Flavonoids: a metabolic network mediating plants adaptation to their real estate

1The multicellular algae that changed their marine environment to the harsh terrestrial had to face numerous challenges including: higher oxygen concentration, desiccation, increasing gravity, damaging heat and UV light, greater diurnal and seasonal fluctuations in temperatures, chances to be infected and eaten by new pathogens and grazers. Decision to stand upright spreading branches horizontally to catch more sun light will require an efficient strategy to fight against gravity and establish a system of waterproof ‘pipes’ pumping water from the roots to the rest of the plants. New inventive strategy recruiting insects for pollination will need development of the first colorful flowers and volatile chemicals. A seed dispersal by first representatives of moving, running and flying vegetarian dinosaurs will associate with an urgent demand for quick-reacting machinery to adapt to new geographical locates. And the fact that plants had to overcome all of these challenges standing on the same place made their evolution perspective a ‘mission impossible.’

2Today, more than 500 million years later, we observe that not only all of these challenges were successfully overcome, but modern plants represent most evolutionally successful eukaryotic species colonizing all parts of our planet, between North and South poles. If the development of unique stem cells machinery contributed to plants’ longevity (some trees can live for 100s and 1000 years, when just few animals reach their 100th birthday; ) it is the evolution of plant-specialized secondary metabolites which made their life ‘safe and comfortable’ in new established real estates. The acquisition of the secondary metabolites including flavonoids represents an important step in the colonization of Earth’s terrestrial environment by vascular plants. Physiological functions of flavonoids and the reasons for their ubiquitous existence have been widely discussed. A short list of these functions includes: (i) protection against insect predation and defense against microbes; (ii) action as sunscreens to absorb UV radiation and strong light, thus replacing mycosporine-like amino acids usually detected in algae; (iii) attraction of insect pollinators through production of colorful anthocyanins, absorbing different spectra of visible light; (iv) action as antioxidants, inhibiting the generation of reactive oxygen species (ROS), by maintaining their concentration within a sub-lethal range; (v) involvement in pollen germination; (vi) involvement in biological communication in the rhizosphere and action as (vii) developmental regulators, involved in auxin transport and catabolism.

3Our understanding of the evolution of flavonoid pathway has considerably increased over the last decade. However, the remaining ‘gaps’ in our understanding of the primary evolutionary roles of flavonoids in early land plants can be explained by the fact that modern plants might differ significantly from ancestors in their priorities for flavonoid’s functions. It’s been widely accepted that elavonoid molecules are a unique ‘invention’ of land plants assuming that algal and cyanobacterial cells are and were ‘flavonoids-free’. However, first a study reported that representatives of streptophyte Nitella, the most closely related algae to terrestrial plants, also contains flavonoids was published in 1969. Later, the phenylpropanoid pathway enzyme, phenylalanine ammonia lyase (PAL) that catalyzes the first and essential step of the general phenylpropanoid pathway, was detected in the cyanobacteria Anabaena variabilis and Nostoc punctiforme and in the green microalga Chlorella pyrenoidosa. Recent extensive algal genomes sequencing program revealed a number of homologs of plant flavonoid genes including, chalcone isomerase (CHI) and isoflavone reductase in Chlamydomonas, dihydrokaempferol-4-reductase and naringenin chalcone synthase (CHS) in Phaeodactylum, and CHI and dihydroflavonol reductase in Ectocarpus. It was, however, suggested that some of these enzymes could be involved in other than flavonoid pathways, for example, fatty acid biosynthesis. Breakthrough in this area was recently made by, who analyzed all known precursors, key intermediates and end products of the flavonoid biosynthetic pathway in representatives of divergent algal lineages (Cyanobacteria, Rhodophyta, Chlorophyta, Haptophyta, Ochrophyta). This research showed that distant microalgae representatives contain a wide range of flavonoids which composition is compatible with the established basic flavonoid pathway observed in higher plants. This implies that the flavonoid biosynthetic pathway arose much earlier in evolution compared to what is generally accepted and could belong to the phylum Plantae, which involves plants, glaucocystophytes, red algae, and green algae.

