Modification of flavonoid biosynthesis in crop plants

Flavonoids comprise the most common group of polyphenolic plant secondary metabolites. In plants, flavonoids play an important role in biological processes. Beside their function as pigments in flowers and fruits, to attract pollinators and seed dispersers, flavonoids are involved in UV-scavenging, fertility and disease resistance. Since they are present in a wide range of fruits and vegetables, flavonoids form an integral part of the human diet. Currently there is broad interest in the effects of dietary polyphenols on human health. In addition to the potent antioxidant activity of many of these compounds in vitro, an inverse correlation between the intake of certain polyphenols and the risk of cardiovascular disease, cancer and other age related diseases has been observed in epidemiological studies. The potential nutritional effects of these molecules make them an attractive target for genetic engineering strategies aimed at producing plants with increased nutritional value. This review describes the current knowledge of the molecular regulation of the flavonoid pathway and the state of the art with respect to metabolic engineering of this pathway in crop plants.     

Flavonoids act as negative regulators of auxin transport in vivo in arabidopsis

Polar transport of the plant hormone auxin controls many aspects of plant growth and development. A number of synthetic compounds have been shown to block the process of auxin transport by inhibition of the auxin efflux carrier complex. These synthetic auxin transport inhibitors may act by mimicking endogenous molecules. Flavonoids, a class of secondary plant metabolic compounds, have been suggested to be auxin transport inhibitors based on their in vitro activity. The hypothesis that flavonoids regulate auxin transport in vivo was tested in Arabidopsis by comparing wild-type (WT) and transparent testa (tt4) plants with a mutation in the gene encoding the first enzyme in flavonoid biosynthesis, chalcone synthase. In a comparison between tt4 and WT plants, phenotypic differences were observed, including three times as many secondary inflorescence stems, reduced plant height, decreased stem diameter, and increased secondary root development. Growth of WT Arabidopsis plants on naringenin, a biosynthetic precursor to those flavonoids with auxin transport inhibitor activity in vitro, leads to a reduction in root growth and gravitropism, similar to the effects of synthetic auxin transport inhibitors. Analyses of auxin transport in the inflorescence and hypocotyl of independent tt4 alleles indicate that auxin transport is elevated in plants with a tt4 mutation. In hypocotyls of tt4, this elevated transport is reversed when flavonoids are synthesized by growth of plants on the flavonoid precursor, naringenin. These results are consistent with a role for flavonoids as endogenous regulators of auxin transport.     

Flavonoids as developmental regulators

Flavonoids, usually regarded as dispensable phytochemicals derived from plant secondary metabolism, play important roles in the biology of plants by affecting several developmental processes. Bioactive flavonoids also signal to microbes, serve as allelochemicals and are important nutraceuticals in the animal diet. Despite the significant progress made in identifying flavonoid pathway genes and regulators, little is currently known about the protein targets of flavonoids in plant or animal cells. Recently, there have been advances in our understanding of the roles that flavonoids play in developmental processes of plants. The multiple cellular roles of flavonoids can reflect their chemical diversity, or might suggest the existence of cellular targets shared between many of these seemingly disparate processes.     

Comprehensive review of antimicrobial activities of plant flavonoids

Flavonoids are one of the largest classes of small molecular secondary metabolites produced in different parts of the plant. They display a wide range of pharmacological and beneficial health effects for humans, which include, among others, antioxidative activity, free radical scavenging capacity, coronary heart disease prevention and antiatherosclerotic, hepatoprotective, anti-inflammatory, and anticancer activities. Hence, flavonoids are gaining high attention from the pharmaceutical and healthcare industries. Notably, plants synthesize flavonoids in response to microbial infection, and these compounds have been found to be a potent antimicrobial agent against a wide range of pathogenic microorganisms in vitro. Antimicrobial action of flavonoids results from their various biological activities, which may not seem very specific at first. There are, however, promising antibacterial flavonoids that are able not only to selectively target bacterial cells, but also to inhibit virulence factors, as well as other forms of microbial threats, e.g. biofilm formation. Moreover, some plant flavonoids manifest ability to reverse the antibiotic resistance and enhance action of the current antibiotic drugs. Hence, the development and application of flavonoid-based drugs could be a promising approach for antibiotic-resistant infections. This review aims to improve our understanding of the biological and molecular roles of plant flavonoids, focusing mostly on their antimicrobial activities.

