Flavonoids – flowers, fruit, forage and the future

ABSTRACT

Flavonoids are plant-specific secondary metabolites that arose early during land-plant colonisation, most likely evolving for protection from UV-B and other abiotic stresses. As plants increased in complexity, so too did the diversity of flavonoid compounds produced and their physiological roles. The most conspicuous are the pigments, including yellow aurones and chalcones, and the red/purple/blue anthocyanins, which provide colours to flowers, fruits and foliage. Anthocyanins have been particularly well studied, prompted by the ease of identifying mutants of genes involved in biosynthesis or regulation, providing an important model system to study fundamental aspects of genetics, gene regulation and biochemistry. This has included identifying the first plant transcription factor, and later resolving how multiple classes of transcription factor coordinate in regulating the production of various flavonoid classes – each with different activities and produced at differing developmental stages. In addition, dietary flavonoids from fruits/vegetables and forage confer human- and animal-health benefits, respectively. This has prompted strong interest in generating new plant varieties with increased flavonoid content through both traditional breeding and plant biotechnology. Gene-editing technologies provide new opportunities to study how flavonoids are regulated and produced and to improve the flavonoid content of flowers, fruits, vegetables and forages.

Introduction

Flavonoids are an incredibly diverse group of plant secondary metabolites, which include the well-known pigments that colour our fruits and flowers, as well as less conspicuous metabolites that provide protection from UV-B radiation, antioxidant activity, or act as signalling molecules for plant-microbe symbioses (Figure 1A).

Figure 1.

Physiological roles of flavonoids in plants and their biosynthesis. A, Flavonoids perform numerous physiological roles in plants. Flavones or flavonols act as UV-B sunscreens and are induced by high irradiance white light as well as UV-B and UV-A. Isoflavonoids are signalling molecules for rhizobia, forming root nodules (nitrogen fixation) in legumes. Anthocyanins provide visual cues to pollinators and seed distributers. Bees have preferences for purple-blue flowers and/or flowers with high contrast patterns with other pigments, including UV-B absorbing flavones or flavonols; bird-pollinated flowers are less attractive to bees and are typically red with low UV-B absorption, while moth-pollinated flowers are typically white with high UV-B absorption. Anthocyanins are often also produced during senescence, or by abiotic stresses such as nutrient deficiency (N, P), high light intensities or cold. Proanthocyanidins are unpalatable, deterring insect pests in vegetative tissues and consumption of immature fruit. B, The flavonoid biosynthetic pathway begins with chalcone synthase (CHS). Many tissues contain multiple classes of flavonoids, which share common biosynthetic steps. For example, CHS, CHI and F3H are common to flavonol (green), proanthocyanidin (brown) and anthocyanin (purple) biosynthesis. Coordinated expression of genes in each these pathways is necessary for metabolite biosynthesis to occur. The activities of flavonoid 3′hydroxylase (F3′H), flavonoid 3′5′hydroxylase (F3′5′H) add hydroxyl groups to the 3 and 5 position of the B-ring of dihydroflavonols which can progress through towards anthocyanins, proanthocyanidins, but also to generate flavonols with different hydroxylation patterns (not shown). The three main anthocyanidins are pelargonidin (Pel), cyanidin (Cy) and delphinidin (Del), which can become glycosylated and decorated to produce anthocyanins, or undergo reduction to become flavan 3-ols for proanthocyanidins. Some biosynthetic steps are not fully resolved (?), such as those involved in converting aurones into auronidins, and for polymerising flavan 3-ols into oligomeric and polymeric proanthocyanidins. Abbreviations: chalcone synthase (CHS), chalcone Isomerase-Like (CHI-L), chalcone isomerase (CHI), flavone synthase (FNS), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′5′-hydroxylase (F3′5′H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS)/leucoanthocyanidin dioxygenase (LDOX), leucoanthocyanidin reductase (LAR), anthocyanidin reductase (ANR), flavonoid 3-O-glucosyltransferase (3GT), flavonoid 5-O-glucosyltransferase (5GT), acyltransferase (AT), methyltransferase (MT), stilbene synthase (STS), aurone synthase (AS), isoflavone synthase (IFS).

The best-known flavonoids are undeniably the anthocyanins, which provide red-purple-blue colours to most groups of plants. Their bright colours provide visual cues to attract pollinators and to signal when fruit are ripe and ready to eat, so seeds may be distributed. Furthermore, their physiological roles extend beyond visual signals, and anthocyanins are produced in groups of plants lacking flowers and fleshy fruits, such as ferns. Anthocyanins accumulate in vegetative tissues in response to a variety of abiotic (cold, high light intensity, nutrient deficiency) and biotic (insect herbivory, disease) stresses. It is generally accepted that anthocyanins increase plant resilience in the presence of stressors; however, the mode of action is not clear. There are several hypotheses for how they function, which are not mutually exclusive. In vegetative tissues anthocyanins are often produced in epidermal or sub-epidermal cell layers, screening light from underlying chloroplasts within the most photosynthetically active cells. During stress conditions, chloroplast function is likely to be impaired and unable to cope with excess quanta without generating significant amounts of reactive oxygen species (ROS). Reduced light results in less ROS. Anthocyanins and other flavonoids also have strong antioxidant activities in vitro, suggesting ROS neutralisation may be another mode of activity within plant cells, in addition to screening roles (Agati et al. 2021; Davies et al. 2018). Other notable examples of anthocyanin induction include wound-induced pigmentation and autumnal colours.

The flavones and flavonols are important UV-B absorbing compounds that act as UV-sunscreens in vegetative tissues (Li et al. 1993; Mehrtens et al. 2005), a function that is conserved between the two most distantly related groups of land plants, the liverworts and flowering plants (Clayton et al. 2018), and may predate the occurrence of anthocyanins (Berland et al. 2019). These compounds can have additional roles in flowers, where they can produce UV-absorbing patterns visible to insect pollinators (Thompson et al. 1972; Davies et al. 2012). Flavones and flavonols are also effective co-pigments, physically interacting with anthocyanin pigments through stacking of their phenolic rings, which increases the intensity and alters the hue (blue-shifted) of the colour from anthocyanins (Tanaka et al. 2008). The effects of co-pigmentation can be truly striking and are responsible for generating true blue colour present in cornflowers with the same base anthocyanin present in red roses (Shiono et al. 2005).

