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

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. Received: 4 November 1996/Accepted: 4 December 1996     

Five Nodulation Mutants of White Sweetclover (Melilotus alba Desr.) Exhibit Distinct Phenotypes Blocked at Root Hair Curling,Infection Thread Development,and Nodule Organogenesis

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.     

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.     

Flavonols stimulate development, germination, and tube growth of tobacco pollen

The effect of anther-derived substances on pollen function was studied using pollen produced by in vitro culture of immature pollen of tobacco (Nicotiana tabacum L.) and petunia (Petunia hybrida). Addition of conditioned medium consisting of diffusates from in situ matured pollen strongly increased pollen germination frequency and pollen tube growth, as well as seed set after in situ pollination. Thin-layer chromatography and depletion of phenolic substances by Dowex treatment indicated that flavonols are present in the diffusate and may be the active compounds. When added to the germination medium, flavonols (quercetin, kaempferol, myricetin) but not other flavonoids strongly promoted pollen germination frequency and pollen tube growth in vitro. The best results were obtained at very low concentrations of the flavonols (0.15-1.5 μm), indicating a signaling function. The same compounds were also effective when added during pollen development in vitro.     

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.     

Regulation of gene expression involved in flavonol and anthocyanin biosynthesis during petal development in lisianthus (Eustoma grandiflorum)

To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full-length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3'5'H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.     

Cell surface and Golgi pools of beta-1,4-galactosyltransferase are differentially regulated during embryonal carcinoma cell differentiation.

beta-1,4-Galactosyltransferase (GalTase) has two functionally distinct subcellular distributions. In the Golgi apparatus, GalTase participates in the glycosylation of secretory and membrane-bound glycoproteins, whereas on the cell surface it mediates specific aspects of intercellular adhesion. For this study, a murine GalTase clone was obtained by screening a lambda gt10 cDNA library made from F9 embryonal carcinoma cells with a heterologous bovine GalTase cDNA probe. The murine GalTase cDNA probe was used in conjunction with assays of GalTase activity to investigate the expression and distribution of GalTase during differentiation of F9 stem cells into secretory endodermal epithelium. During the initial phase of F9 cell differentiation, GalTase mRNA levels remained relatively constant; however, as differentiation progressed, as assayed by expression of the differentiation-specific marker laminin B1, GalTase mRNA levels and enzyme activity rose dramatically. Furthermore, subcellular fractionation of these cells showed that the increased GalTase levels were specifically associated with the Golgi apparatus, whereas GalTase specific activity on the plasma membrane remained constant. These results show that levels of cell surface and Golgi GalTase change relative to one another during F9 cell differentiation and suggest that these functionally distinct pools of GalTase are independently and differentially regulated.     

Flavonols and fertilization in Petunia hybrida: localization and mode of action during pollen tube growth

Flavonols form an important class of flavonoids which serve an essential function during plant reproduction. Flavonoid biosynthesis is initiated by the enzyme chalcone synthase (CHS). A high abundance of flavonols and chs mRNA was demonstrated in male and female reproductive organs of Petunia hybrida. Detailed analyses revealed precise spatial and temporal regulation of the chs promoter and flavonol synthesis in the stigma, style and ovules. Transgenic plants were generated with a complete block of flavonol biosynthesis as the result of anti-sense inhibition of chs gene activity. The absence of flavonols by this dominant mutation rendered these plants self-sterile. Pollination experiments with wild-type and mutant plants revealed that the production of flavonols in either the anthers or the pistils was required for pollen tube growth and seed set. Mutant pollen without flavonols in their exine germinated normally. However, after a short period of in vitro pollen tube growth the tips of these tubes disrupted and the protoplasm was disloaded leading to the death of the pollen grain. Addition of flavonol aglycones but not other flavonoids complemented this phenotype. Confocal laser scanning microscopy revealed the localization of high levels of flavonols throughout the wild-type pollen tube. These compounds were not detected in the exine or cell wall of growing tubes. In addition, it was observed that the flavone apigenin could completely inhibit pollen tube growth. Taken together, it is shown that flavonols play an important role in the growth of the pollen tube and their mode of action is discussed.     

Novel flavonol 2-oxoglutarate dependent dioxygenase: affinity purification, characterization, and kinetic properties

