不同花色菊花品种花色素结构基因的表达特性

对红色、黄色、粉紫色和白色菊花品种不同开放度的花序舌状花中CHS、CHI、DFR、F3H、F3′H和3GT基因的表达量进行了相对定量分析。结果表显示:6个基因的表达因不同花色、不同发育阶段而异。‘钟山红鹰’(红色)中各基因的表达量均较高,且均在Ⅱ(松蕾期)或Ⅲ(半开期)期达到峰值,其中DFR、3GT基因的表达量远高于其他花色品种。‘金陵娇黄’(黄色)中CHS、CHI基因表达量较高,且Ⅰ(紧蕾期)、Ⅱ期表达量高于Ⅲ、Ⅳ(盛开期)期;3GT、DFR基因表达量分别高或低于‘金陵笑靥’(粉紫色)品种中相应基因的表达量,但均比红色品种低;F3H在4个品种中表达量最低,F3′H表达量接近或略低于红色或粉紫色品种,且各阶段表达水平较稳定。‘金陵笑靥’中DFR表达量仅次于‘钟山红鹰’,3GT和CHS表达量低于红色与黄色品种。‘钟山雪桂’(白色)中各基因仅有微量表达,除F3H外各基因的表达量明显低于其他花色品种。研究表明,花色素结构基因DFR、3GT是菊花花色素合成的关键基因,DFR很可能是限速关键基因,一定表达水平的CHS、CHI也是菊花花色素合成所必须的,F3H基因与花色素合成不存在直接相关。     

Flower colour and cytochromes P450

Flavonoids are major constituents of flower colour. Plants accumulate specific flavonoids and thus every species often exhibits a limited flower colour range. Three cytochromes P450 play critical roles in the flavonoid biosynthetic pathway. Flavonoid 3′-hydroxylase (F3′H, CYP75B) and flavonoid 3′,5′-hydroxylase (F3′5′H, CYP75A) catalyze the hydroxylation of the B-ring of flavonoids and are necessary to biosynthesize cyanidin-(red to magenta) and delphinidin-(violet to blue) based anthocyanins, respectively. Pelargonidin-based anthocyanins (orange to red) are synthesized in their absence. Some species such as roses, carnations and chrysanthemums do not have violet/blue flower colour due to deficiency of F3′5′H. Successful expression of heterologous F3′5′H genes in roses and carnations results in delphinidin production, causing a novel blue/violet flower colour. Down-regulation of F3′H and F3′5′H genes has yielded orange petunia and pink torenia colour that accumulate pelargonidin-based anthocyanins. Flavone synthase II (CYP93B) catalyzes the synthesis of flavones that contribute to the bluing of flower colour, and modulation of FNSII gene expression in petunia and tobacco changes their flower colour. Extensive engineering of the anthocyanin pathway is therefore now possible, and can be expected to enhance the range of flower colours.     

乙烯利处理对葡萄花色苷合成相关基因表达的影响

利用荧光定量PCR技术分析‘京优’葡萄果实成熟过程中,花色苷生物合成途径相关酶基因mRNA转录水平的变化以及乙烯利处理对果皮中花色苷含量和关键酶基因转录水平的影响。结果显示,葡萄果实发育进入着色期,花色苷合成过程中主要相关基因(CHSs、CHIs、F3Hs、F3′H、F3′5′H、DFR、LDOX、UFGT、OMT和GST)和转录因子(MybA1和MybA1-2)转录水平都显著提高,其中UFGT、GST、MybA1和CHSs、CHIs、F3Hs基因家族中的CHS3、CHI2、F3H2随着花色苷合成而大量转录;乙烯利处理能够增强花色苷合成相关基因的转录,使其转录时期前移和转录水平提高,其中对GST、UFGT和MybA1转录的促进作用最明显。相关性分析表明,花色苷合成与一些花色苷合成相关基因(CHS3、CHI2、F3H2、F3′5′H、UFGT、GST)和转录因子(MybA1)的转录水平呈显著或极显著正相关;与CHS1、CHS2、CHI1、F3H1、DFR、F3′H、LDOX和OMT转录水平的相关性均不显著。本研究结果为进一步阐明花色苷生物合成机理和花色苷类色素的生产应用提供一定的理论依据。     