Proanthocyanidins (PAs) are produced from a branch off the biosynthetic route to anthocyanins. They range from favan-3-ol monomers (e.g. catechin, epicatechin), to oligomers (dimers and trimers) through to large polymers. They inhibit herbivory from insect pests and herbivores because they have strong protein binding properties – which imparts astringency, dry-mouth sensation when consumed, making them unpalatable. They often form part of the polymers in seed coats to provide strength and resilience, and they are also commonly produced in fruit.

The biosynthesis of flavonoids begins with the aromatic amino acid phenylalanine, and is part of the larger phenylpropanoid biosynthesis pathway, which produces lignin, phenolic acids and volatiles, in addition to flavonoids. The first committed step to flavonoid biosynthesis (Figure 1B, refer for enzyme abbreviations) is catalysed by CHS, after which a series of isomerisation (CHI), hydroxylation (F3H, F3′H, F3′5′H), reduction (DFR, LAR, ANR) or oxidation (FLS, ANS) reactions occur to generate various flavonoid compounds, which are further modified or ‘decorated’ by glycosylation, methylation or acylation. Martin and Gerats (1993) coined the term ‘early’ and ‘late’ flavonoid biosynthesis genes. The ‘early biosynthetic genes’ were steps that did not show substantially reduced expression in anthocyanin regulatory mutants (typically CHS, CHI, F3H), while the ‘late biosynthetic genes’ showed substantial or a complete loss of expression (DFR, ANS, UFGT). This term has been widely adopted in the literature, but is often misinterpreted to suggest the early biosynthetic genes are not targeted by regulators of anthocyanin biosynthesis. More recently, studies have identified genes encoding non-enzymatic biosynthetic proteins, such as CHI-Like and the PR10 proteins, which are necessary for efficient production of flavonoids (Muñoz et al. 2010; Morita et al. 2014; Ban et al. 2018; Clayton et al. 2018; Berland et al. 2019), possibly by binding metabolite intermediates and channelling them to enzymes (e.g. CHS) efficiently, with correct stereochemistry (Dastmalchi 2021). The regulation of different branches of flavonoid production is complex, nuanced, and involves redundant regulation of some biosynthetic genes, particularly genes common to multiple pathways. Central to the regulation of flavonoids are R2R3-MYB transcription factors, which have diversified into sub-groups that have specialised in regulating the production of different metabolites (Figure 2A).

Figure 2.

R2R3 MYB genes regulate flavonoid biosynthesis. A, MrBayes Phylogenetic tree of R2R3-MYB subgroups (posterior probabilities shown), rooted on MpMYB14 from Marchantia polymorpha. Notable examples of each subgroup (SG) are indicated. * indicate subgroups that form MYB-bHLH-WDR (MBW) complexes. B, Several branches of the flavonoid biosynthetic pathway are regulated by R2R3-MYB proteins as part of MBW, in particular those belonging to SG5 (proanthocyanidins) and 6 (anthocyanins) are well characterised. The stoichiometry isn’t clear, but at least two different MYB proteins can be present, bound through their interactions with dimerised bHLH proteins. (1) If all three components are present, they can form a complex, which (2) can then bind the promoters of target genes and activate transcription, ultimately resulting in phenotypes such as metabolite production. (3) bHLH and WDR genes are themselves regulated by MBW complexes (reinforcement). (4) R2R3- and R3-MYB repressor genes are regulated by MBW (feed-back inhibition). They can interfere with the correct assembly of the MBW complex, by competing with MYB activator proteins for bHLHs. R2R3-MYB repressors can also be incorporated into MBW complexes and recruited to promoters where C-terminal motifs (e.g. EAR, TLLFR) inhibit transcription.

In this review, we will focus on key flavonoid metabolites that are important for the quality of flowers, fruits/vegetables and forages, with a particular focus on the regulation of these compounds and ways in which their content and profiles have been improved to date, and might be further enhanced in future.

Flowers

There are a great variety of flower colours in nature, and the majority of these are based on flavonoid pigments (Andersen and Markham 2006). In particular, the anthocyanins account for most pink through to blue colouration. While carotenoids are the basis of most yellow or orange flower colours, there are some species in which these colours are from the production of flavonoids. Specifically, yellow can be produced by the chalcones or aurones, and oranges may be from the anthocyanin pelargonidin (if cellular conditions are appropriate) or the 3-deoxyanthocyanins. Aurones and 3-deoxyanthocyanins are of limited occurrence as flower pigments, but they are important pigments in non-flowering plants (Berland et al. 2019; Davies et al. 2020; Ogutcen et al. 2020), with 3-deoxyanthocyanins being the anthocyanin types founds in ferns and mosses (Güngör et al. 2021).

Ornamental species, in particular Antirrhinum and petunia, alongside maize, were key model species for pioneering discoveries on flavonoids. These include early studies on the fundamental principles of genetic inheritance, the understanding of the chemical basis of pigmentation, discovery of the biosynthetic and regulatory genes, early genetic modification (GM) experiments, and the discovery of RNA-based gene silencing (Davies et al. 2012). In particular, anthocyanin biosynthesis in flowers offered a visual phenotype so non-lethal mutations could be easily identified.

Nearly all of the genes encoding the enzymes required for the production of the common flavonoids found in flowers have now been characterised (Figure 1B). This includes those for many of the secondary modifications of anthocyanins (glycosylation, acylation and methylation) and for some of the other coloured flavonoids, such as aurones (Tanaka et al. 2008). Knowledge gaps include the genetics of some of the complex anthocyanin modifications associated with blue flowers and the anthocyanidin types of rare occurrence, such as the 6- and 8-hydroxyanthocyanins (Andersen and Markham 2006). The absence of such compounds in the classical model species (with the exception of aurones in Antirrhinum) have limited discovery of the associated biosynthetic pathways; however, advances in sequencing technologies and metabolomics are enabling rapid gene discovery in non-model species (Hodgson et al. 2019; Nett et al. 2020; Delli-Ponti et al. 2021; Hawkins et al. 2021).