4Evolutionary, small quantities of flavonoids may be existed in early representatives of algae and plants as side products of enzymes of primary metabolism as a result of the proximity of their substrate binding domains. Stepwise and lengthy development of first flavonoids was a result of a number of evolutionary processes which led to production of first three enzymes, CHS, CHI, and flavanone 3-hydroxylase (F3H) in representatives of bryophytes (mosses), liverworts, and hornworts the earliest plants colonizing Earth. Some of these enzymes could evolve via gene duplication of genes of primary metabolism. Alternatively, key phenylpropanoid enzymes could be acquired via horizontal gene transfer during plant symbioses with bacteria and fungi that are known to be established very early during the first steps of land colonization. It was suggested that PAL was horizontally acquired from PAL homologs existed in sediment/soil bacteria. CHI-like enzymes were also present in fungi, early branching mycetozoa, and in some representatives of proteobacteria and could also be acquired via horizontal gene transfer. Evolution of CHS and F3H was a result of recruitment and gene duplication of representatives of polyketide synthases and the oxoglutarate-dependent dioxygenase, respectively, from primary metabolism. Activities of the CHS, CHI, and F3H led to production of the first three flavonoids, chalcones, flavonols, and flavones which represent the oldest plant flavonoids. These metabolites, remained intact over 500 million years, represent core intermediates for modern pathways producing a massive spectrum flavonoid products with irreducible complexity.

5Two main hypotheses regarding the primary roles of flavonoids proposed that these molecules evolved (i) as an effective sunscreen protecting against UV radiation as plants began colonizing land or (ii) as developmental regulators of auxin transport and catabolism. Protection from damaging UV light as a presumable primary function of ancestral flavonoids does not match well with the observation that representatives of flavonols (quercetin), produced in early land plants do not absorb UV-B wavelengths as efficiently as other flavonoids and phenolics. Moreover, these flavonols have much lesser ability to absorb light wavelengths over the 290–320 nm and UV-B than their ‘counterparts’ from ancestral algae, mycosporine like aminoacid (MAA;;, ). And, since flavonoids exist at nanomolar concentrations it is unlikely they could be immediately effective as an efficient sunscreen from damaging UV penetration into leaf tissues. Moreover, recent research on role of flavonoids in photoprotection showed that flavonoids accumulate not only in epidermal cells but also in the mesophyll cells after exposure to sunlight.

6Involvement of flavonoids in nanomolar concentrations as signal molecules mediating the cascades of oxidative stresses and as regulators of intra-cellular and long-distance movements of multifunctional growth regulators, such as auxins is well investigated. In modern plants quercetin, kaempferol, apigenin were found to be involved as signaling molecules in auxin-regulated cell division. Mutation of flavonoids can trigger pleiotropic effect on overall plants architecture affecting root growth, lateral root density, root hair development and length, shoot/flower organ number, and seed organ density and fertility. It was also recently shown that activated by light auxin can regulate the glycosylation pattern of different flavonoids in response to UV-B significantly contributing to stress-induced morphogenetic responses.

7Nuclear localization of CHS and CHI enzymes and also flavonoids, their interaction with mitotic chromosomes and histone proteins, regulation of chromatin conformation, involvement in epigenetic processes and gene expression attract increasing attention among wide spectrum of scientists. Anti-oxidative role of nuclear flavonoids protects mitotic chromosomes at stages when they are not enveloped by nuclear membrane and are most sensitive to oxidative stresses. Fragmentation of DNA was shown be markedly reduced by monomeric and dimeric catechins through donation of electrons or hydrogens to transcriptionally important basic amino acids of histones, such as lysine, arginine, and histidine, protecting them against oxidative stress. Binding of flavonoids to the histone and none-histone chromatin binding proteins can change chromatin’s conformation affecting gene expression. Changes in concentrations of nuclear flavonoids were observed in response to environmental stresses, such as heat and drought.

8Accumulated data suggest that the oxidative stress-induced signaling and regulatory functions of flavonoids very likely represent their primary roles in early plants. Most of these functions has remained intact over 500 million years of evolution and are conserved across the kingdoms of modern plants. Other functions such as the direct protection of plants from environmental stresses such as UV sunscreen, pigmentation and others were gradually evolved at later stages when concentrations and spectrum of flavonoids were increased and reached levels observed in modern terrestrial plants.