Analysis of the Expression of Anthocyanin Pathway Genes in Developing Vitis vinifera L. cv Shiraz Grape Berries and the Implications for Pathway Regulation

Anthocyanin synthesis in Vitis vinifera L. cv Shiraz grape berries began 10 weeks postflowering and continued throughout berry ripening. Expression of seven genes of the anthocyanin biosynthetic pathway (phenylalanine ammonia lyase [PAL], chalcone synthase [CHS], chalcone isomerase [CHI], flavanone-3-hydroxylase [F3H], dihydroflavonol 4-reductase [DFR], leucoanthocyanidin dioxygen-ase [LDOX], and UDP glucose-flavonoid 3-o-glucosyl transferase [UFGT]) was determined. In flowers and grape berry skins, expression of all of the genes, except UFGT, was detected up to 4 weeks postflowering, followed by a reduction in this expression 6 to 8 weeks postflowering. Expression of CHS, CHI, F3H, DFR, LDOX, and UFGT then increased 10 weeks postflowering, coinciding with the onset of anthocyanin synthesis. In grape berry flesh, no PAL or UFGT expression was detected at any stage of development, but CHS, CHI, F3H, DFR, and LDOX were expressed up to 4 weeks postflowering. These results indicate that the onset of anthocyanin synthesis in ripening grape berry skins coincides with a coordinated increase in expression of a number of genes in the anthocyanin biosynthetic pathway, suggesting the involvement of regulatory genes. UFGT is regulated independently of the other genes, suggesting that in grapes the major control point in this pathway is later than that observed in maize, petunia, and snapdragon.     

Functional characterization of key structural genes in rice flavonoid biosynthesis

Rice is a model system for monocot but the molecular features of rice flavonoid biosynthesis have not been extensively characterized. Rice structural gene homologs encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) were identified by homology searches. Unique differential expression of OsF3H, OsDFR, and OsANS1 controlled by the Pl ^w locus, which contains the R/B-type regulatory genes OSB1 and OSB2, was demonstrated during light-induced anthocyanin accumulation in T65-Plw seedlings. Previously, F3H genes were often considered as early genes co-regulated with CHS and CHI genes in other plants. In selected non-pigmented rice lines, OSB2 is not expressed following illumination while their expressed OSB1sequences all contain the same nucleotide change leading to the T⁶⁴ M substitution within the conserved N-terminal interacting domain. Furthermore, the biochemical roles of the expressed rice structural genes (OsCHS1, OsCHI, OsF3H, and OsF3′H) were established in planta for the first time by complementation in the appropriate Arabidopsis transparent testa mutants. Using yeast two-hybrid analysis, OsCHS1 was demonstrated to interact physically with OsF3H, OsF3′H, OsDFR, and OsANS1, suggesting the existence of a macromolecular complex for anthocyanin biosynthesis in rice. Finally, flavones were identified as the major flavonoid class in the non-pigmented T65 seedlings in which the single-copy OsF3H gene was not expressed. Competition between flavone and anthocyanin pathways was evidenced by the significant reduction of tricin accumulation in the T65-Plw seedlings. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in Anthocyanin, Carotenoid, Chlorophyll, and Protein in Developing Florets of the Chrysanthemum

