Flavonoids: Recent Advances and Applications in Crop Breeding

Abstract

Flavonoids are secondary metabolites that perform a wide range of roles in plants. These include their involvement in plant growth, pigmentation, and UV protection, to a variety of defense and signaling activities. Flavonoids such as chalcones, flavones, flavanols, anthocyanins, and proanthocyanins are widely distributed in the plant kingdom. The metabolic routes of the flavonoids are exploited extensively using several biotech approaches to enhance the crop variety and incorporate varied nutritional benefits. Many flavonoids are key components of medicinal plants and possess nutritional significance. Specific mutations in flavonoid-related genes are typically responsible for the diversity in flavonoids, resulting in quantitative and qualitative variations in metabolic profiles. Thereby numerous attempts have been made to increase flavonoid content in agronomically important species. Flavonoids are also employed in the regulation of inflammation, in arthritis, and in cancer prevention strategies, due to their ubiquity in the human diet. Advances in the comprehension of flavonoid biosynthesis and modulation have prompted a surge in researches aiming at modifying the flavonoid pathway to improve nutritional value, plant defenses against infections and the feeding value of livestock. This chapter briefly discusses the varied role of flavonoids, their biosynthesis, and their distribution over the plant kingdom. Furthermore, it exclusively highlights the several biotech-based trending pieces of research based on introducing flavonoid biosynthesis in commercial crops.

1. Introduction

1.1 What are flavonoids?

Flavonoids are naturally occurring secondary metabolites in plants, possessing a polyphenolic structure. They are widely distributed in the leaves, seeds, barks, flowers, fruits, and vegetables. Over 8000 flavonoids have been identified to date and most of them have been found to be engaged in various biological activities in plants, animals, and bacteria. Apart from imparting pigmentation in plants, flavonoids afford protection against UV radiation, herbivores, and pathogens [1, 2, 3]. In addition, flavonoids have also been found to serve as detoxifying and antimicrobial defense agents in the animal world. They appear to have played a significant part in the effectiveness of ancient medicinal therapies, and since then, their usage has continued to this day. Notably, detailed pieces of evidence from various studies have confirmed flavonoids’ role in growth metabolism and gene regulation as well [4].

1.2 Structure and types of flavonoids

The name ‘Flavonoids’ refers to a group of plant pigments generated mostly from benzo-γ-pyrone (Figure 1). Advanced techniques such as [1H-1H]-correlated spectroscopy, [1H]- and [13C]-NMR spectrometry, X-ray diffraction, mass spectrometry, circular dichroism, and optical rotatory dispersion help us to analyze and elucidate the flavonoid structures and configurations [4]. The class of flavonoids primarily comprises of anthocyanidins, proanthocyanins, flavonols, iso-flavonoids, chromones, flavones, iso-flavones, flavanes, flavanones, flavanols, catechins, aurones, benzo-furones, and coumarins.

Figure 1.

Basic structure of flavonoid.

The variability observed in the flavonoids mainly occurs due to differences in the following features:

1. Changes in the aglycone’s ring structure and state of oxidation/reduction.

2. Variations in the aglycone’s hydroxylation extent and the locations of hydroxyl groups.

3. Various methods of derivatizing the hydroxyl groups, such as using methyl groups, polysaccharides, or isoprenoids [4].

Flavonoids exhibit a range of beneficial effects in human health like

1. anticholinesterase activity and combating neurodegenerative diseases [5].

2. anti-inflammatory activity [6].

3. steroid-genesis modulators [5].

4. xanthine oxidase modulator [7].

5. radical scavenging agent [8].

6. anti-carcinogenic activity [6].

1.3 Role of flavonoids in plants

Flavonoids are responsible for distinct flavor and color, which draw pollinators, as well as characteristic color and fragrance of flowers. Additionally, they facilitate the germination of seeds and spores, as well as the growth and development of seedlings, by fruit dispersal. Plants can be protected from biotic and abiotic challenges by flavonoids, which also operate as UV filters, signal molecules, phytoalexins, detoxifying agents, and antimicrobial defense components. They are recognized for their ability to frequently play a useful role in the capacity of plants to adapt to heat, to cold, to frost, to drought, and to both. Early advances in floral genetics have mostly been made by mutation techniques that impact flower colors that are produced from flavonoids, and it has been proven that plants that are involved in flavonoid production are capable of functional gene silencing [1].

1.3.1 Role in pigmentation

Flavonoids play a prominent role in floral coloration, as well as pollinator attractiveness and UV protection [9, 10]. A study on Papaver flowers has revealed that the spatial occurrence of flavonoids is responsible for its wide-range variation in flower color [11]. Approximately 8000 flavonoids contribute to the vibrant colors seen in fruits, herbs, vegetables, and medicinal plants.

1.3.2 Role as a growth regulator

Flavonoids have the ability to regulate auxin movement and catabolism. Recent research has revealed that flavonoids are capable of modulating protein activity during cell growth [12].

1.3.3 Role in nitrogen metabolism

Flavonoids, through inducing root nodulation, play a significant role in nitrogen metabolism in nitrogen-fixing plants. Dinitrogen-fixing bacteria, such as the Rhizobium strain, exist in symbiosis with leguminous plants and are found in these nodules. The major action of flavonoids is likely to be the stimulation of genes that express proteins necessary by nodule cells, but because they are antioxidative, they are also well adapted to participate in dioxygen removal [4].

1.3.4 Role in combating oxidative stress

As a result of a variety of biotic and abiotic stimuli causing oxidative stress, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced in plants. Nearly all of the increase in flavonoid synthesis in plants comes from oxidative stress. Flavonoids like mono-apigenin and mono-kaempferol and dihydroxy B-ring-substituted (Luteolin and Quercetin) flavonoid glucosides reduce ROS generation, quench ROS once they have formed, and absorb the UV-B and UV-A wavelengths. When early plants went from the water to the land, flavonoids performed key UV-B screening roles. The type of substitution on distinct rings of flavonoids determines the extent of antioxidant capability and ability to absorb UV wavelengths [12].

1.3.5 Role in defense against pathogens and insects

Flavonoids in plants are also helpful in protecting plants from harmful bacteria and fungus. Catechins and other flavanols potentially act as a plant’s defensive mechanism against insects that are damaging to it [13, 14, 15].

2. Flavonoid metabolism and Biosynthesis

2.1 Distribution of Flavonoid subclasses in the plant kingdom

According to study, flavonoids may be found in angiosperms, gymnosperms, and pteridophytes. Due to the wealth of information available on flavonoids in many species, flavonoid subclasses (such as anthocyanins, chalcones, flavones, flavonols, and proanthocyanidins) are present in each subgroup of plants can be identified. Flavone and flavanone are present in all plant groups, with the exception of hornworts. Plant families that produce flavonoid subclasses have evolved and diversified as well. For instance, the angiosperms have the most varied flavonoid aglycones. The liverworts Radula variabilis and Radula spp. contain prenyldihydrochalcone, whereas more than 1000 prenylflavonoids have been found in legumes. These results indicate that either the two plant groups independently evolved the ability to make prenylflavonoids or that many species lost this capacity over evolution. Flavonoid molecules show that plants have genes for the manufacture of flavonoids. Therefore, analytical approaches for identifying flavonoids are necessary to comprehend the evolution of flavonoid metabolism in the plant kingdom [16] (Figure 2).

