An evolutionary view of vanillylamine synthase pAMT, a key enzyme of capsaicinoid biosynthesis pathway in chili pepper.

Pungent capsaicinoid is synthesized only in chili pepper (Capsicum spp.). The production of vanillylamine from vanillin is a unique reaction in the capsaicinoid biosynthesis pathway. Although putative aminotransferase (pAMT) has been isolated as the vanillylamine synthase gene, it is unclear how Capsicum acquired pAMT. Here, we present a phylogenetic overview of pAMT and its homologs. The Capsicum genome contained 5 homologs, including pAMT, CaGABA-T1, CaGABA-T3, and two pseudogenes. Phylogenetic analysis indicated that pAMT is a member of the Solanaceae cytoplasmic GABA-Ts. Comparative genome analysis found that multiple copies of GABA-T exist in a specific Solanaceae genomic region, and the cytoplasmic GABA-Ts other than pAMT are located in the region. The cytoplasmic GABA-T was phylogenetically close to pseudo-GABA-T harboring a plastid transit peptide (pseudo-GABA-T3). This suggested that Solanaceae cytoplasmic GABA-Ts occurred via duplication of a chloroplastic GABA-T ancestor and subsequent loss of the plastid transit signal. The cytoplasmic GABA-T may have been translocated from the specific Solanaceae genomic region during Capsicum divergence, resulting in the current pAMT locus. A recombinant protein assay demonstrated that pAMT had higher vanillylamine synthase activity than those of other plant GABA-Ts. pAMT was expressed exclusively in the placental septum of mature green fruit, whereas tomato orthologs SlGABA-T2/4 exhibit a ubiquitous expression pattern in plants. These findings suggested that both the increased catalytic efficiency and transcriptional changes in pAMT may have contributed to establish vanillylamine synthesis in the capsaicinoid biosynthesis pathway. This study provides insights into the establishment of pungency in the evolution of chili peppers.

Identification of two 6'-deoxychalcone 4'-glucosyltransferase genes in dahlia (Dahlia variabilis).

MAIN CONCLUSION: Two glycosyltransferase genes belonging to UGT88 family were identified to have 6'-deoxychalcone 4'-glucosyltransferase activity in dahlia. 6'-Deoxychalcones (isoliquiritigenin and butein) are important pigments for yellow and orange to red flower color. 6'-Deoxychalcones are glucosylated at the 4'-position in vivo, but the genes encoding 6'-deoxychalcone 4'-glucosyltransferase have not yet been identified. In our previous study, it was indicated that snapdragon (Antirrhinum majus) chalcone 4'-O-glucosyltransferase (Am4'CGT) has isoliquiritigenin 4'-glucosylation activity. Therefore, to identify genes encoding 6'-deoxychalcone 4'-glucosyltransferase in dahlia (Dahlia variabilis), genes expressed in ray florets that shared high homology with Am4'CGT were explored. As a result, c34671_g1_i1 and c35662_g1_i1 were selected as candidate genes for 6'-deoxychalcone 4'-glucosyltransferases in dahlia. We conducted transient co-overexpression of three genes (c34671_g1_i1 or c35662_g1_i1, dahlia aldo-keto reductase1 (DvAKR1) or soybean (Glycine max) chalcone reductase5 (GmCHR5), and chili pepper (Capsicum annuum) MYB transcription factor (CaMYBA)) in Nicotiana benthamiana by agroinfiltration. Transient overexpression of c34671_g1_i1, DvAKR1, and CaMYBA resulted in increase in the accumulation of isoliquiritigenin 4'-glucosides, isoliquiritigenin 4'-O-glucoside, and isoliquiritigenin 4'-O-[6-O-(malonyl)-glucoside]. However, transient overexpression of c35662_g1_i1, DvAKR1, and CaMYBA did not increase accumulation of isoliquiritigenin 4'-glucosides. Using GmCHR5 instead of DvAKR1 showed similar results suggesting that c34671_g1_i1 has isoliquiritigenin 4'-glucosyltransferase activity. In addition, we conducted co-overexpression of four genes (c34671_g1_i1, c35662_g1_i1 or Am4'CGT, DvAKR1 or GmCHR5, CaMYBA, and chalcone 3-hydroxylase from dahlia). Accumulation of butein 4'-O-glucoside and butein 4'-O-[6-O-(malonyl)-glucoside] was detected for c35662_g1_i1, suggesting that c35662_g1_i1 has butein 4'-glucosyltransferase activity. Recombinant enzyme analysis also supported butein 4'-glucosyltransferases activity of c35662_g1_i1. Therefore, our results suggested that both c34671_g1_i1 and c35662_g1_i1 are 6'-deoxychalcone 4'-glucosyltransferases but with different substrate preference.