Florets of a purple cultivar (Fandango) of the horticulturalchrysanthemum (Chrysanthemum morifolium Ramat) were removedfrom flower heads at seven stages of opening (from unopenedbud to dying flower) and segregated into different lengths,each of which was analysed. Wet weight per floret increased from 0.25–1 mg in thebud to about 3 mg (tubular florets) or to 20–40 mg (rayflorets) in the fully open flower. Protein decreased from 6per cent of the wet weight in the bud to about 2 per cent inthe open flower. In the ray florets anthocyanin concentrationreached a maximum in the half-open flower and then decreasedsharply, whereas carotenoid and chlorophyll declined continuouslyfrom the bud stage. Almost no anthocyanin was formed by thetubular florets and chlorophyll declined as in the ray florets,but carotenoid concentration increased to a maximum in the half-openflower and then decreased. In another cultivar (Light Bronze Fandango) the content of anthocyaninwas lower and that of carotenoid higher but similar changesin pigment levels were observed except that carotenoid roseto a maximum in the ray florets. In two other cultivars, Redand Cerise Fandango, the anthocyanin content was the same asin Fandango but the carotenoid concentration was the same orabout half that in Light Bronze Fandango respectively.     

Pigment Production by Cultured Florets of Chrysanthemum morifolium

Individual florets (4–5 mm long) of a purple cultivar(Fandango) of the horticultural chrysanthemum (Chrysanthemummorifolium Ramat) were taken from flower buds just prior toopening and cultured in a sterile liquid medium (containinginorganic salts and sucrose) at 15 °C under a 12-h day.For the first 14 days increase in wet weight was exponential.Anthocyanin appeared on the third day and was then synthesizedrapidly. Chlorophyll and carotenoid were present initially:carotenoid levels rose quickly while chlorophyll remained almostconstant. Highest pigment content and most growth were foundwhen the florets were grown on 3 per cent sucrose. However,the highest anthocyanin concentration was found with 4 per centsucrose, the highest carotenoid concentration with 0.6 per centsucrose. No anthocyanin was produced when the florets were grownat 6 or 30 °C; maximum yield was at 15 °C. Most carotenoidwas formed at 30 °C and most chlorophyll was found at 20–5°C. All florets from 1 to 7 mm long could be cultured. Theseresults are discussed in relation to flower colour and pigmentformation in vivo.     

Heterologous Expression of Two Gentian Cytochrome P450 Genes can Modulate the Intensity of Flower Pigmentation in Transgenic Tobacco Plants

Two different heterologous expression systems, microsomal fractions of Saccharomyces cerevisiae and transgenic tobacco plants, were used to investigate the enzymatic activities of flavonoid 3′-hydroxylase (GtF3′H) and flavone synthase II (GtFSII) homologues isolated from gentian petals. Recombinant GtF3′H expressed in yeast showed hydroxylation activities in the 3′ position with several flavonoid substrates, while recombinant GtFSII was able to produce flavone from flavanone. GtF3′ H-expressing transgenic tobacco plants showed a slight increase in anthocyanin content and flower color intensity, and conversion of the flavonol quercetin from kaempferol. On the other hand, GtFSII-expressing plants showed a remarkable reduction in anthocyanin content and flower color intensity, and additional accumulation of flavone, especially luteolin derivatives. We demonstrated that two cytochrome P450s from gentian petals have F3′H and FSII enzymatic activities both in vitro and in vivo, and might therefore be useful in modification of flower color using genetic engineering.     

The identification of flavonoids and the expression of genes of anthocyanin biosynthesis in the chrysanthemum flowers