9The stepwise emergence, diversity and evolutionary success of flavonoid pathway were triggered by a number of major events: (i) recruitment of enzymes from primary metabolism including the shikimate, the phenylpropanoid and polyketide pathways through the cascades of gene duplication events; (ii) horizontal gene transfer during plant/algal symbioses with bacteria and fungi; (iii) modification of the metabolic enzymes through the changes in regiospecificity (ability to modify different parts of the substrate molecules) and substrate selectivity (ability to bind to different substrates); (iv) the changes in regulatory control of flavonoid genes; (v) plasticity of flavonoid pathways and their ability depending on the demands of the local ecosystem to redirect the flows of intermediate molecules towards biosynthesis of the sophisticated scaffolds of very different chemicals. As a result of these evolutionary events more than 10,000 of these chemicals have been identified in more than 9000 modern plant species making flavonoids one of the most widely spread pathways in modern plants.

10Decades of study of the flavonoid, anthocyanin, proanthocyanidin and lignin pathways have coalesced around understandings of molecular aspects of some of these events including: (i) detailed gene-to-metabolite study of above mentioned metabolic pathways linking spatio-temporal profiles of their gene expression to the functional metabolomics of related products and (ii) analysis of the molecular and metabolic cross-talks between the flavonoid and other pathways in response to stresses.

11Molecular aspects of flavonoid pathway were extensively investigated and reviewed. Plant phenolics are products of the shikimate, phenylpropanoid, flavonoid, anthocyanin, and lignin pathways. The shikimate pathway produces the aromatic amino acids, including phenylalanine, which can be further modified through the sequential of elongation and cyclization steps to form the flavonoids (Figure 1). In general, flavonoids are sub-classified into several families of fifteen-carbon molecules including flavonol, flavone, flavanone, flavan-3-ol, isoflavone, and anthocyanidin. As a first step, phenylalanine gets converted into coumaroyl-CoA by a number of enzymatic reactions involving PAL, cinnamate 4-hydroxylase (C4H) and 4-coumarate:CoA ligase (4CL). The CHS and CHI convert coumaroyl-CoA into naringenin and onward, the pathway diverges forming flavanones, dihydroflavonols, leucoanthocyanins, anthocyanidins, and flavan-3-ols through a series of enzymatic steps shown in Figure 1. These molecules can produce a series of products, flavones and isoflavones, flavonols, anthocyanins, and proanthocyanidins (condensed tannins) existing in the forms of monomers, dimers, and polymers. Three main classes within these molecules are differing only in the extent of B-ring hydroxylation.

12Cell-specific and intracellular localization of flavonoids has indeed been extensively investigated. Flavonoids were found in epidermal cells, including trichomes as well as within palisade and spongy mesophyll cells. Intracellularly flavonoids also occur in various cell compartments such as chloroplasts, vacuole and nucleus.

13Despite the fact that anthocyanin and proanthocyanidin pathways share the same metabolic intermediates they represent the most and the least extensively investigated branches of flavonoid pathway, respectively. Both branches involve the conversion of 4-coumaroyl-CoA and malonyl-CoA to the families of precursors (Figure 1;,;; ). Dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), as well as a range of anthocyanidin-modifying enzymes convert dihydrokaempferols, dihydroquercetins, and dihydromyricetins into anthocyanins with pelargonidin cyanidin and delphinidin backbones, respectively. A list of anthocyanidin-modifying enzymes includes glycosyltransferases, UDP-glucuronosyl/UDP-glycosyltransferases, glutathione transferases, methyltransferases, and anthocyanidin rhamnosyltransferases. In proanthocyanidin-specific pathway dihydroflavonols are converted to cis- and trans-epimeric forms of afzelechins, catechins, and gallocatechins, GCs by the family of DFR, leucoanthocyanidin reductase (LAR), ANS, and anthocyanidin reductase (ANR) enzymes.

14Proanthocyanidins, polymers of flavan-3-ol subunits are best known for their protein-binding ability and are commercially significant because of their antioxidant properties and their potential health benefits when included at a low level in the diets of humans and livestock. Proanthocyanidins are found in the leaves, flowers, fruit, seeds, bark, and roots of many plant species. Legumes offer opportunities for studying proanthocyanidin biosynthesis accumulating them across a broad range of tissues [for reviews, see. Medicago sativa and M. truncatula accumulate a high level of proanthocyanidins in seed coats, but low levels in flowers, stems, roots, and leaves. Members of Lotus species accumulate to the large extent proanthocyanidins in their leaf mesophyll cells (Figure 2; ), which makes them very attractive for ruminants health and production. White clover (Trifolium repens), a major component of temperate pastures worldwide accumulates proanthocyanidins mainly in floral organs and seeds (Figure 2;, ). Accumulation of proanthocyanidins in vegetative tissues (leaves and stems) is restricted to the trichome cells.