BpCYP76AD15 is involved in betaxanthin biosynthesis in bougainvillea callus.

MAIN CONCLUSION: BpCYP76AD15 is involved in betaxanthin biosynthesis in callus, but not in bracts, in bougainvillea. Bougainvillea (Bougainvillea peruviana) is a climbing tropical ornamental tree belonging to Nyctaginaceae. Pigments that are conferring colorful bracts in bougainvillea are betalains, and that conferring yellow color are betaxanthins. In general, for red-to-purple betacyanin biosynthesis, α clade CYP76AD that has tyrosine hydroxylase and DOPA oxygenase activity is required, while for betaxanthin biosynthesis, ß clade CYP76AD that has only tyrosine hydroxylase is required. To date, betaxanthin biosynthesis pathway genes have not been identified yet in bougainvillea. Since bougainvillea is phylogenetically close to four-O-clock (Mirabilis jalapa), and it was reported that ß clade CYP76AD, MjCYP76AD15, is involved in floral betaxanthin biosynthesis in four-O-clock. Thus, we hypothesized that orthologous gene of MjCYP76AD15 in bougainvillea might be involved in bract betaxanthin biosynthesis. To test the hypothesis, we attempted to identify ß clade CYP76AD gene from yellow bracts by RNA-seq; however, we could not. Instead, we found that callus accumulated betaxanthin and that ß clade CYP76AD gene, BpCYP76AD15, were expressed in callus. We validated BpCYP76AD15 function by transgenic approach (agro-infiltration and over-expression in transgenic tobacco), and it was suggested that BpCYP76AD15 is involved in betaxanthin biosynthesis in callus, but not in bracts in bougainvillea. Interestingly, our data also indicate the existence of two pathways for betaxanthin biosynthesis (ß clade CYP76AD-dependent and -independent), and the latter pathway is important for betaxanthin biosynthesis in bougainvillea bracts.

The mutation of CaCKI1 causes seedless fruits in chili pepper (Capsicum annuum).

KEY MESSAGE: The seedless mutant tn-1 in chili pepper is caused by a mutation in CaCKI1 (CA12g21620), which encodes histidine kinase involving female gametophyte development. An amino acid insertion in the receiver domain of CaCKI1 may be the mutation responsible for tn-1. Seedlessness is a desirable trait in fruit crops because the removal of seeds is a troublesome step for consumers and processing industries. However, little knowledge is available to develop seedless chili peppers. In a previous study, a chili pepper mutant tn-1, which stably produces seedless fruits, was isolated. In this study, we report characterization of tn-1 and identification of the causative gene. Although pollen germination was normal, confocal laser microscopy observations revealed deficiency in embryo sac development in tn-1. By marker analysis, the tn-1 locus was narrowed down to a 313 kb region on chromosome 12. Further analysis combined with mapping-by-sequencing identified CA12g21620, which encodes histidine kinase as a candidate gene. Phylogenetic analysis revealed CA12g21620 was the homolog of Arabidopsis CKI1 (Cytokinin Independent 1), which plays an important role in female gametophyte development, and CA12g21620 was designated as CaCKI1. Sequence analysis revealed that tn-1 has a 3-bp insertion in the 6th exon resulting in one lysine (K) residue insertion in receiver domain of CaCKI1, and the sequence nearby the insertion is widely conserved among CKI1 orthologs in various plants. This suggested that one K residue insertion may reduce the phosphorylation relay downstream of CaCKI1 and impair normal development of female gametophyte, resulting in seedless fruits production in tn-1. Furthermore, we demonstrated that virus-induced gene silencing of CaCKI1 reduced normally developed female gametophyte in chili pepper. This study describes the significant role of CaCKI1 in seed development in chili pepper and the possibility of developing seedless cultivars using its mutation.

