Effect of postharvest ethylene treatment on carotenoid accumulation and the expression of carotenoid biosynthetic genes in the flavedo of orange (Citrus sinensis L. Osbeck) fruit

Degreening with ethylene is a common postharvest practice in citrus fruit. In this work we have examined the effect of ethylene treatment on carotenoid content and composition, and on the expression of carotenoid biosynthetic genes in the flavedo of Navelate orange (Citrus sinensis L.) harvested at two ripening stages. The ethylene-induced fruit coloration and carotenoid content in the flavedo increased with the ripening stage of the fruit. Analysis of the changes in individual carotenoids revealed that ethylene stimulated an increase in phytoene, phytofluene, (9Z)-violaxanthin which is the main carotenoid in fully ripened orange peel, and the apocarotenoid β-citraurin, and decreased the concentration of chloroplastic carotenoids. These changes are consistent with the effect of ethylene on the expression of carotenoid biosynthetic genes, since it up-regulated the expression of phytoene synthase, ζ-carotene desaturase and β-carotene hydroxylase genes, sustained or transiently increased accumulation of phytoene desaturase, plastid terminal oxidase, β-lycopene cyclase and zeaxanthin epoxidase mRNAs, and decreased the expression of the ɛ-lycopene cyclase gene. These data indicate that exogenous ethylene reproduces and accelerates the physiological and molecular changes in the carotenoid biosynthesis naturally occurring during maturation of citrus fruit. On the other hand, gibberellic acid, which delays fruit degreening, reduced the ethylene-induced expression of early carotenoid biosynthetic genes and the accumulation of phytoene, phytofluene and β-citraurin.     

Characterization of carotenoid pigments and their biosynthesis in two yellow flowered lines of Sandersonia aurantiaca (Hook)

The basis of the novel cream/yellow flower color found in two Sandersonia aurantiaca lines was examined as part of a project to develop new colors for this cut flower crop in New Zealand. The original color, bright orange, is due to the accumulation of the carotenoid pigments zeaxanthin and β-cryptoxanthin. The cream/yellow lines have much lower levels of total carotenoid pigments (17% and 21%) in their tepal tissue compared to the wild type progenitor. Microscopic analysis of epidermal cells showed alteration in the pigment cluster bodies of tepal tissue of the cream/yellow lines compared to the orange wild type. HPLC analysis of the pigments showed that one cream/yellow line (Y-H) produced the same pigment profile as the wild type (zeaxanthin and β-cryptoxanthin). In comparison, the other cream/yellow line (Y-S) produced the carotenoid profile normally found in green vegetative tissue (β-carotene and lutein). Analysis of carotenoid biosynthetic gene expression in Sandersonia indicated that the cream/yellow Y-H line showed expression patterns similar to the wild type, and gene expression in the Y-S line is decreased relative to the wild type and the Y-H line. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of carotenogenic gene expression in petals and leaves of carnation (Dianthus caryophyllus L.)

Carnation (Dianthus caryophyllus L.) belongs to the family Caryophyllaceae in the order Caryophyllales. Plants in this order do not accumulate carotenoids in petals. To understand how carotenoid accumulation is controlled in carnation petals, we analysed the expression of genes related to carotenoid accumulation. Petals at an early stage of development accumulated small amounts of carotenoids. As petals matured, their carotenoid content decreased to extremely low levels. In contrast, carnation leaves contained substantial amounts of carotenoids that are essential for photosynthesis. Most of the carotenogenic genes were expressed in petals at levels similar to those in leaves, and the expression levels of these genes increased during petal development. Genes encoding phytoene synthase and lycopene ε‐cyclase were exceptions. Their expression levels in petals were very low compared with those in leaves. Expression of the gene encoding carotenoid cleavage dioxygenase 4 was detected in neither leaves nor petals. These data suggest that the low levels of carotenoids in carnation petals are caused not by enzymatic degradation but rather by low rates of carotenoid biosynthesis.     

