Three 2-oxoglutarate-dependent dioxygenase activities of Equisetum arvense L. forming flavone and flavonol from (2S)-naringenin

Equisetum arvense L. (Equisetaceae-horsetail) accumulates various flavones and flavonols in infertile shoot. Enzyme assays conducted with crude extracts of the green tissue revealed chalcone synthase activity and also three further activities assigned to flavonoid biosynthesis and identified as flavone synthase I, flavanone 3β-hydroxylase and flavonol synthase. The latter three activities were characterized as soluble, 2-oxoglutarate-dependent dioxygenases by their typical cofactor requirements and peculiar inhibition. Notably, this is the first report of flavone synthase I which had been considered to be restricted solely to species of the Apiaceae from a distant plant taxon.     

Novel role for pectin methylesterase in Arabidopsis: A new function showing ribosome-inactivating protein (RIP) activity

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.     

7-Rhamnosylated Flavonols Modulate Homeostasis of the Plant Hormone Auxin and Affect Plant Development

Flavonols are a group of secondary metabolites that affect diverse cellular processes. They are considered putative negative regulators of the transport of the phytohormone auxin, by which they influence auxin distribution and concomitantly take part in the control of plant organ development. Flavonols are accumulating in a large number of glycosidic forms. Whether these have distinct functions and diverse cellular targets is not well understood. The rol1-2 mutant of Arabidopsis thaliana is characterized by a modified flavonol glycosylation profile that is inducing changes in auxin transport and growth defects in shoot tissues. To determine whether specific flavonol glycosides are responsible for these phenotypes, a suppressor screen was performed on the rol1-2 mutant, resulting in the identification of an allelic series of UGT89C1, a gene encoding a flavonol 7-O-rhamnosyltransferase. A detailed analysis revealed that interfering with flavonol rhamnosylation increases the concentration of auxin precursors and auxin metabolites, whereas auxin transport is not affected. This finding provides an additional level of complexity to the possible ways by which flavonols influence auxin distribution and suggests that flavonol glycosides play an important role in regulating plant development.     

Flavonols are not essential for fertilization in Arabidopsis thaliana

Flavonols are plant metabolites suggested to serve a vital role in fertilization of higher plants. Petunia and maize plants mutated in their flavonol biosynthesis are not able to set seed after self-pollination. We have investigated the role of these compounds in Arabidopsis thaliana. Like in all other plant species, high levels of flavonols could be detected in pollen of wild-type A. thaliana. No flavonols were detected in reproductive organs of the A. thaliana tt4 mutant in which the chs gene is mutated. Surprisingly, this mutant did set seed after self-fertilization and no pollen tube growth aberrations were observed in vivo. The role of flavonols during fertilization of Arabidopsis is discussed.Abbreviations CHS chalcone synthase - TLC thin-layer chromatography     

Arabidopsis thaliana expresses a second functional flavonol synthase

Arabidopsis thaliana L. produces flavonoid pigments, i.e. flavonols, anthocyanidins and proanthocyanidins, from dihydroflavonol substrates. A small family of putative flavonol synthase (FLS) genes had been recognized in Arabidopsis, and functional activity was attributed only to FLS1. Nevertheless, other FLS activities must be present, because A. thalianafls1 mutants still accumulate significant amounts of flavonols. The recombinant FLSs and leucoanthocyanidin dioxygenase (LDOX) proteins were therefore examined for their enzyme activities, which led to the identification of FLS3 as a second active FLS. This enzyme is therefore likely responsible for the formation of flavonols in the ldox/fls1-2 double mutant. These double mutant and biochemical data demonstrate for the first time that LDOX is capable of catalyzing the in planta formation of flavonols.     

Biochemical Evaluation of the Decarboxylation and Decarboxylation-Deamination Activities of Plant Aromatic Amino Acid Decarboxylases

Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site residues.     

Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera)

We identified two glycosyltransferases that contribute to the structural diversification of flavonol glycosides in grapevine (Vitis vinifera): glycosyltransferase 5 (Vv GT5) and Vv GT6. Biochemical analyses showed that Vv GT5 is a UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (GAT), and Vv GT6 is a bifunctional UDP-glucose/UDP-galactose:flavonol-3-O-glucosyltransferase/galactosyltransferase. The Vv GT5 and Vv GT6 genes have very high sequence similarity (91%) and are located in tandem on chromosome 11, suggesting that one of these genes arose from the other by gene duplication. Both of these enzymes were expressed in accordance with flavonol synthase gene expression and flavonoid distribution patterns in this plant, corroborating their significance in flavonol glycoside biosynthesis. The determinant of the specificity of Vv GT5 for UDP-glucuronic acid was found to be Arg-140, which corresponded to none of the determinants previously identified for other plant GATs in primary structures, providing another example of convergent evolution of plant GAT. We also analyzed the determinants of the sugar donor specificity of Vv GT6. Gln-373 and Pro-19 were found to play important roles in the bifunctional specificity of the enzyme. The results presented here suggest that the sugar donor specificities of these Vv GTs could be determined by a limited number of amino acid substitutions in the primary structures of protein duplicates, illustrating the plasticity of plant glycosyltransferases in acquiring new sugar donor specificities.     