A novel aldo-keto reductase gene is involved in 6'-deoxychalcone biosynthesis in dahlia (Dahlia variabilis).

MAIN CONCLUSION: A novel gene belonging to the aldo-keto reductase 13 family is involved in isoliquiritigenin biosynthesis in dahlia. The yellow pigments of dahlia flowers are derived from 6'-deoxychalcones, which are synthesized via a two-step process, involving the conversion of 3-malonyl-CoA and 4-coumaloyl-CoA into isoliquiritigenin in the first step, and the subsequent generation of butein from isoliquiritigenin. The first step reaction is catalyzed by chalcone synthase (CHS) and aldo-keto reductase (AKR). AKR has been implicated in the isoflavone biosynthesis in legumes, however, isolation of butein biosynthesis related AKR members are yet to be reported. A comparative RNA-seq analysis between two dahlia cultivars, 'Shukuhai' and its butein-deficient lateral mutant 'Rinka', was used in this study to identify a novel AKR gene involved in 6'-deoxychalcone biosynthesis. DvAKR1 encoded a AKR 13 sub-family protein with significant differential expression levels, and was phylogenetically distinct from the chalcone reductases, which belongs to the AKR 4A sub-family in legumes. DNA sequence variation and expression profiles of DvAKR1 gene were correlated with 6'-deoxychalcone accumulation in the tested dahlia cultivars. A single over-expression analysis of DvAKR1 was not sufficient to initiate the accumulation of isoliquiritigenin in tobacco, in contrast, its co-overexpression with a chalcone 4'-O-glucosyltransferase (Am4'CGT) from Antirrhinum majus and a MYB transcription factor, CaMYBA from Capsicum annuum successfully induced isoliquiritigenin accumulation. In addition, DvAKR1 homologous gene expression was detected in Coreopsideae species accumulating 6'-deoxychalcone, but not in Asteraceae species lacking 6'-deoxychalcone production. These results not only demonstrate the involvement of DvAKR1 in the biosynthesis of 6'-deoxychalcone in dahlia, but also show that 6'-deoxychalcone occurrence in Coreopsideae species developed evolutionarily independent from legume species.

Morphological and gene expression characterization of maf-1, a floral chili pepper mutant caused by a nonsense mutation in CaLFY.

Chili peppers are important as vegetables and ornamental crops, because of the variety of fruit shapes and colors. Understanding of flower and fruit development in Capsicum is limited compared with closely related Solanaceae crops such as tomato. This study reports a novel malformed fruit mutant named malformed fruit-1 (maf-1), which was isolated from an ethyl methanesulfonate-induced mutant population of chili pepper. maf-1 exhibited homeotic changes in the floral bud, which were characterized by conversion of petals and stamens into sepal-like and carpel-like organs, respectively. In addition, the indeterminate formation of carpel-like tissue was observed. Genetic analysis demonstrated that the causative gene in maf-1 is a nonsense mutation in CaLFY. This is the first characterization of an lfy mutant in Capsicum. Unlike tomatoes, the CaLFY mutation did not affect the architecture of sympodial unit or flowering time but mainly affected the formation of flower organs. Gene expression analysis suggested that a nonsense mutation in CaLFY led to decreased expression of multiple class B genes, resulting in homeotic changes in the flower and fruit. This maf-1 mutant may provide new insights at the molecular level in understanding flower organ formation and the genetic manipulation of fruit shape in chili peppers. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01304-w.

Post-transcriptional gene silencing of CYP76AD controls betalain biosynthesis in bracts of bougainvillea.

Betalain is one of four major plant pigments and shares some features with anthocyanin; however, no plant has been found to biosynthesize both pigments. Previous studies have reported that anthocyanin biosynthesis in some plants is regulated by post-transcriptional gene-silencing (PTGS), but the importance of PTGS in betalain biosynthesis remains unclear. In this study, we report the occurrence of PTGS in betalain biosynthesis in bougainvillea (Bougainvillea peruviana) 'Thimma', which produces bracts of three different color on the same plant, namely pink, white, and pink-white. This resembles the unstable anthocyanin pigmentation phenotype that is associated with PTGS, and hence we anticipated the presence of PTGS in the betalain biosynthetic pathway. To test this, we analysed pigments, gene expression, small RNAs, and transient overexpression. Our results demonstrated that PTGS of BpCYP76AD1, a gene encoding one of the betalain biosynthesis enzymes, is responsible for the loss of betalain biosynthesis in 'Thimma'. Neither the genetic background nor DNA methylation in the BpCYP76AD1 sequence could explain the induction of PTGS, implying that another locus controls the unstable pigmentation. Our results indicate that naturally occurring PTGS contributes to the diversification of color patterns not only in anthocyanin biosynthesis but also in betalain biosynthesis.