A 2-oxoglutarate-dependent dioxygenase [EC 1.14.11-] that catalyzes the 6-hydroxylation of partially methylated flavonols has been purified to near homogeneity from Chrysosplenium americanum. Enzyme purification was achieved by fast protein liquid chromatography on Superose 12 and Mono Q columns as well as by affinity chromatography on 2-oxoglutarate-Sepharose and immunoaffinity columns. The specific activity of the 6-hydroxylase eluted from Mono Q (97.1 pkat/mg) was enriched 538-fold, with a 0.63% recovery. Both affinity chromatography steps resulted in the elimination of most contaminating proteins, but not without loss of enzyme activity and stability. The molecular mass of both the native and denatured enzyme was found to be 42 and 45 kDa, respectively, suggesting a monomeric protein. The enzyme exhibits strict specificity for position 6 of partially methylated flavonols possessing a 7-methoxyl group, indicating its involvement in the biosynthesis of polymethylated flavonols in this plant. The cofactor dependence of the enzyme is similar to that of other plant dioxygenases, particularly its dependence on ferrous ions for catalytic activity and reactivation. Internal amino acid sequence information indicated its relatedness to other plant flavonoid dioxygenases. The results of substrate interaction kinetics and product inhibition studies suggest an ordered, sequential reaction mechanism (TerTer), where 2-oxoglutarate is the first substrate to bind, followed by O2 and the flavonol substrate. Product release occurs in the reverse order where the hydroxylated flavonol is the first to be released, followed by CO2 and succinate. To our knowledge, this is the first reported 2-oxoglutarate-dependent dioxygenase that catalyzes the aromatic hydroxylation of a flavonoid compound.     

Flavonols accumulate asymmetrically and affect auxin transport in Arabidopsis

Flavonoids represent a class of secondary metabolites with diverse functions in plants including ultraviolet protection, pathogen defense, and interspecies communication. They are also known as modulators of signaling processes in plant and animal systems and therefore are considered to have beneficial effects as nutraceuticals. The rol1-2 (for repressor of lrx1) mutation of Arabidopsis (Arabidopsis thaliana) induces aberrant accumulation of flavonols and a cell-growth phenotype in the shoot. The hyponastic cotyledons, aberrant shape of pavement cells, and deformed trichomes in rol1-2 mutants are suppressed by blocking flavonoid biosynthesis, suggesting that the altered flavonol accumulation in these plants induces the shoot phenotype. Indeed, the identification of several transparent testa, myb, and fls1 (for flavonol synthase1) alleles in a rol1-2 suppressor screen provides genetic evidence that flavonols interfere with shoot development in rol1-2 seedlings. The increased accumulation of auxin in rol1-2 seedlings appears to be caused by a flavonol-induced modification of auxin transport. Quantification of auxin export from mesophyll protoplasts revealed that naphthalene-1-acetic acid but not indole-3-acetic acid transport is affected by the rol1-2 mutation. Inhibition of flavonol biosynthesis in rol1-2 fls1-3 restores naphthalene-1-acetic acid transport to wild-type levels, indicating a very specific mode of action of flavonols on the auxin transport machinery.     

Pollen tubes of flavonol-deficient Petunia show striking alterations in wall structure leading to tube disruption

Despite the vital role that flavonols play in fertilization and pollen tube growth of a number of species such as petunia and maize, their function is still unclear. Pollen tubes of the flavonol-deficient transformant T17.02 of Petunia hybrida L. are able to germinate and start growing in vitro, but eventually disrupt at the tip approximately 2 h after germination. In order to establish the possible role of flavonols in this process, wild-type and flavonol-deficient pollen tubes were subjected to cytological and ultrastructural analyses and screened for differences. The results showed that before disruption of the flavonol-deficient pollen tubes, the structure of the primary wall at the tip dramatically changed from layered to granular. Secretory vesicles at the tip still fused with the wall but lost their capacity to melt into the wall and to form layers. Instead they remained as dark, electron-dense granular structures surrounded by an electron-translucent matrix. Apparently the matrix is not able to sustain the wall's coherence and as a consequence the tube disrupts. No other remarkable cytological or ultrastructural differences between the transformant and the wild-type pollen tubes could be found before tip disruption. Even a morphometric analysis of abundance and distribution of endoplasmic reticulum, dictyosomes and mitochondria did not reveal any significant difference. However, for the first time, obvious morphological differences were observed in the wall of the flavonol-deficient pollen tubes. We conclude that flavonols act on precursors of the pollen tube wall of petunia and interfere with a cross-linking system in the wall, possibly via extensins. Received: 23 February 1998 / Accepted: 13 August 1998     

Über Vorkommen und Lokalisation eines flavonolumwandelnden Enzyms in Pflanzen

Summary The occurrence and distribution of an enzyme converting flavonols to 2,3-dihydroxy flavanones has been measured in various plants and found to occur in all plants tested.In garbanzo bean, Cicer arietinum L., the enzyme is found mainly in roots, hypocotyls, epicotyls and cytoledons while the other organs, rich in flavonols, possess much lower levels of the enzyme. In garbanzo seedlings the enzyme is formed between the second and sixth day after germination and appears parallel to the phenylalanine ammonia-lyase. The data indicate that the enzymes for both flavonol biosynthesis and turnover are formed simultaneously. These results further support earlier observations that regulation of flavonoid formation and regulation of turnover seem to be dependent on each other.The activity of the flavonol-converting enzyme does not increase in Cicer arietinum plants transferred to darkness though under such conditions flavonol turnover is accelerated.In Pisum sativum, Glycine max and Sinapis alba the flavonol converting enzyme is more evenly distributed over all organs, so that a correlation between flavonol content and enzyme is less obvious.The data are discussed with respect to intracellular regulation of flavonol turnover.