Chalcone isomerase in flowers of mutants of Petunia hybrida

The effect of the gene Po on the activity of chalcone isomerase was investigated in Petunia hybrida. Furthermore, isomerase activities isolated from petals were compared with those extracted from anthers. No effect of Po on the pH-dependence of the isomerase and its kinetic properties was observed. With respect to these criteria, the enzyme extracted from anthers behaved in an identical manner to that extracted from petals. Upon chromatofocussing of a petal extract two peaks of activity were present with slightly different isoelectric points (pI 4.8 and pI 5.1). The occurrence of these activities was dependent on the method of enzyme extraction. An isolation procedure using polyvinylpyrrolidone besides Dowex to remove phenolic compounds, followed by (NH₄)₂SO₄ precipitation of the protein, resulted in only one peak of isomerase at a pI of 5.3. This observation was independent of Po and did not occur in anthers. In anthers one peak of enzyme activity with a pI of 4.5 was present. The moleuclar weight of the isomerase from flowers (62,500 dalton in Po-dominant and Po-recessive plants) differed from the molecular weight of the anther enzyme (44,000 dalton). In Po-recessive mutants the isomerase activity in mature flowers was low compared with Po-dominant mutants, indicating that the mutation in Po either reflects a temporal mutation in the expression of chalcone isomerase or an increased degradation of the enzyme.     

A novel purification procedure for chalcone flavanone isomerase from Petunia hybrida and the use of its antibodies to characterize the Po mutation

We have purified chalcone flavanone isomerase (CHI) from flowerbuds of Petunia hybrida to high purity. We made use of an affinity matrix consisting of Sepharose-bound Dextran Blue that is known to bind proteins containing the dinucleotide fold [S. T. Thompson, K. H. Cass, and E. Stellwagen (1975) Proc. Natl. Acad. Sci. USA 72, 669-672]. The final step, consisting of preparative elution from a denaturing acrylamide gel, yielded an approximately 2000-fold purified CHI protein. The enzyme is a single polypeptide with Mr = 29,000, and highly specific antiserum was raised against it. Using this antiserum it was shown that corolla and anther tissues express different forms of the enzyme as judged by pI. Furthermore, the absence of immunoreactive CHI was demonstrated in a mutant of P. hybrida (genotype popo) which accumulates 2',4,4',6'-tetrahydroxy-chalcone in anthers as a consequence of lack of enzyme activity.     

一种花色突变雄性不育油菜的发现

在甘蓝型油菜杂交种C022(其母本为由3对基因控制育性的隐性细胞核雄性不育系9012A)不育株开放受粉的后代中,发现一种稀有的黄白双色嵌合花瓣的甘蓝型油菜突变体991S。其具有3个形态特征:(1)4片花瓣每片中央均为条带状黄色色斑,而两侧为白色,为嵌合双色花瓣;(2)4片花萼也可发生中间条带状白化;(3)目前只在不同群体的雄性不育株中出现,与同源的黄色花不育株形态相似,植株纤细矮小,花器也较小,花瓣较为平整,雌蕊弯曲,雄蕊退化,花药干缩。通过对其材料来源及后代花色表型分析,初步认为黄白双色性状由可局部表达的隐性白化基因控制。     

Pollen- and anther-specific chi promoters from petunia: tandem promoter regulation of the chiA gene.

We have analyzed the spatial and temporal activities of chalcone flavanone isomerase (chi) A and B gene promoters from petunia. To study the tandem promoter regulation of chiA, various chiA promoter fragments were fused with the beta-glucuronidase (GUS) reporter gene. Analysis of transgenic plants containing these chimeric genes provided definitive proof that the chiA coding region is regulated by two distinct promoters (designated PA1 and PA2). We also showed that both promoters can function independently and that the chiA PA1 promoter is expressed in limb (epidermal and parenchyma cells), tube (inner epidermal and parenchyma cells), seed (seed coat, endosperm, and embryo), sepal, leaf, and stem. The use of chiA and chiB promoters in the regulation of anther- and pollen-specific gene expression has been studied. By analyzing transgenic plants containing chimeric genes consisting of chiA and B promoter fragments and the GUS reporter gene, we were able to identify a 0.44-kilobase chiA PA2 promoter fragment that drives pollen-specific gene expression and a 1.75-kilobase chiB PB promoter fragment that confers anther-specific (pollen and tapetum cells) expression to the GUS gene.     