Elucidating anthocyanin structures and how they behave to confer specific colours in the chemical environment of the vacuole has been the subject of a great deal of research. Anthocyanins can transition between chemical forms with differing colours or that are colourless, largely determined by pH. The influence of pH is readily apparent on isolated anthocyanins (Houghton et al. 2021) but can also be seen directly in some flowers. Notably, petals of Japanese morning glory (Ipomoea tricolor) start as reddish-purple in the buds but become bright blue in open flowers, accompanying a pH change from 6.6 to 7.7 generated by a tonoplast-located Na⁺/H⁺ exchanger (Yoshida et al. 1995; Fukada-Tanaka et al. 2000). At the physiological pH typical of the vacuole (about pH 5.5), simple 3-O-glucosylated anthocyanins would shift towards colourless or pale yellow forms. To encourage anthocyanins to form stable coloured forms, plants have evolved mechanisms to change the local environment around the chromophore to reduce hydration. The most significant mechanisms are secondary modifications to the anthocyanin itself and surrounding the molecule with co-pigments: typically colourless flavonoids such as flavonols or flavones. This has been an evolutionary driver for anthocyanin structural diversity, and more than 1000 different anthocyanin structures have been reported (Trouillas et al. 2016). There has been a particular, long-standing, interest in understanding how blue colours are formed in flowers. The chemistry and structural diversity of anthocyanins and how they behave in solution or with co-pigments is an area of much current interest. Not only is it a fascinating area of biology, combining chemistry, molecular biology and ecology, but it also has relevance to colour stability in food products, such as wine, and to the development of effective natural food colourants (Trouillas et al. 2016; Dangles and Fenger 2018; Houghton et al. 2021; Trunschke et al. 2021).

Because of the visual phenotypes and the well characterised biosynthetic genes, the flavonoid pathway was one of the first targets for genetic modification (GM). In some cases, it was also thought that GM ornamental crops would have easier regulatory and consumer acceptance. Two notable examples are GM petunia modified to accumulate pelargonidin and has associated orange flower colours, and the extensive GM experiments for generating blue flower colour through the introduction of the F3′5′H, notably for the production of a blue rose. These examples also illustrate the intended and unintended commercial release of GM products.

One of the earliest examples of engineering of a plant trait was the introduction into petunia of the maize DFR gene (the A1-DFR) (Meyer et al. 1987). The maize DFR can use any of the three dihydroflavonols as substrates, but the petunia DFR does not accept dihydrokaempferol, preventing the production of pelargonidin-based anthocyanins. The experiment was successful in conferring significant pelargonidin production in petunia transgenics, and the initial lines were used to breed many attractive new varieties. However, these were not commercialised because of the barriers to market entry for GM products (Oud et al. 1995). Nevertheless, in the mid-2010s petunia varieties with strong orange flower colours started appearing for sale around the world, with significant uptake by consumers in the USA and Europe. In 2015 and 2016, researchers who had worked on GM of the flavonoid pathway noticed the orange-coloured petunias in flower boxes decorating the Helsinki railway station in Finland. When they analysed these, they found them to be direct descendants of the first GM experiments that introduced the maize A1-DFR sequence (Bashandy and Teeri 2017). As these were unapproved GM products, a worldwide recall of the plant lines followed. Recently, the orange petunias have made a reappearance. In January 2021 the US Department of Agriculture deregulated 15 A1-DFR petunia lines (APHIS 2021), and varieties are now for sale that openly declare they contain the ‘A1DFR gene’ (e.g. Westflowers 2021).

Many ornamental species lack production of delphinidin-based anthocyanins, and associated purple-blue flower colours, including the three most valuable cut flowers: rose, carnation and chrysanthemum. These species lack the F3′5′H for generating the substrates necessary for delphinidin formation. Japanese and Australian researchers started a project in 1990 that identified the F3′H and F3′5′H genes and subsequently introduced them into rose, carnation and chrysanthemum (Katsumoto et al. 2007; Tanaka and Brugliera 2013; Noda 2018). The amount of delphinidin-based anthocyanins produced as a percentage of total anthocyanins varied among the transgenics, as did the resultant flower colours. Additionally, even with delphinidin production, other cellular factors need to be suitable to generate flowers with strong blue colours. These include appropriate vacuolar pH, appropriate co-pigments, and/or extensive secondary modifications to the core anthocyanidin, specifically, glycosylation and then acylation on the sugar residues (Andersen and Markham 2006; Houghton et al. 2021; Lu et al. 2021). For carnation, there were target genotypes available that contained mutations in endogenous anthocyanin biosynthetic genes that enabled the engineering of strong delphinidin production and excellent flower colour outcomes (Tanaka and Brugliera 2013). These transgenic carnations were commercialised in many regions of the world in the late-1990s, and have been joined by more cultivars with additional GM: more than 25 million GM carnations are now sold world-wide every year (Tanaka and Brugliera 2013). Similar approaches generated transgenic chrysanthemums with violet-blue flower colours (Brugliera et al. 2013). For rose, achieving blue flower colours from the introduction of the F3′5′H transgene proved challenging, even though cultivars that had relatively higher pH and no F3′H activity were identified as the recipients for the transgenes (Tanaka and Brugliera 2013). Trials of alternative F3′5′H transgenes and modification of other pathway factors did result in transgenics with almost 100% delphinidin production and mauve-blue petal colours, and the Suntory’s blue rose ‘Applause’ was commercialised in 2009 (Tanaka and Brugliera 2013). Addition of transgenes for the anthocyanin secondary modification activities (e.g. Kallam et al. 2017; Lu et al. 2021) could encourage a shift towards the blue spectrum. This was illustrated by the production of chrysanthemum with blue flower colours by the introduction of an uridine diphosphate (UDP)–glucose: anthocyanin 3′,5′-O-glucosyltransferase transgene along with the F3′5′H (Noda et al. 2017). Effective gene technologies for engineering floral vacuolar pH have proved elusive. Improving the blue flower colour of GM ornamentals thus remains an area of interest, as does conferring aurone production for yellow colours (Hoshino et al. 2019), and the possibilities offered for potential non-GM plant lines from gene editing using CRISPR technologies.