Figure 2.

Distribution of flavonoids in plant kingdom and their respective structures.

2.2 Evolution of Flavonoid metabolism

The enzymes, 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 were created as a result of several evolutionary processes in representatives of bryophytes (mosses), liverworts, and hornworts. CHI-like enzymes were discovered in certain proteobacteria and fungi, and they may have been acquired by horizontal gene transfer. Contrarily, the recruitment and gene duplication of polyketide synthases and oxoglutarate-dependent dioxygenases from primary metabolism, respectively, led to the evolution of CHS and F3H. The first three flavonoids, chalcones, flavanols, and flavones, were created as a result of the CHS, CHI, and F3H activities. These metabolites, which have not altered in 500 million years, are essential intermediates in today’s irreducibly complicated flavonoid manufacturing pathways in plants [17, 18].

The number of key events that sparked the flavonoid pathway’s gradual rise, variety, and evolutionary successes are:

• the recruitment of enzymes from fundamental metabolisms, such as the polyketide, phenylpropanoid, and shikimate pathways

• horizontal gene transfer between bacteria and fungi in plant/algal symbioses

• variations in the substrate selectivity and regiospecificity (the ability to change specific regions of the substrate molecules) of metabolic enzymes (ability to bind to different substrates)

• modifications in the regulation of the flavonoid gene

• the flexibility of flavonoid pathways and their capacity to shift intermediate molecule fluxes towards the production of complex scaffolds of quite varied chemicals depending on the needs of the local ecosystem.

More than 10,000 of these compounds have been found in over 9000 current plant species as a result of these evolutionary processes, making flavonoids one of the most extensively distributed routes in plants today [16, 19].

2.3 The flavonoid biosynthetic pathways

From a genetic standpoint, a lot of work has been done to decipher the flavonoids’ biosynthesis routes. Flavonoid synthesis mutants have been discovered in a variety of plant species. The first important experimental models in this system were snapdragon (Antirrhinum majus), petunia (Petunia hybrida), and maize (Zea mays), which led to further discovery of several structural and regulatory flavonoid genes. Arabidopsis (Arabidopsis thaliana) has recently contributed to the study of flavonoid pathway regulation and subcellular localization [20].

2.3.1 Following are the biosynthetic pathways of some major flavonoids

The Figure shows the eight branches of flavonoid biosynthetic pathway (showed in the eight different colored boxes) and four important intermediate metabolites (represented by the green boxes) (Figure 3). The abbreviated forms of enzyme names and flavonoid compounds mentioned in the figure are as follows: (i) ANR: anthocyanidin reductase; (ii) ACCase: acetyl-CoA carboxylase; (iii) AS: aureusidin synthase; (iv) 4CL: 4-coumarate: CoA ligase; (v) CHS: chalcone synthase; (vi) CHI: chalcone isomerase; (vii) CHR: chalcone reductase; (viii) C4H: cinnamic acid 4-hydroxylase; (ix) CH2′GT: chalcone 2′-glucosyltransferase; (x) CH4′GT: chalcone 4′-O-glucosyltransferase; (xi) ANS: anthocyanidin synthase; (xii) CLL-7: cinnamate–CoA ligase; (xiv) FNS: flavone synthase; (xv) F6H: flavonoid 6-hydroxylase; (xvi) IFS: isoflavone synthase; (xvii) HID: 2-hydroxyisoflavanone dehydratase; (xviii) FNR: flavanone 4-reductase; (xix) F8H: flavonoid 8-hydroxylase; (xx) F3’5’H: flavanone 3′,5′-hydroxylase; (xxi) F3H: flavanone 3-hydroxylase; (xxii) DHK: dihydrokaempferol; (xxiii) DHM: dihydromyricetin; (xxiv) DFR: dihydroflavonol-4-reductase; (xxv) DHQ: dihydroquercetin; (xxvi) FLS: flavonol synthase; (xxvii) OMT: O-methyl transferases; (xxviii) PAL: phenylalanine ammonia lyase; (xxix) UFGT: UDP-glucose flavonoid 3-Oglucosyltransferase; (xxx) LAR: leucoanthocyanidin reductase [21].

Figure 3.

The branches of flavonoid biosynthetic pathway.

2.3.1.1 Phenylpropanoid pathway

The phenylpropanoid route produces flavonoids from phenylalanine, whereas the shikimate pathway produces phenylalanine [22, 23]. The general phenylpropanoid route refers to the first three steps of the phenylpropanoid pathway [24]. The aromatic amino acid phenylalanine is transformed to p-coumaroyl-CoA in this route. The typical phenylpropanoid route begins with the deamination of phenylalanine to trans-cinnamic acid, which is catalyzed by the enzyme phenylalanine ammonia lyase (PAL) [25]. In plants, PAL also has a significant role in controlling the transfer of carbon from primary to secondary metabolism [26]. The second step in the general phenylpropanoid route is catalyzed by the activity of C4H, a cytochrome P450 monooxygenase found in plants that hydroxylates trans-cinnamic acid to produce p-coumaric acid [27]. The quantity of lignin, a crucial phenylpropanoid metabolite, in Populus trichocarpa and Arabidopsis thaliana, is connected to the degree of C4H expression [24, 28]. In the third step, 4-coumarate (4CL) catalyzes the production of p-coumararoyl-CoA by incorporating a coenzyme A (CoA) unit into p-coumaric acid [29].

2.3.1.2 Chalcone biosynthesis

Specific flavonoid synthesis, which starts with chalcone formation, is initiated with the entry of p-coumaroyl-CoA into the flavonoid biosynthesis pathway [30]. One molecule of p-coumaroyl-CoA and three molecules of malonyl-COA are converted into naringenin chalcone (4,2′,4′,6′-tetrahydroxychalcone [THC]) by the action of CHS (produced from acetyl-CoA) [31]. CHS, a polyketide synthase, is the main and first rate-limiting enzyme in the flavonoid biosynthesis pathway [32]. An intermediate of the CHS reaction is subjected to action by the aldo-keto reductase superfamily member chalcone reductase (CHR), which catalyzes its C-6′ dehydroxylation to produce isoliquiritigenin (4,2′,4′-trihydroxychalcone [deoxychalcone]) [33]. In one of the studies, the amount of anthocyanin decreased, when the Lotus japonicus CHR1 gene was overexpressed in petunia [21, 34]. Chalcones are recognized as the first important intermediate metabolite in the production of flavonoids, and are also considered as a crucial yellow pigment in plants [35].

2.3.1.3 Flavanones biosynthesis

The intramolecular cyclization of chalcones by CHI, which occurs in the cytoplasm to produce flavanones and the heterocyclic ring C, is a step in the flavonoid pathway [30]. According to the substrate used, CHIs in plants may often be split into two classes. Type I CHIs, which are present throughout the entire vascular plant, transform THC into naringenin. Type II CHIs may manufacture naringenin and liquiritigenin utilizing either THC or isoliquiritigenin and are primarily found in leguminous plants [36]. More than these two forms, there are two other variants of CHI (type III and type IV) that retain the catalytic activity of the CHI fold but lack its ability to cycle chalcones [37]. Additionally, flavanones are a frequent substrate for the downstream flavonoid pathway as well as the flavone, isoflavone, and phlobaphene branches [38, 39].