Capsaicinoid biosynthesis in the pericarp of chili pepper fruits is associated with a placental septum-like transcriptome profile and tissue structure.

KEY MESSAGE: CAP biosynthesis in the pericarp of chili pepper fruits occurs with an ambiguous boundary in the placental septum and pericarp. Capsaicinoid (CAP) is a pungent ingredient of chili pepper fruits. Generally, CAP biosynthesis is limited to the placental septum of fruits, but it has been reported that its biosynthesis occurs even in the pericarp of some extremely pungent varieties, resulting in a substantial increase in total content. To examine the mechanism of CAP biosynthesis in the pericarp, comparative transcriptome analysis of a variety that produces CAP in the pericarp (MY) and a variety that does not (HB) was carried out. RNA-seq revealed that 2264 genes were differentially expressed in the MY pericarp compared with the HB pericarp. PCA analysis and GO enrichment analysis indicated that the MY pericarp has a gene expression profile more like placental septum than the HB pericarp. The gene expression of CAP biosynthesis-related genes in the MY pericarp changed coordinately with the placental septum during fruit development. In most Capsicum accessions including HB, the distribution of slender epidermal cells producing CAP was limited to the placental septum, and the morphological boundary between the placental septum and pericarp was clear. In some extremely pungent varieties such as MY, slender epidermal cells ranged from the placental septum to the pericarp region, and the pericarp was morphologically similar to the placental septum, such as the absence of large sub-epidermal cells and abundant spaces in the parenchymal tissue. Our data suggest that CAP biosynthesis in the pericarp occurred with an ambiguous boundary in the placental septum and pericarp. These findings contribute to further enhancement of CAP production in chili pepper fruits.

Post-transcriptional silencing of chalcone synthase is involved in phenotypic lability in petals and leaves of bicolor dahlia (Dahlia variabilis) 'Yuino'.

MAIN CONCLUSION: Post-transcriptional gene silencing (PTGS) of a chalcone synthase ( DvCHS2 ) occurred in the white part of bicolor petals and flavonoid-poor leaves; however, it did not in red petals and flavonoid-rich leaves. Petal color lability is a prominent feature of bicolor dahlia cultivars, and causes plants to produce not only original bicolor petals with colored bases and pure white tips, but also frequently single-colored petals without white tips. In this study, we analysed the molecular mechanisms that are associated with petal color lability using the red-white bicolor cultivar 'Yuino'. Red single-colored petals lose their white tips as a result of recover of flavonoid biosynthesis. Among flavonoid biosynthetic genes including four chalcone synthase (CHS)-like genes (DvCHS1, DvCHS2, DvCHS3, and DvCHS4), DvCHS1 and DvCHS2 had significantly lower expression levels in the white part of bicolor petals than in red petals, while DvCHS3, DvCHS4, and other flavonoid biosynthetic genes had almost the same expression levels. Small RNAs from the white part of a bicolor petal were mapped onto DvCHS1 and DvCHS2, while small RNAs from a red single-colored petal were not mapped onto any of the four CHS genes. A relationship between petal color and leaf flavonoid accumulation has previously been demonstrated, whereby red petal-producing plants accumulate flavonoids in their leaves, while bicolor petal-producing plants tend not to. The expression level of DvCHS2 was down-regulated in flavonoid-poor leaves and small RNAs from flavonoid-poor leaves were mapped onto DvCHS2, suggesting that the down-regulation of DvCHS2 in flavonoid-poor leaves occurs post-transcriptionally. Genomic analysis also suggested that DvCHS2 is the key gene involved in bicolor formation. Together, these results suggest that post-transcriptional gene silencing of DvCHS2 plays a key role in phenotypic lability in this bicolor dahlia.