15In white clover flowers the epidermal cells of petals and inner organs, such as stamen filaments, carpels, ovules, and immature embryos were intensively investigated by our group because of spatial co-localization of the anthocyanin and proanthocyanidin pathways. Biosynthesis of proanthocyanidins in epidermal cells of these organs starts in cells located on adaxial side followed by their production on abaxial sides (Figure 2). Accumulation of the 2,3-trans-flavan 3-ols (gallocatechins, GC) and 2,3-cis-flavan 3-ols (epigallocatechins, EGC), the building blocks of proanthocyanidins is developmentally regulated being active at the early stages of flowers development (stages 1–3) followed by their sharp down-regulation at the later stages of development (stages 4–6; Figure 3; ). This suggests that in floral organs biosynthesis of proanthocyanidins is not light-regulated and is active at the stages when proanthocyanidin-producing organs are well protected from light by sepals. Exposure of the petals to light at the stages 4–6 correlates with significant increase of glycosylated forms of flavonols, representing all three B-ring hydroxylated variants, with an abundance of myricetin glycosides (m/z 479; R3# = OH, R5# = OH) and quercetin glycosides (m/z 463; m/z 505; R3# = OH, R5# = H) and the onset of light-induced anthocyanins with a predominance of delphinidins is synthesized. A low level of kaempferol-based anthocyanins (m/z 447; R3# = H, R5# = H) and virtually no pelargonidin-based anthocyanins were found in flowers. Developmentally regulated profiles of proanthocyanidin and anthocyanin biosynthesis were confirmed by developmentally regulated profiles of expression of known flavonoid-related genes. Generated gene-to-metabolite data suggest that these pathways, are potentially recruiting different isoforms of the same flavonoid biosynthetic enzymes, represented by distinct members of multigene families (Figure 3).

16Cross talk between members of proanthocyanidin- and anthocyanin-specific branches of the flavonoid pathways was shown in a number of plant species. Ectopic expression of ANR gene in tobacco down-regulated anthocyanin production and resulted in the biosynthesis of proanthocyanidins in flower petals. Ectopic expression of ANR in M. truncatula plants resulted in a decrease of approximately 50% in anthocyanin production and up to a threefold increase in proanthocyanidin production in a subset of leaf cells. Re-direction of anthocyanin pathway into proanthocyanidin pathway in Nicotiana tabacum, M. sativa, and T. repens plants was achieved by ectopic expression of TaMYB14 from T. arvense. In white clover plants, expression of TaMYB14 led to accumulation of proanthocyanidins in leaves up to 1.8% dry matter. Silencing of TaMYB14 resulted in almost complete cessation of proanthocyanidin biosynthesis in T. arvense. Production of commercial white clover cultivars with enhanced levels of foliar condensed tannins could significantly contribute to pasture productivity via increased production, forage quality and decreases in adverse environmental impacts.

17Redirection of flavonoid biosynthesis from proanthocyanidin into anthocyanin biosynthesis was firstly shown as production of red-colored seed coats in the Arabidopsis banyuls mutant. Down-regulation of ANR in this mutant led to the precocious accumulation of anthocyanins in proanthocyanidin-producing tissues. Down-regulation of the TrANR gene in white clover plants and associated reducing level of EGCs have triggered changes in accumulation profiles of delphinidin-based anthocyanins as a result of the diversion of intermediates from flavan 3-ol to anthocyanin production (Figure 4; ). This triggered accumulation of anthocyanins in all epidermal cells which normally produced proanthocyanidins in wild-type plants (Figure 4). Moreover, light-independent biosynthesis of anthocyanins in red-flowered TrANRhp lines is developmentally regulated, with most intense red coloration produced at the stage 3 when proanthocyanidin pathway is most active in wild-type flowers. Metabolic redirection of intermediate molecules in these plants is associated with significant changes in expression profiles of the late flavonoid genes involved in anthocyanin production, the members of the glycosyltransferases, UDP-glucuronosyl/UDP-glycosyltransferases, glutathione transferases, methyltransferases, and anthocyanidin rhamnosyltransferases. The most striking results of this experiment was the fact that down-regulation of the ANR gene has triggered significant changes in level and profiles of early flavonoids which biosynthesis occurs in the upstream part of flavonoid pathway. This can be explained by changes in the expression profiles of the gene families functioning upstream of TrANR. A list of affected upstream genes includes members of the early flavonoid pathway namely CHS, CHI, F3H, DFR, ANS, CHR as well as genes involved in isoflavone biosynthesis, IF3#H-, IFOMT-, and VR-like genes. Global changes in expression profiles of flavonoid genes associated with up- and down-regulation of the members of transcription factors in TrANRhp lines is providing further support for transcriptional regulation of this pathway.

