Metabolome-based discrimination of chrysanthemum cultivars for the efficient generation of flower color variations in mutation breeding.

INTRODUCTION: The color variations of ornamental flowers are often generated by ion-beam and gamma irradiation mutagenesis. However, mutation rates differ significantly even among cultivars of the same species, resulting in high cost and intensive labor for flower color breeding. OBJECTIVES: We aimed to establish a metabolome-based strategy to identify biomarkers and select promising parental lines with high mutation rates using Chrysanthemum as the case study. METHODS: The mutation rates associated with flower color were measured in 10 chrysanthemum cultivars with pink, yellow, or white flowers after soft X-ray irradiation at the floret-formation stage. The metabolic profiles of the petals of these cultivars were clarified by widely targeted metabolomics and targeted carotenoid analysis using liquid chromatography-tandem quadrupole mass spectrometry. Metabolome and carotenoid data were subjected to an un-supervised principal component analysis (PCA) and a supervised logistic regression with least absolute shrinkage and selection operator (LASSO). RESULTS: The PCA of the metabolic profile data separated chrysanthemum cultivars according to flower color rather than mutation rates. By contrast, logistic regression with LASSO generated a discrimination model to separate cultivars into two groups with high or low mutation rates, and selected 11 metabolites associated with mutation rates that can be biomarkers candidates for selecting parental lines for mutagenesis. CONCLUSION: This metabolome-based strategy to identify metabolite markers for mutation rates associated with flower color might be applied to other ornamental flowers to accelerate mutation breeding for generating new cultivars with a wider range of flower colors.

Comparison of petunia and calibrachoa in carotenoid pigmentation of corollas.

Petunia (Petunia hybrida) is an important ornamental plant with a wide range of corolla colors. Although pale-yellow-flowered cultivars, with a low amount of carotenoids in their corollas, are now available, no deep-yellow-flowered cultivars exist. To find why petunia cannot accumulate enough carotenoids to have deep-yellow flowers, we compared carotenoid profiles and expression of carotenoid metabolic genes between pale-yellow-flowered petunia and deep-yellow-flowered calibrachoa (Calibrachoa hybrida), a close relative. The carotenoid contents and the ratios of esterified xanthophylls to total xanthophylls in petunia corollas were significantly lower than those in calibrachoa, despite similar carotenoid components. A lower esterification rate of trans-xanthophylls than of cis-xanthophylls in petunia suggests that petunia xanthophyll esterase (XES) has low substrate specificity for trans-xanthophylls, which are more abundant than cis-xanthophylls in petunia corolla. The expression of genes encoding key enzymes of carotenoid biosynthesis was lower and that of a carotenoid catabolic gene was higher in petunia. XES expression was significantly lower in petunia. The results suggest that low biosynthetic activity, high cleavage activity, and low esterification activity cause low carotenoid accumulation in petunia corollas.

Alteration of flower colour in Ipomoea nil through CRISPR/Cas9-mediated mutagenesis of carotenoid cleavage dioxygenase 4.

Japanese morning glory, Ipomoea nil, exhibits a variety of flower colours, except yellow, reflecting the accumulation of only trace amounts of carotenoids in the petals. In a previous study, we attributed this effect to the low expression levels of carotenogenic genes in the petals, but there may be other contributing factors. In the present study, we investigated the possible involvement of carotenoid cleavage dioxygenase (CCD), which cleaves specific double bonds of the polyene chains of carotenoids, in the regulation of carotenoid accumulation in the petals of I. nil. Using bioinformatics analysis, seven InCCD genes were identified in the I. nil genome. Sequencing and expression analyses indicated potential involvement of InCCD4 in carotenoid degradation in the petals. Successful knockout of InCCD4 using the CRISPR/Cas9 system in the white-flowered cultivar I. nil cv. AK77 caused the white petals to turn pale yellow. The total amount of carotenoids in the petals of ccd4 plants was increased 20-fold relative to non-transgenic plants. This result indicates that in the petals of I. nil, not only low carotenogenic gene expression but also carotenoid degradation leads to extremely low levels of carotenoids.

Molecular mechanisms underlying the diverse array of petal colors in chrysanthemum flowers.

Chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the most important floricultural crops in the world. Although the origin of modern chrysanthemum cultivars is uncertain, several species belonging to the family Asteraceae are considered to have been integrated during the long history of breeding. The flower color of ancestral species is limited to yellow, pink, and white, and is derived from carotenoids, anthocyanins, and the absence of both pigments, respectively. A wide range of flower colors, including purplish-red, orange, red, and dark red, has been developed by increasing the range of pigment content or the combination of both pigments. Recently, green-flowered cultivars containing chlorophylls in their ray petals have been produced, and have gained popularity. In addition, blue/violet flowers have been developed using a transgenic approach. Flower color is an important trait that influences the commercial value of chrysanthemum cultivars. Understanding the molecular mechanisms that regulate flower pigmentation may provide important implications for the rationale manipulation of flower color. This review describes the pigment composition, genetics, and molecular basis of ray petal color formation in chrysanthemum cultivars.

