The diacylglycerol forming pathways differ among floral organs of Petunia hybrida

The origin of diacylglycerol, a substrate for membrane lipid biosynthesis, is not fully understood. Here, we report that Petunia hybrida floral organs contain large amounts of diacylglycerol. Our data suggest that in stamens and pistils diacylglycerol is supplied both from phosphatidylcholine by non-specific phospholipase C activity and de novo via the Kennedy pathway and phosphatidic acid phosphatase, whereas in petals the two-step pathway catalyzed by phospholipase D and phosphatidic acid phosphatase predominates. Therefore, the pathways that supply diacylglycerol differ among floral reproductive organs, although large amounts of diacylglycerol are commonly accumulated in these organs.     

Genetic control of dihydroflavonol 4-reductase gene expression in Petunia hybrida

The functions of four loci ( An1, An2, An4 , and An6 ) which control pigmentation in flowers of Petunia hybrida have been characterized. Linkage-analysis and molecular complementation experiments showed that the An6 locus contains the structural dfrA gene, encoding the enzyme dihydroflavonol 4-reductase (DFR). Analysis of gus gene expression driven by the dfrA promoter in transgenic plants showed that the dfrA promoter is highly active in the flower corolla, the anthers and seeds and, at a lower level, in ovules and the flower stem. These data are discussed in relation to the expression of other pigmentation genes and the accumulation pattern of anthocyanins. The expression of the dfrA-gus transgene was dependent on the genes an1 (in every tissue), an2 (in the flower limb only) and an4 (in anthers), demonstrating that these genes encode regulatory factors that control dfrA promoter activity.     

Effects of cor15a-IPT gene expression on leaf senescence in transgenic Petunia x hybrida and Dendranthema x grandiflorum

To prevent leaf senescence of young transplants or excised shoots during storage under dark and cold conditions, the cytokinin biosynthetic gene isopentenyl transferase (ipt) was placed under the control of a cold-inducible promoter cor15a from Arabidopsis thaliana and introduced into Petunia x hybrida 'Marco Polo Odyssey' and Dendranthema x grandiflorum (chrysanthemum) 'Iridon'. Transgenic cor15a-ipt petunia and chrysanthemum plants and excised leaves remained green and healthy during prolonged dark storage (4 weeks at 25 degrees C) after an initial exposure to a brief cold-induction period (4 degrees C for 72 h). However, cor15a-ipt chrysanthemum plants and excised leaves that were not exposed to a cold-induction period, senesced under the same dark storage conditions. Regardless of cold-induction treatment, leaves and plants of non-transformed plants senesced under prolonged dark storage. Analysis of ipt expression indicated a marked increase in gene expression in intact transgenic plants as well as in isolated transgenic leaves exposed to a short cold-induction treatment prior to dark storage. These changes correlated with elevated concentrations of cytokinins in transgenic leaves after cold treatment. Cor15a-ipt transgenic plants showed a normal phenotype when grown at 25 degrees C.     

Modifying the acylation of flavonols in Petunia hybrida

The potential for chemically-regulating the acylation of natural products in whole plants has been determined by treating petunia leaves with phenylpropanoid acyl donors supplied as the respective methyl esters. Treatment with derivatives of the naturally-occurring acylating species caffeic acid resulted in a general increase in flavonol derivatives, notably caffeoylated quercetin-3-O-diglucoside (QDG) and kaempferol-3-O-diglucoside (KDG). Similarly, methyl ferulate increased the content of feruloylated KDG 40-fold. Treatment with methyl coumarate resulted in the appearance of a coumaroylated derivative of quercetin-3-O-glucuronyl-glucoside (QGGA). When the feeding studies were repeated with the equivalent phenylpropanoid isosubstituted with fluorine groups a semi-synthetic 4-fluorocinnamoyl ester of QGGA was observed. Our results demonstrate the potential to regulate the acylation of flavonols and potentially other natural products by treating whole plants with methyl esters of natural and unnatural acyl donors.     

