone synthase A (CHSA) inhibits anthocyanin biosynthesis, resulting in marginal white tissue formation. In this study, we found that a low molecular mass compound, fluacrypyrim, inhibits PTGS of CHSA, and we explored the site-specific PTGS mechanism of operation. Fluacrypyrim treatment abolished the picotee pattern and eliminated site-specific differences in the levels of anthocyanin-related compounds, CHSA expression, and CHSA small interfering RNA (siRNA). In addition, fluacrypyrim abolished the petunia star-type pattern, which is also caused by PTGS of CHSA. Fluacrypyrim treatment was effective only at the early floral developmental stage and predominantly eliminated siRNA derived from CHS genes; i.e. siRNA derived from other genes remained at a comparable level. Fluacrypyrim probably targets the induction of PTGS that specifically operates for CHS genes in petunia picotee flowers, rather than common PTGS maintenance mechanisms that degrade mRNAs and generate siRNA. Upon treatment, the proportion of colored tissue increased due to a shift of the border between white and colored sites toward the margin in a time- and dose-dependent manner. These findings imply that the fluacrypyrim-targeted PTGS induction is completed gradually and its strength is attenuated from the margins to the center of petunia picotee petals.

[Long-Term Survival in a Patient with Abdominal Wall Metastasis of Early Gastric Cancer-A Case Report].

A 76-year-old man was diagnosed with type 1 early gastric cancer. Attempted ESD on the lesion resulted in perforation, and distal gastrectomy with D1+dissection was performed. After 1 year and 6 months, a mass measuring 2.4 cm appeared in the abdominal wall. Cytological examination revealed adenocarcinoma, and the patient was diagnosed with abdominal wall metastasis of gastric cancer. There were no evidences of recurrence in the other organs, and extraction was performed. After 6 months, 1 year, and 2 years, the same metastases were found in the abdominal wall, and repeated extractions were performed. All 4 masses had resulted from the metastasis of gastric cancer, but the patient has been alive without recurrence for 1 year and 6 months after the surgery.

Application of the CRISPR/Cas9 system for modification of flower color in Torenia fournieri.

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.

Development of basic technologies for improvement of breeding and cultivation of Japanese gentian.

Japanese gentians are the most important ornamental flowers in Iwate Prefecture and their breeding and cultivation have been actively conducted for half a century. With its cool climate and large hilly and mountainous area, more than 60% of gentian production in Japan occurs in Iwate Prefecture. Recent advances in gentian breeding and cultivation have facilitated the efficient breeding of new cultivars; disease control and improved cultivation conditions have led to the stable production of Japanese gentians. Molecular biology techniques have been developed and applied in gentian breeding, including the diagnosis of viral diseases and analysis of physiological disorders to improve gentian production. This review summarizes such recent approaches that will assist in the development of new cultivars and support cultivation. More recently, new plant breeding techniques, including several new biotechnological methods such as genome editing and viral vectors, have also been developed in gentian. We, therefore, present examples of their application to gentians and discuss their advantages in future studies of gentians.

Carnation I locus contains two chalcone isomerase genes involved in orange flower coloration.

Carnations carrying a recessive I gene show accumulation of the yellow pigment chalcononaringenin 2'-glucoside (Ch2'G) in their flowers, whereas those with a dominant I gene do accumulation the red pigment, anthocyanin. Although this metabolic alternative at the I gene could explain yellow and red flower phenotypes, it does not explain the development of orange flower phenotypes which result from the simultaneous accumulation of both Ch2'G and anthocyanin. The carnation whole genome sequencing project recently revealed that two chalcone isomerase genes are present, one that is consistent with the I gene (Dca60979) and another (Dca60978) that had not been characterized. Here, we demonstrate that Dca60979 shows a high level of gene expression and strong enzyme activity in plants with a red flower phenotype; however, functional Dca60979 transcripts are not detected in plants with an orange flower phenotype because of a dTdic1 insertion event. Dca60978 was expressed at a low level and showed a low level of enzyme activity in plants, which could catalyze a part of chalcone to naringenin to advance anthocyanin synthesis but the other part remained to be catalyzed chalcone to Ch2'G by chalcone 2'-glucosyltransferase, resulting in accumulation of anthocyanin and Ch2'G simultaneously to give orange color.

Repressed expression of a gene for a basic helix-loop-helix protein causes a white flower phenotype in carnation.

In a previous study, two genes responsible for white flower phenotypes in carnation were identified. These genes encoded enzymes involved in anthocyanin synthesis, namely, flavanone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR), and showed reduced expression in the white flower phenotypes. Here, we identify another candidate gene for white phenotype in carnation flowers using an RNA-seq analysis followed by RT-PCR. This candidate gene encodes a transcriptional regulatory factor of the basic helix-loop-helix (bHLH) type. In the cultivar examined here, both F3H and DFR genes produced active enzyme proteins; however, expression of DFR and of genes for enzymes involved in the downstream anthocyanin synthetic pathway from DFR was repressed in the absence of bHLH expression. Occasionally, flowers of the white flowered cultivar used here have red speckles and stripes on the white petals. We found that expression of bHLH occurred in these red petal segments and induced expression of DFR and the following downstream enzymes. Our results indicate that a member of the bHLH superfamily is another gene involved in anthocyanin synthesis in addition to structural genes encoding enzymes.