Biochemical and genetic characterization of Arabidopsis flavanone 3beta-hydroxylase

Flavanone 3beta-hydroxylase (F3H; EC 1.14.11.9) is a 2-oxoglutarate dependent dioxygenase that catalyzes the synthesis of dihydrokaempferol, the common precursor for three major classes of 3-hydroxy flavonoids, the flavonols, anthocyanins, and proanthocyanidins. This enzyme also competes for flux into the 3-deoxy flavonoid branch pathway in some species. F3H genes are increasingly being used, often together with genes encoding other enzymes, to engineer flavonoid synthesis in microbes and plants. Although putative F3H genes have been cloned in a large number of plant species, only a handful have been functionally characterized. Here we describe the biochemical properties of the Arabidopsis thaliana F3H (AtF3H) enzyme and confirm the activities of gene products from four other plant species previously identified as having high homology to F3H. We have also investigated the surprising "leaky" phenotype of AtF3H mutant alleles, uncovering evidence that two related flavonoid enzymes, flavonol synthase (EC 1.14.11.23) and anthocyanidin synthase (EC 1.14.11.19), can partially compensate for F3H in vivo. These experiments further indicate that the absence of F3H in these lines enables the synthesis of uncommon 3-deoxy flavonoids in the Arabidopsis seed coat.     

Hrp?mutant bacteria as biocontrol agents: Toward a sustainable approach in the fight against plant pathogenic bacteria

Sustainable agriculture necessitates development of environmentally safe methods to protect plants against pathogens. Among these methods, application of biocontrol agents has been efficiently used to minimize disease development. Here we review current understanding of mechanisms involved in biocontrol of the main Gram-phytopathogenic bacteria-induced diseases by plant inoculation with strains mutated in hrp (hypersensitive response and pathogenicity) genes. These mutants are able to penetrate plant tissues and to stimulate basal resistance of plants. Novel protection mechanisms involving the phytohormone abscisic acid appear to play key roles in the biocontrol of wilt disease induced by Ralstonia solanacearum in Arabidopsis thaliana. Fully understanding these mechanisms and extending the studies to other pathosystems are still required to evaluate their importance in disease protection.     

Flavonoid genes of pear (<Emphasis Type="Italic">Pyrus communis</Emphasis>)

Pear (Pyrus sp.) is a major fruit crop of temperate regions with increasing extent of cultivation. Pear flavonoids contribute to its fruit color, pathogen defense, and are health beneficial ingredients of the fruits. Comparative Southern analyses with apple (Malus x domestica) cDNAs showed comparable genomic organization of flavonoid genes of both related genera. A homology-based cloning approach was used to obtain the cDNAs of most enzymes of the main flavonoid pathway of Pyrus: phenylalanine ammonia lyase, chalcone synthase, chalcone isomerase, flavanone 3β-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, leucoanthocyanidin reductase 1 and 2, anthocyanidin synthase, anthocyanidin reductase, and UDP-glucose : flavonoid 7-O-glucosyltransferase. The substrate specificities of the recombinant enzymes expressed in yeast were determined for physiological and non-physiological substrates and found to be in general agreement with the characteristic pear flavonoid metabolite pattern of mainly B-ring dihydroxylated anthocyanins, flavonols, catechins, and flavanones. Furthermore, significant differences in substrate specificities and gene copy numbers in comparison to Malus were identified. Cloning of the cDNAs and studying the enzymes of the Pyrus flavonoid pathway is an essential task toward a comprehensive knowledge of Pyrus polyphenol metabolism. It also elucidates evolutionary patterns of flavonoid/polyphenol pathways in the Rosaceae, which allocate several important crop plants.     

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.     

Cloning,characterization and functional analysis of a flavonol synthase from <Emphasis Type="Italic">Vaccinium corymbosum</Emphasis>

Key message

VcFLS from Vaccinium corymbosum promoted myricetin biosynthesis in Arabidopsis thaliana and VcFLS expression was induced by salicylic acid.