2.3.1.4 Aurone biosynthesis

Aurones, a family of flavonoids produced from chalcone, are significant yellow pigments in plants. Aurone pigments generate a stronger yellow hue than chalcones and are responsible for the golden coloration of numerous common ornamental plants. Snapdragon, sunflowers, and coreopsis are only a few of the plant species that contain aurones [40, 41]. Aurone production requires THC as a direct substrate [42]. In the cytoplasm of the plant cells, chalcone 4′-O-glucosyltransferase catalyzes the production of THC 4′-O-glucoside from THC. The former is transferred to the vacuole by aureusidin synthase (AS), where it is converted into aureusidin 6-O-glucoside (aurone) [43].

2.3.1.5 Flavone biosynthesis

In all higher plants, flavone production is an essential branch of the flavonoid pathway. Flavone synthase (FNS) converts flavanones into flavones (FNS) [44, 45]. When present in flavanones, FNSI and FNSII encourage the formation of a double bond between C-2 and C-3 positions of the ring C [46]. FNS is a crucial enzyme in the production of flavones. Both naringenin and eriodictyol can be used as substrates by Morus notabilis FNSI to produce flavones [47]. Overexpression of Pohlia nutans FNSI causes apigenin accumulation in A. thaliana [48]. FNSII expression levels in flower buds of Lonicera japonica were shown to be congruent with flavone accumulation patterns [46]. Flavanones can be transformed into C-glycosyl flavones as well [21].

2.3.1.6 Isoflavone biosynthesis

Leguminous plants serve as the primary source of isoflavones [49]. Isoflavone synthase (IFS) transports flavanone to the isoflavone route [50] and appears to be able to convert liquiritigenin and naringenin into 2,7,4′-trihydroxyisoflavanone and 2-hydroxy-2,3-dihydrogenistein, respectively [51, 52]. Under the action of hydroxyisoflavanone dehydratase (HID), they are further transformed to the isoflavones genistein and daidzein [53]. Additionally, HID, IFS, and isoflavanone O-methyl transferase can catalyze the conversion of liquiditigenin to 6,7,4′-trihydroxyflavanone, which can then be converted to glycitein (an isoflavone) [54]. IFS and HID catalyze two processes that result in the formation of isoflavone: the formation of a double bond between C-2 and C-3 positions of ring C and the transfer of ring B from C-2 position to C-3 position of ring C [55, 56]. The isoflavone production route begins with IFS, a cytochrome P450 hydroxylase. The accumulation of the isoflavone genistein in invitro tissues was caused by Glycine max IFS overexpression in Allium cepa [57]. The use of CRISPR/Cas9 to knock off the expression of the IFS1 gene resulted in a considerable drop in isoflavones like genistein [44].

2.3.1.7 Flavanol biosynthesis

Flavonols are flavonoid metabolites that have had their ring C-3 hydroxylated [38]. Because their C-3 position is very susceptible to glycosidation, they frequently occur in glycosylated forms in plant cells. Flavonol synthase (FLS) converts the dihydroflavonols like dihydroquercetin (DHQ), dihydrokaempferol (DHK), and dihydromyricetin (DHM) to the flavonols quercetin, kaempferol, and myricetin, respectively [58]. Through the activity of enzymes such as GTs, methyltransferases, and acyltransferase (AT), quercetin, kaempferol, and myricetin are further changed to numerous flavonol derivatives [59]. A C-2 and C-3 double bonds are formed in ring C via the desaturation of dihydroflavonol, which is catalyzed by FLS, a FeII/2-oxoglutarate-dependent dioxygenase. In the flavonol biosynthesis pathway, FLS is considered the key rate-limiting enzyme [21].

2.3.1.8 Anthocyanin and Leucoanthocyanidin Biosynthesis

Major enzyme in flavonoid metabolism in the anthocyanidin and proanthocyanidin pathways is dihydroflavonol-4-reductase (DFR). A hydroxyl group is produced at C-4 position of ring C by the NADPH-dependent reductase known as DFR [60, 61, 62]. Dihydroflavonols, DHQ , DHK, and DHM are reduced by DFR to produce leucocyanidin, leucopelargonidin, leucoanthocyanidins, and leucodelphinidin [63]. DFR, for example, transforms DHK to leucopelargonidin in Vitis vinifera [64]. The direct synthetic precursor of anthocyanidin and proanthocyanidin. Leucoanthocyanidin, is a crucial intermediary by-product in the flavonoid pathway. The colorless leucopelargonidin, leucocyanidin, and leucodelphinidin are converted into the equivalent anthocyanidins under the catalysis of anthocyanidin synthase (ANS) (the colored pelargonidin, cyanidin, and delphinidin) [65, 66]. An alternative name for ANS is leucoanthocyanidin dioxygenase (LDOX). Similar to FNSI, F3H, and FLS, ANS/LDOX is a FeII/2-oxoglutarate-dependent dioxygenase that stimulates the dehydroxylation of C-4 and formation of a double bond in ring C [67]. In Strawberries, anthocyanin content has been found to get enhanced when ANS is overexpressed [68].

2.3.1.9 Proanthocyanidin biosynthesis

Condensed tannins, also known as proanthocyanidins, are a form of flavonoid made up of leucoanthocyanidins and anthocyanidins [69]. The primary proanthocyanidin units are cis-flavan-3-ols, trans-flavan-3-ols, and flavan-3-ols. Proanthocyanidins are produced when flavan-3-ols are polymerized (or condensed) [70, 71]. To make colored tannins (yellow to brown), polyphenol oxidase (PPO) converts colorless proanthocyanidins into plant vacuoles [72]. The major and rate-limiting enzymes in proanthocyanidin production are leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR). Studies have revealed that overexpression of putative leucoanthocyanidin reductase gene (PtrLAR3) significantly elevates proanthocyanidin levels in Populus tomentosa [73]. Additionally, in alfalfa (Medicago sativa), overexpression of OvBAN, an ANR gene obtained from Onobrychis viviaefolia, increases the concentration of proanthocyanidin and the activity of the ANR enzyme [74]. However, proanthocyanidin and anthocyanin biosynthesis pathways have a competitive relationship since they utilize the same substrates [75].

2.4 Flavonoid biosynthesis in plants is regulated by transcriptional regulation

In the modification of flavonoid production, transcriptional control is crucial. The major transcriptional regulator in flavonoid biosynthesis is the MBW complex, which consists of WD40, bHLH, and MYB. The MYB domain at the N-terminus of MYB transcription factors (TFs) is needed for DNA binding and interaction with other proteins [76]. According to the amount and location of MYB domain repeats, MYB proteins is categorized into four groups: 3R-MYB, 4R-MYB, R2R3-MYB, and 1R-MYB/MYB-related. Among the four, R2R3-MYB members are mostly engaged in flavonoid metabolism regulation [21].

