Sanae Kishimoto

Carotenoid Cleavage Dioxygenase (CmCCD4a) Contributes to White Color Formation in Chrysanthemum Petals

The white petals of chrysanthemum (Chrysanthemum morifolium Ramat.) are believed to contain a factor that inhibits the accumulation of carotenoids. To find this factor, we performed polymerase chain reaction-Select subtraction screening and obtained a clone expressed differentially in white and yellow petals. The deduced amino acid sequence of the protein (designated CmCCD4a) encoded by the clone was highly homologous to the sequence of carotenoid cleavage dioxygenase. All the white-flowered chrysanthemum cultivars tested showed high levels of CmCCD4a transcript in their petals, whereas most of the yellow-flowered cultivars showed extremely low levels. Expression of CmCCD4a was strictly limited to flower petals and was not detected in other organs, such as the root, stem, or leaf. White petals turned yellow after the RNAi construct of CmCCD4a was introduced. These results indicate that in white petals of chrysanthemums, carotenoids are synthesized but are subsequently degraded into colorless compounds, which results in the white color.

Modification of flower color in torenia (Torenia fournieri Lind.) by genetic transformation

We modified flower color in torenia (Torenia fournieri Lind.) by transferring the chalcone synthase (CHS) or the dihydroflavonol-4-reductase (DFR) gene in sense or antisense orientation by Agrobacterium-mediated gene transfer. The modification patterns of flower color among the transformants formed three groups: (1) same color as the wild-type plant; (2) whole corolla changed to a uniformly light color; and (3) with greater degree of lightening in the tube than in the lip. Transformants incorporating antisense transgene(s) tended to become group 2 types, with no plants becoming group 3 type. Transformants harboring sense transgene(s) tended to become group 3 types, rather than group 2 types. Sense genes and antisense genes seemed to have different potential for changing the flower color. We also produced transformants with new characters in torenia flower color; for example, lines with pastel flowers, wavy patterned flowers and parti-colored flowers. We regard this system to be useful for flower color breeding in torenia and for studying gene expression.

Analysis of Carotenoid Composition in Petals of Calendula (Calendula officinalis L.)

Nineteen carotenoids were identified in extracts of petals of orange- and yellow-flowered cultivars of calendula (Calendula officinalis L.). Ten carotenoids were unique to orange-flowered cultivars. The UV–vis absorption maxima of these ten carotenoids were at longer wavelengths than that of flavoxanthin, the main carotenoid of calendula petals, and it is clear that these carotenoids are responsible for the orange color of the petals. Six carotenoids had a cis structure at C-5 (C-5′), and it is conceivable that these (5Z)-carotenoids are enzymatically isomerized at C-5 in a pathway that diverges from the main carotenoid biosynthesis pathway. Among them, (5Z,9Z)-lycopene (1), (5Z,9Z,5′Z,9′Z)-lycopene (3), (5′Z)-γ-carotene (4), and (5′Z,9′Z)-rubixanthin (5) has never before been identified. Additionally, (5Z,9Z,5′Z)-lycopene (2) has been reported only as a synthesized compound.

Carotenoid composition and carotenogenic gene expression during Ipomoea petal development

Japanese morning glory (Ipomoea nil) is a representative plant lacking a yellow-flowered cultivar, although a few wild Ipomoea species contain carotenoids in their petals such as Ipomoea sp. (yellow petals) and I. obscura (pale-yellow petals). In the present study, carotenoid composition and the expression patterns of carotenogenic genes during petal development were compared among I. nil, I. obscura, and Ipomoea sp. to identify the factors regulating carotenoid accumulation in Ipomoea plant petals. In the early stage, the carotenoid composition in petals of all the Ipomoea plants tested was the same as in the leaves mainly showing lutein, violaxanthin, and β-carotene (chloroplast-type carotenoids). However, in fully opened flowers, chloroplast-type carotenoids were entirely absent in I. nil, whereas they were present in trace amounts in the free form in I. obscura. At the late stage of petal development in Ipomoea sp., the majority of carotenoids were β-cryptoxanthin, zeaxanthin, and β-carotene (chromoplast-type carotenoids). In addition, most of them were present in the esterified form. Carotenogenic gene expression was notably lower in I. nil than in Ipomoea sp. In particular, β-ring hydroxylase (CHYB) was considerably suppressed in petals of both I. nil and I. obscura. The CHYB expression was found to be significantly high in the petals of Ipomoea sp. during the synthesis of chromoplast-type carotenoids. The expression levels of carotenoid cleavage genes (CCD1 and CCD4) were not correlated with the amount of carotenoids in petals. These results suggest that both I. obscura and I. nil lack the ability to synthesize chromoplast-type carotenoids because of the transcriptional down-regulation of carotenogenic genes. CHYB, an enzyme that catalyses the addition of a hydroxyl residue required for esterification, was found to be a key enzyme for the accumulation of chromoplast-type carotenoids in petals.