In order to provide additional information on the coloration of chrysanthemum flowers, the flavonoid composition and the expression of six structural genes involved in anthocyanin pathway in the ray florets of a pink flowering (cv. H5) and two white flowering (cvs. Keikai and Jinba) Chrysanthemum grandiflorum cultivars were examined. HPLCDAD/ESI-MSⁿ analysis showed that cyanidin 3-O-(6″-O-malonylglucoside) and cyanidin 3-O-(3″,6″-O-dimalonylglucoside) were the two major flavonoids presented in H5, while white flowering cultivars contained flavones instead of anthocyanins. Nine flavone derivatives were detected in the three cultivars, the amount of each flavone varied upon cultivars, and seven of these were identified as luteolin 7-O-arabinosylglucuronide, apigenin 7-O-glucoside, luteolin 7-O-malonylglucoside, apigenin 7-O-malonylglucoside, chrysoeriol 7-O-malonylglucoside, acacetin 7-O-rutinoside and acacetin 7-O-malonylglucoside. The two white flowering cultivars showed similar total flavonoid content, which was about two fold higher than that in H5. A high expression of the genes encoding dihydroflavonol 4-reductase and 3-O-glucosyltransferase was detected only in H5 but not in Keikai or Jinba. Chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, and flavonoid 3′-hydroxylase were expressed in all flowers, suggesting that the lack of anthocyanin in white flowering cultivars cannot be due to any blockage of their expression.     

Functional analysis of <Emphasis Type="Italic">Antirrhinum kelloggii</Emphasis> flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase genes; critical role in flower color and evolution in the genus <Emphasis Type="Italic">Antirrhinum</Emphasis>

The enzymes flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) play an important role in flower color by determining the B-ring hydroxylation pattern of anthocyanins, the major floral pigments. F3′5′H is necessary for biosynthesis of the delphinidin-based anthocyanins that confer a violet or blue color to most plants. Antirrhinum majus does not produce delphinidin and lacks violet flower colour while A. kelloggii produces violet flowers containing delphinidin. To understand the cause of this inter-specific difference in the Antirrhinum genus, we isolated one F3′H and two F3′5′H homologues from the A. kelloggii petal cDNA library. Their amino acid sequences showed high identities to F3′Hs and F3′5′Hs of closely related species. Transgenic petunia expressing these genes had elevated amounts of cyanidin and delphinidin respectively, and flower color changes in the transgenics reflected the type of accumulated anthocyanidins. The results indicate that the homologs encode F3′H and F3′5′H, respectively, and that the ancestor of A. majus lost F3′5′H activity after its speciation from the ancestor of A. kelloggii.     

Cloning, Functional Identification and Sequence Analysis of Flavonoid 3′-hydroxylase and Flavonoid 3′,5′-hydroxylase cDNAs Reveals Independent Evolution of Flavonoid 3′,5′-hydroxylase in the Asteraceae Family

Flavonoids are ubiquitous secondary plant metabolites which function as protectants against UV light and pathogens and are involved in the attraction of pollinators as well as seed and fruit dispersers. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of the vast and versatile cytochrome P450 protein (P450) family, the flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H). Phylogenetic analysis of known sequences of F3′H and F3′5′H indicated that F3′5′H was recruited from F3′H before the divergence of angiosperms and gymnosperms. Seven cDNAs were isolated from species of the Asteraceae family, all of which were predicted to code for F3′Hs based on their sequences. The recombinant proteins of four of the heterologously in yeast expressed cDNAs exhibited the expected F3′H activity but surprisingly, three recombinant proteins showed F3′5′H activity. Phylogenetic analyses indicated the independent evolution of an Asteraceae-specific F3′5′H. Furthermore, sequence analysis of these unusual F3′5′H cDNAs revealed an elevated rate of nonsynonymous substitutions as typically found for duplicated genes acquiring new functions. Since F3′5′H is necessary for the synthesis of 3′,4′,5′-hydroxylated delphinidin-derivatives, which normally provide the basis for purple to blue flower colours, the evolution of an Asteraceae-specific F3′5′H probably reflects the adaptive value of efficient attraction of insect pollinators.     