2.5 Plasticity of flavonoid pathway

Flavonoids have been discovered in epidermal cells such as trichomes, palisade, and spongy mesophyll. Moreover, flavonoids are found intracellularly in numerous cell compartments such as chloroplasts, vacuoles, and the nucleus [77, 78, 79]. The shikimate, phenylpropanoid, flavonoid, anthocyanin, and lignin pathways produce plant phenolics. The aromatic amino acids, including phenylalanine, are produced through the shikimate pathway, and the flavonoids are formed by a series of elongation and cyclization stages. Flavonoids get divided into numerous 15-carbon families, including flavanone, flavonol, flavone, flavan-3-ol, anthocyanidin, and isoflavone. It is evident that the level of B-ring hydroxylation is the sole difference between most of the main molecules [80].

2.5.1 Anthocyanin-proanthocyanidin pathway cross-talk

Despite the fact that the anthocyanin and proanthocyanidin routes use identical biochemical intermediates, they are the most and least studied flavonoid processes, respectively. Both branches include the formation of precursors from 4-coumaroyl-CoA and malonyl-CoA. ANS, DFR, and a variety of anthocyanidin-modifying enzymes transform dihydromyricetins, dihydroquercetins, and dihydrokaempferols into anthocyanins. Anthocyanidin rhamnosyltransferases, UDP-glucuronosyl/UDP-glycosyltransferases, methyltransferases, glutathione transferases, and Glycosyltransferases are among the anthocyanidin-modifying enzymes. On the other hand, the family of DFR, LAR, and ANR enzymes convert dihydroflavonols to trans- and cis-epimeric forms of gallocatechins, catechins, and afzelechins in the proanthocyanidin-specific pathway [19, 72, 81, 82].

In a number of plant species, cross-talk between members of the flavonoid pathways’ anthocyanin- and proanthocyanidin-specific branches has been seen. Studies have revealed that overexpression of the ANR gene in tobacco has suppressed anthocyanin production and induced proanthocyanidin biosynthesis in flower petals. Meanwhile, upregulation of ANR has caused a subset of leaf cells in Medicago truncatula plants to produce three times more proanthocyanidin and cut anthocyanin synthesis by half [83, 84].

2.5.2 Lignin-flavonoid pathway cross-talk

Chemical scaffolds of lignin polymers originated and evolved to offer mechanical support to plants, shield them from UV damage and pathogen invasion, as well as increase the hydrophobicity of their vasculature. As a result, these metabolites have played a critical role in the development of land plants and also, in the colonization of various geographical and ecological environments. Similar to flavonoids, this route assisted the manufacture of H and G lignin in early terrestrial plants by enlisting enzymes from primary metabolism [85].

Redirecting metabolic fluxes between the lignin and flavonoid pathways showed molecular and metabolic cross-talks in a variety of plants with down-regulated genes implicated in the phenylpropanoid, lignin, and flavonoid processes. The flow from feruloyl-CoA to G and S units is lessened when the CCR gene is silenced in tobacco, tomato, and poplar, which resulted in a decrease in the amount of phenolic chemicals which are particular to lignin [86, 87]. The quantities and composition of several stress-related flavonoid intermediates and derivatives, on the other hand, were significantly increased in these transgenic lines.

3. Effect of gene regulation and modification in flavonoid research and production in crop breeding: recent advances and applications

3.1 Engineering of flavonoid pathway

The flavonoid pathway has been extensively employed in the industry with the goal of accumulating compounds on purpose. Plant species like gerbera, petunia, rose, lisianthus, torenia, and carnation have been genetically modified for the production of novel flower colors. This was achieved by modification of the flavonoid biosynthesis pathway, either via transcriptional down-regulation, inactivation of key anthocyanin pathway enzymes, or by heterologous expression of key enzymes.

There are two significant, possible ways for improving flavonoid biosynthesis. The first is based on the discovery of TFs as a viable alternative to multi-step engineering, while the second is based on the use of inducible promoters to avoid the negative consequences of a constitutive production system. Virus-induced gene silencing has also been proven to be a simple and rapid method of functionalizing TF genes [88].

A few years ago, it was discovered that the pathway to pelargonidin might be opened by transferring a gene encoding DFR from a species where the enzyme does not really exhibit substrate selectivity into a petunia line Lacking F3′5′H activity. In another study, Brick red petunia flowers were produced using the maize gene A1 and petunia lines with vivid orange blooms obtained from an ornamental plant Gerbera hybrida [89]. Now various initiatives are being undertaken to boost anthocyanin concentrations beyond those found naturally. According to a previous short study undertaken, the high-anthocyanin tomatoes have been found to slow tumor development in cancer-prone rats. However, the consequences on human health still require additional research. In this subject, basic proof-of-concept research has been undertaken on a variety of vegetable and fruit species, including apple, grape, tomato, and cauliflower.

3.2 Natural flavonoids variation in horticultural species and horticulture breeding

The molecular basis of various flavonoid production focuses on the activation of genes along with respective pathways by diverse means. The majority of the important structural enzymes, part of the central flavonoid metabolism is encoded by single-copy genes, although some, such as PAL, CHS, F3H, or FLS, are encoded by several genes. The expression of biosynthetic (structural) genes varies significantly between species [19].

Activation of flavonoid and anthocyanin biosynthetic genes in response to light has been reported in most horticultural types. Accumulation of anthocyanin is regarded as one of the most investigated mechanisms in potatoes. This is because colored potato varieties are considered to be a strong source of phytochemicals at levels similar to cranberries, blackberries, blueberries, and grapes. Potato, like other species, has numerous genetic loci that influence anthocyanin production. StAN1, StAN2, StMYBA1, and StMYB113 are important regulators of the phenylpropanoid and anthocyanin pathways. However, bHLH co-factors also play a role, since StAN1 and StAN2 associates with StbHLH1 and StJAF13 in diverse organs, such as the tuber and leaf. A WD40-repeat gene, namely StAN11 has been recently postulated as a regulator of the system via modulating the expression of DFR among other TFs encoding genes, impacting anthocyanin accumulation in potatoes [90]. Apart from potatoes, few other horticulture species have also been exploited due to their antioxidant property via molecular plant breeding techniques [15].

Recently, a variety of red-fleshed and high-flavonoid containing apple genetic resources had been embodied in the complexities of the control of flavonoid production. In one of the studies, red-fleshed apple flavonoid metabolism has been found to get influenced by both hereditary and environmental variables. Numerous flavonoid biosynthesis cascade genes have also been discovered and cloned, so as to identify the flavonoid metabolism that get affected by several environmental factors and genetic variabilities [91].

All these current researches make it evident that flavonoids play a significant role in both food and primary agriculture development and will soon be an intriguing target for molecular plant breeding.

3.3 Flavonoids in tomato breeding

Tomatoes (Solanum lycopersicum) are the most abundant dietary source of carotenoids (lycopene), polyphenols, and flavonoids, which are key bioactive compounds favorable to human health. The flavonoids that are mainly produced in tomatoes are predominantly produced mostly in the peels. Naringenin chalcone and rutin (quercetin-rutinoside) are the two primary flavonoids found in tomato fruit so far [92, 93]. To date, three approaches have been made to engineer the flavonoid pathway in tomatoes (S. lycopersicum) with the goal to alter its agronomical traits such as its nutritional value, its flower and fruit color as well as its ability to build resistance against insects [94]. They are as follows:

1. Use of structural or regulatory genes to increase endogenous tomato flavonoids; Structural genes are the genes that encode enzymes that directly engage in the synthesis of flavonoids. On the other hand, regulatory genes are the ones that influence the expression of structural genes.