     

Plant flavonoids in cancer chemoprevention: role in genome stability

Carcinogenesis is a multistage process that involves a series of events comprising of genetic and epigenetic changes leading to the initiation, promotion and progression of cancer. Chemoprevention is referred to as the use of nontoxic natural compounds, synthetic chemicals or their combinations to intervene in multistage carcinogenesis. Chemoprevention through diet modification, i.e., increased consumption of plant-based food, has emerged as a most promising and potentially cost-effective approach to reducing the risk of cancer. Flavonoids are naturally occurring polyphenols that are ubiquitous in plant-based food such as fruits, vegetables and teas as well as in most medicinal plants. Over 10,000 flavonoids have been characterized over the last few decades. Flavonoids comprise of several subclasses including flavonols, flavan-3-ols, anthocyanins, flavanones, flavones, isoflavones and proanthocyanidins. This review describes the most efficacious plant flavonoids, including luteolin, epigallocatechin gallate, quercetin, apigenin and chrysin; their hormetic effects; and the molecular basis of how these flavonoids contribute to the chemoprevention with a focus on protection against DNA damage caused by various carcinogenic factors. The present knowledge on the role of flavonoids in chemoprevention can be used in developing effective dietary strategies and natural health products targeted for cancer chemoprevention.     

Nuclear localization of flavonoid enzymes in Arabidopsis

Flavonoids represent one of the oldest, largest, and most diverse families of plant secondary metabolites. These compounds serve a wide range of functions in plants, from pigmentation and UV protection to the regulation of hormone transport. Flavonoids also have interesting pharmacological activities in animals that are increasingly being characterized in terms of effects on specific proteins or other macromolecules. Although flavonoids are found in many different locations both inside and outside the cell, biosynthesis has long been believed to take place exclusively in the cytoplasm. Recent reports from a number of different plant species have documented the presence of flavonoids in nuclei, raising the possibility of novel mechanisms of action for these compounds. Here we present evidence that not only flavonoids, but also at least two of the biosynthetic enzymes, are located in the nucleus in several cell types in Arabidopsis. This is the first indication that differential targeting of the biosynthetic machinery may be used to regulate the deposition of plant secondary products at diverse sites of action within the cell.     

Flavonoids and auxin transport: modulators or regulators?

Flavonoids are polyphenolic compounds found in all vascular and non-vascular plants. Although nonessential for plant growth and development, flavonoids have species-specific roles in nodulation, fertility, defense and UV protection. Flavonoids have been shown to modulate transport of the phytohormone auxin in addition to auxin-dependent tropic responses. However, flavonoids are not essential regulators of these processes because transport and tropic responses occur in their absence. Flavonoids modulate the activity of auxin-transporting P-glycoproteins and seem to modulate the activity of regulatory proteins such as phosphatases and kinases. Phylogenetic analysis suggests that auxin transport mechanisms evolved in the presence of flavonoid compounds produced for the scavenging of reactive oxygen species and defense from herbivores and pathogens.     

Regulation, evolution, and functionality of flavonoids in cereal crops

Flavonoids are plant secondary metabolites that contribute to the adaptation of plants to environmental stresses, including resistance to abiotic and biotic stress. Flavonoids are also beneficial for human health and depress the progression of some chronic diseases. The biosynthesis of flavonoids, which belong to a large family of phenolic compounds, is a complex metabolic process with many pathways that produce different metabolites, controlled by key enzymes. There is limited knowledge about the composition, biosynthesis and regulation of flavonoids in cereals. Improved understanding of the accumulation of flavonoids in cereal grains would help to improve human nutrition through these staple foods. The biosynthesis of flavonoids, scope for altering the flavonoid composition in cereal crops and benefits for human nutrition are reviewed here.     

