Specific expression of the vacuolar iron transporter, TgVit, causes iron accumulation in blue-colored inner bottom segments of various tulip petals

Several flowers of Tulipa gesneriana exhibit a blue color in the bottom segments of the inner perianth. We have previously reported the inner-bottom tissue-specific iron accumulation and expression of the vacuolar iron transporter, TgVit1, in tulip cv. Murasakizuisho. To clarify whether the TgVit1-dependent iron accumulation and blue-color development in tulip petals are universal, we analyzed anthocyanin, its co-pigment components, iron contents and the expression of TgVit1 mRNA in 13 cultivars which show a blue color in the bottom segments of the inner perianth accompanying yellow- and white-colored inner-bottom petals. All of the blue bottom segments contained the same anthocyanin component, delphinidin 3-rutinoside. The flavonol composition varied with cultivar and tissue part. The major flavonol in the bottom segments of the inner perianth was rutin. The iron content in the upper part was less than that in the bottom segments of the inner perianth. The iron content in the yellow and white petals was higher in the bottom segment of the inner perianth than in the upper tissues. TgVit1 mRNA expression was apparent in all of the bottom tissues of the inner perianth. The result of a reproduction experiment by mixing the constituents suggests that the blue coloration in tulip petals is generally caused by iron complexation to delphinidin 3-rutinoside and that the iron complex is solubilized and stabilized by flavonol glycosides. TgVit1-dependent iron accumulation in the bottom segments of the inner perianth might be controlled by an unknown system that differentiated the upper parts and bottom segments of the inner perianth.     

Genes encoding the vacuolar Na+/H+ exchanger and flower coloration

Vacuolar pH plays an important role in flower coloration: an increase in the vacuolar pH causes blueing of flower color. In the Japanese morning glory (Ipomoea nil or Pharbitis nil), a shift from reddish-purple buds to blue open flowers correlates with an increase in the vacuolar pH. We describe details of the characterization of a mutant that carries a recessive mutation in the Purple (Pr) gene encoding a vacuolar Na+/H+ exchanger termed InNHX1. The genome of I. nil carries one copy of the Pr (or InNHX1) gene and its pseudogene, and it showed functional complementation to the yeast nhx1 mutation. The mutant of I. nil, called purple (pr), showed a partial increase in the vacuolar pH during flower-opening and its reddish-purple buds change into purple open flowers. The vacuolar pH in the purple open flowers of the mutant was significantly lower than that in the blue open flowers. The InNHX1 gene is most abundantly expressed in the petals at around 12 h before flower-opening, accompanying the increase in the vacuolar pH for the blue flower coloration. No such massive expression was observed in the petunia flowers. Since the NHX1 genes that promote the transport of Na+ into the vacuoles have been regarded to be involved in salt tolerance by accumulating Na+ in the vacuoles, we can add a new biological role for blue flower coloration in the Japanese morning glory by the vacuolar alkalization.     

Perianth bottom-specific blue color development in Tulip cv. Murasakizuisho requires ferric ions

The entire flower of Tulipa gesneriana cv. Murasakizuisho is purple, except the bottom, which is blue. To elucidate the mechanism of the different color development in the same petal, we prepared protoplasts from the purple and blue epidermal regions and measured the flavonoid composition by HPLC, the vacuolar pH by a proton-selective microelectrode, and element contents by the inductively coupled plasma (ICP) method. Chemical analyses revealed that the anthocyanin and flavonol compositions in both purple and blue colored protoplasts were the same; delphinidin 3-O-rutinoside (1) and major three flavonol glycosides, manghaslin (2), rutin (3) and mauritianin (4). The vacuolar pH values of the purple and blue protoplasts were 5.5 and 5.6, respectively, without any significant difference. However, the Fe(3+) content in the blue protoplast was approximately 9.5 mM, which was 25 times higher than that in the purple protoplasts. We could reproduce the purple solution by mixing 1 with two equimolar concentrations of flavonol with lambda(vismax) = 539 nm, which was identical to that of the purple protoplasts. Furthermore, addition of Fe(3+) to the mixture of 1-4 gave the blue solution with lambda(vismax) = 615 nm identical to that of the blue protoplasts. We have established that Fe(3+) is essential for blue color development in the tulip.     

