Leaf-Mediated Light Responses in Petunia Flowers

In the present work we studied the role of light in the regulation of flavonoid gene expression and anthocyanin synthesis in petunia (Petunia hybrida) corollas. We found that light is required for chalcone synthase gene (chs) expression, anthocyanin synthesis, and growth of detached and attached petunia corollas. Although direct illumination induced chs expression, pigmentation, and elongation of the detached corollas, irradiation of green leaves or sepals played the main role in the attached corollas. The duration, intensity, and spectrum of the light reaction suggest that phytochrome-mediated high-irradiance reactions are involved in the regulation of corolla development. Using the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, we showed that photosynthesis does not significantly contribute to the leaf-mediated light responses. When sepals were removed or covered. [14C]sucrose up-take by the corolla of detached intact flowers was inhibited. The results of this study suggest that light is perceived by leaves and sepals and enhances corolla sink activity, elongation, pigmentation, and chs expression. The role of leaves and sepals in the light regulation of petunia corolla development is discussed.     

Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility.

Inhibition of flower pigmentation in transgenic petunia plants was previously accomplished by expressing an antisense chalcone synthase (chs) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. This chimeric gene was not effective in inhibiting pigmentation in anthers, presumably because the viral CaMV 35S promoter was insufficiently expressed in cell types of this organ in which the pigments are produced. Insertion of the anther box, a homologous sequence found in other genes expressed in anthers, resulted in a modified expression pattern driven by this promoter, as monitored by the beta-glucuronidase (gus) gene. In addition to the basic CaMV 35S expression pattern in anthers, GUS activity was observed in tapetum cells when the modified promoter was fused to the gus gene. This promoter construct was subsequently used to drive an antisense chs gene in transgenic petunia, which led to the inhibition of pigment synthesis in anthers of five of 35 transformants. Transgenic plants with white anthers were male sterile due to an arrest in male gametophyte development. This finding indicated that flavonoids play an essential role in male gametophyte development.     

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.     

Cloning and Analysis of Promoter Regions of Flavonoid Biosynthesis Genes in Safflower

The flower of the safflower plant Carthamus tinctorius L. has been widely used in traditional Chinese medicine because of its ability to improve cerebral blood flow. Flavonoids are the main active ingredient of safflower plants. Analyses of the regulation of flavonoids are of great significance for the artificial regulation of their biosynthesis, and previous studies have mainly focused on their metabolic synthesis in safflower. For a better understanding of the regulation of flavonoid biosynthesis in safflower, promoters of flavonoid biosynthesis genes in safflower were cloned and analysed. First, the expression of the flavonoid genes in safflower was analysed. Twenty-four genes involved in flavonoid biosynthesis exhibited large differences. Four promoters of flavonoid genes that were mainly expressed in the flowers of safflower (termed pctCHI-1, pctCHS-2, pctF3H-2 and pctGT-1) were cloned and analysed. Elements that respond to light, i.e. phytohormones and the MYB regulator, were identified. The promoter activities were analysed using a protoplast system. All four promoters had activities in the flower protoplast, and the pctCHS-2 activity was the highest. Our results also demonstrate that pctCHS-2 is a flower-specific promoter. All of these results provide a basis for improved analyses of flavonoid regulation in safflower.     

The 5[prime] Flanking Regions of Vicilin and Napin Storage Protein Genes Are Down-Regulated by Desiccation in Transgenic Tobacco

Drying of seeds, when imposed prematurely, elicits a switch in metabolism; events unique to development, such as synthesis of storage protein, are terminated, whereas syntheses associated with germination and growth are initiated. To determine the role of desiccation in down-regulating the expression of genes for storage proteins, the desiccation responsiveness of the 5[prime] and 3[prime] regulatory regions of the genes encoding the pea storage protein vicilin and the Brassica napus storage protein napin was tested in transgenic tobacco seed. Chimeric genes were introduced into tobacco; these genes consisted of the coding region of the reporter gene for [beta]-glucuronidase (GUS) and 5[prime] and/or 3[prime] regions from the vicilin or napin genes or, as controls, the same regions derived from constitutively expressed genes, presumed to be desiccation insensitive. In transgenic seed expressing the gene constructs containing the vicilin or napin promoters, GUS activities declined during late seed development, and more dramatically, after imbibition of mature dry seed or prematurely dried seed. In contrast, GUS activities increased after seed rehydration when the constitutive viral promoter replaced the storage-protein gene 5[prime] region. Transient expression assays support the hypothesis that premature drying down-regulates the expression of the storage-protein gene promoter. Following desiccation, this region may become insensitive to positive controlling factors; alternatively, changes to trans-acting factors may occur.     