2. Use of RNA interference methods to block particular stages in the flavonoid pathway.

3. Introduction of new flavonoid pathways to produce novel tomato flavonoids [93].

Knowing the fact that there is a lack of flavonoid expression in tomatoes, to date, several attempts have been previously undertaken to generate transgenic tomatoes (Table 1).

Crop Variety/Name	Gene responsible	Name of Flavonoid Induced	Reference
Tomato	FNS-II gene of Gerbera	N/A	[93]
Tomato	SlANT1 and SlAN2 (coding for R2R3-MYB)	Anthocyanin	[92]
Tomato	CHI gene from red onion by transgenesis	Flavanol and Anthocyanin	[94]
Tomato	Del/Ros1 gene from snapdragon by transgenesis	Flavanol and Anthocyanin	[94]

Table 1.

Genes responsible for different flavonoid production in tomato.

For example:

1. Heterologous expression of the FNS-II gene obtained from Gerbera was employed in an attempt to create flavones in tomato. This seemed to have been a good approach to get novel flavone-derived metabolites into tomato fruit [93].

2. Several components of the MBW complex in tomatoes have recently been discovered and partially described as an anthocyanin biosynthesis regulator. Anthocyanins1 (SlAN1) and Anthocyanins2 (SlAN2), are the two paralog genes that encode for homologous R2R3-MYB TFs. Both of these paralogs are found in tomatoes on chromosome 10. Thereby, SIAN1 and SIAN2 expression in transgenic tomato lines have been found to be responsible for anthocyanin production in multiple organs [92].

3. To establish hairy root cultures (HRCs), Agrobacterium rhizogenes was used to transfer a construct for PhAN4 expression into the micro tomato genotype MicroTom. HRCs were created to serve as a testbed for whole-plant engineering methodologies that might enhance attributes for space culture [95].

4. Using TFs to improve a broad spectrum of flavonoids. In addition to the elevated number of flavonoids in tomato fruit caused by DET1 silencing, the transcription regulator AtMYB12 expression in tomatoes has been found to trigger flavanol production as well as the caffeoylquinic acid biosynthetic pathway [96].

5. The CHI gene from red onion and the Del/Ros1 gene from snapdragon has been employed to boost the flavanol and anthocyanin content of tomatoes, respectively, via transgenic techniques [94].

6. In one of the studies, simultaneous overexpression of the two maize TFs C1 and Lc have resulted in a 60-fold upregulation in kaempferol glycosides in tomato flesh tissue [96].

In one of the studies, it was concluded that despite having intense debate over the advantages, disadvantages, and risks of genetically modified food, around 96% of the customers showed interest in purchasing high flavonoid containing tomatoes. It is considered that this changing mindset of people will prove to be crucial for the development of transgenic vegetables in the future [94]. Moreover, various other studies are in process to make transgenic tomato breeding more productive and nutritional in the coming future.

3.4 Flavonoids in rice breeding

Rice is staple food in many Asian countries. Even though white rice is the most popular, Asian cuisine often includes colored rice. Several pieces of evidence reveal that pigmented rice has important biological properties, including antioxidants, anti-allergic, and neuro-protective properties. The rich flavonoid and nutritional content of colored rice warrants enhancement flavonoid content in rice by implementation of different breeding strategies (Table 2).

Crop Variety/Name	Gene responsible	Codes for	Name of Flavonoid Induced	Reference
Black Rice	Kala3	R2R3-Myb	N/A	[97]
Black Rice	Kala4	bHLH	Anthocyanin	[97]
Red Rice	Rc	bHLH	Proanthocyanidin	[97]
Rice	OsCOP1 gene introduced via CRISPR-Cas9	N/A	N/A	[98]

Table 2.

Genes responsible for different flavonoid production in rice.

The functional activities of TFs influence the color of rice grains. In black rice, the Kala3 gene, which codes for R2R3-Myb, and the Kala4 gene, which codes for basic helix–loop–helix (bHLH), activate the flavonoid biosynthesis genes ANS CHS, and DFR resulting in anthocyanin pigment buildup in the grain. In red rice, the Rc gene expressing bHLH activates CHS, DFR, and LAR, resulting in the buildup of proanthocyanidin pigment in the grain. The promoter of Kala4 in white rice differs from that in pigmented rice, and loss of 14 base pairs inside the Rc open reading frame, resulted in lack of color in the grain. In addition, the gene CYP75B3 is strongly expressed in pigmented rice grains, along with other flavonoid pathway genes. This explains why leucocyanidin-derived anthocyanin and proanthocyanidin pigments are abundant in colored rice grains [97].

Recently, in order to create flavone, isoflavone, and flavonol in rice grain, the flavonol (AtF3H/AtFLS), isoflavone (GmIFS), and flavone (PoFNSI/GmFNSII) biosynthetic enzyme genes, as well as OsPAL and OsCHS, were expressed in a seed-specific way. These biosynthetic genes were expressed in seed using the GluB-1 promoter and the 18-kDa oleosin promoter [99]. In another study, the OsCOP1 gene, an ortholog of Arabidopsis thaliana constitutive photomorphogenic 1 (COP1) was introduced in rice using CRISPR-Cas9. This not only turned the pericarp of the rice variety yellowish but also caused embryonic death. Moreover, this also reduced the size of the transgenic seeds [98]. According to a study, a total of 82 flavonoids have been chemically identified in transgenic rice seeds. Moreover, exogenous enzymes produced flavonoids in rice seeds that were later altered by endogenous enzymes and transported, causing persistent accumulation in PB-I and/or PB-II. Based on these results, the heterologous and ectopic expression of biosynthetic enzymes in rice seeds not only serves as a productive platform for the production of flavonoids but can also be used to broaden the structural diversity of flavonoids and hence open up a new, untapped source of bioactive substances [100].

Increasing the flavonoid biosynthesis and its accumulation in rice have been found to contribute to the enhanced heat tolerance under stress, as well as plays a regulatory role in the activation of the antioxidant enzyme system [101].

3.5 Flavonoid research in maize

Obtaining security of grain supply in the twenty-first century with limited arable land is a big challenge because of the constantly changing environment and increasing global population [102, 103, 104]. Maize plays a very important role in global grain production. Drought is a significant factor restricting plant development and productivity. Drought stress affects growth and development of plants, which is directly related to yield. Under drought stress, doi57 gene is observed to play an important role in maintaining the plant to grow and survive in order to give good yield. doi57 gene is one of the key genes involved in biosynthesis of flavonoid. With less soil water content (SWC), doi57 guard cells can accumulate more flavonols and less hydrogen peroxide (H₂O₂). Furthermore, under drought conditions, doi57 seedling extracts had a stronger potential to scavenge oxygen free radicals than B73 maize genome. Moreover, in terms of transpiration rates, photosynthetic rates, water consumption efficiency, and stomatal conductance, doi57 seedlings outperformed B73, resulting in high biomass and enhanced root/shoot ratios in doi57 mutant plants [105].