矮牵牛新种质的花色表型及花色素分析

以27个上海交通大学自育矮牵牛新种质为研究材料,对花色这一重要观赏性状及其花色素进行了系统研究。用RHSCC比色和色差仪测色方法描述了矮牵牛的花色表型,通过特征显色反应初步判断了矮牵牛的花色素类型,以标准曲线法和pH示差法等方法测定了矮牵牛3类花色素的含量。研究表明：这27个矮牵牛种质的花色可归于5个色系,以紫红色和红色为主;矮牵牛花色在CIELab表色系统中分布较广,而且不同色系花色参数的区分度较大。矮牵牛花瓣中含有类黄酮和花色苷,不含或含少量类胡萝卜素。13个被测种质的花瓣类黄酮含量在2.5～12.2 mg·/g–1 ·FW之间,花色苷含量在0.08～3.88 mg·g–1 FWmg/g·FW之间,而类胡萝卜素在矮牵牛花瓣中含量很低,远远低于类黄酮含量,在7个被测种质中,最高仅为0.216 mg·g–1 FWmg/g·FW,最低为0.004 mg·g–1 FWmg/g·FW。以上结果显示,5个色系矮牵牛所含花色素种类不尽相同,含量也有明显差异,其中紫红色系和红色系花瓣大多不含或含极少量类胡萝卜素,黄色系、白色系和紫色系花瓣的类黄酮含量较高,紫色系和紫红色系花瓣花色苷含量较高。     

Gibberellin regulates post-microsporogenesis processes in petunia anthers

Previous studies have suggested that gibberellins (GAs) are produced in petunia anthers and transported to the corolla to induce growth and pigmentation. In this work, we studied the role of GA in the regulation of anther development. When petunia plants were treated with the GA-biosynthesis inhibitor paclobutrazol, anther development was arrested. Microscopic analysis of these anthers revealed that paclobutrazol inhibits post-meiotic developmental processes. The treated anthers contained pollen grains but the connective tissue and tapetum cells were degenerated. A similar phenotype was obtained when the Arabidopsis GA-signal repressor, SPY, was over-expressed in transgenic petunia plants, i.e. anther development was arrested following microsporogenesis. The expression of the GA-induced gene, GIP , can be used in petunia as a molecular marker to study GA responses. GA₃ treatment of young anthers promoted, and paclobutrazol inhibited, GIP expression, suggesting that the hormone controls the natural activation of the gene in the anthers. Analyses of GIP expression during anther development revealed that the gene is induced only after microsporogenesis. This observation further suggests a role for GA in the regulation of post-meiotic processes during petunia anther development.     

Flavonoid components and flower color change in transgenic tobacco plants by suppression of chalcone isomerase gene

A cDNA encoding chalcone isomerase (CHI) was isolated from the petals of Nicotiana tabacum and the effect of its suppression on flavonoid biosynthesis was analyzed in transgenic tobacco plants. CHI-suppression by RNA interference (RNAi) showed reduced pigmentation and change of flavonoid components in flower petals. The plants also accumulated high levels of chalcone in pollen, showing a yellow coloration. Our results first demonstrated that suppression of CHI by genetic transformation is possible in higher plants. This suggests that CHI plays a major part in the cyclization reaction from chalcone to flavanone, and that spontaneous reactions are few, if any, in tobacco plants.     

稀有植物小丛红景天花部综合特征与繁育系统

对稀有物种生物学特征及繁育系统的研究将有利于我们对稀有物种的保育。该文通过野外定位观测稀有植物小丛红景天(Rhodiola dumulosa)的花部综合特征,并运用杂交指数、花粉-胚珠比的方法首次对其繁育系统进行初步测定。结果表明:该物种单花花期一般为5~6 d,开花过程中花柱逐渐伸长并向外弯曲,子房开裂并逐渐伸长,花药散粉具有先后顺序,一般外轮对萼花药先散粉,其后1 d左右对瓣花药开始散粉。单花花期依照其形态和散粉时间可分为6个阶段:花蕾期、内轮散粉期、内轮散粉末期、外轮散粉期、外轮散粉末期和凋谢期。而种群花期一般可持续30 d以上,不同海拔观测地点有所不同,海拔2 202 m处的观测地点B开花物候最为滞后。由于其杂交指数不小于4,花粉-胚珠比在700~1 000之间, 根据Dafni(1992)和Cruden(1977)的标准,初步判定该物种的繁育系统属于异交为主,部分自交亲和,传粉过程需要传粉者。     