Carotenoid accumulations and carotenogenic gene expressions in the petals of Eustoma grandiflorum

Eustoma grandiflorum is one of the leading cut‐flowers in Japan. There are market demands for cultivars with deep‐yellow flowers, but they have never been bred successfully. By investigating the carotenoid accumulation and carotenogenic gene expressions, this study attempted to explore the reasons that block the formation of deep‐yellow colour in Eustoma. High performance liquid chromatography analysis showed that the carotenoid compositions in petals were similar to those in leaves, accumulating mainly lutein, violaxanthin and β‐carotene. The total carotenoid contents decreased as the petals matured in all the cultivars tested. Quantitative real‐time PCR analysis showed that the expression levels of PSY, LCYB, ZDS and LCYE showed significant differences between white and pale‐yellow petals or between petals and leaves, indicating that these enzymes may play a key role in the carotenoid biosynthesis in E. grandiflorum. The expression levels of CCD4 were high in both pale‐yellow and white petals during development, suggesting that carotenoid degradation activity is high in the petals. We then conclude that the total carotenoid accumulation level could be determined by the balance between carotenoid biosynthesis and degradation activities.     

棉花八氢番茄红素脱氢酶GhPDS1基因的克隆与表达谱分析

八氢番茄红素脱氢酶(PDS)在胡萝卜素生理代谢和病毒诱导外源基因沉默中起着非常重要的作用.本研究通过同源克隆方法,分析已报道植物八氢番茄红素脱氢酶氨基酸保守区,设计筒并引物,扩增出编码棉花PDS基因cDNA中间片段.通过RACE技术成功地克隆了棉花PDS基因的全长cDNA,命名为GhPDS1(GenBank No.HQ...     

Diversity in the carotenoid profiles and the expression of genes related to carotenoid accumulation among citrus genotypes

Carotenoids are not only important to the plants themselves but also are beneficial to human health. Since citrus fruit is a good source of carotenoids for the human diet, it is important to study carotenoid profiles and the accumulation mechanism in citrus fruit. Thus, in the present paper, we describe the diversity in the carotenoid profiles of fruit among citrus genotypes. In regard to carotenoids, such as β-cryptoxanthin, violaxanthin, lycopene, and β-citraurin, the relationship between the carotenoid profile and the expression of carotenoid-biosynthetic genes is discussed. Finally, recent results of quantitative trait locus (QTL) analyses of carotenoid contents and expression levels of carotenoid-biosynthetic genes in citrus fruit are shown.     

Control of ripening in transgenic tomatoes

Major progress has been made over the last few years in the identification and regulation of tomato ripening genes. At least 25 genes showing elevated expression during ripening have been cloned and several, including polygalacturonase, which modifies fruit textures, have been shown to be ripening-specific. In addition, genes have been cloned for ACC synthase and ACC oxidase, which control the synthesis of ethylene, which plays a critical role in ripening. Inhibition of expression of polygalacturonase, pectinesterase, ACC synthase, ACC oxidase and phytoene synthase has been achieved in transgenic plants, using antisense technology. The expression of several genes has also been inhibited by sense gene suppression. New traits caused by these transgenes are stably inherited. Antisense tomatoes with reduced polygalacturonase have improved textural qualities which are being exploited commercially for the fresh and processed markets. Overexpression of phytoene synthase has been shown to restore carotenoid production in the yellow flesh mutant and can be used to enhance colour in other cultivars. Antisense fruit in which ACC synthase or ACC oxidase are inhibited show slower ripening and reduced over-ripening. ACC oxidase antisense genes have also been shown to delay leaf senescence. It is to be expected that further genes determining other quality traits will be identified and manipulated soon.     

‘皮球桃’及其突变体类胡萝卜素积累及相关关键基因分析

本研究以‘皮球桃’及其芽变桃不同发育阶段的果肉与叶片中脉为试材,采用HPLC法测定两种桃中类胡萝卜素的组分构成与含量积累,并对类胡萝卜素生物合成及代谢过程中关键基因的转录水平进行实时荧光定量PCR分析。结果显示,芽变桃中类胡萝卜素总含量极显著高于‘皮球桃’,β-隐黄质、α-胡萝卜素和β-胡萝卜素的积累差异是造成两种桃呈色不同的主要原因。实时荧光定量PCR结果表明,CCD4在果肉和叶片中脉中的表达规律完全相同,且与类胡萝卜素的积累模式一致,因此CCD4的表达差异可能是导致芽变桃果肉及叶片中脉突变为黄色的重要原因。本研究利用‘皮球桃’及其自然突变体作为探讨类胡萝卜素合成及代谢调控的理想材料,以期从生理及分子层面揭示变异黄桃的形成机理,为桃的类胡萝卜素调控基因开发、变异的早期鉴定、认识和选育富含类胡萝卜素桃品种提供理论基础。     

Thin-layer chromatographic analysis of flower color phenotypes in Dendranthema grandiflorum Ramatuelle inbreds and clonal cultivars