3.6 Flavonoid profile in millets breeding

Millet polyphenols protect the neurological system by lowering oxidative stress, and vitexin is a crucial component of millet polyphenols. Vitexin, a flavonoid derived from millet, is present in many foods such as millet, mung bean, and others. It is also known chemically as apigenin-8-C-glucoside. According to research, vitexin contains potent free radical scavenging and antioxidant enzyme protection properties that may protect cells from oxidative damage [106].

3.7 Flavonoids in olive breeding

With almost 1200 olive varieties listed, the olive has a great genetic diversity. High heterozygosity, prolonged juvenile phase, and a paucity of information on trait heritability have all been major limiting factors in olive breeding. Attempts to obtain new varieties have concentrated on improving olive response to varied growing situations through systematic breeding. New olive breeding methods uses two varieties “Picual” and “Arbequina” as controls along with other varieties. The phenolic compound metabolism in the olive tree is quite complex, and is controlled by environmental and genetic factors that regulate the final phenolic composition of olive fruits. The unique breeding selection UCI2–68 demonstrated an optimal phenolic profile, resulting in good agronomic performance [107].

3.8 Flavonoids in soybean breeding

In adverse conditions, soybean (Glycine max) productivity drops significantly. Soybean breeding might benefit from discovering regulatory components that impart stress tolerance. HSFB2b, a class B heat shock factor, enhances salt tolerance by activating one subset of flavonoid biosynthesis-related genes and blocking the repressor GmNAC2 to release another collection of flavonoid biosynthesis-related genes. Silencing GmFNSII, the principal flavone-producing gene, reduces flavone levels and increases salt sensitivity in hairy soybean roots [108]. Leaf-chewing insects are severe pests of soybeans, lowering seed quality and limiting output (G. max). The CRISPR/Cas9 expression vector was introduced into the soybean cultivar via agrobacterium-mediated transformation, resulting in Glyma.07 g110300-gene mutants. A 33-bp deletion and a single-bp insertion in the GmUGT coding domain increased resistance to Cotton bollworm (Helicoverpa armigera) and Tobacco cutworm (Spodoptera litura). Furthermore, GmUGT overexpression made soybean species susceptible to H. armigera and S. litura [109].

3.9 Molecular breeding of peanut with high flavonoid content

Peanuts include a variety of bioactive substances in addition to helpful fatty acids and minerals. Flavonoids found in peanuts include flavonol, dihydroquercetin, C-glycoside flavone, dihydroflavonol, flavonone, and 5,7-dimethoxyisoflavone. BARI2011 is the most drought-tolerant of the peanut cultivars. According to one study, BARI2011 retained more water. MYB123 encodes an R2R3 MYB domain-containing TF that has been demonstrated to upregulate flavonoid production and is a critical determinant in proanthocyanidin accumulation. Correlational investigation of TF expression with flavonoid biosynthesis and accumulation of phenolics, flavanols, and anthocyanins in peanuts revealed that TFs coregulated flavonoid production under water stress [110].

3.10 Flavonoid content in mustard

The seed coat color of Brassica crops is an essential horticultural feature. Seeds with a yellow seed coat have higher oil quality, more protein, and less fiber. As a result, the yellow seed coat color is seen as a good characteristic in Brassica juncea, Brassica rapa, and Brassica napus hybrids. The majority of seed coat color is produced by the accumulation of proanthocyanidins, the ultimate product of the flavonoid biosynthetic pathway, which is mostly deposited in the innermost cell layer of testa (chalaza, micropyle, and endothelium) [111].

3.11 Flavonoid content in lettuce breeding

Lettuce (Lactuca sativa) is one of the world’s most important vegetables. The GWAS identified 5311 expression quantitative trait loci (eQTL) that influence the expression of 4105 genes, including nine eQTLs that regulate flavonoid biosynthetic genes. GWAS has found six candidate loci for anthocyanin variation in lettuce leaves [112].

UV-A supplementation increased flavonoids, anthocyanin, and polyphenol levels and the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical scavenging rate. UV-A can modify plant phenolic contents and flavonoid metabolism by increasing the expression of associated genes such as CHS and MYB in the flavonoid pathway and PAL in the propane metabolic pathway. Under additional UV-A and FR light, anthocyanin levels in lettuce seedlings were 11% higher and 40% lower, respectively [113] (Table 3).

Crop Variety/Name	Gene used	Name of Flavonoid Induced	Method Used	Reference
Lettuce	rol C	N/A	Agrobacterium-mediated transformation	[114]
Lettuce	rol ABC	N/A	Agrobacterium-mediated transformation	[115]

Table 3.

Genes responsible for different flavonoid production in Lactuca sativa L.

Transformation of L. sativa L. with rol C gene inducing an increase in total flavonoid contents [114].

The gene expression of rol ABC genes was used to boost secondary metabolites in L. sativa L. (cv. Grand Rapids), particularly antioxidants such as phenolics and flavonoids. A. tumefaciens GV3101 and the rol ABC genes were used to transform Lactuca sativa L. (cv. Grand Rapids). The transformation increased the secondary metabolites in lettuce and also induced free radical inhibitor effect and lipid peroxidation scavenging properties [115].

Thereby, the three key categories that is used to group the goals of current lettuce breeding programmes include: (1) Improvement in horticultural traits such as quality and resistance to early bolting, (2) resilience to diseases and pests, and (3) to attain higher yield and uniformity [116].

3.12 Flavonoid content in buckwheat

Buckwheat (Fagopyrum esculentum) is an annual crop that is planted all over the world. Buckwheat seeds, leaves, and stems are high in flavonoids such as rutin and proanthocyanidins (PAs). The discovery of the ANR and LAR buckwheat genes could result in the development of buckwheat cultivars with different PA levels. In one investigation, one gene sequence (AT1, Fes sc0002933.1.g000003.aua.1) encoding ANR was found, along with three gene sequences encoding LARs (LT1, Fes sc0001063.1.g000007.aua.1; LT2, Fes sc0016501.1.g000002.aua.1; and LT3, Fes sc0010963.1.g000003.aua.1) [117].

The flavonoid contents in buckwheat sprouts were commonly in the following order: rutin > quercetin > isovitexin > vitexin > isoorientin > kaemferol [118]. Tartary buckwheat grain contains orientin, vitexin, rutin, and quercetin and is used to make drinks and biscuits. Because of its high content of flavonoids and other phenolic compounds, tartary buckwheat is resistant to pests, plant diseases, and UV-B radiation damage. As a result, Tartary buckwheat can be cultivated organically and without synthetic fertilizers or chemical treatments [119].

3.13 Distinctive flavonoid profiles in legume

Legumes have sparked a lot of attention because of their health benefits and their polyphenolic compounds. The xanthine oxidase XO inhibitory activity appears to be reduced when a glycoside or a methyl group is substituted for the hydroxyl groups at C7 and C3 of the basic flavonoid structure [120].

Recently, isolation of genes encoding the critical enzymes of various phenylpropanoid branch pathways has opened the door for engineering crucial agricultural plants like alfalfa for:

1. improving the forage digestibility through lignin composition and content,

2. enhancing the disease resistibility via introducing novel phytoalexins, or by changing transcriptional regulator expressions,

3. improving nodulation efficiency by overproduction of flavonoid nod gene inducers [121].