Natural variation in petal color in Lycoris longituba revealed by anthocyanin components

Lycoris longituba is one of the species belonging to the Amaryllidaceae family. Despite its limited distribution, endemic to central eastern China, this species displays an exceptionally wide diversity of flower colors from purple, red, orange, to yellow, in nature. We study the natural variation of floral color in L. longituba by testing the components of water-soluble vacuolar pigments--anthocyanins--in its petals using high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization mass spectrometry. Four anthocyanins were identified, cyanidin-3-sophoroside (Cy3So), cyanidin-3-xylosylglucoside (Cy3XyGlc), cyanidin-3-sambubioside (Cy3Sa), and pelargonidin-3-xylosylglucoside (Pg3XyGlc), which occur at various amounts in L. longituba petals of different colors. A multivariate analysis was used to explore the relationship between pigments and flower color. Anthocyanins have been thought to play a major role in acting as a UV screen that protects the plant's DNA from sunlight damage and attracting insects for the purpose of pollination. Thus, knowledge about the content and type of anthocyanins determining the petal coloration of Lycoris longituba will help to study the adaptive evolution of flowers and provide useful information for the ornamental breeding of this species.     

The involvement of tonoplast proton pumps and Na+(K+)/H+ exchangers in the change of petal color during flower opening of Morning Glory, Ipomoea tricolor cv. Heavenly Blue

The petal color of morning glory, Ipomoea tricolor cv. Heavenly Blue, changes from purplish red to blue during flower opening. This color change is caused by an unusual increase in vacuolar pH from 6.6 to 7.7 in the colored adaxial and abaxial cells. To clarify the mechanism underlying the alkalization of epidermal vacuoles in the open petals, we focused on vacuolar H+-ATPase (V-ATPase), H+-pyrophosphatase (V-PPase) and an isoform of Na+/H+ exchanger (NHX1). We isolated red and blue protoplasts from the petals in bud and fully open flower, respectively, and purified vacuolar membranes. The membranes contained V-ATPase, V-PPase and NHX1, which were immunochemically detected, with relatively high transport activity. NHX1 could be detected only in the vacuolar membranes prepared from flower petals and its protein level was the highest in the colored petal epidermis of the open flower. These results suggest that the increase of vacuolar pH in the petals during flower opening is due to active transport of Na+ and/or K+ from the cytosol into vacuoles through a sodium- or potassium-driven Na+(K+)/H+ exchanger NXH1 and that V-PPase and V-ATPase may prevent the over-alkalization. This systematic ion transport maintains the weakly alkaline vacuolar pH, producing the sky-blue petals.     

Ferric ions involved in the flower color development of the Himalayan blue poppy, Meconopsis grandis

The Himalayan blue poppy, Meconopsis grandis, has sky blue-colored petals, although the anthocyanidin nucleus of the petal pigment is cyanidin. The blue color development in this blue poppy involving ferric ions was therefore studied. We analyzed the vacuolar pH, and the organic and inorganic components of the colored cells. A direct measurement by a proton-selective microelectrode revealed that the vacuolar pH value was 4.8. The concentrations of the total anthocyanins in the colored cells were around 5mM, and ca. three times more concentrated flavonols were detected. Fe was detected by atomic analysis of the colored cells, and the ratio of Fe to anthocyanins was ca. 0.8 eq. By mixing the anthocyanin, flavonol and metal ion components in a buffered aq. solution at pH 5.0, we were able to reproduce the same blue color; the visible absorption spectrum and CD were identical to those in the petals, with Fe(3+), Mg(2+) and flavonol being essential for the blue color. The blue pigment in Meconopsis should be a new type of metal complex pigment that is different from a stoichiometric supramolecular pigment such as commelinin or protocyanin.     

Characterization of a novel Na+/H+ antiporter gene InNHX2 and comparison of InNHX2 with InNHX1, which is responsible for blue flower coloration by increasing the vacuolar pH in the Japanese morning glory

The reddish-purple buds of the wild-type Japanese morning glory (Ipomoea nil) change into blue open flowers, and the shift in the flower coloration correlates with an increase in the vacuolar pH of the flower epidermal cell. In the mutant deficient in the InNHX1 gene for the vacuolar Na(+)/H(+) antiporter, the vacuolar alkalization occurs only partially, and reddish-purple buds become purple open flowers. While most of the plant NHX genes characterized are generally expressed in leaves, stems and roots and induced by NaCl treatment, the InNHX1 gene is expressed predominantly in the flower limbs at around 12 h before flower opening. It is expressed very sparsly in leaves, stems and roots, and no induction occurs in response to NaCl treatment. Here, we identified a novel vacuolar Na(+)/H(+) antiporter gene InNHX2, which is expressed in leaves, stems and roots and is induced in response to NaCl treatment. In addition, relatively higher expression of InNHX2 was observed in the flower limbs shortly before flower opening. We also discovered that both the InNHX1 and InNHX2 proteins can catalyze both Na(+) and K(+) transport into vacuoles. These results suggest that InNHX2 performs dual functions: to confer salt tolerance on the plant and to promote partial vacuolar alkalization in the petals. The implication is that the InNHX2 protein is probably one of the components responsible for converting reddish-purple buds into purple open flowers by partially increasing the vacuolar pH in the absence of major InNHX1 activity.     