Tobacco streak virus (strain dahlia) suppresses post-transcriptional gene silencing of flavone synthase II in black dahlia cultivars and causes a drastic flower color change.

MAIN CONCLUSION: Tobacco streak virus suppressed post-transcriptional gene silencing and caused a flower color change in black dahlias, which supported the role of cyanidin-based anthocyanins for black flower appearance. Black flower color of dahlia (Dahlia variabilis) has been attributed, in part, to the high accumulation of cyanidin-based anthocyanins that occurs when flavone synthesis is reduced because of post-transcriptional gene silencing (PTGS) of flavone synthase II (DvFNS). There are also purple-flowering plants that have emerged from a black cultivar 'Kokucho'. We report that the purple color is not caused by a mutation, as previously thought, but by infection with tobacco streak virus (TSVdahlia), which suppresses the PTGS of DvFNS. When TSVdahlia was eliminated from the purple-flowering 'Kokucho' by leaf primordia-free shoot apical meristem culture, the resulting flowers were black. TSVdahlia-infected purple flowers had lower numbers of siRNAs to DvFNS than black flowers, suggesting that TSVdahlia has a silencing suppressor. The graft inoculation of other black cultivars with TSVdahlia altered their flower color drastically except for 'Fidalgo Blacky', a very deep black cultivar with the highest amount of cyanidin-based anthocyanins. The flowers of all six TSVdahlia-infected cultivars accumulated increased amounts of flavones and reduced amounts of cyanidin-based anthocyanins. 'Fidalgo Blacky' remained black despite the change in pigment accumulation, and the amounts of cyanidin-based anthocyanins in its TSVdahlia-infected plants were still higher than those of other cultivars. We propose that black flower color in dahlia is controlled by two different mechanisms that increase the amount of cyanidin-based anthocyanins: DvFNS PTGS-dependent and -independent mechanisms. If both mechanisms occur simultaneously, the flower color will be blacker than if only a single mechanism is active.

Endogenous post-transcriptional gene silencing of flavone synthase resulting in high accumulation of anthocyanins in black dahlia cultivars.

Black color in flowers is a highly attractive trait in the floricultural industry, but its underlying mechanisms are largely unknown. This study was performed to identify the bases of the high accumulation of anthocyanidins in black cultivars and to determine whether the high accumulation of total anthocyanidins alone leads to the black appearance. Our approach was to compare black dahlia (Dahlia variabilis) cultivars with purple cultivars and a purple flowering mutant of a black cultivar, using pigment and molecular analyses. Black cultivars characteristically exhibited low lightness, high petal accumulation of cyanidin and total anthocyanidins without flavones, and marked suppression of flavone synthase (DvFNS) expression. A comparative study using black and purple cultivars revealed that neither the absence of flavones nor high accumulation of total anthocyanidins is solely sufficient for black appearance, but that cyanidin content in petals is also an important factor in the phenotype. A study comparing the black cultivar 'Kokucho' and its purple mutant showed that suppression of DvFNS abolishes the competition between anthocyanidin and flavone synthesis and leads to accumulation of cyanidin and total anthocyanidins that produce a black appearance. Surprisingly, in black cultivars the suppression of DvFNS occurred in a post-transcriptional manner, as determined by small RNA mapping.

A basic helix-loop-helix transcription factor DvIVS determines flower color intensity in cyanic dahlia cultivars.

The study was aimed to identify the factors that regulate the intensity of flower color in cyanic dahlia (Dahlia variabilis), using fifteen cultivars with different color intensities in their petals. The cultivars were classified into three groups based on their flavonoid composition: ivory white cultivars with flavones; purple and pink cultivars with flavones and anthocyanins; and red cultivars with flavones, anthocyanins, and chalcones. Among the purple, pink, and ivory white cultivars, an inverse relationship was detected between lightness, which was used as an indicator for color intensity and anthocyanin content. A positive correlation was detected between anthocyanin contents and the expression of some structural genes in the anthocyanin synthesis pathway that are regulated by DvIVS, a basic helix-loop-helix transcription factor. A positive correlation between anthocyanin content and expression of DvIVS was also found. The promoter region of DvIVS was classified into three types, with cultivars carrying Type 1 promoter exhibited deep coloring, those carrying Type 2 and/or Type 3 exhibited pale coloring, and those carrying Type 1 and Type 2 and/or Type 3 exhibited medium coloring. The transcripts of the genes from these promoters encoded full-length predicted proteins. These results suggested that the genotype of the promoter region in DvIVS is one of the key factors determining the flower color intensity.