Colored floral organs influence pollinator behavior and pollen transfer in Commelina communis (Commelinaceae)

Visual floral guides such as colored anthers, lines, dots, and UV-absorption patterns on petals are commonly observed in insect-pollinated angiosperms. Floral guides that are known to enhance foraging efficiency of visitors on flowers thus promote return visits (foraging facilitation hypothesis, which predicts that visitors will discriminate against flowers with inferior floral guides). In this study, we experimentally examined the hypothesis that floral guides also prevent pollen-theft behavior by floral visitors (theft prevention hypothesis), which has rarely been tested. Nectarless flowers of Commelina communis have three types of brightly colored floral organs: large blue petals, rewarding yellow anthers, and nonrewarding yellow anthers. Colored floral organs were removed artificially from plants in two natural populations of C. communis. Removal of the nonrewarding yellow anthers diminished hoverings in front of flowers and tended to reduce the number of total floral visitor landings, supporting the foraging facilitation hypothesis. Additionally, removal of the rewarding yellow anther decreased the frequency of legitimate landings on flowers and the legitimate landing-to-total landing ratio, which is consistent with the theft prevention hypothesis. The nonrewarding anthers and the rewarding yellow anthers were shown to play an important role in increasing visitor landings and orienting floral visitors toward a landing point appropriate for pollination, respectively. We also showed that the absence of yellow anthers decreased both pollen dispatch from brown anthers and receipt by stigmas in C. communis. These findings support both the foraging facilitation hypothesis and the theft prevention hypothesis.     

The pollination ecology of buzz-pollinated Rhexia virginica (Melastomataceae)

We examined the function of floral traits associated with buzz pollination through studies of Rhexia virginica (Melastomataceae) in the Muskoka region of Ontario, Canada. Controlled pollinations demonstrated that the species is self-compatible, but dependent on insects for pollen transfer. Bumble bees made 82 and 90% of observed insect visits to R. virginica in 1996 and 1997, respectively, and effectively buzzed flowers. Buzz pollination did not appear to be highly "specialized" since various species of bumble bee were capable of pollination, and pollen transfer efficiency appeared to be relatively low. Experimental manipulations provided little support for the hypothesis that the yellow color of melastome anthers mimics abundant pollen, thereby deceiving pollinators to visit regardless of whether most pollen has been removed. Fruit set averaged 52.6% among populations, owing largely to infrequent pollinator visits and pollen limitation. Flowers of R. virginica were infertile after a single day of anthesis, but petals were subsequently maintained for 1-2 d and stamens underwent a color change from bright yellow to red. Second-day flowers may function to increase floral display size and hence fertility, without a concomitant increase in pollen discounting. Studies of bumble bee foraging behavior and correlates of seed set provided indirect support for this hypothesis.     

Excision of transposable elements from the chalcone isomerase and dihydroflavonol 4-reductase genes may contribute to the variegation of the yellow-flowered carnation (Dianthus caryophyllus)

In the "Rhapsody" cultivar of the carnation, which bears white flowers variegated with red flecks and sectors, a transposable element, dTdic1, belonging to the Ac/Ds superfamily, was found within the dihydroflavonol 4-reductase (DFR) gene. The red flecks and sectors of "Rhapsody" may be attributable to a reversion to DFR activity after the excision of dTdic1. The yellow color of the carnation petals is attributed to the synthesis and accumulation of chalcone 2'-glucoside. In several of the carnation cultivars that bear yellow flowers variegated with white flecks and sectors, both the chalcone isomerase (CHI) and DFR genes are disrupted by dTdic1.     