The recognition of flavonoids in legume root exudates by the bacterium, originates in the rhizosphere and activates a particular set of genes involved in bacterial nodulation (Nod). These nodulations produce and secretes a very specific signal known as lipochito-oligosaccharides or Nod factors. The formation of rhizobia-infected root nodules is the result of a variety of host responses brought on by the perception of Nod factors by the plant LysM receptors, including bacterial invasion of the root hairs, root hair curling, cortical cell divisions and induced expression of the host symbiotic genes [122].

4. Other functions and applications of flavonoids

4.1 Effects and influence of flavanol rich cocoa on cognitive performance

The potential of flavonoids to alter signaling pathways enhancing neuronal function and brain connection appears to be one of the processes underlying the health benefits of frequent cocoa consumption [123].

4.2 Role for polyphenols in the prevention of degenerative diseases

Flavonoids protect the brain cells in a multitude of ways, such as by strengthening the functional neurons or by promoting neuronal regeneration. After 6-hydroxydopamine lesioning, it has been found that the citrus flavanone tangeretin preserves nigro-striatal integrity and functioning in the context of Parkinson’s disease [124].

4.3 Flavonoids in decorticated sorghum grains exert antioxidant and antidiabetic activities

Flavonoids are said to be helpful in preventing metabolic disorders including type 2 diabetes, obesity, hypertension, and several malignancies. They are primarily abundant in the bran portion (pericarp, testa, and aleurone tissues) of the plants. According to the findings, decorticated sorghum grains contain significant amounts of flavonoids, which may inhibit glucose hydrolyzing enzymes and lessen the symptoms of diabetes and its companion disorders [125].

4.4 Chrysoeriol7: a natural chemical and repellent, against brown planthopper in rice

Brown Planthopper (BPH), a rice pest, causes severe damage such as viral infections, leaf blights, nutrition loss, and tissue death, all of which have a direct impact on rice productivity. Chrysoeriol7, a secondary metabolite, has been reported to be effective against BPH by establishing resistance due to the presence of an aromatic group from the flavonoid family that emanates a distinct scent that repels BPH [126].

4.5 Quinoa-derived polyphenols regulate glucose and lipid metabolism: protecting against chronic human illnesses

Quinoa is known as the “golden grain” because of its excellent protein profile, high polyphenol and vitamin content, and health advantages such as anti-diabetic, antioxidant, and anti-obesogenic properties. Furthermore, quinoa leaves are high in phenolic compounds, which can help reduce the risk of cardiovascular disease, neurological illnesses, and diabetes. Quinoa extracts contains significant quantities of sinapinic, ferulic, and gallic acids, isorhamnetin, kaempferol, and rutin. These chemicals are associated directly to a reduction in prostate cancer cell growth and motility [127, 128].

4.6 B. juncea (L.) Czern. leaves show high flavonoid content: reducing rheumatoid arthritis caused by adjuvants

The leaves of B. juncea contain certain medicinal components that have been found to reduce the synovial inflammation as well as treat the damage caused by rheumatoid arthritis [129].

4.7 Nutritional value of Tartary buckwheat for humans and activity evaluation of its major flavonoids

Tartary buckwheat, which is produced mostly in northern India, Nepal, Bhutan, China, and central Europe, has been found to be more cold resistant and drought tolerant than regular ones. The phenolic compounds, resistant starch, and protein contained in grains, as well as interactions between these constituents, are largely responsible for reduction of the risk of a number of chronic illnesses such as cardiovascular disease, obesity, hypertension, and gallstone formation. Tartary buckwheat is resistant to pests, plant diseases, and UV-B radiation because it contains flavonoids such as rutin, vitexin, orientin, and quercetin, as well as other phenolic compounds [119].

4.8 Using purple tomato anthocyanins as new antioxidants to improve human health

It is generally known that tomatoes help to lessen the risk of developing cancer since they contain carotenoids and polyphenols. According to several recent research, anthocyanins can have a variety of impacts on the health of the eye.

Recently, in order to examine the potential impacts of genetically modified “Indigo” tomatoes on the host gut microbiota, inflammatory reactions and the signs of inflammatory bowel diseases (IBDs) were tested in an unexpected ulcerative colitis mouse model. In addition, consumption of anthocyanins, in particular, has shown a favorable correlation with a decrease in cardiovascular risk, as well enhanced the vascular health and prevented the formation of atherosclerotic plaque [130].

5. Effect of gene regulation and modification in Flavonoid production

5.1 A citrus cytochrome P450 gene, CsCYT75B1 helping to induce drought tolerance by antioxidant Flavonoid accumulation

Cytochrome P450 gene present in Citrus sinensis (CsCYT75B1) is linked with flavonoid metabolism and reported to be notably induced after drought stress. CsCYT75B1 gene when overexpressed in A. thaliana significantly increased total flavonoid content. It also enhanced antioxidant activity in the transgenic Arabidopsis plant. Diverse genes responsible for flavonoids biosynthesis showed increased induction (2–12 folds) as a result of CsCYT75B1 gene overexpression in these transgenic Arabidopsis lines. After induction of draught stress, these plants showed enhanced drought tolerance along with antioxidant flavonoids accumulation, lower level of ROS and superoxide radicals when compared to wild type plants. These transgenic lines also exhibited significantly lower levels of electrolytic leakage than wild types [131].

5.2 Selected mutagenesis of GmUGT augmented soybean resistance against leaf-chewing insects via flavonoids Biosynthesis

Seed quality and yield in Soybeans (G. max) is negatively affected by Leaf-chewing insects. Minimization of insecticide use and loss reduction can be achieved by breeding Leaf-chewing insects-resistant soybean varieties. Marker genes for QTL-M, Glyma.07 g110300 (LOC100775351) encoding UDP-glycosyltransferase (UGT) is the major deciding factor for resistance against leaf-chewing insects in soybean. It manifests loss of function in insect-resistant germplasms of soybean. Zhang Y et al. reported a study, where they have introduced CRISPR/Cas9 expression vector into the soybean cultivar Tianlong No. 1 using Agrobacterium-mediated transformation to generate Glyma.07 g110300-gene mutants. A 33-bp deletion and a single-bp insertion in the GmUGT coding region obtained from this experiment resulted in enhanced resistance to Helicoverpa armigera and S. litura. Soybean varieties further sensitive to H. armigera and S. litura was generated by upregulation of GmUGT. This particular coding sequence is also involved in providing resistance to leaf-chewing insects via alteration of flavonoid content and gene expression pattern related to flavonoid biosynthesis and defense [109].

5.3 Transferability and polymorphism of simple sequence repeats (SSRs) In the Flavonoid pathway genes of strawberry (Fragaria) and Rasberry (Rubus Sp.)

Fragaria and Rubus are two extremely popular crops, whose breeding programs are increasingly reliant on the use of functional DNA markers. Improvement in the nutritional quality and disease resistance abilities are essential challenges in breeding programs of these crops. There is a total of 118 microsatellite (simple sequence repeat-SSR) loci in the nucleotide sequences of genes that are involved in flavonoid production and pathogenicity and a count of 24 SSR markers that represent some of these structural and regulatory genes have yet been discovered. Using these markers, 48 specimens of Fragaria and Rubus, comprising unusual cultivars and wild species were examined in one of the studies to determine their overall genetic diversities. It is believed that the enhancement of anthocyanin-related phenotypes in strawberry and raspberry breeding programs may get benefited from the use of SSR markers collection as a molecular tool [132].

