Anthocyanin synthesis in a white flowering mutant of Petunia hybrida

In flower buds of the white flowering mutant W19 of Petunia hybrida four biologically active dihydroflavonol intermediates-dihydroquercetin-7-glucoside, dihydroquercetin-4-glucoside, dihydroquercetin, and dihydrokaempferol-7-glucoside-are accumulated. When dihydroquercetin was supplied to in vitro cultured corollas of the white flowering mutant W18, a mixture of cyanidin and delphinidin glycosides was produced, cyanidin-3-glucoside being the major pigment. The quantity of dihydroquercetin accumulated in W19 is very small, but this compound appears to be a more direct precursor of anthocyanins than the glucosides of dihydrokaempferol and dihydroquercetin. The conditions for pigment synthesis in W18 were optimalized. The quantitative uptake of dihydroquercetin was also studied. It was demonstrated that ca. 1/3 of the quantity present in the culture solution entered the corolla. From the absorbed dihydroquercetin only 14% was converted into anthocyanins. Complementation experiments to determine the biosynthetic sequence of the anthocyanin genes An1, An2, and An3 indicated that the genes An1 and An2 are indistinguishable by this technique.Abbreviation DHQ (+) dihydroquercetin     

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.     

一个新的玉米黄绿叶突变体ygl-m的初步研究

在田间选育系谱过程中发现了一份黄绿叶突变体ygl-m,该突变体叶片在苗期自发地表现黄绿色,待植株长到6周大左右植株叶片开始恢复绿色,最后整个植株叶片都恢复正常的绿色。苗期ygl-m与野生型植株B73相比,叶片总叶绿素、叶绿素a、叶绿素b含量均显著下降,叶绿素a/b比值显著升高;苗期叶片叶绿体中基粒类囊体片层较少,排列不规则,结构松散。遗传分析表明,突变体ygl-m的黄绿叶表型由隐性单基因控制。本研究将为开展ygl-m基因的分子标记定位和进一步探讨其利用潜力奠定基础。     

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.     

Cloning and expression analysis of a Petunia hybrida flower specific mitotic-like cyclin

A cyclin cDNA clone (Pethy;CycB1;1) was isolated from a Petunia hybrida ovary specific cDNA library. Sequence comparison revealed that Pethy;CYCB1;1 protein is highly homologous to mitotic B1 cyclins. Northern analysis and in situ hybridisation experiments showed that its expression is developmentally regulated and restricted to flower organs. We have attempted to define some of the cell division patterns which contribute to shaping each floral organ by analysing Pethy;CycB1;1 expression on Petunia flower sections. While in sepals, epidermis and parenchyma cell division patterns were comparable, there were two distinct cell division patterns in petals. In the epidermis, Pethy;CYCB1;1 expression was found both at the petal tip and along epidermis, whereas in the parenchyma only at the petal tips. In reproductive organs cell divisions were detected only in sporophytic tissues. No signals were detected inside meiotic cells.     

Identification of a S-ribonuclease-binding protein in Petunia hybrida

To investigate protein-protein interactions in gametophytic self-incompatibility, we used a yeast two-hybrid assay to identify proteins that could interact with the S-ribonuclease protein. These assays identified a pollen-expressed protein, which we have named PhSBP1, that appears to bind with a high degree of specificity to the Petunia hybrida S-ribonuclease. Although PhSBP1 activates reporter gene expression only when expressed in tandem with a S-RNAse bait protein, binding is not allele-specific. Sequence analysis demonstrated that PhSBP1 contained a C-terminal cysteine-rich region that includes a RING-HC domain. Because many RING-finger domain proteins appear to function as E3 ubiquitin ligases, our results suggest that ubiquitination and protein degradation may play a role in regulating self-incompatibility interactions. Together, these results suggest that PhSBP1 may be a candidate for the recently proposed general inhibitor (RI) of self-incompatibility ribonucleases.     