Anthocyanin regulation and the ‘MBW’ complex

The transcription factors activating anthocyanin biosynthesis in flowers were among the first to be characterised for plants, and the flavonoid transcriptional regulatory systems for flowers are now well characterised for many species. Initial studies were for Antirrhinum and petunia, but many other systems have since been added to these. Notable among these are Mimulus (Erythranthe spp.), which has become a model for the genetics of plant-pollinator interactions (Yuan 2019), and Lilium and Phalaenopsis (Yamagishi et al. 2014; Hsu et al. 2015; Yamagishi and Sakai 2020), for which a body of research has developed explaining mechanisms regulating patterning of pigmentation. The identification of regulatory mutants for genes encoding R2R3-MYB and bHLH transcription factors, and a WD-Repeat protein were key discoveries. The strong genetic data from flower colour models were reconciled when protein interaction studies in maize and Arabidopsis suggested these transcription factors act together within an MYB-bHLH-WDR (MBW) complex (Figure 2B). The Antirrhinum and petunia research contributed to establishing the following underpinning principles of the model that has developed for the regulatory system for anthocyanins:

1. The central components of the direct MBW regulatory module are R2R3-MYBs, bHLH and WDR proteins. Identification and analysis of mutants for these genes demonstrate that all three components are necessary for regulating anthocyanin biosynthesis (Goodrich et al. 1992; de Vetten et al. 1997; Quattrocchio et al. 1999; Spelt et al. 2000). The role of the WDR protein may be structural, providing a scaffold to stabilise the complex. These MBW complexes bind promoters of target genes and activate transcription.

2. R2R3-MYBs provide specificity to the complex, acting with bHLH and WDR proteins that are common to other regulatory pathways. R2R3-MYBs of sub-group 6 (SG6) typically activate anthocyanin production, while those belonging to SG5 and MYBPA1 clades regulate PA production. MBW complexes containing other R2R3-MYB SGs control vacuolar acidification (pH), seed coat mucilage and, in brassicas, trichome and root hair development (Koes et al. 2005; Ramsay and Glover 2005).

3. The R2R3-MYBs are the key determinants of the spatial and temporal occurrence of anthocyanins in flowering plants. Ectopic MYB expression introduces pigmentation to tissues where it was previously absent (e.g. vegetative tissues), with numerous examples from transgenic studies and naturally occurring gene activations in ornamental, crop and model species (Borevitz et al. 2000; Schwinn et al. 2006; Espley et al. 2007; Butelli et al. 2012; Zhou et al. 2014; Zheng et al. 2021). In contrast, overexpressing bHLH genes alone enhances the existing patterns that are determined by endogenous MYBs (Quattrocchio et al. 1998; Robbins et al. 2003; Albert et al. 2009).

4. Small gene families are present for the SG6 R2R3-MYBs genes controlling anthocyanins, and these have diversified in activation strength and targets and gene expression domains. Gene duplication has played an important role in generating additional genes that can adopt new expression domains. Control of floral patterning by multiple spatially and temporally distinct SG6 R2R3-MYBs is widely conserved in flowering plants. Each gene typically controls a single pattern, e.g. vein-associated anthocyanins, spots, anthers/pollen and full petal colourations, and another associated with light/stress-regulated vegetative anthocyanin production (Quattrocchio et al. 1999; Schwinn et al. 2006; Yamagishi et al. 2010; Albert et al. 2011, 2015; Yuan et al. 2014; Zhang et al. 2021).

5. The bHLH genes may contribute to major spatial domains. This includes epidermal specificity, localising anthocyanins to conical cells in petals, and determining domains (e.g. tube/lobe) in flowers. Antirrhinum with mutations of Delila (Del) have bicoloured flowers lacking anthocyanins in the tube but have full-coloured lobes, which is regulated by a second bHLH, Incolorata I (Inc I) (Albert et al. 2021). Additionally, expression of bHLH genes in petals (e.g. AmDel, PhAN1) is restricted to epidermal cells (Goodrich et al. 1992; Quattrocchio et al. 2006), and overlapping expression with R2R3-MYBs genes can generate 2D patterns such a vein-associated pigmentation (venation) (Shang et al. 2011; Davies et al. 2012).

6. Two major sub-clades of bHLH genes contribute to anthocyanin and PA regulation. Both sub-clades belong to bHLH subgroup IIIf, but further separated into what have become known as the bHLH1 and bHLH2 subclades (Albert et al. 2014). bHLH1 genes include JAF13, Del, GL3/EGL3 and R/B, while bHLH2 includes AN1, Inc I, TT8 and In, from petunia, Antirrhinum, Arabidopsis and maize, respectively. While both clades participate in MBW complexes and can activate flavonoid genes (e.g. DFR), in some plants (e.g. Solanaceae) bHLH1 alone are not sufficient to regulate anthocyanins, and bHLH2 activity is also required (Montefiori et al. 2015). bHLH2 genes are also essential for regulating PAs and vacuole acidification, demonstrating subfunctionalised roles for these regulators (Nesi et al. 2000; Spelt et al. 2002).

7. Differential interaction strengths occur for individual bHLHs with the WDR protein. Enhanced activation activity against a DFR promoter was observed when AmWDR1 was co-infiltrated with Inc I (bHLH2) but not Del (bHLH1), demonstrating differential requirements for the WDR (Albert et al. 2021). Furthermore, wdr mutants in maize and rice have reduced rather than an absence of anthocyanins (Carey et al. 2004; Yang et al. 2021), possibly because the bHLH1 regulators are still capable of forming MYB-bHLH complexes.

8. Hierarchical regulation occurs between the different components of MBW regulatory network. The MBW complex promotes expression of bHLH2 and MYB repressor genes (R3-MYB and SG4 R2R3-MYB) (Baudry et al. 2006; Albert et al. 2014). In species where bHLH1 genes are not sufficient to regulate anthocyanins alone, their role may be primarily to activate or enhance bHLH2 expression. A recent example in Antirrhinum demonstrates that despite bHLH1 (Del) being an efficient anthocyanin regulator, it also regulates bHLH2 (Inc I), expanding expression into the flower tube (Albert et al. 2021).

9. R2R3-MYBs and R3-MYBs with repressive actions towards the MBW activation complex fine-tune anthocyanin regulation and contribute to determining floral pigment patterning. Initially described for anthocyanin production (Aharoni et al. 2001; Albert et al. 2014; Gates et al. 2018), repressors also contribute to PA regulation (Huang et al. 2014; Albert 2015; Jun et al. 2015). Hierarchical regulation of repressors by MBW complexes provides feedback regulation. Differential regulation of individual MYB repressor genes by anthocyanin or PA MYBs can result in specialised roles (Albert 2015). For example, R2R3-MYB repressors act cell autonomously, while many R3-MYB can move between cells and suppress MBW activity in adjacent cells (Albert et al. 2014). Combined with their hierarchical regulation, this allows for the establishment of complex patterning through reaction/diffusion-based models (Meinhardt and Gierer 1974; Davies et al. 2012; Albert et al. 2014), e.g. spot formation in Mimulus (Ding et al. 2020).