Genetic analysis of instability in Petunia hybrida

Summary In crossing experiments with Petunia hybrida, new mutations, some unstable, have been found in descendants of plants having an unstable allele of the anthocyanin gene An1. One of the unstable mutations affecting the new anthocyanin gene An11 was genetically analyzed, and it was subsequently established in which step of anthocyanin synthesis that An11 is involved. The discovery of new, unstable mutations at other loci indicates that in Petunia also a relation exists between unstable mutations and the presence of transposable elements in the genome. It was demonstrated that reverted alleles (an1^+/+) originating from unstable An1 alleles are less stable than the original wild-type allele An1, and that reversions do not increase the chances of occurrence of new, stable or unstable mutations at other loci. These results provide additional arguments in favour of the hypothesis posed in an earlier paper that reversions of unstable An1 alleles are not the result of excision of the inserted transposable element, but are due to the repair of secondary mutations induced by the insert in the regulatory region of the locus. Consequently, a reverted allele still contains the inserted element that may again induce mutations leading to inactivation of An1.     

Genetic analysis of instability in Petunia hybrida

Summary The effect of environmental factors on the reversion rates of several unstable alleles in Petunia hybrida was investigated. It is demonstrated that the reversion frequency of three unstable alleles, viz. an allele of gene An1 and of gene An11, both involved in anthocyanin synthesis, and of gene Yg3 for leaf colour, is drastically reduced when the temperature is raised from 18 °C to 25 °C. For two of the alleles it was established that this temperature effect is reversible. Changing the light period or light intensity did not have an effect on the reversion rate of the unstable allele of gene An11 at 18 °C or at 25 °C. The results found are in contrast with those obtained in earlier experiments, in which a rise in temperature resulted in an increase in the reversion rate of another unstable allele of gene An1.     

A standard karyogram of Petunia hybrida hort

An improved technique, viz., maceration with cellulase and pectinase, was applied in order to obtain chromosome preparations of botanical material suitable for fluorescence microscopy. The application of this method to Petunia hybrida allows the individual distinction of all chromosomes in mitosis. The following three criteria are required for this distinetion: the centromere index, the relative length, and the fluorescence intensity pattern after staining with Quinacrine. A standard karyogram is drawn up to render possible a comparison with other Petunia material.     

The Importance of the Stigma in Flower Senescence in Petunia (Petunia hybrida)

Senescence of flowers of Petunia hybrida Vilm. cv Gypsy is characterizedby colour changes, wilting and abscission. In emasculated detachedflowers the onset of these processes is hastened by any treatmentwhich reduces the vigour of the stigma. Thus pricking it, excisingsegments, or freezing with liquid nitrogen all reduce the timeto morphological changes associated with corolla senescence.Removal of the stigma has the most dramatic effect, reducinglifespan of the flower by about 50 per cent, to 3 d. This reductioncan be lessened if IAA or 2,4-D is applied to the cut surfaceof the style. In intact flowers, the style may usually be implicatedin the production of a stimulus leading to corolla abscission,but abscission will also occur in the absence of the style.Some senescence acceleration takes place not only in the completeabsence of the style, but also when the upper part of the ovaryhas been excised in addition. The speeding up of senescenceand of corolla abscission cannot be due solely to damage perse since when the corolla limb was excised, leaving only thecorolla tube, the tube abscised at about the same time as thecontrols, despite the quite extensive wounding. This also impliesthat the distal parts of the corolla do not play a major rolein the development of the abscission zone at the base of thecorolla tube. A healthy, undamaged stigma appears to be very important incorolla longevity and one of its roles may be to prevent theproduction of an abscission/wilting stimulus by some other componentof the flower. Possibly auxins in the stigma are important inthat either they are mobile and protect the abscission zoneor they create a sink for other substances which are implicatedin flower senescence. Petunia hybrida, abscission, auxins (IAA, 2,4-D), corolla, flower senescence, stigma, style, wilting     

Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes

In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays.The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively).Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.     