5.4 B. napus L.’s efficient oil production is controlled by targeted mutation of BnTT8 homologs

B. napus is an important oil crop, but despite its importance, no naturally occurring or artificially produced yellow seed germplasms have been discovered yet. Recently, in one of the studies, CRISPR/Cas9 system has been used to produce yellow mutant rapeseeds (Brassica napus). The targeted alterations of the BnTT8 gene were persistently passed down through generations, and a variety of homozygous mutants with damaged alleles of the target genes were acquired for phenotyping. BnA09.TT8 and BnC09.TT8b are the two targeted mutants of BnTT8 which were able to restore the yellow-seeded phenotype. These mutants generated seeds with increased protein and oil content, improved fatty acid (FA) composition, and no significant abnormalities in yield-related parameters [133].

6. Mediterranean crop modification techniques: from the laboratory to the field

6.1 Marker-assisted breeding (MAB)

This method/strategy selects plants and animals for breeding programs early in their development by exploiting DNA markers linked with desirable features. Thus, it significantly shortens the time required in a breeding cycle, to locate/identify variations or breeds that display the desired trait. Two separate studies in tomatoes recorded before, one using a mutant inbred line and the other using an interspecific Solanum Chmielewski population, discovered that the colorless-peely mutant on chromosome 1 is controlled by a SlMYB12-regulated transcriptional network that controls the accumulation of yellow-colored flavonoid (naringenin chalcone) in the fruit epidermis [134, 135].

6.2 Crop plants that have undergone genetic modification

Overexpression and competition with the target route, overriding rate-limiting steps, preventing the catabolism pathway of the desired product, and blocking other pathways are all part of the process of maximizing the synthesis of specialized target molecules [136]. For example, the principal flavonoid metabolic route has been studied to optimize these critical molecules in Solanum lycopersicum. Flavonoids (such as naringenin, chalcone, and rutin) are predominantly found in tomato peel, with just trace levels found in tomato flesh [137]. Ectopic expression of a single structural gene (CHI) or many structural genes (CHS, CHI, F3H, and FLS) increased the number of flavonols (quercetin- and kaempferol- glycosides) in tomato peel and flesh. The co-expression of onion CHI in the purple tomato Delila bHLH and Rosea1 R2R3-MYB recently transformed flavonoid to flavanol and boosted anthocyanin concentration [138].

6.3 Modern breeding methods (MBTs)

The rapid growth and usage of genome editing tools has opened up new ways for introducing change and influencing gene expression at multiple levels, including transcription, mRNA processing, and mRNA translation. CRISPR/Cas9 technology has primarily been used to study plant biosynthetic potential by blocking competing biosynthetic pathways and changing metabolite flux towards target chemical synthesis. One such example, the Salvia miltiorrhiza rosmarinic acid synthase (SmRAS) gene was edited using the CRISPR/Cas9 technology [139]. This mutation led to a decrease in phenolic acid content, such asrosmarinic acid, and an increase in its precursor, 3,4-dihydroxyphenyl lactic acid, especially in the homozygous variety. Another study in S. miltiorrhiza employed CRISPR/Cas9 to knock out the SmCPS1 gene, which codes for a diterpene synthase involved in tanshinone synthesis. This was done to investigate the feasibility of encouraging the accumulation of the substrate for taxol synthesis as tanshinones and taxol sharing the same precursor (Geranylgeranyl Pyrophosphate) [140].

6.4 Regulation of specific metabolism and transcription factor modulation

The functional characterization of TFs involved in the control of anthocyanin metabolism is an outstanding example of how transcriptional regulatory research can be carried out to fine-tune specialized metabolic pathways. The WD-repeat/bHLH/MYB complex regulates anthocyanin accumulation in plants by positively regulating the gene expression of DFR, anthocyanin synthase, and glucosyltransferase. This molecular pathway, conserved across many species via orthologous TFs, which is essential in the color determination of flowers and fruits such as those of apples, grapes, and oranges [141].

6.5 A target for molecular biology-based breeding and other biotechnological approaches: metabolism of plant glandular Trichomes

Trichomes are specialized biosynthetic, storage structures composed of epidermal extensions found on the surface of aerial plant parts. They can exist in both non-glandular and glandular trichrome forms and are widespread throughout the plant kingdom. Glandular trichomes can produce, store, and release exudates containing a wide range of chemo-diverse compounds such as essential oils, oleoresins, phenols, glycerids, and extremely complex terpenes [142]. Efficient isolation techniques such as laser microdissection pressure catapulting (LMPC) has aided with the separation and enrichment of specific cell types, such as multicellular glandular trichomes allowing chemical, transcriptional, and biosynthetic studies to focus solely on specialized glandular metabolites [143].

7. Future prospect

Flavonoid-rich diets have been shown to delay the onset of dementia-related diseases and prevent age-related cognitive decline. Alterations in cerebral blood flow caused by flavonoids, progenitor cells, quantitative changes in brain stem cells and gray matter density, as well as electrophysiological anomalies, can all be examined utilizing imaging and spectroscopic tools such as NMR and MRI. All of these initiatives will result in mechanism-based linkages between flavonoid medication and brain activities, as well as therapeutic dosage data. Flavonoids have been shown to concentrate in the brain and activate Akt-CREB and ERK-CREB mediated memory, making them interesting therapeutic candidates for memory enhancement. Flavonoid-loaded nanoparticles, liposomes, or other nanocarriers can pave the way for flavonoids in the future by increasing the half-life of flavonoids in organisms, drug delivery strategies boost their effects. Moreover, since flavonoids are naturally occurring dietary components, therapeutically effective amounts of flavonoids can have varying lethal effects on cancer cell lines as well as tissues due to cancer heterogeneity. Ototoxicity is a side effect of chemotherapeutic medicines that patients experience during chemotherapy. According to one study, epigallocatechin-3-gallate protects patients against ototoxicity, which can also pave the door for flavonoids to be utilized as additive treatments to reduce the side effects of chemotherapy drugs. It is predicted that addressing difficulties like as bioavailability and metabolism, building physiologically acceptable in vitro models, determining the effects of processing, standard measuring methodologies, and adequate clinical biomarkers will surely influence the future of flavonoid research [144, 145, 146].

8. Conclusion

The use of phytochemicals, particularly flavonoids, in disease prevention and treatment is well documented. Each flavonoid discovered in nature has distinct chemical, physical, and physiological features. The structure-function link of flavonoids is the pinnacle of major biological functions. Incorporation of flavonoid in various horticulture species and the practice of molecular plant breeding will bring in a great revolution in the coming future. This will elevate numerous therapeutic potentials of several plant species and would thereby provide aid in the advancement of the food, pharmaceutical, floricultural, and chemical industries. Additional accomplishments will bring in other newer insights and will almost probably usher in a new era of flavonoid-based pharmacological agents for the treatment of numerous infectious and degenerative disorders.