Copigmentation gives bluer flowers on transgenic torenia plants with the antisense dihydroflavonol-4-reductase gene

When anthocyanins in plants make complexes with copigments such as flavones or flavonols (copigmentation), the visible absorption maximum of the flowers is shifted so that it becomes longer: that is, the flowers look bluer. In an earlier study, our group reported the modification of flower color in torenia (Torenia fournieri Lind.) by re-introduction of the dihydroflavonol-4-reductase (DFR) gene or the chalcone synthase (CHS) gene. Our initial observation of torenia transformants was that plants with the antisense DFR gene produced bluer flowers than plants with the antisense CHS gene. In the present study we found that inactivation of the DFR gene by genetic transformation caused the accumulation of flavones - possible copigments - and that the resulting copigmentation likely to make the torenia flowers bluer. This method could be applied to other plant species to produce bluer flowers.

Identification of the carotenoid modifying gene PALE YELLOW PETAL 1 as an essential factor in xanthophyll esterification and yellow flower pigmentation in tomato (Solanum lycopersicum).

Xanthophylls, the pigments responsible for yellow to red coloration, are naturally occurring carotenoid compounds in many colored tissues of plants. These pigments are esterified within the chromoplast; however, little is known about the mechanisms underlying their accumulation in flower organs. In this study, we characterized two allelic tomato (Solanum lycopersicum L.) mutants, pale yellow petal (pyp) 1-1 and pyp1-2, that have reduced yellow color intensity in the petals and anthers due to loss-of-function mutations. Carotenoid analyses showed that the yellow flower organs of wild-type tomato contained high levels of xanthophylls that largely consisted of neoxanthin and violaxanthin esterified with myristic and/or palmitic acids. Functional disruption of PYP1 resulted in loss of xanthophyll esters, which was associated with a reduction in the total carotenoid content and disruption of normal chromoplast development. These findings suggest that xanthophyll esterification promotes the sequestration of carotenoids in the chromoplast and that accumulation of these esters is important for normal chromoplast development. Next-generation sequencing coupled with map-based positional cloning identified the mutant alleles responsible for the pyp1 phenotype. PYP1 most likely encodes a carotenoid modifying protein that plays a vital role in the production of xanthophyll esters in tomato anthers and petals. Our results provide insight into the molecular mechanism underlying the production of xanthophyll esters in higher plants, thereby shedding light on a longstanding mystery.

Agrobacterium tumefaciens-mediated transformation of Cyclamen persicum Mill.

Abstract A method for Agrobacterium -mediated transformation of Cyclamen persicum Mill. is reported. Etiolated petiole segments were infected with Agrobacterium tumefaciens strain AGL0 or LBA4404. These strains have a binary vector plasmid, pIG121Hm, that includes the β-glucuronidase (GUS) gene with an intron as reporter gene, and the neomycin phosphotransferase II gene and the hygromycin phosphotransferase gene as selection markers. Explants were cultured on Murashige and Skoog medium supplemented with 1.0 mg/l thidiazuron, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, 300 mg/l ticarcillin, and 5 mg/l hygromycin or 100 mg/l kanamycin (selection medium) for regeneration. Transformation was confirmed by histochemical assays of GUS activity in plant tissues, and by Southern blot analysis of the GUS gene. Through five experiments, 103 independent GUS-positive plants were obtained from 920 explants.