Glucosylation of flavonoids in petals of Petunia hybrida

During the biosynthesis of anthocyanins in Petunia hybrida, the 3-hydroxyl group is glucosylated. Their supposed biosynthetic precursors, the dihydroflavonols, are glucosylated at the 7 or 4 positions. The question arose of whether these glucosides or the aglucones act as a substrate in anthocyanin synthesis. Using isolated flower buds of white flowering mutants that were blocked in an earlier step of biosynthesis, it was found that anthocyanin-3-glucosides and dihydroquercetin-7-glucoside were synthesized if dihydroquercetin, dihydroquercetin-7-glucoside, or dihydroquercetin-4-glucoside were used as precursors in these experiments. Intracellular dihydroquercetin-glucosides were not used as a substrate for anthocyanin synthesis. The results are explained by deglucosylation of dihydroquercetin-glucosides during uptake by isolated flower limbs. Dihydroquercetin-7-glucoside, formed intracellularly, is not available as a precursor for anthocyanins. We conclude that the aglucone form of dihydroquercetin acts as a substrate in anthocyanin biosynthesis.Abbreviations dHO dihydroquercetin - dHQ-7=g dihydroquercetin-7-glucoside - dHQ-4-g dihydroquercetin-4-glucoside     

The morphology and anatomy of the stigma of Petunia hybrida

Summary A mature stigma of Petunia hybrida ready for pollination shows 4-6 large shining drops of the exudate along with numerous smaller ones. A developing style and stigma have a columnar tissue that flares at the top. In the stigma there can be distinguished a secretory and a storage zone. In the former, schizogenous cavities are formed which are filled with the exudate. The mode of formation and secretion of the drop has been studied with light and electron microscope. The nature of reserves has been studied histochemically.The exudation takes place in two stages. In the first stage, the epidermal and papillae cells release out the oily exudate upon rupture of the cuticle. The second phase of exudation begins with anthesis. The exudate from the schizogenous cavities is released between the epidermal cells. There are distinct loci on the stigma surface where more exudate is given out than at other places.

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Zusammenfassung Bei der Durchsicht der einschlägigen Literatur ergab sich, daß die Kenntnisse über die Entwicklungsgeschichte und den Sekretionsmechanismus des Narben-Schleimes äußerst lückenhaft sind, obgleich dieser im Verlauf des Bestäubungs-Vorganges bei vielen Pflanzen eine bedeutende Rolle spielt. Die vorliegende Untersuchung versuchte daher eine detaillierte Einsicht in die Morphologie und Anatomie der Narbe von Petunia hybrida zu geben, da diese Art als Objekt für zahlreiche physiologische Untersuchungen auf dem Gebiet der Befruchtungs-inkompatibilität dient.Die Blüten von Petunia sind zwittrig, zygomorph und fünfzählig. Sie besitzen fünf epipetale Antheren mit drei verschiedenen Filament-Längen; diese öffnen sich zu verschiedenen Zeitpunkten. Die reife Narbe zeigt zum Zeitpunkt der Bestäubung 4-6 große sowie zahlreiche kleinere, transparente Flüssigkeits-Tropfen.Der sich entwickelnde Griffel mit der Narbe besteht aus einem säulenartigen Gewebe, das sich an der Spitze verbreitert. Bei der Narbe kann eine Sekretions-und eine Reservematerial-Zone unterschieden werden. In der Sekretionszone werden schizogen Hohlräume gebildet, die sich mit dem Exsudat füllen. Die Bildung und Sekretion der Narbenflüssigkeit wurde licht-und elektronenmikroskopisch untersucht. Mittels histochemischer Methoden wurden die Substanzen der Reservematerial-Zone näher charakterisiert.Die Freisetzung der Narben-Flüssigkeit findet in zwei Schritten statt: Während der ersten Phase wird das ölige Exsudat von den epidermalen Zellen und den Papillen der Narbe nach Zerreißen der Narben-Cuticula freigesetzt. In der zweiten Phase jedoch tritt das Exsudat aus den schizogenen Hohlräumen durch die Zwischenräume in der Narben-Epidermis aus. Auf der Narben-Oberfläche gibt es Bezirke, welche offensichtlich in reichlicherem Maße Flüssigkeit produzieren als andere Bezirke.

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Dedicated to the memory of the late Prof. P. Maheshwari FRS, our teacher (R. N. K.) and friend (H. F. L.) who read through the Ms in April at Paris, a month before his death.     