Phosphorus starvation induces post-transcriptional CHS gene silencing in Petunia corolla.

KEY MESSAGE: The corolla of Petunia 'Magic Samba' exhibits unstable anthocyanin expression depending on its phosphorus content. Phosphorus deficiency enhanced post-transcriptional gene silencing of chalcone synthase - A in the corolla. Petunia (Petunia hybrida) 'Magic Samba' has unstable red-white bicolored corollas that respond to nutrient deficiency. We grew this cultivar hydroponically using solutions that lacked one or several nutrients to identify the specific nutrient related to anthocyanin expression in corolla. The white area of the corolla widened under phosphorus (P)-deficient conditions. When the P content of the corolla grown under P-deficient conditions dropped to <2,000 ppm, completely white corollas continued to develop in >40 corollas until the plants died. Other elemental deficiencies had no clear effects on anthocyanin suppression in the corolla. After phosphate was resupplied to the P-deficient plants, anthocyanin was restored in the corollas. The expression of chalcone synthase-A (CHS-A) was suppressed in the white area that widened under P-suppressed conditions, whereas the expression of several other genes related to anthocyanin biosynthesis was enhanced more in the white area than in the red area. Reddish leaves and sepals developed under the P-deficient condition, which is a typical P-deficiency symptom. Two genes related to anthocyanin biosynthesis were enhanced in the reddish organs. Small interfering RNA analysis of CHS-A showed that the suppression resulted from post-transcriptional gene silencing (PTGS). Thus, it was hypothesized that the enhancement of anthocyanin biosynthetic gene expression due to P-deficiency triggered PTGS of CHS-A, which resulted in white corolla development.

Temperature-sensitive phenotype caused by natural mutation in Capsicum latescent in two tropical regions.

Plants in tropical regions experience temperature fluctuation only in non-extreme ambient temperatures. Thus, moderate changes in temperatures, which they never experience in their local environments, might be sufficient to manifest the locally hidden phenotype caused by natural mutation. To validate this hypothesis, temperature-treating experiments were performed on Capsicum accessions collected from tropical regions. Thirty-six Capsicum accessions, collected from Caribbean countries, were screened for temperature sensitivity. Similarities in their temperature sensitivities were compared with Sy-2 (C. chinense) from Seychelles, which was previously found to be a temperature-sensitive accession. Tr-13 from Trinidad & Tobago exhibited developmental abnormalities at temperatures below 24 °C. Expression of defense-related genes was induced, and salicylic acid, which is a key molecule in the plant's defense response, accumulated in Tr-13 at temperatures below 24 °C. Tr-13 and Sy-2 appeared normal when they were grown at temperatures simulating those in Trinidad and Seychelles, respectively. Crossing Tr-13 with No. 3341 or Sy-2 revealed that the temperature-sensitive phenotype of Tr-13 was caused by a genetic mutation in the same locus as Sy-2. Plants having a temperature-sensitive phenotype that is caused by natural mutations evade artificial selection and exist as crops in specific environments, such as tropical regions.

Defense response of a pepper cultivar cv. Sy-2 is induced at temperatures below 24°C.

Temperature is one of the most important environmental factors that influence plant growth and development. Recent studies imply that plants show various responses to non-extreme ambient temperatures. Previously, we have found that a pepper cultivar cv. Sy-2 (Capsicum chinense) shows developmental defects at temperatures below 24°C. In this study, to gain new insights into the temperature sensitivity of cv. Sy-2, temperature-sensitive genes were screened using microarray techniques. At restrictive temperature of 20°C, almost one-fourth of the 411 up-regulated genes were defense related or predicted to be defense related. Further expression analyses of several defense-related genes showed that defense-related genes in cv. Sy-2 were constitutively expressed at temperatures below 24°C. Moreover, accumulation of high level of salicylic acid (SA) in cv. Sy-2 grown at 20°C suggests that the defense response is activated in the absence of pathogens. To confirm that the defense response is induced in cv. Sy-2 below 24°C, we evaluated the resistance to biotrophic bacterial pathogen Xanthomonas campestris pv. vesicatoria and necrotrophic fungal pathogen Cercospora capsici. Cv. Sy-2 showed enhanced resistance to X. campestris pv. vesicatoria, but not to C. capsici.