Summary Four glasshouse and 13 garden chrysanthemum cultivars or inbred seedlings from the color groups white, yellow, pink/purple, and orange/red/bronze were analyzed with thin-layer chromatography (TLC) for qualitative and quantitative pigment constituents. TLC confirmed the presence of eight carotenoids in yellow and bronze cultivars. Qualitative differences exised only for spots 5–8. Rf values for lutein matched those of spot 4. Flavonoid analysis of purples and bronzes revealed two anthocyanidins, one of which was cyanidin, one flavone, and two phenylpropanoids. White cultivars had four phenylpropanoids, two of which were caffeic and ferulic acids. In most cases, inbred and non-inbred cultivars within a given phenotypic class did not exhibit qualitative differences. Color analysis could be pursued at any level of inbreeding, but inheritance studies would be severely hampered using non-inbred genotypes of this hexaploid floricultural crop.     

Identification of the fourth allele of the ANS (anthocyanidin synthase) gene and its effect on red color intensity in onions (Allium cepa)

Bulb color in onions (Allium cepa) is an important trait, and homogenous red coloration is desirable in red onion cultivars. The gene encoding anthocyanin synthase (ANS) is required for anthocyanin biosynthesis in onions. We have previously described three different alleles of the ANS gene. Here we report identification of the fourth allele of ANS, ANS-h1, found in a dark red doubled haploid line. ANS-h1 is similar to a non-functional allele found in Brazilian yellow cultivars except that it has several point mutations and indels throughout the promoter and coding regions, none of which are predicted to inactivate enzymatic activity. F₂ and backcross populations originating from the crosses between wild-type (ANS-L) allele-containing red and pink (ANS-p) allele-containing white or yellow parents show a discrete segregation ratio of 3 red to 1 light pink, indicating that the wild-type allele is completely dominant over the pink allele. In contrast, segregating populations derived from the crosses between ANS-h1 allele-containing red and the same white or yellow parents show a gradient of red intensity from light pink to dark red, suggesting that other genetic factors may affect expression of ANS-h1. A newly developed PCR-based marker and two previously developed markers for allelic selection of the ANS gene were used to examine allele composition in fifty-six breeding lines and commercial cultivars. Most lines are heterogeneous for the ANS gene with two or three alleles detected. The frequency of the pink allele is low in red breeding lines, but it is predominant in white and yellow lines.     

Postharvest ultraviolet-C irradiation suppressed Psy 1 and Lcy-β expression and altered color phenotype in tomato (Solanum lycopersicum) fruit

Mature green cherry tomato fruit were harvested and treated with ultraviolet-C (UV-C) irradiation at a predetermined dose of 4.2 kJ m⁻², and stored at 18 °C for 35 days. The effects of UV-C treatment on color change, pigment contents, and the expression of major genes involved in carotenoid metabolism, including Psy 1, Pds, Lcy-β, and Lcy-ɛ, encoding phytoene synthase, phytoene desaturase, lycopene β-cyclase and lycopene ɛ-cyclase, respectively, were examined. The UV-C treated fruit developed a pink red color in contrast to the normal orange red color of control fruit. Lycopene accumulation during ripening in UV-C treated fruit was significantly inhibited but its final content was not affected. However, both accumulation and final content of β-carotene were significantly suppressed in UV-C treated fruit. The lower content of β-carotene, leading to a higher lycopene to β-carotene ratio, is probably responsible for the altered color phenotype in UV-C treated fruit. Psy 1, a major gene involved in lycopene synthesis was inhibited by UV-C irradiation. Significantly suppressed expression of Lcy-β gene was also observed in UV-C treated fruit. Thus it is possible that the lower transformation from lycopene to carotenes contributed to the relatively stable content of lycopene.     

Introgression of antioxidant activity into cassava (Manihot esculenta C.): an effective technique for extending fresh storage roots shelf life

Physiological postharvest deterioration (PPD) of cassava is the main constraint affecting its nutritional and economical values. PPD is induced by wounds when detaching storage roots from mother plant during harvesting. It is accelerated by the reactive oxygen species (ROS) such as oxygen ion (O₂) and peroxide (O₂)^?2. The carotenoid content and its antioxidant property can help in extending shelf life of cassava storage roots. The primary mode of action of carotenoids as antioxidant is to quench singlet oxygen. Cassava breeding was reported to successfully introgress and improve carotenoid content into cassava. The two types of phytoene synthase (PSY) enzymes (PSY1 and PSY2) are key regulators of carotenoids accumulation in cassava. Carotenoids formation and accumulation in cassava storage roots are induced by a single nucleotide polymorphism in PSY2 which causes a non‐conservative amino acid exchange. This single nucleotide polymorphism in PSY gene is co‐segregated with β‐carotene in cassava storage roots, a phenomenon that could help to unravel the mechanism of introgression of carotenoids into cassava. This article investigates breeding feasibility for improving quality of cassava landraces in developing countries.     