18The emergence and evolution of chemical scaffolds of lignin polymers provided plants with mechanical support, protected them from pathogen invasion, damaging UV, and enhanced the hydrophobicity of their vasculature. Thus these metabolites have been of paramount importance for the evolution of land plants and their colonization of the distinct geographical and ecological locales. Similar to flavonoids also this pathway recruited enzymes from primary metabolism to promote the biosynthesis of H and G lignin in early terrestrial plants. Evolutional advantage of S lignin in adaptation to environment was a result of the selective structural alterations of the ring modification enzymes such as ferulate 5-hydroxylase and caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) at later stages of evolution. Lignification consequently transformed phenylpropanoid metabolism into a major sink for carbon in plants estimated to represent as much as 30% of the total biomass produced in the biosphere.

19Over last year’s research in our group was focused on (i) functional analysis of the lignin specific genes in C3 and C4 grasses; (ii) understanding the roles, and substrate promiscuity, of catalytic enzymes of COMT family in shaping the composition of lignin polymers; and (iii) metabolic reprogramming of lignin pathway redirecting metabolic fluxes between S and G- pathways (changing S/G ratio) and between lignin and flavonoid pathways under reverse genetics conditions. Downregulation of LpCCR1, the first lignin-specific gene in Lolium perenne resulted in a decreased level of the downstream products in the lignin biosynthesis pathway, such as H, G, and S units, caffeoyl aldehyde, and coniferaldehyde (these compounds are shown in red in Figure 5). This suggests that coumaroyl-CoA and caffeoyl-CoA, along with feruloyl CoA, may serve as the primary substrates for CCR1 in perennial ryegrass. This also indicates that coniferaldehyde, a common intermediate of both G- and S-lignin biosynthesis, may be synthesized from feruloyl-CoA or from caffeyl aldehyde. The latter scenario correlates with predicted roles of LpOMT1 in methylation of 3-OH in caffeoyl aldehyde, suggesting an additional, caffeoyl aldehyde-mediated branch in both G- and S-related monolignol biosynthesis (;; Figure 5).

20Molecular and metabolic cross-talks through redirection of metabolic fluxes between lignin and flavonoid pathways were shown for a variety of species with down regulated genes involved in phenylpropanoid, lignin, and flavonoid pathways. Silencing of the CCR gene in tobacco, tomato, and poplar results in decreased flux from feruloyl-CoA to G and S units reducing the level of lignin-specific phenolic molecules. By contrast, the levels and composition of the some of the stress-related flavonoid intermediates and derivatives were strongly enhanced in these transgenic lines. Most of the accumulating flavonoid metabolites are modified through quination and glucosylation by p-hydroxycinnamoyl-CoA:D quinate and a family of glycosyl transferases, respectively. Some molecules, such as coumaroyl-CoA, can be also conjugated with shikimate producing p-coumaroyl-shikimate by p-hydroxycinnamoyl-CoA:shikimate p-hydroxycinnamoyltransferase (Figure 5;;; ).