10. Additional TFs may join the core MBW complex. Recent studies suggest that WRKY transcription factors may act as co-activators. The best example is for vacuole acidification in petunia (Verweij et al. 2016), but this may occur more widely (Lloyd et al. 2017; Paauw et al. 2019).

CRISPR-mutagenesis and anthocyanins production in ornamental crops

The knowledge accumulated for the genetic basis of anthocyanin production in flowers provides a tremendous resource for breeding programmes to improve flower colour phenotypes in commercial ornamental species. Marker Assisted Selection (MAS) is established for some ornamental species, but application of genomic sequence-based approaches has been slower than for the major food crops, and can be challenging for ornamental crops that are a mix of several sexually compatible species. However, progress is being made. Whole Genome Sequences are appearing at a rapid rate for ornamental crops, or their diploid relatives, with sequences published for at least 15 species (Giovannini et al. 2021). Moreover, GWAS has been used successfully for identifying SNPs associated with floral traits in chrysanthemum, an autohexaploid (Chong et al. 2016). Lack of genomic information and challenging genetic backgrounds of some ornamental crops currently provide limitations for CRISPR approaches. However, there are already several examples of gene editing directed at altering flavonoid production to change flower colour, including targeting DFR in Ipomoea nil (Watanabe et al. 2017) and the F3′H in Torenia fournieri (Nishihara et al. 2018). The types of novel flavonoid-based flower phenotypes deliverable by current gene editing approaches (knockouts) are more limited than those offered by GM approaches. The GM plants for sale with dramatic novel orange and blue phenotypes require gain of function transgenes. However, some opportunities, such as generating white flowers, altering co-pigmentation, or knockout of the F3′H and F3′5′H to encourage the production of cyanidin or pelargonidin-based anthocyanins should be possible. Indeed, proof of concept of changes to anthocyanin types and accompanying flower colour in Torenia hybrida, Cyclamen persicum and gentian (Gentiana triflora × Gentiana scabra) transgenics has already been demonstrated (Ueyama et al. 2002; Boase et al. 2010; Nakatsuka et al. 2010; Nishihara et al. 2018). CRISPR has also been used to mutate the F3′H gene in poinsettia (Euphorbia pulcherrima) to shift the colour from red towards orange-red (Nitarska et al. 2021), albeit the coloured organs here are bracts rather than the flowers themselves. Other knockout applications could remove branches of the flavonoid pathway that compete for anthocyanin substrate flow, alter co-pigmentation, or remove transcription factors with a repressive action of the pathway. CRISPR will also have many non-pigmentation-related applications in ornamentals, such as generating sterile flowers to prevent unintended spread of pollen (Shinoyama et al. 2020).

Fruit

Fruit are rich sources of phytochemicals beneficial to human and animal health.

This includes essential vitamins, such as vitamin C and pro-vitamin A/β-carotene (a carotenoid pigment), as well as flavonoids (PAs, anthocyanins, flavonols) and polyphenolics (chlorogenic acid, stilbenes). The presence of multiple polyphenolic metabolites from similar biosynthetic routes means their production is tightly coordinated and regulated, both spatially and temporally.

Proanthocyanidins

Proanthocyanidins (PAs) (also known as condensed tannins) are colourless polymers present in vegetative tissues, seed coats and unripe fruit. They are responsible for the bitter and astringent flavour of unripe fruit, deterring consumption by herbivores until ripening, when seeds are mature and ready for dispersal. Many domesticated fruit, vegetable, grain and pulse varieties have been selected for reduced PA content, because of the unpalatability high concentrations of PAs confer (Paauw et al. 2019; Albert and Allan 2021), despite PAs being associated with numerous health benefits (Aron and Kennedy 2008). Common freshly consumed fruit with notable PA content include blueberries, grapes, apples and pomegranates, as well as fruit that are processed into juices, cider, wine, coffee and chocolate (Gu et al. 2004). In wine grapes, PAs are a quality trait conferring depth and structure of taste and mouthfeel into complex wines that will mature and age well. As such, grape research has made a substantial contribution to our understanding of PA biosynthesis and regulation in fruit.

PA biosynthesis is regulated by an MBW complex, similar to anthocyanins (Baudry et al. 2004) (Figure 2B). The key difference is the type of MYB gene included in the complex; SG5 R2R3-MYB for PA instead of SG6 for anthocyanins, which alters the target specificity of the complex. In Arabidopsis, the MBW complex for PA regulation consists of TRANSPARENT TESTA 2 (TT2) (SG5 MYB), TT8 (bHLH) and TRANSPARENT TESTA GLABRA1 (TTG1: WDR), which regulate ANR and LAR, in addition to genes shared with anthocyanin biosynthesis (Figure 1B) (Walker et al. 1999; Nesi et al. 2000, 2001; Baudry et al. 2004). Homologous SG5 MYB genes (sometimes referred to as MYBPA2) have subsequently been identified as key PA regulators in many fruit crops (Terrier et al. 2009; Akagi et al. 2010; Schaart et al. 2013; Gesell et al. 2014).

In many species, full regulation of PA biosynthesis appears to require SG5/MYBPA2 regulators as well as additional classes of MYB. This includes MYBPA1 (Bogs et al. 2007) and PAR/MYBPA3 genes (Verdier et al. 2012; Karppinen et al. 2021), which are both absent in Arabidopsis, and MYB5/PH4 clade genes (Figure 2A), which have some members able to regulate core genes within the flavonoid pathways (Deluc et al. 2008; Gonzalez et al. 2009; Liu et al. 2014), while others control vacuole acidification (Quattrocchio et al. 2006; Cavallini et al. 2014; Zhang et al. 2019). The MYBPA1 is particularly interesting because early studies from grape showed VvMYBPA1 could complement the Arabidopsis tt2 mutant (Bogs et al. 2007). Functional studies of MYBPA1 regulators from a variety of fruit species show they regulate F3′5′H and ANR (Bogs et al. 2007; Akagi et al. 2010; Zifkin et al. 2012; James et al. 2017; Lafferty et al. 2022), but they are usually not sufficient to induce PA accumulation in tissues where they are not normally produced (Karppinen et al. 2021; Lafferty et al. 2022), contrasting with SG5 MYBs, which typically can (Mellway et al. 2009; Hancock et al. 2012). Resolving this has been challenging, since mutants of MYBPA1 genes have not been identified or generated, and because some of their target genes are redundantly regulated by other MYBs. However, recent findings in Vaccinium spp. suggest that MYBPA1 proteins may have important roles regulating genes in common to both PA and anthocyanin biosynthesis, acting as co-regulators that boost expression of key bottlenecks (e.g. CHS, F3′5′H, ANS) (Günther et al. 2020; Karppinen et al. 2021), roles also suggested for MYB5. Hierarchical regulation of MYBPA1 by SG5/MYBPA2 genes is conserved between grape, poplar and blueberry (Terrier et al. 2009; James et al. 2017; Lafferty et al. 2022), resulting in co-regulation that allows MYBPA1 to assist with activating core flavonoid genes, while SG5/MYBPA2 MYBs must also regulate additional genes necessary for PA biosynthesis, transport or polymerisation (Lafferty et al. 2022).