矮牵牛"Wafe"的组织培养及快速繁殖

1 植物名称矮牵牛(Petunia hybrida) 美国进口垂吊品种:“Wafe“. 　　2 材料类别叶片. 　　3 培养条件以MS为基本培养基.(1)启动培养基:MS 6-BA 2.0 mg*L-1 (单位下同) NAA 0.2; (2)增殖继代培养基:MS 6-BA 3.0 NAA 0.1; (3)生根培养基:MS NAA 2.0 6-BA 0.1. 以上培养基(1)、(2)均附加3%蔗糖,培养基(3)附加2%蔗糖,0.25%活性炭,pH 6.0.培养温度为有光照25℃,无光照20℃,光照时间12 h*d-1,光照度1 500～2 000 lx. 　　……     

Dissection of floral pollination syndromes in Petunia

Animal-mediated pollination is essential in the reproductive biology of many flowering plants and tends to be associated with pollination syndromes, sets of floral traits that are adapted to particular groups of pollinators. The complexity and functional convergence of various traits within pollination syndromes are outstanding examples of biological adaptation, raising questions about their mechanisms and origins. In the genus Petunia, complex pollination syndromes are found for nocturnal hawkmoths (P. axillaris) and diurnal bees (P. integrifolia), with characteristic differences in petal color, corolla shape, reproductive organ morphology, nectar quantity, nectar quality, and fragrance. We dissected the Petunia syndromes into their most important phenotypic and genetic components. They appear to include several distinct differences, such as cell-growth and cell-division patterns in the basal third of the petals, elongation of the ventral stamens, nectar secretion and nectar sugar metabolism, and enzymatic differentiation in the phenylpropanoid pathway. In backcross-inbred lines of species-derived chromosome segments in a transposon tagging strain of P. hybrida, one to five quantitative trait loci were identified for each syndrome component. Two loci for stamen elongation and nectar volume were confirmed in introgression lines and showed large allelic differences. The combined data provide a framework for a detailed understanding of floral syndromes from their developmental and molecular basis to their impact on animal behavior. With its molecular genetic tools, this Petunia system provides a novel venue for a pattern of adaptive radiation that is among the most characteristic of flowering plants.     

Physiology and biochemistry of the stigmatic fluid of Petunia hybrida

Summary Production of stigma exudate per flower of Petunia hybrida is about 200 g. The effect of light, temperature, metabolic poison and emasculation on the production of the exudate at different ages of the bud has been studied. The presence of a thin film of water below the stigmatic exudate has been demonstrated. Physical properties of the exudate such as relative viscosity and surface tension have also been determined. Chemical analysis of the stigmatic fluid showed that it consists primarily of an oil, sugars and amino acids. No protein could be detected. It also contains no acid phosphatase.Behaviour of the pollen from its deposition on the stigmatic fluid until it germinates on the stigma surface has been studied in vivo and also with the aid of an artificial stigma.The role of the stigmatic fluid in pollination has been determined.

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Zusammenfassung Die Narbenflüssigkeit, welche von zahlreichen Pflanzen ausgeschieden wird zu einem Zeitpunkt, da die Blüte reif ist zur Bestäubung, spielt eine wichtige Rolle bei der Fixierung des Pollens. Um so überraschender ist die Tatsache, daß hinsichtlich der Physiologie und Biochemie des Narbenschleimes so wenig detaillierte Informationen zur Verfügung stehen.Bei Petunia wird je Blüte im Mittel 200 g Narben-Flüssigkeit produziert. Die Intensität der Narbenschleimproduktion ist abhängig von der Temperatur und der Länge der Lichtperiode. Hingegen wird die Ausscheidung der Narbenflüssigkeit durch Hemmung der Atmung und durch partielle Kastration nicht beeinflußt.Die physikalischen Eigenschaften (Dichte, relative Viscosität und Oberflächenspannung) werden bestimmt. Bei der chemischen Analyse des Narbenschleimes ergab sich, daß dieser hauptsächlich aus einem öligen Fett besteht, dessen Fettsäure-Zusammensetzung ermittelt wurde. Hingegen werden nur sehr geringe Spuren von Zuckern und Aminosäuren gefunden. Der Narbenschleim ist frei von Proteinen und zeigt keine enzymatische Aktivität.Das Verhalten des Pollens bei der Landung in einem Tropfen der Narbenflüssigkeit wird in vivo und mit Hilfe einer künstlichen Modell-Narbe untersucht.Die Bedeutung der Narbenflüssigkeit im Zusammenhang mit der Bestäubung und den einleitenden Stadien der Pollenkeimung wird diskutiert.

     

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.