Metabolic engineering of flavonoids in tomato (Solanum lycopersicum): the potential for metabolomics

Flavonoids comprise a large and diverse group of polyphenolic plant secondary metabolites. In plants, flavonoids play important roles in many biological processes such as pigmentation of flowers, fruits and vegetables, plant-pathogen interactions, fertility and protection against UV light. Being natural plant compounds, flavonoids are an integral part of the human diet and there is increasing evidence that dietary polyphenols are likely candidates for the observed beneficial effects of a diet rich in fruits and vegetables on the prevention of several chronic diseases. Within the plant kingdom, and even within a single plant species, there is a large variation in the levels and composition of flavonoids. This variation is often due to specific mutations in flavonoid-related genes leading to quantitative and qualitative differences in metabolic profiles. The use of such specific flavonoid mutants with easily scorable, visible phenotypes has led to the isolation and characterisation of many structural and regulatory genes involved in the flavonoid biosynthetic pathway from different plant species. These genes have been used to engineer the flavonoid biosynthetic pathway in both model and crop plant species, not only from a fundamental perspective, but also in order to alter important agronomic traits, such as flower and fruit colour, resistance, nutritional value. This review describes the advances made in engineering the flavonoid pathway in tomato (Solanum lycopersicum). Three different approaches will be described; (I) Increasing endogenous tomato flavonoids using structural or regulatory genes; (II) Blocking specific steps in the flavonoid pathway by RNA interference strategies; and (III) Production of novel tomato flavonoids by introducing novel branches of the flavonoid pathway. Metabolite profiling is an essential tool to analyse the effects of pathway engineering approaches, not only to analyse the effect on the flavonoid composition itself, but also on other related or unrelated metabolic pathways. Metabolomics will therefore play an increasingly important role in revealing a more complete picture of metabolic perturbation and will provide additional novel insights into the effect of the introduced genes and the role of flavonoids in plant physiology and development.     

On the natural diversity of phenylacylated-flavonoid and their in planta function under conditions of stress

Plants contain light signaling systems and undergo metabolic perturbation and reprogramming under light stress in order to adapt to environmental changes. Flavonoids are one of the largest classes of natural phytochemical compounds having several biological functions conferring stress defense to plants and health benefits in animal diets. A recent study of phenylacylated-flavonoids (also called hydroxycinnamoylated-flavonoids) of natural accessions of Arabidopsis suggested that phenylacylation of flavonoids relates to selection under different natural light conditions. Phenylacylated-flavonoids which are decorated with hydroxycinnamoyl units, namely cinnamoyl, 4-coumaroyl, caffeoyl, feruloyl and sinapoyl moieties, are widely distributed in the plant kingdom. Currently, more than 400 phenylacylated flavonoids have been reported. Phenylacylation renders enhanced phytochemical functions such as ultraviolet-absorbance and antioxidant activity, although, the physiological role of phenylacylation of flavonoids in plants is largely unknown. In this review, we provide an overview of the occurrence and natural diversity of phenylacylated-flavonoids as well as postulating their biological functions both in planta and with respect to biological activity following their consumption.     

Perception and modification of plant flavonoid signals by rhizosphere microorganisms

Flavonoids are a diverse class of polyphenolic compounds that are produced as a result of plant secondary metabolism. They are known to play a multifunctional role in rhizospheric plant-microbe and plant-plant communication. Most familiar is their function as a signal in initiation of the legume-rhizobia symbiosis, but, flavonoids may also be signals in the establishment of arbuscular mycorrhizal symbiosis and are known agents in plant defence and in allelopathic interactions. Flavonoid perception by, and impact on, their microbial targets (e.g. rhizobia, plant pathogens) is relatively well characterized. However, potential impacts on 'non-target' rhizosphere inhabitants ('non-target' is used to distinguish those microorganisms not conventionally known as targets) have not been thoroughly investigated. Thus, this review first summarizes the conventional roles of flavonoids as nod gene inducers, phytoalexins and allelochemicals before exploring questions concerning 'non-target' impacts. We hypothesize that flavonoids act to shape rhizosphere microbial community structure because they represent a potential source of carbon and toxicity and that they impact on rhizosphere function, for example, by accelerating the biodegradation of xenobiotics. We also examine the reverse question, 'how do rhizosphere microbial communities impact on flavonoid signals?' The presence of microorganisms undoubtedly influences the quality and quantity of flavonoids present in the rhizosphere, both through modification of root exudation patterns and microbial catabolism of exudates. Microbial alteration and attenuation of flavonoid signals may have ecological consequences for below-ground plant-microbe and plant-plant interaction. We have a lack of knowledge concerning the composition, concentration and bioavailability of flavonoids actually experienced by microbes in an intact rhizosphere, but this may be addressed through advances in microspectroscopic and biosensor techniques. Through the use of plant mutants defective in flavonoid biosynthesis, we may also start to address the question of the significance of flavonoids in shaping rhizosphere community structure and function.     