21Reduced flux from coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA to H, G, and S units, as a result of downregulation of LpCCR1 in L. perenne led to enhanced accumulation of coumaric acid, cinnamic acid, and ferulic acid. As a result, an elevation in the levels of p-coumaroyl quinic acid, caffeoyl quinic acid, and caffeoyl shikimic acid were occurring in hpCCR1-1 plants. Accumulated caffeoyl shikimate and caffeoyl quinate can be stored or, alternatively, converted back into caffeoyl-CoA and feruloyl-CoA. Deficiency of CCR1 activity also diverts coumaroyl CoA esters into the flavonoid biosynthetic pathway, significantly increasing level of some of its components: coumaroyl quinic acid (up 14.1-fold), caffeoyl shikimic acid (up 5.9-fold), caffeoyl quinic acid (14.1-fold), rutin-7-glycoside (up 5.1-fold), kaempferol-O-rutinoside (up 4.3-fold), kaempferol-7,3-glycoside ferulic acid (up 3.9-fold), kaempferol diglucoside malonate (up 3.6-fold), rutin (up 3.1-fold), and kaempferol-O-glycoside (up 2.9-fold; ). Dramatic decrease in the levels of total lignin and S, G, and H subunits associated with enhanced levels of flavonol glycosides and acylated anthocyanins was shown for poplar, tobacco, and tomato transgenic plants. Consequent modification of accumulated flavonols through glycosylation and quinylation in these plants enhanced detoxiflcation of the phenylpropanoid intermediates. and found that reduced lignin content is strongly associated with the changes in pigmentation of seed coat traits in the species of Brassicaceae representatives. The silencing of a lignin biosynthetic gene hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase in A. thaliana led to both, lignin synthesis repression and the redirection of the metabolic flux into flavonoids increasing CHS activity in transgenic plants. Reverse genetics approach was successfully implemented also to show redirection of the carbon flow from lignin to cellulose. Downregulation of 4-coumarate-CoA ligase in aspen (Populus tremuloides) resulted in a 45% decrease in lignin content and a concomitant 15% increase in cellulose content. These figures were further increased to a 52% reduction in lignin content and a 30% increase in cellulose content when coniferaldehyde 5-hydroxylase was also down-regulated. Interaction between MYB75 with another secondary cell wall regulator, the KNOX transcription factor KNAT7, contributes to the regulation of secondary cell wall deposition in the Arabidopsis inflorescence stem re-directing the metabolic flux between the lignin, fiavonoid, and polysaccharide pathways. Down-regulation of another member of MYB-family in Zea mays reduced expression of several genes involved in the synthesis of monolignols, which significantly reduced lignin content in transgenic plants. These plants also showed induced expression of stress-related proteins, suggesting redirection of carbon flux towards the biosynthesis of flavonoids.

22Redirection of metabolic fluxes in opposite direction – from flavonoid into lignin pathway was observed in genetically modified strawberry (Fragaria × ananassa). Down-regulation of the members of CHS diverted the carbon flux from anthocyanin precursors into lignin pathway triggering accumulation of 4-coumaroyl-CoA-derived metabolites, such as 4-coumaryl alcohol and 4-coumaryl acetate. Simultaneous overexpression of eugenol synthase showed the enhanced production of the phenolic volatiles. Similar results were shown recently by who showed that silencing of CHS in strawberry leads to the loss of pigmentation, which was accompanied by a significant increase in lignin content and, in turn, with enhanced firmness of the fruits.

23Longer average life span, decreasing physical activity, and most importantly, the increasing consumption of unhealthy foods have been identified by the World Health Organization as the main triggers of chronic disease in the decade 2005–2015. Plant biochemistry and metabolomics have significantly contributed to the wellbeing of human and animal health through identifying and understanding the health-promoting components of food, known as nutraceuticals. The best-described property of almost every group of flavonoids is their capacity to act as antioxidants protecting the body against ROS. The antioxidant activities (TEAC) per 100 g fresh weight, uncooked and portion size, are: strawberry >> raspberry = red plum >> red cabbage >>> grapefruit = orange > spinach > broccoli > green grape > onion > green cabbage > pea > apple > cauliflower > tomato peach = leek > banana = lettuce. Common dietary flavan-3-ols present in fresh fruits, vegetables, and nuts are catechin, catechin gallate, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, gallocatechin, and gallocatechin gallate. Dietary flavonoids also include the anthocyanins cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin together with the flavanones hesperetin and naringenin, the flavones apigenin and luteolin, and the flavonols myricetin, kaempferol, and quercetin. Apart from antioxidant activities, food-derived flavonoids were shown to reduce the incidence of atherosclerosis, cancer, cardiovascular diseases, diabetes, thrombosis, inflammation in arthritis, neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases, obesity, hyperlipidemia, nerve injury and hypertension.