Studies on the inheritance of root color and carotenoid content in red × yellow and red × white crosses of carrot,Daucus carota L.

Summary In red × yellow crosses of carrot (Daucus carota L.) three major genes were found to be segregating: Y ₂, inhibiting the synthesis of carotenoids, L, stimulating lycopene synthesis and A ₁, the action of which was not very clear. It is assumed that the dominant allele A ₁ enhances the formation of beta- and alpha-carotene at the expense of lycopene resulting in a more orange color instead of red, however, minor genes, modifiers and various interactions were obviously also involved.At least two inhibitor genes (Y and Y ₂) were segregating in red × white crosses. Evidence was found for a third inhibitor gene (Y ₁) in some crosses but this was not clear-cut.In F₂ progenies of red × white crosses a new phenotype was detected, i.e. tinge yellow-red, the xylem of which had a higher total carotenoid content than the phloem. Nothing is yet known about the genetics of this phenotype; tentatively it is suggested that one of the Y-genes might be less effective in the xylem than in the phloem regarding the suppression of lycopene synthesis.Much variation in pigment composition was found in F₂ generations of red × yellow and red × white crosses. Lycopene and beta-carotene were the predominant pigments in red and orange roots; zeta-carotene and phytofluene were generally shown to be present in smaller amounts while the presence of gamma-carotene and neurosporene could only be demonstrated in a limited number of roots. White and yellow roots were low in total carotenoids and consequently no or only a few specific carotenoids were detected in these roots.Research supported by the College of Agricultural and Life Sciences and by a grant from the Campbell Soup Company, Camden, New Jersey. The investigation is a portion of a thesis submitted in 1978 as partial fulfillment of the requirements of the PhD degree.     

Diversity of flower colours in Rhododendron simsii Planch. and prospects for breeding

Summary A survey of the flower colours present in evergreen azaleas, Rhododendron simsii Planch is given. The following colours occur: white, red, carmine red, pink, purple and lilac. Crosses can lead to greater diversity although no really new shades have resulted. Quantitative anthocyanin and flavonol determinations for the cultivars enable us to gain an insight into the possiblities of increasing these contents and thus obtaining new colours. The prospects of breeding for intensely red, yellow and blue cultivars are also reviewed.     

Watermelon lycopene β-cyclase: promoter characterization leads to the development of a PCR marker for allelic selection

In the carotenoid biosynthetic pathway, lycopene β-cyclase (LCYB) catalyzes the cyclization that converts lycopene into β-carotene. Only a single copy of LCYB was identified and was suggested to encode a chromoplast-specific LCYB (CYCB type) in watermelon [Citrullus lanatus (Thunb.), Matsum & Nakai]. Splicing variants in the 5′-untranslated region were identified, but alternative splicing did not provide an explanation of the regulation of carotenoid accumulation in watermelon flesh. A quantitative assay using real time-PCR showed that differential expression was not detected between red- and canary yellow-fleshed watermelon cultivars. LCYB promoter regions were isolated and characterized, and a sequence difference was identified in the promoter region between red and canary yellow LCYB alleles. This polymorphism did not change the expression of LCYB, but does provide a reliable marker for discriminating LCYB alleles for red and canary yellow flesh. To develop a PCR-based marker to distinguish between the two LCYB alleles, we designed primers flanking the polymorphic region. The newly developed marker, designated Clcyb.600, co-segregated perfectly with flesh color phenotypes and single nucleotide polymorphism (SNP) markers developed in our previous study. Moreover, the Clcyb.600 marker offers easier discrimination of LCYB alleles than SNP or cleaved amplified polymorphic sequence markers, as it does not require restriction enzyme digestion for genotyping. Genotyping of LCYB promoter alleles in various commercial cultivars and plant introductions indicated that watermelon cultivars can be classified into two groups, those carrying a red LCYB allele or a canary yellow LCYB allele.     