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。     

Genetic and linkage analysis of purple-blue flower in soybean

Flower color of soybean is primarily controlled by genes W1, W3, W4, Wm, and Wp. In addition, the soybean gene symbol W2, w2 produces purple-blue flower in combination with W1. This study was conducted to determine the genetic control of purple-blue flower of cultivar (cv). Nezumisaya. F(1) plants derived from a cross between Nezumisaya and purple flower cv. Harosoy had purple flowers. Segregation of the F(2) plants fitted a ratio of 3 purple:1 purple-blue. F(3) lines derived from F(2) plants with purple-blue flowers were fixed for purple-blue flowers, whereas those from F(2) plants with purple flowers fitted a ratio of 1 fixed for purple flower:2 segregating for flower color. These results indicated that the flower color of Nezumisaya is controlled by a single gene whose recessive allele is responsible for purple-blue flower. Complementation analysis revealed that flower color of Nezumisaya is controlled by W2. Linkage mapping revealed that W2 is located in molecular linkage group B2. Sap obtained from banner petals of cvs. with purple flower had a pH value of 5.73-5.77, whereas that of cvs. with purple-blue flower had a value of 6.07-6.10. Our results suggested that W2 is responsible for vacuolar acidification of flower petals.     

Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin

Flower color is mainly determined by anthocyanins. Rosa hybrida lacks violet to blue flower varieties due to the absence of delphinidin-based anthocyanins, usually the major constituents of violet and blue flowers, because roses do not possess flavonoid 3',5'-hydoxylase (F3'5'H), a key enzyme for delphinidin biosynthesis. Other factors such as the presence of co-pigments and the vacuolar pH also affect flower color. We analyzed the flavonoid composition of hundreds of rose cultivars and measured the pH of their petal juice in order to select hosts of genetic transformation that would be suitable for the exclusive accumulation of delphinidin and the resulting color change toward blue. Expression of the viola F3'5'H gene in some of the selected cultivars resulted in the accumulation of a high percentage of delphinidin (up to 95%) and a novel bluish flower color. For more exclusive and dominant accumulation of delphinidin irrespective of the hosts, we down-regulated the endogenous dihydroflavonol 4-reductase (DFR) gene and overexpressed the Irisxhollandica DFR gene in addition to the viola F3'5'H gene in a rose cultivar. The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding. Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations.     

Mechanism of dusky reddish-brown "kaki" color development of Japanese morning glory,Ipomoea nil cv. Danjuro

The mechanism of dusky reddish-brown "kaki" color development of morning glory, Ipomoea nil cv. Danjuro, was studied. Three major known anthocyanins were isolated as glucosylated pelargonidin derivatives. Measurement of the vacuolar pH with proton-selective microelectrodes revealed the vacuolar pH of the colored cell of open flowers to be 6.8, while that of buds was 5.8. Mixing of the three anthocyanins according to the composition ratio in petals at pH 6.8 allowed the identical color to that of petals to be reproduced. The typical "kaki" color development was mostly caused by 5-OH free acylated anthocyanins, which have two lambdamax around 435 and 535 nm in the visible region.     

基于花青素代谢培育蓝色花卉的研究进展

花色是植物吸引昆虫传播花粉的主要因素,对于植物在自然界的生存必不可少,也是观赏植物最重要的性状之一。在蓬勃发展的花卉产业中,色彩各异花卉的培育,可以弥补自然花色的匮乏,但是令人垂涎的蓝色花比较难培育。花色的多样性主要是由花青素及其衍生物的种类和含量等因素决定的,飞燕草色素的合成是形成蓝色花的关键因素,许多植物体内缺少合成飞燕草色素的结构基因。近年来,利用基因工程技术培育蓝色花的研究也时有报道。文中以常见的观赏植物为例,基于花青素代谢调控,从影响飞燕草色素合成的关键因素和不同分子改良途径培育蓝色花等几个方面对植物花朵呈色的机制进行了综述,并展示不同分子育种策略可能在其他领域的应用,为其他植物或经济作物的色泽改良如彩色棉蓝色纤维的培育等提供参考和技术支持。     

Analysis of flavonoids in flower petals of soybean near-isogenic lines for flower and pubescence color genes