Flower Colour Modification of Chrysanthemum by Suppression of F3'H and Overexpression of the Exogenous Senecio cruentus F3'5'H Gene

Chrysanthemum (Chrysanthemum × morifolium) is one of the most important ornamental plants in the world. They are typically used as cut flowers or potted plants. Chrysanthemum can exhibit red, purple, pink, yellow and white flowers, but lack bright red and blue flowers. In this study, we identified two chrysanthemum cultivars, C × morifolium ‘LPi’ and C × morifolium ‘LPu’, that only accumulate flavonoids in their ligulate flowers. Next, we isolated seven anthocyanin biosynthesis genes, namely CmCHS, CmF3H, CmF3’H, CmDFR, CmANS, CmCHI and Cm3GT in these cultivars. RT-PCR and qRT-PCR analyses showed that CmF3′H was the most important enzyme required for cyanidin biosynthsis. To rebuild the delphinidin pathway, we downregulated CmF3’H using RNAi and overexpressed the Senecio cruentus F3′5′H (PCFH) gene in chrysanthemum. The resultant chrysanthemum demonstrated a significantly increased content of cyanidin and brighter red flower petals but did not accumulate delphinidin. These results indicated that CmF3′H in chrysanthemum is important for anthocyanin accumulation, and Senecio cruentus F3′5′H only exhibited F3′H activity in chrysanthemum but did not rebuild the delphinidin pathway to form blue flower chrysanthemum.     

Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries

Water deficits consistently promote higher concentrations of anthocyanins in red winegrapes and their wines. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth. Early (ED) and late (LD) season water deficits, applied before or after the onset of ripening (veraison), were imposed on field grown Vitis vinifera “Cabernet Sauvignon”, and the responses of gene expression in the flavonoid pathway and their corresponding metabolites were determined. ED accelerated sugar accumulation and the onset of anthocyanin synthesis. Both ED and LD increased anthocyanin accumulation after veraison. Expression profiling revealed that the increased anthocyanin accumulation resulted from earlier and greater expression of the genes controlling flux through the anthocyanin biosynthetic pathway, including F3H, DFR, UFGT and GST. Increases in total anthocyanins resulted predominantly from an increase of 3′4′5′-hydroxylated forms through the differential regulation of F3′H and F3′5′H. There were limited effects on proanthocyanidin, other flavonols, and on expression of genes committed to their synthesis. These results demonstrate that manipulation of abiotic stress through applied water deficits not only modulates compositional changes during berry ripening, but also alters the timing of particular aspects of the ripening process.     

非洲菊盘状花雄蕊发育与舌状花生长着色的对应关系

非洲菊(Gerbern hybrida)头状花序由外轮舌状花和内轮盘状花构成。通过观察内轮盘状花雄蕊花粉囊和花粉粒的形态结构与发育顺序,和测定外轮舌状花花瓣的长度、宽度、花色素苷含量等,对它们之间的对应关系进行了研究。花序外轮舌状花花瓣开始着色时为P3期,此时第1轮盘状花出现成熟花粉粒。研究明确了内轮盘状花花粉粒发育与外轮舌状花生长时期和花色素苷积累的对应关系。     

Genes up-regulated during red coloration in UV-B irradiated lettuce leaves

Molecular analysis of gene expression differences between green and red lettuce leaves was performed using the SSH method. BlastX comparisons of subtractive expressed sequence tags (ESTs) indicated that 7.6% of clones encoded enzymes involved in secondary metabolism. Such clones had a particularly high abundance of flavonoid-metabolism proteins (6.5%). Following SSH, 566 clones were rescreened for differential gene expression using dot-blot hybridization. Of these, 53 were found to overexpressed during red coloration. The up-regulated expression of six genes was confirmed by Northern blot analyses. The expression of chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), and dihydroflavonol 4-reductase (DFR) genes showed a positive correlation with anthocyanin accumulation in UV-B-irradiated lettuce leaves; flavonoid 3′,5′-hydroxylase (F3′,5′H) and anthocyanidin synthase (ANS) were expressed continuously in both samples. These results indicated that the genes CHS, F3H, and DFR coincided with increases in anthocyanin accumulation during the red coloration of lettuce leaves. This study show a relationship between red coloration and the expression of up-regulated genes in lettuce. The subtractive cDNA library and EST database described in this study represent a valuable resource for further research for secondary metabolism in the vegetable crops.