The bHLH and WDR components are highly conserved between species, likely because they have shared functions with other pathways (anthocyanins, vacuolar pH, seed coat mucilage). Yet in cultivated crops, selections for mutations in these genes has occurred, despite producing pleiotropic effects. In Citrus spp., mutant alleles of the bHLH gene Noemi result in ‘acid-less’ fruit (perceived sweeter), but also eliminates anthocyanins and PAs. This is because Noemi (bHLH2) acts within multiple MBW complexes, containing distinct MYB genes for each pathway (Butelli et al. 2019; Strazzer et al. 2019; Zhang et al. 2019). Similarly, mutation in Mendel’s ‘A’ gene in pea (Pisum sativum), which encodes a bHLH2 protein, or ‘A2’, encoding a WDR protein, results in loss of floral anthocyanins and seed coat pigmentation (likely PAs) (Hellens et al. 2010), while in Sorghum, mutations in the WDR are responsible for the loss of PAs and anthocyanins (Wu et al. 2012).

Anthocyanins

Anthocyanins in fruit provide important visual signals to animals that fruit is ripe, a reward to enable distribution of viable mature seeds. As such, anthocyanin pigments often accumulate rapidly at the onset of the ripening processes, regardless of whether the fruit is climacteric or non-climacteric.

The regulation of anthocyanins in fruit share many similarities with that described from flower colour models (MBW complex, multi-gene families etc.). Early observations from fruit suggested that differences existed for those genes considered ‘late’ biosynthetic genes, or more properly, those that appeared to limit anthocyanin accumulation – usually glucosyltransferases (UFGTs) (Boss et al. 1996; Takos et al. 2006; Montefiori et al. 2011). This likely reflected that many of the flavonoid biosynthesis genes were already expressed, as part of regulatory modules regulating flavonols and PAs (Bogs et al. 2007; Czemmel et al. 2009; Terrier et al. 2009).

Just as PA regulation includes multiple MYBs to fully regulate the pathway, this may also be true for anthocyanin regulation in many fruit species. Silencing MYBPA1 in bilberry (Vaccinium myrtillus) resulted in a loss of anthocyanins and reduction of several flavonoid biosynthesis genes, despite expression of a SG6 MYB (VmMYBA1) (Karppinen et al. 2021), and MYBA1/MYBPA1 having tightly correlated expression in blueberries and bilberry (Günther et al. 2020; Lafferty et al. 2022). This is because MYBA1 activates the expression of MYBPA1, ensuring both MYBs are present to coordinately regulate the flavonoid pathway. It is likely this hierarchical regulation of MYBPA1 genes by SG6 MYB proteins also occurs in other fruit species, such as grape and apple which also show MYBPA1 expression correlating with anthocyanins and PAs (Bogs et al. 2007; Wang et al. 2018).

Studies from fruit and other crops have identified interesting examples of gene activations or knockouts, resulting in diverse pigmentation phenotypes that have been actively selected for, as ornamental landraces (e.g. apples), or valued heirloom varieties. Loss of MYB or bHLH gene activity is commonly associated with loss of anthocyanins in fruit and vegetable crops. White wine grapes are probably the best-known example, where mutations in VviMYBA1 and 2 result in a loss of ripening-associated fruit colour (Kobayashi et al. 2005; Walker et al. 2007), and similar disruptions in MYB genes contribute to anthocyaninless (acyanic) phenotypes in diverse fruit crops (Butelli et al. 2017; Yan et al. 2020; Karppinen et al. 2021).

Fruit crops have undergone substantial selection for varieties with altered anthocyanin patterns, usually the result of altered expression of SG6 MYB genes. Many gene activations result in ectopic expression throughout the plant, resulting in red/purple foliage, as well as intensely coloured fruit. This can arise from promoter variations, such as cis-element replication and autoregulation e.g. red flesh in apple (Espley et al. 2007, 2009) and Teinturier grapes (Röckel et al. 2020), and transposable element (TE)-mediated activation e.g. pepper (Jung et al. 2019) and brassicas (Chiu et al. 2010; Yan et al. 2019). In other examples, TE activations provide new localised expression patterns, or they act as enhancers. Blood oranges are characterised by anthocyanins in fruit flesh, which is induced by cold temperatures. This is because transcription initiates from a cold-responsive retrotransposon within the promoter of the SG6 MYB Ruby (Butelli et al. 2012). In octoploid strawberry, a TE insertion within the promoter of FaMYB10-2 enhances expression, conferring red-flesh, compared with alleles lacking the TE. This may be because the TE contains additional MYB binding cis-elements, allowing autoregulation, or additional ripening-related cis-elements (e.g. abscisic acid response elements) (Castillejo et al. 2020). Altered expression of upstream regulators of SG6 MYBs can also generate increased anthocyanin phenotypes. The red-flesh trait in blood peaches arises because of ectopic expression (TE gene activation) of a NAC TF, PpBL, which activates the expression of PpMYB10.1 (Zhou et al. 2015; Hara-Kitagawa et al. 2019). Thus, altered expression of SG6 MYBs is commonly the basis of high anthocyanin traits in fruit crops.