矮牵牛叶片体细胞胚胎发生发育的组织学观察

以矮牵牛生根试管苗的叶片为外植体,在培养基MS NAA0.1mg/L 6-BA1.6mg/L上诱导体细胞胚胎直接发生。从接种后第一天开始观察叶片愈伤组织发生、发育的外部形态变化,从接种后第七天开始,每隔3天取变化明显的叶片组织块切片观察其胚状体的组织细胞学连续变化。组织切片观察表明,矮牵牛叶片体细胞胚胎发生类似于合子胚的发育过程;矮牵牛体细胞胚起源于叶肉细胞,胚性细胞与非胚性细胞染色明显不同,体细胞胚胎与周边其它组织有明显界线;体细胞胚胎的发育经历胚性细胞、多细胞原胚、球形胚、梨形胚、心形胚、鱼雷胚、类子叶胚等几个阶段。     

Brassinolide affects the rate of cell division in isolated leaf protoplasts of Petunia hybrida

Brassinosteroids are known to promote cell elongation in a wide range of plant species but their effect on cell division has not been as extensively studied. We examined the effect of brassinolide on the kinetics and final division frequencies of regenerating leaf mesophyll protoplasts of Petunia hybrida Vilm v. Comanche. Under optimal auxin and cytokinin conditions, 10–100 nM brassinolide accelerated the time of first cell division by 12 h but had little effect on the final division frequencies after 72–120 h of culture. One micromolar brassinolide showed the same acceleration of first cell division but inhibited the final division frequency by approximately 20%. Under sub-optimal auxin conditions, 10–100 nM brassinolide both accelerated the time of first cell division and dramatically increased the 72- to 120-h final division frequencies. Isolated protoplasts may provide a useful model system to investigate the molecular mechanisms of brassinosteroid action on cell proliferation. Received: 1 December 1997 / Revision received: 13 February 1998 / Accepted: 24 April 1998     

Regulation of gene action in Petunia hybrida: Unstable alleles of a gene for flower colour

Summary In a progeny of a selfed individual of the dark red-flowered cultivar Roter Vogel some white-flowered plants appeared as the result of a mutation of the genetic factor Anl involved in anthocyanin synthesis. The white flowers of these plants had red spots owing to back-mutations in the dermal cells of the young corolla.Owing to a striking unstability of the new allele of Anl, a number of mutants originated which differ mutually in the frequency of reversion, which expressed itself in the very substantial differences in the spot density of the limb of the corolla. Between a mean number of less than one spot per cm² of the limb and a mean number of over 10.000 spots/cm², a series of transitions was found.The reversions did not remain restricted to the young epidermis but also occurred in sporogenous tissues. This resulted in the appearance of selfcoloured red descendants of plants with red-spotted white flowers. There is a positive correlation between the spot density of the parent plants and the percentage of plants with completely red corollas.The red spots on the corolla usually have the same colour as the wild type (Roter Vogel), but occasionally mutants occur with paler spots, the colour varying from a very pale pink to a red nearly as deep as in the wild type. The selfcoloured descendants of such mutants also show this colour variation from pale pink to red.On the grounds of these observations a theory was formulated which postulates that the Anl locus consists of a structural gene responsible for an enzyme active during anthocyanin synthesis and a regulatory element built up from intermediate repetitive DNA. This regulatory element in turn is built up of two components, one of which, the mutator, decides the activation of the structural gene while the other, the expressor, modifies the rate of activation. The mutations must be considered representative of larger or smaller deletions within one or both of these components. Reversions are the result of the restoration of the deletions by means of an amplification of the repetitive DNA in dividing cells of the developing flower buds.     

Centromeric localization of an S-RNase gene in Petunia hybrida Vilm.