Correction to: Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology.

After publication of the original article [1] the authors noted that the following errors had occurred.

Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology.

BACKGROUND: Chrysanthemum morifolium is one of the most economically valuable ornamental plants worldwide. Chrysanthemum is an allohexaploid plant with a large genome that is commercially propagated by vegetative reproduction. New cultivars with different floral traits, such as color, morphology, and scent, have been generated mainly by classical cross-breeding and mutation breeding. However, only limited genetic resources and their genome information are available for the generation of new floral traits. RESULTS: To obtain useful information about molecular bases for floral traits of chrysanthemums, we read expressed sequence tags (ESTs) of chrysanthemums by high-throughput sequencing using the 454 pyrosequencing technology. We constructed normalized cDNA libraries, consisting of full-length, 3'-UTR, and 5'-UTR cDNAs derived from various tissues of chrysanthemums. These libraries produced a total number of 3,772,677 high-quality reads, which were assembled into 213,204 contigs. By comparing the data obtained with those of full genome-sequenced species, we confirmed that our chrysanthemum contig set contained the majority of all expressed genes, which was sufficient for further molecular analysis in chrysanthemums. CONCLUSION: We confirmed that our chrysanthemum EST set (contigs) contained a number of contigs that encoded transcription factors and enzymes involved in pigment and aroma compound metabolism that was comparable to that of other species. This information can serve as an informative resource for identifying genes involved in various biological processes in chrysanthemums. Moreover, the findings of our study will contribute to a better understanding of the floral characteristics of chrysanthemums including the myriad cultivars at the molecular level.

Transcriptome analysis in petals and leaves of chrysanthemums with different chlorophyll levels.

BACKGROUND: Chlorophylls (Chls) are magnesium-containing tetrapyrrole macromolecules responsible for the green color in plants. The Chl metabolic pathway has been intensively studied and nearly all the enzymes involved in the pathway have been identified and characterized. Synthesis and activity of these enzymes are tightly regulated in tissue- and developmental stage-specific manners. Leaves contain substantial amounts of Chls because Chls are indispensable for photosynthesis. In contrast, petals generally contain only trace amounts of Chls, which if present would mask the bright petal color. Limited information is available about the mechanisms that control such tissue-specific accumulation of Chls. RESULTS: To identify the regulatory steps that control Chl accumulation, we compared gene expression in petals and leaves of chrysanthemum cultivars with different Chl levels. Microarray and quantitative real-time PCR analyses showed that the expression levels of Chl biosynthesis genes encoding glutamyl-tRNA reductase, Mg-protoporphyrin IX chelatase, Mg-protoporphyrin IX monomethylester cyclase, and protochlorophyllide oxidoreductase were well associated with Chl content: their expression levels were lower in white petals than in green petals, and were highest in leaves. Among Chl catabolic genes, expression of STAY-GREEN, encoding Mg-dechelatase, which is a key enzyme controlling Chl degradation, was considerably higher in white and green petals than in leaves. We searched for transcription factor genes whose expression was well related to Chl level in petals and leaves and found three such genes encoding MYB113, CONSTANS-like 16, and DREB and EAR motif protein. CONCLUSIONS: From our transcriptome analysis, we assume that a low rate of Chl biosynthesis and a high rate of Chl degradation lead to the absence of Chls in white chrysanthemum petals. We identified several candidate transcription factors that might affect Chl accumulation in chrysanthemum petals. Functional analysis of these transcription factors will provide a basis for future molecular studies of tissue-specific Chl accumulation.

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.

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

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.

Genetic engineering of novel bluer-colored chrysanthemums produced by accumulation of delphinidin-based anthocyanins.

Chrysanthemums (Chrysanthemum morifolium Ramat.) have no purple-, violet- or blue-flowered cultivars because they lack delphinidin-based anthocyanins. This deficiency is due to the absence of the flavonoid 3',5'-hydroxylase gene (F3'5'H), which encodes the key enzyme for delphinidin biosynthesis. In F3'5'H-transformed chrysanthemums, unpredictable and unstable expression levels have hampered successful production of delphinidin and reduced desired changes in flower color. With the aim of achieving delphinidin production in chrysanthemum petals, we found that anthocyanin biosynthetic gene promoters combined with a translational enhancer increased expression of some F3'5'H genes and accompanying delphinidin-based anthocyanin accumulation in transgenic chrysanthemums. Dramatic accumulation of delphinidin (up to 95%) was achieved by simple overexpression of Campanula F3'5'H controlled by a petal-specific flavanone 3-hydroxylase promoter from chrysanthemum combined with the 5'-untranslated region of the alcohol dehydrogenase gene as a translational enhancer. The flower colors of transgenic lines producing delphinidin-based anthocyanins changed from a red-purple to a purple-violet hue in the Royal Horticultural Society Colour Charts. This result represents a promising step toward molecular breeding of blue chrysanthemums.