MYB repressors are increasingly being recognised as important for conditioning anthocyanin content in fruit, acting epistatically with SG6 MYB activators. SG4 R2R3- and R3-MYB TFs are two well characterised types of repressors with distinct modes of action. The SG4 MYB repressor FaMYB1 was initially reported in strawberry (Aharoni et al. 2001), but proteins with a similar action have now been identified and characterised in many fruit and vegetable crops (Cavallini et al. 2015). Such repressors are recruited to target genes by binding to existing MBW complexes, where they inhibit transcription via C-terminal repression motifs (EAR, TLLLFR), but they can also competitively bind bHLH proteins (Aharoni et al. 2001; Matsui et al. 2008; Albert et al. 2014). A notable example is purple cabbage, in which full colouration requires active alleles of SG6 MYB genes but also secondary mutations in the MYBL2 repressor (Song et al. 2018). The R3-MYBs are small proteins that bind bHLH proteins, acting as competitive inhibitors with SG6 MYBs, preventing assembly of MBW complexes. Their roles in anthocyanin regulation have been best characterised in plants from the Solanaceae (Kroon 2004; Albert et al. 2014; Liu et al. 2015 Liu et al. 2019; Andrea et al. 2020), and underlie the basis of the atroviolacea (atv) locus in tomato, which conditions increased anthocyanin content when combined with active Anthocyaninfruit (Aft) (SG6 MYB) alleles, in varieties such as ‘Indigo Rose’ (Cao et al. 2017; Colanero et al. 2018).

Engineering flavonoids in fruit

Insights into anthocyanin regulation have led to advancements in the development of novel fruit varieties with enhanced flavonoid content via genetic engineering. Early attempts to introduce anthocyanins into tomato used the maize anthocyanin regulator genes C1 (SG5 MYB) and Lc (bHLH1), which resulted in high concentrations of flavonols, but not anthocyanins (Bovy et al. 2002). This was because these maize regulators failed to activate F3′5′H, which is key for producing the di/trihydroxylated substrates required by DFR enzymes in the Solanaceae (Butelli et al. 2008). Early attempts at overexpressing SlANT1 or AtPAP1 (SG6 MYBs) from a constitutive promoter increased anthocyanin content in some tissues, though this was variable (Mathews et al. 2003; Zuluaga et al. 2008).

A major breakthrough came when Butelli et al. (2008) introduced the Del (bHLH1) and Ros1 (SG6 MYB) TFs from Antirrhinum into tomato, under the control of a fruit-specific, ripening-induced promoter (E8). This overcame pleiotropic effects due to high concentrations of anthocyanins in vegetative tissues (e.g. reduced growth), producing them late in development as fruit matured, generating dark purple fruit with anthocyanin throughout the skin and flesh, at concentrations comparable to berryfruit. While some heirloom/specialty tomatoes produce anthocyanins in fruit (e.g. Aft⁺ atv^-, such as ‘Indigo Rose’), this is limited to the skin and is at low concentrations (Jones et al. 2003; Colanero et al. 2020). The Ros/Del tomato fruit were then used in health studies, matched with near-isogenic controls, providing strong evidence for the protective effects of anthocyanins against chronic diseases. This approach for determining the health benefits of anthocyanins augments other excellent work that makes use of natural mutants/variants for anthocyanins, such as maize and oranges (Cappellini et al. 2021).

The transgenic tomato platform established by Cathie Martin and Eugenio Butelli allows different types of metabolites to be produced in tomato fruit, for use in controlled health studies. This includes tomatoes accumulating high concentrations of flavonols, by expressing AtMYB12 (SG7 MYB) (Luo et al. 2008). Combining transcription factors (Ros/Del or MYB12) with biosynthetic mutations or transgenes has led to the development of tomato fruit producing anthocyanins (including specific types), isoflavonoids, stilbenes and combinations of multiple metabolites (Zhang et al. 2015; Butelli et al. 2021). These provide valuable resources for investigating health effects of metabolites, including effects of multiple phytochemicals. An exciting example was the synergistic effect of stilbenes and anthocyanins in substantially reducing disease symptoms in an inflammatory bowel disease model. The effects observed using a controlled transgenic tomato food source were able to be replicated with other foods containing similar contents of anthocyanins and stilbenes (Liso et al. 2018).

Forage

Just as flavonoid compounds provide quality traits and health benefits to humans, they also contribute to quality in pasture and forage crops for animal consumption. Many pastoral systems include a perennial forage legume in mixed swards with grasses. In New Zealand, these are exemplified by perennial ryegrass (Lolium perenne) and white clover (Trifolium repens). The legume provides numerous benefits, including nitrogen fixation (through rhizobia), is highly digestible as well as being protein-rich. Legumes also have interesting profiles of flavonoids, including flavonols, isoflavonoids and PAs (condensed tannins/CTs).

One consequence of being a rich source of protein is that it contributes to a condition in ruminant animals called pasture-, legume- or frothy-bloat, which if unmanaged can result in death. When protein is released during digestion, it can form stable foams, trapping gases produced by microbial fermentation, resulting in distention or bloating (Jacobson et al. 1957; Waghorn and Jones 1989). While this is linked to consumption of legume forages such as white clover and alfalfa/lucerne (Medicago sativa), legumes such as Lotus (Lotus spp. including L. pedunculatus, L. corniculatus, L. japonicus) and sainfoin (Onobrychis viciifolia) are bloat-safe (Jones and Lyttleton 1971). The basis for this is the presence of high concentrations of foliar PAs. PAs are best known for their astringency, or drying mouth-feel, arising from their protein binding properties – humans experience this when drinking red wine or black tea (Hagerman and Butler 1981; Dixon et al. 2005). It is these same properties that prevent bloat in ruminants, disrupting the formation of stable protein-foams, and also protecting protein from microbial digestion (Waghorn et al. 1987) resulting in increased weight gains and/or productivity (Aerts et al. 1999; Barry and McNabb 1999; Waghorn 2008). These bloat-safe forages can also provide additional animal health and production benefits, including resistance to parasites (Niezen et al. 1998; Hoste et al. 2006) and reduced methane and nitrous oxide emissions (Carulla et al. 2005; Tavendale et al. 2005; Eckard et al. 2010).