Pollen competition between morphs in a pollen‐color dimorphic herb and the loss of phenotypic polymorphism within populations

Flower color polymorphism is relatively uncommon in natural flowering plants, suggesting that maintenance of different color morphs within populations is difficult. To address the selective mechanisms shaping pollen‐color dimorphism, pollinator preferences and reproductive performance were studied over three years in Epimedium pubescens in which some populations had plants with either green or yellow pollen (and anthers). Visitation rate and pollen removal and receipt by the bee pollinator (Andrena emeishanica) did not differ between the two color morphs. Compared to the green morph, siring success of the yellow morph's pollen was lower, but that of mixtures of pollen from green and yellow morphs was lowest. This difference, corresponding to in vivo and ex vivo experiments on pollen performance, indicated that pollen germination, rather than tube growth, of the green morph was higher than that of the yellow morph and was seriously constrained in both morphs if a pollen competitor was present. A rare green morph may invade a yellow‐morph population, but the coexistence of pollen color variants is complicated by the reduced siring success of mixed pollinations. Potential pollen competition between morphs may have discouraged the maintenance of multiple phenotypes within populations, a cryptic mechanism of competitive exclusion.     

Pollen and stamen mimicry: the alpine flora as a case study

Many melittophilous flowers display yellow and UV-absorbing floral guides that resemble the most common colour of pollen and anthers. The yellow coloured anthers and pollen and the similarly coloured flower guides are described as key features of a pollen and stamen mimicry system. In this study, we investigated the entire angiosperm flora of the Alps with regard to visually displayed pollen and floral guides. All species were checked for the presence of pollen- and stamen-imitating structures using colour photographs. Most flowering plants of the Alps display yellow pollen and at least 28% of the species display pollen- or stamen-imitating structures. The most frequent types of pollen and stamen imitations were (mostly yellow and UV-absorbing) colour patches on petals (65% of species displaying imitations), patterns of inflorescences (18%), stamen-like pistils (10%), and staminodes (6%), as well as three-dimensional structures such as convex lower lips and filamental hairs (<5%). Dichogamous and diclinous species display pollen- and stamen-imitating structures more often than non-dichogamous and non-diclinous species, respectively. The visual similarity between the androecium and other floral organs is attributed to mimicry, i.e. deception caused by the flower visitor’s inability to discriminate between model and mimic, sensory exploitation, and signal standardisation among floral morphs, flowering phases, and co-flowering species. We critically discuss deviant pollen and stamen mimicry concepts and evaluate the frequent evolution of pollen-imitating structures in view of the conflicting use of pollen for pollination in flowering plants and provision of pollen for offspring in bees.     

表达核糖核酸酶基因的雄性不育油菜的获得

从细菌Ｂａｃｉｌｌｕｓａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ染色体ＤＮＡ中克隆了ＲＮａｓｅ（ｂａｒｎａｓｅ）基因,构建了ＴＡ－２９基因５＇调控区（－１３００－＋３）与ｂａｒｎａｓｅ基因的嵌合基因,通过农杆菌介导的遗传转化,获得了“双低”甘蓝型油菜“中双８２１”的转基因植株。转化植株与末转化植株在高度、生长速度、花器形态、花色等方面基本相同,但转化植株花丝短小、花药干瘪、没有花粉;自花授粉或以其为父本进行的异花授粉均不能结实,表现为完全的雄性不育。花药的横向解剖结构表明：转基因油菜雄性不育与绒毡层细胞的破坏有关     

Cloning and expression of flavonol synthase from Petunia hybrida

Flavonols are important co-pigments in flower colour and are also essential for pollen tube growth. In petunia, flavonol synthesis is controlled by the Fl locus. Flavonol synthase (FLS) belongs to the 2-oxoglutarate-dependent dioxygenase family. Dioxygenase gene fragments were amplified by PCR on cDNA made from FlFl and flfl flowers using degenerate primers designed from conserved dioxygenase sequences. A petunia petal cDNA library was screened for clones that hybridized more strongly to the Fl PCR products than the fl PCR products. A full-length cDNA clone identified by this screening exhibited FLS activity when expressed in yeast. FLS gene expression is developmentally regulated during flower development. Antisense expression of an FLS cDNA clone in petunia markedly reduced flavonol synthesis in petals. RFLP mapping showed that the FLS gene is linked to Fl , suggesting that Fl is the structural gene for FLS.