S-RNase has been identified to be an S-allele-specific stylar determinant contributing to the self-incompatibility response in Solanaceae. In order to examine the physical location of the S-RNase gene, multi-color fluorescence in situ hybridization (FISH) using the S ^B1 -RNase cDNA probe and ribosomal RNA gene (rDNA) probe was performed on an S ^B1 S ^B2 heterozygote of Petunia hybrida. The S ^B1 -RNase gene was detected as a doublet signal close to the centromere of chromosome III. Next, we performed FISH using a large genome probe prepared from a λSB1–311 clone (20 kb) which contains the S ^B1 -RNase gene and its 3′ flanking region. This probe hybridized to the centromeric regions of all P. hybrida chromosomes. Sequence analysis of the λSB1–311 clone revealed the presence of a repetitive sequence consisting of a novel 666 bp unit sequence. A subclone (pBS-SB1B5) containing this unit sequence also hybridized to all of the centromeric regions, confirming that this unit is the centromeric specific repetitive sequence. These data suggested that the S ^B1 -RNase gene is located very close to (within a distance of 12 kb from) the centromeric-specific repetitive sequence. Likewise, the pBS-SB1B5 probe hybridized to the centromeric regions of all chromosomes in P. littoralis, another Petunia species. However, the probe did not hybridize to the centromere of the chromosomes from other species in Solanaceae. These results suggested that this centromeric repetitive sequence might be a genus-specific one. Received: 3 December 1998 / Accepted: 8 December 1998<@head-com-p1a.lf>Communicated by F. Mechelke     

Intraspecific gametosomatic hybridisation in Petunia hybrida

Following PEG and high pH induced fusion of haploid tetrad protoplasts of a normal purple flowered variety of P. hybrida with cell suspension protoplasts of a nuclear albino mutant of the variety Blue Lace, triploid gametosomatic hybrid plants were recovered. These hybrids possessed an intermediate floral morphology and the expected chromosome number of 2n=3x=21. Selection was based on the fact that pollen tetrad protoplasts failed to divide in culture and that, following complementation to chlorophyll proficiency in the gametosomatic hybrid, the hybrid cells were visualised against a background of albino cells of the variety Blue Lace. The production of such gametosomatic hybrid plants in Petunia has shown that the concept of gametosomatic hybridisation can be extended to genera other than Nicotiana and that alternative selection strategies are available.Abbreviations BAP 6-benzylaminopurine - IAA 3-indole acetic acid - NAA naphthalene acetic acid - Z zeatin - ABN bromonaphthalene - MS Murashige and Skoog (1962) - MW molecular weight - PEG polyethylene glycol     

Differential responses of Brassica napus and Petunia hybrida to leaf protoplast isolation stress

Changes in the response to abiotic stress during the isolation of leaf protoplasts were compared between a recalcitrant species of Brassica napus and regenerating species of Petunia hybrida . Initially, levels of soluble free putrescine (put), spermidine (spd) and spermine (spm) in leaves and protoplasts were determined. The sum of these three polyamines increased in petunia and B. napus leaf protoplasts by 1.6-fold and 1.1-fold, respectively. The soluble free fraction of spd and spm decreased in B. napus but not in petunia protoplasts. During the isolation of leaf protoplasts from B. napus , the ratio of soluble free put to the total PAs almost doubled, but that of spd and spm declined significantly. Petunia leaf protoplasts treated with cyclohexylamine (CHA), an inhibitor of spermidine synthase, accumulated ammonia and soluble putrescine, but lost the soluble spermidine. The soluble polyamine levels of CHA-treated petunia leaf protoplasts corresponded with those in B. napus . Leaves were subjected to abiotic stress during the isolation of protoplasts, namely wounding and osmotic stress which changed soluble free polyamine levels in B. napus and petunia, respectively. Both B. napus and petunia leaf protoplasts showed an increase in ammonia, but total free amino acid content and activation of proteases were only enhanced in B. napus leaf protoplasts. These results suggest that in B. napus wounding initiated senescence of leaf protoplasts during their isolation, leading to a constant production of ethylene early in the culture.