色素万寿菊中叶黄素合成相关基因的表达分析

本项研究以生产中用于杂交制种的8个色素万寿菊品种资源的花蕾期(Ⅰ)、鲜花半开期(Ⅱ)、鲜花盛开期(Ⅲ)的花瓣以及盛开期时的叶子(Ⅳ)为材料,采用实时荧光定量PCR方法,检测了叶黄素合成过程中3个相关基因lcyb(lycopene b-cyclase)、lcye(lycopene e-cyclase)和zds(ζ-carotene desaturase)在8个供试品种的不同发育时期的相对表达量。结果表明:3个基因的表达量在叶片中依次为lcyb>zds>lcye,花瓣中依次为lcye>zds>lcyb。lcye在花瓣中的表达量随花瓣的开放程度呈现先升高后降低的趋势,并在Ⅱ期达到最大值,且均高于叶片中lcye的表达量。Zds的表达趋势为Ⅱ>Ⅳ>Ⅰ>Ⅲ。Lcyb在叶中的表达高于花瓣中的任何时期。父本花瓣Ⅲ期的lcye表达量高于其他品种,且它的叶片中的lcyb的表达也高于其它品种。本项研究结果将有助于进一步阐明lcyb、lcye和zds基因与色素万寿菊叶黄素合成积累之间的关系,为进行色素万寿菊的基因工程育种奠定基础。     

大白菜的类胡萝卜素种类鉴定

为建立大白菜类胡萝卜素鉴定方法,利用反相高效液相色谱（RP-HPLC）,根据标样和二极管阵列检测器的检测结果,对不同类型大白菜材料的类胡萝卜素种类进行鉴定。结果表明：利用C30柱从大白菜中共分离出48类胡萝卜素,根据保留时间和吸收谱特征鉴定出其中的30种成分,链孢红素、玉米黄质、六氢番茄红素、ζ-胡萝卜素、γ-胡萝卜素和紫黄质均为首次在大白菜中报道。橙色大白菜中的类胡萝卜素种类最多,与其他大白菜存在很大差异。说明该鉴定方法适用于大白菜的类胡萝卜素种类研究。     

Significance of <Emphasis Type="Italic">CmCCD4a</Emphasis> orthologs in apetalous wild chrysanthemum species,responsible for white coloration of ray petals

Most chrysanthemum (Chrysanthemum morifolium Ramat.) flowers have a central capitulum, composed of many disc florets that is surrounded by ray petals. CmCCD4a, a gene that encodes a carotenoid cleavage dioxygenase (CCD), is expressed specifically in the ray petals of chrysanthemum cultivars, and its expression leads to white ray petals as a result of carotenoid degradation. Here, we show that wild chrysanthemums with white ray petals have CmCCD4a orthologs, whereas those with yellow ray petals lack these orthologs, as is the case in chrysanthemum cultivars. CmCCD4a orthologs also exist in some lines of Chrysanthemum pacificum and Chrysanthemum shiwogiku, even though these species lack ray petals. Interspecific hybridization between C. shiwogiku and a yellow-flowered chrysanthemum cultivar showed that the CmCCD4a orthologs from C. shiwogiku lead to the development of white ray petals. This indicates that the translation products of the CmCCD4a orthologs maintain enzymatic activity that can degrade carotenoids in chrysanthemums, irrespective of whether or not the ray petals that CmCCD4a expression actually occurred.     

Biofortification of taro (Colocasia esculenta) through breeding for increased contents in carotenoids and anthocyanins

Biofortification of taro (Colocasia esculenta) has never been studied. The aim of the present study is to compare the chemical compositions and individual constituent contents for major compounds (starch, sugars, cellulose, proteins, minerals), carotenoids and anthocyanins between parents and hybrids selected first for their agronomic performance and second for their corm characteristics (flesh color, quality and taste). For major compounds, 45 selected hybrids were compared to 66 cultivars and for carotenoids and anthocyanins, 34 selected hybrids were compared to 79 cultivars. Total sugars, cellulose and mineral contents presented moderate increases in hybrids. Carotenoids and anthocyanins contents were not correlated with corm flesh colors, most likely in relation with the wide range of the observed variability. Anthocyanin contents could not be increased in the selected hybrids, while total carotenoid contents were increased by more than fourfolds. The results of this study indicate that carotenoid contents can be rapidly improved by selecting plants of good agronomic performance and corm shape with increased density of yellow and orange colors. Potential applications to taro breeding programs are discussed.