W1, W3, W4, and Wm genes control flower color, whereas T and Td genes control pubescence color in soybean. W1, W3, Wm, and T are presumed to encode flavonoid 3'5'-hydroxylase (EC 1.14.13.88), dihydroflavonol 4-reductase (EC 1.1.1.219), flavonol synthase (EC 1.14.11.23), and flavonoid 3'-hydroxylase (EC 1.14.13.21), respectively. The objective of this study was to determine the structure of the primary anthocyanin, flavonol, and dihydroflavonol in flower petals. Primary component of anthocyanin in purple flower cultivars Clark (W1W1 w3w3 W4W4 WmWm TT TdTd) and Harosoy (W1W1 w3w3 W4W4 WmWm tt TdTd) was malvidin 3,5-di-O-glucoside with delphinidin 3,5-di-O-glucoside as a minor compound. Primary flavonol and dihydroflavonol were kaempferol 3-O-gentiobioside and aromadendrin 3-O-glucoside, respectively. Quantitative analysis of near-isogenic lines (NILs) for flower or pubescence color genes, Clark-w1 (white flower), Clark-w4 (near-white flower), Clark-W3w4 (dilute purple flower), Clark-t (gray pubescence), Clark-td (near-gray pubescence), Harosoy-wm (magenta flower), and Harosoy-T (tawny pubescence) was carried out. No anthocyanins were detected in Clark-w1 and Clark-w4, whereas a trace amount was detected in Clark-W3w4. Amount of flavonols and dihydroflavonol in NILs with w1 or w4 were largely similar to the NILs with purple flower suggesting that W1 and W4 affect only anthocyanin biosynthesis. Amount of flavonol glycosides was substantially reduced and dihydroflavonol was increased in Harosoy-wm suggesting that Wm is responsible for the production of flavonol from dihydroflavonol. The recessive wm allele reduces flavonol amount and inhibits co-pigmentation between anthocyanins and flavonols resulting in less bluer (magenta) flower color. Pubescence color genes, T or Td, had no apparent effect on flavonoid biosynthesis in flower petals.     

Soluble and insoluble invertase activity in elongating Tulipa gesneriana flower stalks

Previously 'frozen' Tulipa gesneriana L. bulbs cv. Apeldoorn, were planted and grown at higher temperatures to study the role of invertase (EC 3.2.1.26) in the cold-induced elongation of the flower stalk internodes. After planting, flower stalks were left intact, or, the leaves and flower bud were both removed to inhibit internode elongation. In intact flower stalks, elongation of the internodes was accompanied by an accumulation of glucose and an initial decrease in the sucrose content g,⁻¹ dry weight. Insoluble invertase activity g,⁻¹ dry weight hardly changed, but soluble invertase activity showed a peak pattern, that was related, at least for the greater part, to the changes in the sugar contents. Peak activities of soluble invertase were found during (lower- and uppermost internodes) or around the onset of the rapid phase of internode elongation (middle internodes). Internode elongation and glucose accumulation immediately ceased when the leaves and flower bud were removed. Insoluble invertase activity g,⁻¹ dry weight remained at its initial level (lowermost internode) or increased more towards the upper internodes. Soluble invertase activity did not further increase (uppermost internode) or decreased abruptly to a low level. It is concluded that soluble invertase may be one of the factors contributing to glucose accumulation and internode elongation in the tulip flower stalk.     

Molecular characterization of an anthocyanin-related glutathione S-transferase gene in cyclamen

Anthocyanins are a subclass of flavonoids and are a major contributor to flower colors ranging from red to blue and purple. Previous studies in model and ornamental plants indicate a member of the glutathione S-transferase (GST) gene family is involved in vacuolar accumulation of anthocyanins. In order to identify the anthocyanin-related GST in cyclamen, degenerate PCR was performed using total RNA from immature young petals. Four candidates of GSTs (CkmGST1 to CkmGST4) were isolated. Phylogenetic analysis indicated that CkmGST3 was closely related to PhAN9, an anthocyanin-related GST of petunia, and this clade was clustered with other known anthocyanin-related GSTs. Expression analysis at different developmental stages of petals revealed that CkmGST3 was strongly expressed in paler pigmented petals than in fully pigmented petals, in contrast to the constitutive expression of the other three candidates during petal development. This expression pattern of CkmGST3 was correlated with those of other anthocyanin biosynthetic genes such as CkmF3'5'H and CkmDFR2. Molecular complementation of Arabidopsis tt19, a knockout mutant of an anthocyanin-related GST gene, demonstrated that CkmGST3 could complement the anthocyanin-less phenotype of tt19. Transgenic plants that expressed the other three CkmGSTs did not show anthocyanin accumulation. These results indicate CkmGST3 functions in anthocyanin accumulation in cyclamen.     