Flavonoids and Melanins: A Common Strategy across Two Kingdoms

Ultraviolet (UV) radiations alter a number of metabolic functions in vivant. They produce damages to lipids, nucleic acids and proteins, generating reactive oxygen species such as singlet oxygen (O₂), hydroxyl radical (HO) and superoxide anion (O₂^-). Plants and animals, after their water emersion, have developed biochemical mechanisms to protect themselves from that environmental threat through a common strategy. Melanins in animals and flavonoids in plants are antioxidant pigments acting as free radical scavenging mechanisms. Both are phenol compounds constitutively synthesized and enhanced after exposure to UV rays, often conferring a red-brown-dark tissue pigmentation.Noteworthy, beside anti-oxidant scavenging activity, melanins and flavonoids have acquired secondary functions that, both in plants and animals, concern reproductions and fitness. Plants highly pigmented are more resistant to biotic and abiotic stresses. Darker wild vertebrates are generally more aggressive, sexually active and resistant to stress than lighter individuals. Flavonoids have been associated with signal attraction between flowers and insects and with plant-plant interaction. Melanin pigmentation has been proposed as trait in bird communication, acting as honest signals of quality.This review shows how the molecular mechanisms leading to tissue pigmentation have many functional analogies between plants and animals and how their origin lies in simpler organisms such as Cyanobacteria. Comparative studies between plant and animal kingdoms can reveal new insight of the antioxidant strategies in vivant.     

Flavonoid–membrane interactions: Involvement of flavonoid–metal complexes in raft signaling

Flavonoids are polyphenolic compounds produced by plants and delivered to the human body through food. Although the epidemiological analyses of large human populations did not reveal a simple correlation between flavonoid consumption and health, laboratory investigations and clinical trials clearly demonstrate the effectiveness of flavonoids in the prevention of cardiovascular, carcinogenic, neurodegenerative and immune diseases, as well as other diseases. At present, the abilities of flavonoids in the regulation of cell metabolism, gene expression, and protection against oxidative stress are well-known, although certain biophysical aspects of their functioning are not yet clear. Most flavonoids are poorly soluble in water and, similar to lipophilic compounds, have a tendency to accumulate in biological membranes, particularly in lipid rafts, where they can interact with different receptors and signal transducers and influence their functioning through modulation of the lipid-phase behavior. In this study, we discuss the enhancement in the lipophilicity and antioxidative activity of flavonoids after their complexation with transient metal cations. We hypothesize that flavonoid–metal complexes are involved in the formation of molecular assemblies due to the facilitation of membrane adhesion and fusion, protein–protein and protein–membrane binding, and other processes responsible for the regulation of cell metabolism and protection against environmental hazards.     

Occurrence and distribution of free flavonoid aglycones in plants

Flavonoids are widely present in plants as water-soluble glycosides but the lipophilic free aglycones are far less abundant. The 462 flavonoids reported so far to be present in the free state and their plant sources are listed. Evaluation of these data reveals a correlation in most cases between the occurrence of flavonoid aglycones, the presence of secretory structures and the production of other lipophilic plant products. Their accumulation in some plant organs and in certain taxa is discussed. Special attention is given to their occurrence in materials deposited externally on leaves and buds.     

植物查尔酮异构酶研究进展

黄酮类化合物属于多酚类次生代谢物,具有广泛的药用价值。查尔酮异构酶（CHI）是黄酮类代谢途径中的一个关键酶,催化分子内环化反应,使双环的查尔酮转化为有生物学活性的三环（2S）-黄烷酮。植物体内的CHI活性与类黄酮物质的合成有着密切联系,CHI转基因研究对于提高植物类黄酮含量有重要意义。简要概述了查尔酮异构酶的结构特点、催化反应机理以及CHI转基因的研究进展。     

