Loverine P. Taylor

Purification, Cloning, and Heterologous Expression of a Catalytically Efficient Flavonol 3-O-Galactosyltransferase Expressed in the Male Gametophyte of Petunia hybrida

Flavonols are plant-specific molecules that are required for pollen germination in maize and petunia. They existin planta as both the aglycone and glycosyl conjugates. We identified a flavonol 3-O-galactosyltransferase (F3GalTase) that is expressed exclusively in the male gametophyte and controls the formation of a pollen-specific class of glycosylated flavonols. Thus an essential step to understanding flavonol-induced germination is the characterization of F3GalTase. Amino acid sequences of three peptide fragments of F3GalTase purified from petunia pollen were used to isolate a full-length cDNA clone. RNA gel blot analysis and enzyme assays confirmed that F3GalTase expression is restricted to pollen. Heterologous expression of the F3GalTase cDNA inEscherichia coli yielded active recombinant enzyme (rF3GalTase) which had the identical substrate specificity as the native enzyme. Unlike the relatively nonspecific substrate usage of flavonoid glycosyltransferases from sporophytic tissues, F3GalTase uses only UDP-galactose and flavonols to catalyze the formation of flavonol 3-O-galactosides. Kinetic analysis showed that thek cat/K m values of rF3GalTase, using kaempferol and quercetin as substrates, approaches that of a catalytically perfect enzyme. rF3GalTase catalyzes the reverse reaction, generation of flavonols from UDP and flavonol 3-O-galactosides, almost as efficiently as the forward reaction. The biochemical characteristics of F3GalTase are discussed in the context of a role in flavonol-induced pollen germination.

Pollination- or Wound-Induced Kaempferol Accumulation in Petunia Stigmas Enhances Seed Production.

Flavonols are essential for pollen germination and tube growth in petunia and can be supplied by either the pollen or stigma at pollination. HPLC analysis and a sensitive bioassay demonstrated that both pollination and wounding induce flavonol accumulation, especially kaempferol, in the outer cell layers and exudate of the stigma. Pollination and wounding induced nearly identical flavonol kinetics and patterns of accumulation in the same target tissue, suggesting that they share elements of a common signal transduction pathway. The wound response was systemic, because kaempferol accumulated in the stigma when distal tissues, such as the corolla, stamens, or sepals, were wounded. We have exploited the germination requirement for flavonols and the high level of kaempferol that accumulates after wounding to enhance plant fecundity. Seed set was significantly increased by mechanically wounding the corolla and stamens prior to the application of pollen to the stigma. A reproductive role for a plant secondary metabolite and the specific function of stigmatic kaempferol are discussed from an evolutionary perspective.

Chalcone Synthase and Flavonol Accumulation in Stigmas and Anthers of Petunia hybrida

Flavonol aglycones are required for pollen germination in petunia (Petunia hybrida L.). Mutant plants lacking chalcone synthase (CHS), which catalyzes the first committed step in flavonoid synthesis, do not accumulate flavonols and are self-sterile. The mutant pollen can be induced to germinate by supplementing it with kaempferol, a flavonol aglycone, either at the time of pollination or by addition to an in vitro germination system. Biochemical complementation occurs naturally when the mutant, flavonol-deficient pollen is crossed to wild-type, flavonoid-producing stigmas. We found that successful pollination depends on stigma maturity, indicating that flavonol aglycone accumulation may be developmentally regulated. Quantitative immunoblotting, in vitro and in vivo pollen germination, and high-performance liquid chromatographic analyses of stigma and anther extracts were used to determine the relationship between CHS levels and flavonol aglycone accumulation in developing petunia flowers. Although substantial levels of CHS were measured, we detected no flavonol aglycones in wild-type stigma or anther extracts. Instead, the occurrence of a conjugated form (flavonol glycoside) suggests that a mechanism may operate to convert glycosides to the active aglycone form.