Agrobacterium tumefaciens-mediated transformation of Elatior Begonia (Begonia×hiemalis Fotsch)

Abstract We report a method for Agrobacterium-mediated transformation of Elatior Begonia (Begonia×hiemalis Fotsch). Young leaf discs were infected with Agrobacterium tumefaciens strains AGL0 and LBA4404. Each strain has a binary vector plasmid, pIG121Hm that includes the β-glucuronidase (GUS) gene with an intron as a reporter gene, and both the neomycin phosphotransferase II and the hygromycin phosphotransferase genes as selection markers. Explants were cultured on modified MS medium supplemented with 1.0 mg/l BA, 0.5 mg/l IAA, 300 mg/l ticarcillin, and either 100 mg/l kanamycin and 5 mg/l hygromycin, or 300 mg/l kanamycin for selection and regeneration. Out of 500 explants infected with AGL0, 16 plantlets were regenerated, and out of 628 explants infected with LBA4404, two plantlets were regenerated after 4 months of culture. Transformation was confirmed by Southern blot analysis of the GUS gene and by histochemical assays of GUS activity in plant tissues. Ten in vitro transgenic plants were obtained from AGL0 infected explants only.

Carotenoid isomerase is key determinant of petal color of Calendula officinalis.

Background: Reddish 5-cis-carotenoids accumulate in the orange but not yellow petals of calendula. Results: A CRTISO in orange petals of calendula lacks an isomerase activity. Conclusion: CRTISO activity is a key factor in determining calendula petal color. Significance: Cys-His-His at position 462 and Gly at position 450 of CoCRTISO are important for the isomerase activity. Orange petals of calendula (Calendula officinalis) accumulate red carotenoids with the cis-configuration at the C-5 or C-5′ position (5-cis-carotenoids). We speculated that the orange-flowered calendula is a carotenoid isomerase (crtiso) loss-of-function mutant that impairs the cis-to-trans conversion of 5-cis-carotenoids. We compared the sequences and enzyme activities of CRTISO from orange- and yellow-flowered calendulas. Four types of CRTISO were expressed in calendula petals. The deduced amino acid sequence of one of these genes (CoCRTISO1) was different between orange- and yellow-flowered calendulas, whereas the sequences of the other three CRTISOs were identical between these plants. Analysis of the enzymatic activities of the CoCRTISO homologs showed that CoCRTISO1-Y, which was expressed in yellow petals, converted carotenoids from the cis-to-trans-configuration, whereas both CoCRTISO1-ORa and 1-ORb, which were expressed in orange petals, showed no activity with any of the cis-carotenoids we tested. Moreover, the CoCRTISO1 genotypes of the F2 progeny obtained by crossing orange and yellow lines linked closely to petal color. These data indicate that CoCRTISO1 is a key regulator of the accumulation of 5-cis-carotenoids in calendula petals. Site-directed mutagenesis showed that the deletion of Cys-His-His at positions 462–464 in CoCRTISO1-ORa and a Gly-to-Glu amino acid substitution at position 450 in CoCRTISO1-ORb abolished enzyme activity completely, indicating that these amino acid residues are important for the enzymatic activity of CRTISO.

Generation of blue chrysanthemums by anthocyanin B-ring hydroxylation and glucosylation and its coloration mechanism

Coexpression of two anthocyanin modification genes elicits blue flower coloration through interaction with colorless flavonoids. Various colored cultivars of ornamental flowers have been bred by hybridization and mutation breeding; however, the generation of blue flowers for major cut flower plants, such as roses, chrysanthemums, and carnations, has not been achieved by conventional breeding or genetic engineering. Most blue-hued flowers contain delphinidin-based anthocyanins; therefore, delphinidin-producing carnation, rose, and chrysanthemum flowers have been generated by overexpression of the gene encoding flavonoid 3′,5′-hydroxylase (F3′5′H), the key enzyme for delphinidin biosynthesis. Even so, the flowers are purple/violet rather than blue. To generate true blue flowers, blue pigments, such as polyacylated anthocyanins and metal complexes, must be introduced by metabolic engineering; however, introducing and controlling multiple transgenes in plants are complicated processes. We succeeded in generating blue chrysanthemum flowers by introduction of butterfly pea UDP (uridine diphosphate)–glucose:anthocyanin 3′,5′-O-glucosyltransferase gene, in addition to the expression of the Canterbury bells F3′5′H. Newly synthesized 3′,5′-diglucosylated delphinidin-based anthocyanins exhibited a violet color under the weakly acidic pH conditions of flower petal juice and showed a blue color only through intermolecular association, termed “copigmentation,” with flavone glucosides in planta. Thus, we achieved the development of blue color by a two-step modification of the anthocyanin structure. This simple method is a promising approach to generate blue flowers in various ornamental plants by metabolic engineering.