Phenolic acids act as signaling molecules in plant-microbe symbioses

Phenolic acids are the main polyphenols made by plants. These compounds have diverse functions and are immensely important in plant-microbe interactions/symbiosis. Phenolic compounds act as signaling molecules in the initiation of legumerhizobia symbioses, establishment of arbuscular mycorrhizal symbioses and can act as agents in plant defense. Flavonoids are a diverse class of polyphenolic compounds that have received considerable attention as signaling molecules involved in plant-microbe interactions compared to the more widely distributed, simple phenolic acids; hydroxybenzoic and hydroxycinnamic acids, which are both derived from the general phenylpropanoid pathway. This review describes the well-known roles attributed to phenolic compounds as nod gene inducers of legume-rhizobia symbioses, their roles in induction of the GmGin1 gene in fungus for establishment of arbuscular mycorrhizal symbiosis, their roles in inducing vir gene expression in Agrobacterium, and their roles as defense molecules operating against soil borne pathogens that could have great implications for rhizospheric microbial ecology. Amongst plant phenolics we have a lack of knowledge concerning the roles of phenolic acids as signaling molecules beyond the relatively well-defined roles of flavonoids. This may be addressed through the use of plant mutants defective in phenolic acids biosynthesis or knock down target genes in future investigations.Key words: Agrobacterium sp., flavonoids, legume-rhizobium symbioses, phenolic acids, plant defense, vesicular arbuscular mycorrhiza     

Flavonoids-potent and versatile biologically active compounds interacting with cytochromes P450.

Flavonoids represent a group of phytochemicals exhibiting a wide range of biological activities arising mainly from their antioxidant properties and ability to modulate several enzymes or cell receptors. Flavonoids have been recognized to exert anti-bacterial and anti-viral activity, anti-inflammatory, anti-angionic, analgesic, anti-allergic effects, hepatoprotective, cytostatic, apoptotic, estrogenic and anti-estrogenic properties. However, not all flavonoids and their actions are necessarily beneficial. Some flavonoids have mutagenic and/or prooxidant effects and can also interfere with essential biochemical pathways. Among the proteins that interact with flavonoids, cytochromes P450 (CYPs), monooxygenases metabolizing xenobiotics (e.g. drugs, carcinogens) and endogenous substrates (e.g. steroids), play a prominent role. Flavonoid compounds influence these enzymes in several ways: flavonoids induce the expression of several CYPs and modulate (inhibit or stimulate) their metabolic activity. In addition, some CYPs participate in metabolism of flavonoids. Flavonoids enhance activation of carcinogens and/or influence the metabolism of drugs via induction of specific CYPs. On the other hand, inhibition of CYPs involved in carcinogen activation and scavenging reactive species formed from carcinogens by CYP-mediated reactions can be beneficial properties of various flavonoids. Flavonoids show an estrogenic or anti-estrogenic activity owing to the structural similarity with the estrogen skeleton. Mimicking natural estrogens, they bind to estrogen receptor and modulate its activity. They also block CYP19, a crucial enzyme involved in estrogen biosynthesis. Flavonoids in human diet may reduce the risk of various cancers, especially hormone-dependent breast and prostate cancers, as well preventing menopausal symptoms. For these reasons the structure-function relationship of flavonoids is extensively studied to provide an inspiration for a rational drug and/or chemopreventive agent design of future pharmaceuticals.     

Variation in flavonoid pattern in leaves and flowers of Primula veris of different origin and impact of UV-B

Flavonoids can serve as chemotaxonomic markers and play an important role in protection against ultraviolet (UV) radiation. Primula veris originating from two natural field sites in Albania and one cultivar from Austria were used to investigate whether flavonoid pattern may differ between populations and to determine their response to UV. Plants were grown in a common environment and shortly before flowering transferred in two greenhouses with 80% and 4% UV-B transmission, respectively. After two weeks, young leaves and open flowers were harvested and flavonoids analyzed by high performance liquid chromatography. The flavonoid profiles of leaves and flowers were highly distinct for each population, with certain flavonoids occurring only in plants of particular field sites. These flavonoids may be useful biomarkers to identify the origin of plant material. The differences in UV-treatment at that stage had no effect on the total flavonoid contents of both leaves and flowers. However, individual flavonoids of both leaves and flowers responded sensitively to UV, suggesting that they may be involved in protection against UV.