Flavonol 3-O-Glycosyltransferases Associated with Petunia Pollen Produce Gametophyte-Specific Flavonol Diglycosides

Wild-type petunia pollen accumulates high levels of flavonol 3-O-glycosides. Pollen from conditionally male-fertile petunia has no flavonols and is unable to germinate. Pollen function is restored both in vivo and in vitro by providing flavonol aglycones, but not flavonol glycosides, to the pollen. In the present study, incubation of an in vitro suspension of conditionally male-fertile pollen with kaempferol or quercetin resulted in the accumulation of kaempferol and quercetin 3-O-glycosides in the pollen. We identified two glycosyltransferase activities associated with the intact pollen grain that catalyze the formation of a gametophyte-specific class of flavonol glycosides. Feeding studies showed that product formation was highly specific for flavonols with an unsubstituted 3-hydroxyl group and was not dependent on an external source of UDP-hexose. Ultraviolet spectral analysis, fast atom bombardment mass spectrometry, 1H-nuclear magnetic resonance, and 13C-nuclear magnetic resonance identified the products as kaempferol and quercetin 3-O-(2”-O-[beta]-D-glucopyranosyl)-[beta]-D-galactopyranoside, identical with the flavonol 3-O-glycosides present in wild-type pollen. The sugars are linked in a 1->2 configuration that results in a pollen-specific class of compounds. To retain both glycosyltransferase activities in a cell-free extract, it was necessary to add Triton X-100, suggesting that one or both of the proteins may be associated with a pollen membrane. A model for flavonol glycoside biosynthesis and uptake into the pollen is discussed in terms of the germination requirement for flavonols.

The structural requirements of flavonols that induce pollen germination of conditionally male fertile Petunia

Abstract Flavonols are essential for pollen germination and sustained tube growth in Petunia hybrida. An in vitro bioassay, based on biochemical complementation of flavonol-deficient pollen, was used to compare how modifications at different sites on the basic flavonol molecule affect the efficiency of pollen germination. This structural-activity analysis using methylated or glycosylated derivatives showed that only flavonols with unsubstituted hydroxyl groups at positions 3 and 7 could induce rapid pollen germination. In addition, the enhanced germination frequency associated with a hydroxyl group at position 5 was abolished by substituting a methyl group. Increased hydroxylation of the flavonol B-ring had an inhibitory effect on germination, but methylation of the same hydroxyl groups promoted germination. Additional hydroxyl groups within the A-ring at carbon 6 had a mixed effect, but a methoxyl group at position 6 enhanced germination in all cases. Substitutions at position 8 were somewhat inhibitory and introduction of an isoprenyl group into ring A was toxic to both mutant and wild type pollen.

Conditional male fertility in maize

This study compares conditional male fertility (CMF) in maize and petunia. CMF is a reversible defect in pollen germination or tube growth; pollen is nonfunctional in self-crosses but fully functional in outcrosses or when supplied with specific flavonol aglycones at pollination. CMF occurs in maize and petunia mutants that lack chalcone synthase (CHS) activity and therefore do not synthesize flavonols. In maize CMF seedlings and developing male florets, CHS transcripts accumulate to high levels, yet western blot analysis using an anti-CHS antiserum does not detect any CHS protein. This is in contrast to CMF petunia, where no CHS RNA is detected (Vogt et al. 1994). While CMF petunia pollen requires flavonols to germinate, CMF maize pollen germinates and grows both in vivo and in vitro without the addition of flavonols. However, pollen tubes abort after 12 h of growth which explains the lack of seed set in self crosses (Mo et al. 1992). Pollen tubes of CMF maize have an unusual morphology in vivo, with heavy callose deposits throughout the tube and tips that burst within the silk. Normal tube morphology and seed set are restored by adding flavonols to the silks at pollination. As previously shown with petunia, fecundity (seed set) may be enhanced in maize by adding quercetin and kaempferol at pollination.

Synthesis of flavonol derivatives as probes of biological processes

Abstract Two synthetic derivatives of the flavonol kaempferol were prepared in order to probe the biological mechanism of action of this natural product. Efficient synthetic routes to both a benzophenone-containing derivative and an amine-containing derivative are reported herein.