Vicilin and Napin Storage-Protein Gene Promoters Are Responsive to Abscisic Acid in Developing Transgenic Tobacco Seed but Lose Sensitivity following Premature Desiccation

In transgenic tobacco (Nicotiana tabacum L.) seed, expression of chimeric [beta]-glucuronidase (GUS) genes containing the vicilin or napin storage-protein gene promoters is responsive to premature drying and declines upon rehydration (L. Jiang, W.L. Downing, C.L. Baszczynski, A.R. Kermode [1995] Plant Physiol 107: 1439-1449). Desiccation may cause changes in the content of or sensitivity to abscisic acid (ABA), partially or wholly removing the effects of this "modulator" of developmental gene expression. We studied the enhancement of GUS reporter enzyme activities in transgenic tobacco by exogenous ABA before and after drying. A racemic mixture of ABA at 10 [mu]M caused a 2- to 3-fold increase in GUS activity in developing transgenic seed expressing chimeric genes containing the vicilin or napin gene promoters. However, when these seeds were prematurely dried prior to their culture on ABA medium, enhancement of GUS activity was virtually abolished. Use of optically pure ABAs revealed that the enhancement in GUS activity in developing fresh seed was due largely to the natural (+) form of ABA. Chimeric constructs containing a viral 35S promoter did not respond to ABA whether or not premature drying was applied. Thus, vicilin and napin chimeric genes, initially sensitive to ABA, become relatively insensitive to the hormone following drying. A decline in ABA sensitivity may be an important factor in the cessation of storage-protein gene expression.     

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.     

Organ- and Tissue-specific Biosynthesis of Flavonoids in Seedlings of Oenothera odorata (Onagraceae)

In developing Oenothera odorata seedlings, phytochrome-mediated accumulation of various flavonoids (mainly glycosides of cyanidin and quercetin) is detectable, subsequent to a transient induction of the key enzymes of the general phenylpropanoid metabolism, L-phenylalanine ammonia lyase (PAL) and of flavonoid biosynthesis, chalcone synthase (CHS). Organ- and tissue-specific distribution of these enzymes and of the flavonoid end products was investigated in seedlings, irradiated with continuous far-red light. Anthocyanins and quercetin glycosides are mainly localized in both the upper and lower epidermis of the cotyledons and to a lesser extent also in the epidermal cell layer of the hypocotyl. An obvious organ-specific distribution was observed for the anthocyanins: cyanidin-3,5-O-diglucoside accumulates in the epidermal cells of the cotyledons, whereas cyanidin-3-O-glucoside is restricted to the epidermis of the hypocotyl. By contrast the pattern of quercetin glycosides is the same in the cotyledons and in the hypocotyl. The methylated flavonol aglycone 3-0-methylquercetin was found to be localized in the seed coat. According to this organ- and tissue-specific pattern of flavonoids, immunochemical and immunohistochemical detection of PAL and CHS revealed a predominant localization of theenzymes in the epidermal layers of the cotyledons and the hypocotyl but also in the cells surrounding the vascular bundles. The role of compartmentation in regulation of flavonoid biosynthesis and putative functions of flavonoid compounds are discussed.     

Structural characteristics of two wheat histone H2A genes encoding distinct types of variants and functional differences in their promoter activity

To investigate the regulation of plant histone H2A gene expression, we isolated two H2A genes (TH254 and TH274) from wheat, which encode two variants of H2A. Both genes had an intron in the coding region. In the promoters, some characteristic sequences, such as Oct and Nona motifs, which are conserved among plant histone genes, were located in a short region (about 120 bp) upstream from the putative TATA box. Transient expression analyses of promoter activity with H2A–GUS fusion genes using tobacco protoplasts revealed novel types of positive cis/-acting sequences in the TH254 promoter: a direct repeat of a 13 bp sequence (AGTTACATTATTG) and a stretch composed of an AT-rich sequence (ATATAGAAAATTAAAA) and a G-box (CACGTG). Quantitative S1 assay of the mRNA amounts from the TH254/GUS and TH274/GUS chimeric genes in stably transformed and cell cycle-synchronized tobacco cell lines showed that the promoters of both genes contained at least one cis/-acting element responsible for S phase-specific expression. Histochemical analysis of transgenic tobacco plants carrying the chimeric genes showed that the promoters of the two H2A genes were active in developing seedlings and flower organs but were regulated in a different manner.     

The Flavonoid Pathway Regulates the Petal Colors of Cotton Flower

Although biochemists and geneticists have studied the cotton flower for more than one century, little is known about the molecular mechanisms underlying the dramatic color change that occurs during its short developmental life following blooming. Through the analysis of world cotton germplasms, we found that all of the flowers underwent color changes post-anthesis, but there is a diverse array of petal colors among cotton species, with cream, yellow and red colors dominating the color scheme. Genetic and biochemical analyses indicated that both the original cream and red colors and the color changes post-anthesis were related to flavonoid content. The anthocyanin content and the expression of biosynthesis genes were both increased from blooming to one day post-anthesis (DPA) when the flower was withering and undergoing abscission. Our results indicated that the color changes and flavonoid biosynthesis of cotton flowers were precisely controlled and genetically regulated. In addition, flavonol synthase (FLS) genes involved in flavonol biosynthesis showed specific expression at 11 am when the flowers were fully opened. The anthocyanidin reductase (ANR) genes, which are responsible for proanthocyanidins biosynthesis, showed the highest expression at 6 pm on 0 DPA, when the flowers were withered. Light showed primary, moderate and little effects on flavonol, anthocyanin and proanthocyanidin biosynthesis, respectively. Flavonol biosynthesis was in response to light exposure, while anthocyanin biosynthesis was involved in flower color changes. Further expression analysis of flavonoid genes in flowers of wild type and a flavanone 3-hydroxylase (F3H) silenced line showed that the development of cotton flower color was controlled by a complex interaction between genes and light. These results present novel information regarding flavonoids metabolism and flower development.