Simultaneous post-transcriptional gene silencing of two different chalcone synthase genes resulting in pure white flowers in the octoploid dahlia.

Garden dahlias (Dahlia variabilis) are autoallooctoploids with redundant genes producing wide color variations in flowers. There are no pure white dahlia cultivars, despite its long breeding history. However, the white areas of bicolor flower petals appear to be pure white. The objective of this experiment was to elucidate the mechanism by which the pure white color is expressed in the petals of some bicolor cultivars. A pigment analysis showed that no flavonoid derivatives were detected in the white areas of petals in a star-type cultivar 'Yuino' and the two seedling cultivars 'OriW1' and 'OriW2' borne from a red-white bicolor cultivar, 'Orihime', indicating that their white areas are pure white. Semi-quantitative RT-PCR showed that in the pure white areas, transcripts of two chalcone synthases (CHS), DvCHS1 and DvCHS2 which share 69% nucleotide similarity with each other, were barely detected. Premature mRNA of DvCHS1 and DvCHS2 were detected, indicating that these two CHS genes are silenced post-transcriptionally. RNA gel blot analysis revealed that small interfering RNAs (siRNAs) derived from CHSs were produced in these pure white areas. By high-throughput sequence analysis of small RNAs in the pure white areas with no mismatch acceptance, small RNAs were mapped to two alleles of DvCHS1 and two alleles of DvCHS2 expressed in 'Yuino' petals. Therefore, we concluded that simultaneous siRNA-mediated post-transcriptional gene silencing of redundant CHS genes results in the appearance of pure white color in dahlias.

A bHLH transcription factor, DvIVS, is involved in regulation of anthocyanin synthesis in dahlia (Dahlia variabilis).

Dahlias (Dahlia variabilis) exhibit a wide range of flower colours because of accumulation of anthocyanin and other flavonoids in their ray florets. Two lateral mutants were used that spontaneously occurred in 'Michael J' (MJW) which has yellow ray florets with orange variegation. MJOr, a bud mutant producing completely orange ray florets, accumulates anthocyanins, flavones, and butein, and MJY, another mutant producing completely yellow ray florets, accumulates flavones and butein. Reverse transcription-PCR analysis showed that expression of chalcone synthase 1 (DvCHS1), flavanone 3-hydroxylase (DvF3H), dihydroflavonol 4-reductase (DvDFR), anthocyanidin synthase (DvANS), and DvIVS encoding a basic helix-loop-helix transcription factor were suppressed, whereas that of chalcone isomerase (DvCHI) and DvCHS2, another CHS with 69% nucleotide identity with DvCHS1, was not suppressed in the yellow ray florets of MJY. A 5.4 kb CACTA superfamily transposable element, transposable element of Dahlia variabilis 1 (Tdv1), was found in the fourth intron of the DvIVS gene of MJW and MJY, and footprints of Tdv1 were detected in the variegated flowers of MJW. It is shown that only one type of DvIVS gene was expressed in MJOr, whereas these plants are likely to have three types of the DvIVS gene. On the basis of these results, the mechanism regulating the formation of orange and yellow ray florets in dahlia is discussed.

Tissue culture-induced flower-color changes in Saintpaulia caused by excision of the transposon inserted in the flavonoid 3', 5' hydroxylase (F3'5'H) promoter.