Abstract

Flavonoids are polyphenols with important functions in pigmentation, UV filtration, and symbiotic nitrogen fixation. Flavonols are a class of flavonoids that are produced by the desaturation of dihydroflavanols in a reaction that is catalyzed by flavonol synthase (FLS). In the study reported here, we cloned the full-length cDNA of FLS (designated as VcFLS) from Vaccinium corymbosum (blueberry) using rapid amplification of cDNA ends (RACE). The cDNA contained a 1005-bp open reading frame that encoded a 334-amino acid protein. Phylogenetic analysis showed that VcFLS was closely related to FaFLS, a flavonol synthase that catalyzed the formation of kaempferol and had little effect on the formation of quercetin. Quantitative RT-PCR analysis demonstrated that VcFLS was expressed in all of the tissues tested, with particularly high expression in the petals and young leaves (both green and red). The flavanols myricetin and quercetin also occurred in all of these tested tissues, with the highest levels detected in mature leaves. The expression of VcFLS was not consistent with the accumulation of quercetin and myricetin in different tissues, nor were the expressions of VcFLS, VcPAL, VcCHS, VcF3H, and VcF3′5′H consistent with the accumulation of the quercetin during fruit development. However, the change in the trend of VcCHS and VcF3H expression was similar with myricetin accumulation during fruit development. Expression profiling analysis revealed that VcFLS expression was induced by salicylic acid, a phytohormone involved in plant defense against pathogens, and was suppressed by gibberellic acid, a phytohormone involved in various aspects of plant development. Heterologous expression of VcFLS in Arabidopsis thaliana increased the content of myricetin, but did not affect quercetin content. Thus, we conclude that VcFLS is a key enzyme in the flavonol biosynthetic pathway and would appear to be involved in the plant defense response.

     

Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin,proanthocyanidin, and flavonol levels during bilberry fruit development

The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented.     

Novel features of radiation-induced bystander signaling in Arabidopsis thaliana demonstrated using root micro-grafting

Radiation-induced bystander effects (RIBE) have been well demonstrated in whole organisms, as well as in single-cell culture models in vitro and multi-cellular tissues models in vitro, however, the underlying mechanisms remain unclear, including the temporal and spatial course of bystander signaling. The RIBE in vivo has been shown to exist in the model plant Arabidopsis thaliana (A. thaliana). Importantly, the unique plant grafting provides a delicate approach for studying the temporal and spatial course of bystander signaling in the context of whole plants. In our previous study, the time course of bystander signaling in plants has been well demonstrated using the root micro-grafting technique. In this study, we further investigated the temporal cooperation pattern of multiple bystander signals, the directionality of bystander signaling, and the effect of bystander tissues on the bystander signaling. The results showed that the bystander response could also be induced efficiently when the asynchronously generated bystander signals reached the bystander tissues in the same period, but not when they entered into the bystander tissues in an inversed sequence. The absence of bystander response in root-inversed grafting indicated that the bystander signaling along roots might be of directionality. The bystander signaling was shown to be independent of the bystander tissues.     

Inhibition of saturated very-long-chain fatty acid biosynthesis by mefluidide and perfluidone,selective inhibitors of 3-ketoacyl-CoA synthases

The trifluoromethanesulphonanilides mefluidide and perfluidone are used in agriculture as plant growth regulators and herbicides. Despite the fact that mefluidide and perfluidone have been investigated experimentally for decades, their mode of action is still unknown. In this study, we used a cascade approach of different methods to clarify the mode of action and target site of mefluidide and perfluidone. Physiological profiling using an array of biotests and metabolic profiling in treated plants of Lemna paucicostata suggested a common mode of action in very-long-chain fatty acid (VLCFA) synthesis similar to the known 3-ketoacyl-CoA synthase (KCS) inhibitor metazachlor. Detailed analysis of fatty acid composition in Lemna plants showed a decrease of saturated VLCFAs after treatment with mefluidide and perfluidone. To study compound effects on enzyme level, recombinant KCSs from Arabidopsis thaliana were expressed in Saccharomyces cerevisiae. Enzyme activities of seven KCS proteins from 17 tested were characterized by their fatty acid substrate and product spectrum. For the KCS CER6, the VLCFA product spectrum in vivo, which consists of tetracosanoic acid, hexacosanoic acid and octacosanoic acid, is reported here for the first time. Similar to metazachlor, mefluidide and perfluidone were able to inhibit KCS1, CER6 and CER60 enzyme activities in vivo. FAE1 and KCS2 were inhibited by mefluidide only slightly, whereas metazachlor and perfluidone were strong inhibitors of these enzymes with IC₅₀ values in μM range. This suggests that KCS enzymes in VLCFA synthesis are the primary herbicide target of mefluidide and perfluidone.