Uptake and metabolism of flavonols during in-vitro germination of Petunia hybrida (L.) pollen

Abstract. Flavonol-deficient petunia pollen [conditionally male fertile (CMF) pollen] is unable to germinate but application of nanomolar concentrations of flavonol aglycones completely restores function (Mo et al. 1992). In this study a chemically synthesized radioactive flavonol, [4′-O-14C]kaempferide, was used as a model compound to study the metabolism of flavonols during the first few hours of pollen germination. [4′-O-14C] Kaempferide was as efficient at inducing CMF pollen germination as kaempferol and quercetin, the aglycone form of the endogenous flavonols in petunia pollen. Analysis by high-performance liquid chromatography (HPLC) of extracts from both in-vitro-germinated pollen and the germination medium showed that more than 95% of the applied radioactivity was recovered as three kaempferide 3-O-glycosides and unmetabolized kaempferide; no flavonol catabolites were detected. Only HPLC fractions that contained the aglycone, or produced it upon acid hydrolysis, could induce CMF pollen germination in vitro. Structurally diverse flavonols could be classified according to how efficiently the aglycone was internalized and glycosylated during pollen germination. The ability of an individual flavonol to restore germination correlated with the total uptake of flavonols but not with the amount of glycoside formed in the pollen. Thus this study reinforces the conclusion that flavonol aglycones are the active compound for inducing pollen germination.

The Role of Glycosylation in Flavonol-Induced Pollen Germination

Flavonols are small (C15) plant-specific molecules that are required for petunia and maize pollen to germinate. They exist in two chemical forms: the aglycone or glycosyl conjugates. Flavonol-deficient pollen is biochemically complemented by flavonol aglycones but not by the glycosylated forms that accumulate in wild type (WT) pollen. Coincident with the biochemical induction of germination, the added flavonol aglycone is rapidly converted to a galactoside and then to a glucosyl galactoside (diglycoside) that is identical to the compound present in WT pollen. A flavonol 3-O-galactosyltransferase (F3GalTase) activity has been identified that controls the formation of glycosylated flavonols in pollen. Importantly, this enzyme also catalyzes the reverse reaction, i.e. the production of the flavonol aglycone from the galactoside and UDP (Fig. 1). F3GalTase/RevGalTase therefore has the potential to control the level of the bioactive flavonol species and as a result, pollen germination.

Conservation in divergent solanaceous species of the unique gene structure and enzyme activity of a gametophytically-expressed flavonol 3-O-galactosyltransferase.

Flavonol 3-O-galactosyltransferase (F3GalTase) is a pollen-specific enzyme which glycosylates the flavonols required for germination in petunia. The highly restricted tissue-specific expression and substrate usage make F3GalTase unique among all other flavonoid glycosyltransferases (GTs) described to date, including the well characterized Bronze1 (Bz1) gene of maize. RFLP mapping, DNA gel blot, and sequence analyses showed that the single copy F3galtase gene has a different genomic organization than Bz1. Within the promoter of F3galtase are potential regulatory motifs which may confer pollen-specific gene expression and activation by Myb and bHLH transcription factors. However, we provide evidence that F3galtase is not regulated by An4, which encodes a Myb factor known to regulate anthocyanin accumulation in petunia anthers. An unexpected feature of the F3galtase promoter was the presence of large blocks of chloroplast and mitochondrial DNA. Gel blot analyses of genomic DNA from the progenitors of Petunia hybrida, as well as from Nicotiana tabacum, indicated that migration of organellar DNA into the F3galtase gene was an ancient event that occurred prior to speciation of the Solanaceae. Together with enzyme assays and HPLC analyses of pollen extracts from tobacco, tomato, and potato, these results confirmed that the unique F3galtase gene structure, enzyme activity, and pollen-specific flavonol glucosylgalactosides are conserved throughout the Solanaceae.