24The beneficial effects of proanthocyanidins on animal health and farm production have been extensively investigated. One of the most beneficial traits of important forage legumes is the level and composition of proanthocyanidins in their edible parts, leaves and stems. Ruminants fed on forages containing moderate amounts of proanthocyanidins show reduced methane gas emission, which decreases the chances of a digestive disorder, known as bloat. Bloat occurs when grazing ruminants consume large quantities of leguminous plants (e.g., alfalfa or clover). The gases produced in the rumen during fermentation cannot be released in the normal way since they are trapped in the foam caused by the rapid release of soluble proteins during chewing and ruminal degradation. Low concentrations of proanthocyanidins (around 5 mg/g dry matter) can be beneficial, reducing protein fermentation to ammonia in the rumen and methane gas emissions. The mechanisms by which tannins reduce ruminal degradation of different dietary components are not entirely clear. Among the most accepted are substrate privation, enzyme inhibition and direct action on rumen microorganisms. With respect to milk production, reported an increase of 21% during mid and late lactation in sheep fed Lotus corniculatus (44.5 g CT kg-1 DM). They, and others, also reported significant increases in the efficiency of milk production, increased protein and lactose production, and a decrease in the fat content of the milk. Enhanced amounts of foliar condensed tannins (2–4%) would also significantly contribute to forage quality via increased animal health and production and decreases in adverse environmental impacts.

25Representatives of Lotus species, L. pedunculatus, L. corniculatus and L. tenuis contain up to 7.2, 4.6, and 0.8% of dry matter of proanthocyanidins in their leaves, respectively with L. corniculatus containing the most desirable concentration for feeding ruminants. In spite of their low concentration of proanthocyanidins, L. tenuis varieties are more tolerant to waterlogging, alkaline and salt conditions than any commercial varieties of L. corniculatus. crossed a L. tenuis cultivar with a diploid, wild accession of L. corniculatus rich in proanthocyanidins and the L. tenuis × L. corniculatus F1 hybrid population obtained showed successful introgression of several traits from both parents, including the optimal concentration of proanthocyanidins in the herbage. Enhanced levels of proanthocyanidins in the hybrids correlated with increased expression levels of the R2R3MYB transcription factor TT2 and a number of the key structural genes involved in biosynthesis of the building blocks of proanthocyanidins, epicatechin, and catechin. This exciting result highlights the importance of wild relatives as donor of useful traits and the power of traditional breeding to produce genetic pool for breeding Lotus varieties with superior nutritional value.

26Medicago sativa and T. repens, the predominant pasture legume species contain no or very low levels of proanthocyanidins in foliar tissues. Only two Trifolium species, namely T. arvense and T. afflne, are known to accumulate significant levels of proanthocyanidins in leaves. Screening of the cDNA library from T. arvense led to the isolation of the family of R2R3-MYB transcription factors. Silencing of TaMYB14 resulted in almost complete cessation of proanthocyanidin biosynthesis in T. arvense. Ectopic expression of TaMYB14 in M. sativa and T. repens led to accumulation of proanthocyanidins in leaves up to 1.8% dry matter.

27Recent progresses in genomics and functional genomics of flavonoid genes, coupled to extensive metabolomic studies, led to the development of efficient molecular tools to obtain crops with balance levels of flavonoids and related value products beneficial to human and animal health and for the exploitation of crops for the production of renewable energy. In general, engineering of flavonoid pathways in plants can be achieved using three commonly used strategies: (i) up-regulation of endogenous genes (enzymes and transcription factors), directing carbon flow toward the target products; (ii) down-regulation of the genes involved in competing pathways, and (iii) expression of exogenous genes targeting intermediate molecules towards biosynthesis of new products. Strategies to improve market acceptance of transgenic crops include elimination of sequences from non-plant origins and the generation of selectable marker-free events through co-expression of the genes of interest and selectable markers.

28The engineering of the flavonoid pathway started more than 20 years ago with modification of anthocyanins level and composition in ornamental flowers through expression and/or silencing of the key flavonoid genes, CHS, F3H, F3′5′H, DFR. Today a list of ornamental plants with modified floral colors include petunias, gerberas, roses, carnations, lisianthus, lotus, chrysanthemums, and torenia. Most recent papers in this area describe reprogramming of the anthocyanin pathway in transgenic chrysanthemum and camellia plants. Enhanced production of delphinidins was shown in floral petals of chrysanthemum after expression of the F3′5′H gene from pansy regulated by CHS promoter from rose. The additional silencing the endogenous F3′H gene resulted in more intensive blue coloration of floral petals (Figure 6). Expression of flavonol synthase from yellow flower of Camellia nitidissima, in N. tabacum altered floral color into white or light yellow, and metabolic analysis showed significant increasing of flavonols and reducing of anthocyanins in transgenic plants. Shelf-life is one of the most important agronomic traits for tomato (Solanum lycopersicum) and is determined by their susceptibility to opportunistic pathogens, Botrytis cinerea. Over-production of anthocyanins in skins of genetically modified tomato was sufficient to reduce the susceptibility of fruit to B. cinerea.