CCC1 is a transporter that mediates vacuolar iron storage in yeast

The budding yeast Saccharomyces cerevisiae can grow for generations in the absence of exogenous iron, indicating a capacity to store intracellular iron. As cells can accumulate iron by endocytosis we studied iron metabolism in yeast that were defective in endocytosis. We demonstrated that endocytosis-defective yeast (Delta end4) can store iron in the vacuole, indicating a transfer of iron from the cytosol to the vacuole. Using several different criteria we demonstrated that CCC1 encodes a transporter that effects the accumulation of iron and Mn(2+) in vacuoles. Overexpression of CCC1, which is localized to the vacuole, lowers cytosolic iron and increases vacuolar iron content. Conversely, deletion of CCC1 results in decreased vacuolar iron content and decreased iron stores, which affect cytosolic iron levels and cell growth. Furthermore Delta ccc1 cells show increased sensitivity to external iron. The sensitivity to iron is exacerbated by ectopic expression of the iron transporter FET4. These results indicate that yeast can store iron in the vacuole and that CCC1 is involved in the transfer of iron from the cytosol to the vacuole.     

Anthocyanic vacuolar inclusions--their nature and significance in flower colouration

The petals of a number of flowers are shown to contain similar intensely coloured intravacuolar bodies referred to herein as anthocyanic vacuolar inclusions (AVIs). The AVIs in a blue-grey carnation and in purple lisianthus have been studied in detail. AVIs occur predominantly in the adaxial epidermal cells and their presence is shown to have a major influence on flower colour by enhancing both intensity and blueness. The latter effect is especially dramatic in the carnation where the normally pink pelargonidin pigments produce a blue-grey colouration. In lisianthus, the presence of large AVIs produces marked colour intensification in the inner zone of the petal by concentrating anthocyanins above levels that would be possible in vacuolar solution. Electron microscopy studies on lisianthus epidermal tissue failed to detect a membrane boundary in AVI bodies. AVIs isolated from lisianthus cells are shown to have a protein matrix. Bound to this matrix are four cyanidin and delphinidin acylated 3,5-diglycosides (three, new to lisianthus), which are relatively minor anthocyanins in whole petal extracts where acylated delphinidin triglycosides predominate. Flavonol glycosides were not bound. A high level of anthocyanin structural specificity in this association is thus implied. The specificity and effectiveness of this anthocyanin "trapping" is confirmed by the presence in the surrounding vacuolar solution of only delphinidin triglycosides, accompanied by the full range of flavonol glycosides. "Trapped" anthocyanins are shown to differ from solution anthocyanins only in that they lack a terminal rhamnose on the 3-linked galactose. The results of this study define for the first time the substantial effect AVIs have on flower colour, and provide insights into their nature and their specificity as vacuolar anthocyanin traps.     

Effects of exogenous gibberellins and paclobutrazol on floral stalk growth of tulip sprouts isolated from cooled and non-cooled tulip bulbs

The involvement of gibberellins (GAs) in the regulation of floral stalk elongation and flower development has been studied in tulip. The biological activity of GA₄ and GA₉, both endogenous in tulip bulb sprouts, and GA₁, was tested in vitro on sprouts of cooled and non-cooled tulip bulbs ( Tulipa gesneriana L. cv. Apeldoorn), in the presence or absence of the GA biosynthesis inhibitor paclobutrazol. At early starting dates of incubation, floral stalks from both cooled and non-cooled bulbs hardly showed any elongation in the absence of exogenous GA. Paclobutrazol had no effect on floral stalk elongation, and the response to GAs of sprouts from cooled bulbs was greater than that of sprouts from non-cooled bulbs. At later starts of incubation, considerable floral stalk elongation occurred without GA application. Paclobutrazol inhibited this floral stalk elongation, and the growth of sprouts from both cooled and non-cooled bulbs was stimulated by GA application. The effect of paclobutrazol was reversed by simultaneous application of GA₄ or GA₉. Application of GA with and without paclobutrazol resulted in the same elongation of the floral stalk, indicating the absence of substantial side effects of the inhibitor. The isolated sprouts did not develop a full-grown flower without the addition of GA. GA₄ was more effective than GA₉ in stimulating this flower development. The results demonstrate that both sprouts from cooled and non-cooled bulbs are responsive to exogenous GAs in vitro, and may be a site of GA biosynthesis.