The variegated Saintpaulia cultivar Thamires (Saintpaulia sp.), which has pink petals with blue splotches, is generally maintained by leaf cuttings. In contrast, tissue culture-derived progeny of the cultivar showed not only a high percentage of mutants with solid-blue petals but also other solid-color variants, which have not been observed from leaf cuttings. Solid-color phenotypes were inherited stably by their progeny from tissue culture. Petals from each solid-color variant were analyzed by high-performance liquid chromatography and shown to contain different proportions of three main anthocyanin derivatives: malvidin, peonidin, and pelargonidin. Analysis of flavonoid 3', 5'-hydroxylase (F3'5'H) sequences showed no differences in the coding region among the variants and variegated individuals. However, a transposon belonging to the hAT superfamily was found in the promoter region of variegated individuals, and the presence of transposon-related insertions or deletions correlated with the observed flower-color phenotypes. Solid-blue flower mutants contained 8-base pair (bp) insertions (transposon excision footprints), while solid-pink mutants had 58- to 70-bp insertions, and purple- and deep-purple mutants had 21- and 24-bp deletions, respectively. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis showed that F3'5'H expression levels correlated with insertions and deletions (indels) caused by hAT excision, resulting in flower-color differences. Our results showed that tissue culture of Saintpaulia 'Thamires' elicits transposon excision, which in turn alters F3'5'H expression levels and flower colors.

Comparative Analysis of Chrysanthemum Stunt Viroid Accumulation and Movement in Two Chrysanthemum (Chrysanthemum morifolium) Cultivars with Differential Susceptibility to the Viroid Infection.

Chrysanthemum stunt viroid (CSVd) was inoculated into two chrysanthemum (Chrysanthemum morifolium) cultivars, the CSVd-susceptible cultivar Piato and the CSVd-resistant cultivar Mari Kazaguruma. For CSVd inoculation, grafting and Agrobacterium-mediated inoculation were used. In grafting experiments, CSVd was detectable in Mari Kazaguruma after grafting onto infected Piato, but after removal of infected rootstocks, CSVd could not be detected in the uppermost leaves. In agroinfection experiments, CSVd systemic infection was observed in Piato but not in Mari Kazaguruma. However, agro-inoculated leaves of Mari Kazaguruma accumulated circular CSVd RNA to levels equivalent to those in Piato at 7 days post-inoculation. In situ detection of CSVd in inoculated leaves revealed that CSVd was absent in phloem of Mari Kazaguruma, while CSVd strongly localized to this site in Piato. We hypothesize that CSVd resistance in Mari Kazaguruma relates not to CSVd replication but to CSVd movement in leaves.

Histogen Layers Contributing to Adventitious Bud Formation Are Determined by their Cell Division Activities.

Saintpaulia ionantha is propagated by adventitious buds in horticulture, and periclinal chimeral cultivars are usually difficult to propagate. However, some periclinal chimeral cultivars can be propagated with adventitious buds, and the mechanism of which has been unknown. Striped flower cultivars "Kaname," "Concord," and "Monique" were used to investigate what causes flower color separation in adventitious shoot-derived plants by tissue culture. These cultivars were revealed to have mutated flavonoid 3', 5' hydroxylase (SiF3'5'H), WDR1 (SiWDR1), or flavonoid 3 hydroxylase (SiF3H), respectively, in their L1 layer. From our previous study using "Kaname," all flowers from adventitious shoots were colored pink, which was the epidermal color of mother plants' flowers. We used "Concrd" and "Monique" from which we obtained not only monochromatic-colored plants the same as the epidermal color of mother plants, but also plants with a monochromatic colored plants, same as the subepidermal color, and a striped flower color the same as mother plants. Histological observations revealed that epidermal cells divided actively at 14 d after culture and they were involved in the formation of adventitious shoots in the cultured leaf segments of "Kaname." On the other hand, in "Concord" and "Monique," the number of divided cells in the subepidermis was rather higher than that of epidermal cells, and subepidermal cells were sometimes involved in shoot formation. In addition, the plant and leaf size of L1-derived plants from "Concord" and "Monique" were non-vigorous and smaller than those derived from the subepidermal layer. In conclusion, periclinal chimeral cultivars of Saintpaulia can be divided into two types. One type has a high cell division activity in the L1 layer, from which only single flower-colored plants derived from L1 can be obtained as adventitious shoots. Another type has a low cell division activity in the L1 layer, from which striped flower-colored plants the same as mother plants derived from several layers including L1 can be obtained as adventitious shoots. In the periclinal chimeral cultivar capable of propagation with adventitious shoots, the possibility was shown that cells in the L2 layer could form shoots by involving cells of the L1 layer with a low division activity.