29Unexpected link between distant flavonoid and terpenoid pathway was recently shown when expression of the wild-type CHI gene complemented not only anthocyanin deficiency in anthocyanin free (af) tomato mutant but also terpenoid production in glandular trichomes.. These can be explained by the fact that these pathways have obvious complementary roles in plants protecting them from insect herbivores and microbial pathogens as well as mediating plant communication with symbiotic bacteria, natural enemies of arthropod herbivores, and parasitic plants.

30Plant genetics and metabolic engineering have been extensively used to make human foods that differ in their content of specific and healthy phytonutrients (designer food). Recently, different transgenic strategies have been taken to increase flavonoid levels in tomato fruit by overexpressing either the heterologous structural or regulatory genes involved in the biosynthetic pathway. Expression of two transcription factors from snapdragon, a bHLH-type (Delila) and R2R3 MYB-type (Rosea1) under the control of a fruit-specific promoter in tomato (cv. MicroTom) showed the strong visible purple pigmentation with the final concentration of anthocyanins averaged 3 mg/g fresh weight, which is the highest value obtained so far in this species. In these plants expression of Delila and Rosea1 has stimulated the transcription of cascade of the structural genes involved in the biosynthetic pathway, including PAL, CHI and F3′5′H directing flavonoid intermediates towards the anthocyanin products. A pilot animal test with a group of cancer susceptible mice that were fed with transgenic purple tomatoes showed a significant extension of their average life span compared with cancer-susceptible mice. This demonstrates beneficial role of these anthocyanin-rich tomatoes in human health. Other researches also showed up to 100-fold increase in anthocyanin concentration in transgenic tomato with up-regulated anthocyanin pathway.

31Another example of engineering the flavonoid pathway to boost the accumulation of metabolites of pharmacological value for human health includes the modification of the pathway of the isoflavonoid phenolic acids. The production of isoflavones is restricted almost exclusively to the family Fabaceae. These metabolites are known to exhibit anticancerous and anti-osteoporotic activities in animal systems. enhanced isoflavone production in soybean hairy roots by down-regulating two flavonoid genes, the F3H and the flavones synthase II′′. In tobacco, showed a substantial amount of genistein glycoconjugates as a result of the co-expression of the transcription factor AtMYB12 with the soybean IFS gene”. The rare wild species of snow lotus, Saussurea involucrata is a commonly used medicinal herb with great pharmacological value for human health, resulting from its uniquely high level of phenylpropanoid compound production. Transgenic S. involucrata co-expressing PAP1 and Lc genes showed activation of most of the phenylpropanoid pathway genes, and increased accumulation of chlorogenic acid, syringin, cyanrine, and rutin greatly increasing its antioxidant capacity.

32Stilbenes play the important protective roles as antifungal phytoalexins and phytoanticipins and in mitigating UV-induced damages. The biosynthetic pathway for stilbenes diverges from the general phenylpropanoid pathway through the action of stilbene synthases, utilizing p-coumaroyl-CoA, and cinnamoyl-CoA starter units for the generation of the parent stilbene resveratrol and pinosylvin, respectively. Redirection of flavonoid pathway toward production of the stilbenes through co-expression of stilbene O-methyltransferase with a stilbene synthase genes from peanut in tobacco and Arabidopsis plants, resulted in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in tobacco because of substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated confirmed redirection of flavonoid intermediates into stilbene biosynthesis.

33Systemic biological approaches, combining genomics, proteomics, and metabolomics of flavonoids in plants, have provided new insights into the understanding of the origins of flavonoid biosynthesis and how they have evolved historically from Earth’s beginnings into one of the most widely spread pathways in modern plants. Recent progress in engineering of flavonoid pathways offers potential breakthroughs in the modification of secondary metabolite pathways of plants in ways not possible through conventional plant breeding. Remarkable plasticity of plant secondary metabolism could be exploited for the production of novel high-value pharmaceutical compounds not detected in wild type plants. Ability of flavonoids to evolve and modify constantly in response to new stressful environments was shown in response to ozone depletion that has become a major environmental problem over the last three decades. Stress-inducing generation of more and more sophisticated cascades of protecting molecules will keep flavonoids on the top of most important plant metabolites in 21 century and beyond.

34The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.