While bloat-safe, high-PA legumes exist, they tend to have poor performance and persistence within pastures compared with white clover and alfalfa. Breeding efforts to introduce PAs into the foliage of these forages have had limited success and have not substantially altered the distribution of PAs, although gains have been made in increasing PA content in floral tissues where they are normally produced (Burggraaf et al. 2006). Metabolic engineering high PA content into forage has therefore been a target for at least 20 years (Gruber et al. 1999). Early efforts to engineer PA biosynthesis into plants involved similar approaches to modifying flower colour, initially by overexpressing the PA biosynthetic gene ANTHOCYANIDIN REDUCTASE (ANR). This was successful at introducing PAs into tobacco (Nicotiana tabacum), but only in floral tissues that already produced anthocyanins (Xie et al. 2003). Similarly, overexpression of MtANR in Medicago (M. truncatula) increased PA content, particularly in tissues where anthocyanins were produced (Xie et al. 2006). This demonstrated that coordinated regulation of the core flavonoid biosynthetic genes shared with anthocyanin biosynthesis was necessary to provide substrates for producing flavan-3-ols. This key finding informed subsequent strategies, which combined overexpressing the anthocyanin regulator AtPAP1 (SG6 MYB) with ANR, which then allowed ectopic PA accumulation throughout the vegetative tissues (Xie et al. 2006).

The discovery of PA regulatory genes provided new opportunities for metabolic engineering of forage crops. As described earlier for fruit, PA biosynthesis is regulated by MBW complexes, but contain SG5 or MYBPA1-clade R2R3-MYBs and SGIIIf-2 (bHLH2) proteins. However, it has become clear that PA regulation and biosynthesis in leaves is not as simple as anthocyanin regulation in flowers. The Arabidopsis SG5 MYB gene, AtTT2, was identified as a regulator of PAs in the seed coat (Nesi et al. 2001), acting within an MBW complex with AtTT8 (bHLH) and AtTTG1 (WDR) (Baudry et al. 2004). Overexpression of AtTT2 in Arabidopsis resulted in ectopic expression of ANR in vegetative tissues, but this was not sufficient to induce ectopic PA accumulation without co-transforming AtPAP1 (SG6 anthocyanin MYB) (Sharma and Dixon 2005). This suggested that co-operative regulation by multiple MYBs might be necessary for full expression of the PA biosynthesis, transport and polymerisation genes.

Major advances in developing high-PA forage legumes have come from understanding the genetic basis of foliar PA accumulation in Lotus and Trifolium arvense. In Lotus, this trait has arisen from a series of tandem gene duplication events, which have altered the normal expression pattern of the LjTT2 genes (Yoshida et al. 2008; Escaray et al. 2017). In T. arvense a distinct MYB gene, TaMYB14, is expressed in leaves and is necessary for PA accumulation in these tissues (Hancock et al. 2012). The white clover orthologue (TrMYB14) is expressed in inflorescences, where PAs accumulate, but is not detectable in leaves (Albert 2015), suggesting that TaMYB14 is ectopically expressed in T. arvense. Furthermore, 35Spro:TaMYB14 alone could confer high foliar PA production to both white clover and alfalfa (Hancock et al. 2012), demonstrating that it is possible to develop high-PA forage legumes. Commercial development of genetically modified ‘high CT’ white clover and alfalfa lines is progressing, based on overexpression of TaMYB14 (Hancock et al. 2014). Modified white clover lines have achieved PA concentrations of ∼2% (dry matter) in field conditions, similar to that of L. corniculatus, and the PAs were effective at binding protein. However, the production of PAs results in a reduced biomass yield (Woodfield et al. 2019). This may yet be overcome with breeding efforts, or as demonstrated with the Ros/Del tomatoes, refined by using more specific promoters to restrict the tissues where PAs accumulate.

The future

Looking to the future, it is clear that plant phytochemicals, including flavonoids, will be important to meeting the challenges posed by climate change, new growing systems and increasing focus on food quality and security. New breeding techniques, such as CRISPR mutagenesis, provide opportunities to address remaining questions about the biosynthesis and regulation of flavonoids, and to make rapid genetic improvements in crops and non-model plants.

Crops are increasingly being grown under covered production, to extend the growing season, manage diseases, and to ameliorate damage or crop losses due to adverse weather events (Demchak 2009; Lang 2009; Rogers et al. 2016). Furthermore, indoor production is rapidly expanding, with a long-term view to extend from leafy greens or tomatoes towards higher value perennial fruit crops (Lang 2009; O’Sullivan et al. 2020). Both of these scenarios have implications for the phytochemical content (quality) of fruits and vegetables, and potentially the resilience of plants to stress, because these conditions usually alter the light environment (He et al. 2021). For example, removing UV-B robs plants of an important environmental cue used to induce responses that confer resilience to a variety of abiotic and biotic stressors (Wargent et al. 2011; McLay et al. 2020; Escobar-Bravo et al. 2021). Plants have evolved a specific UV-B receptor (UVR8), signalling pathways (HY5/COP1/RUP) and responses that are highly conserved in land plant lineages, which includes the specific induction of flavonoids and polyphenols that confer stress tolerance (Heijde and Ulm 2012; Clayton et al. 2018). If quality and health properties of fruits and vegetables are to be maintained or improved, growing systems need to either work with the underlying biology, by creating environmental conditions that promote the production of flavonoids (e.g. lighting, temperature), or identify genetic approaches to overcome them.

The increasing temperatures brought about by global warming pose numerous threats to food production, but also to quality – affecting the flavonoid content of fruits and vegetables that provide attractive colours and health properties. Anthocyanin pigmentation is enhanced with conditions of high light intensities and cooler temperatures, but their biosynthesis is inhibited by high temperatures, and anthocyanins may be degraded under these conditions. For example, apples grown in warmer climates produce insipidly coloured apples with low anthocyanin content, while the same cultivars grown in temperate climates produced bright red fruit (Lin-Wang et al. 2011). Similar inhibitory effects of high temperatures have been observed in red-fleshed kiwifruit (Actinidia chinensis) and grapes (Mori et al. 2007; Man et al. 2015). High temperatures also reduce anthocyanin pigmentation in potato tubers, which is due to the elevation of a repressor MYB, StMYB44 (Liu et al. 2019). Recently, a new apple cultivar ‘HOT84A1’ has been developed that retains bright red skin pigmentation when grown under high temperatures (Plant & Food Research 2020; T&G 2020). The genetic basis for this is currently unknown, but understanding this could provide useful insights for improving other crops.

The ability to induce targeted mutation using CRISPR in non-model plants, provides opportunities to enhance the flavonoid content in crops and ornamentals. Prime candidates are genes with repressive or inhibitory activity (Albert et al. 2014; Albert and Allan 2021), and removing these can lower the threshold of activator components (MBW) required to achieve intense pigmentation. This extends beyond the MYB repressors already discussed and includes non-coding RNAs (e.g. miR828 and miR858, tas4, lncRNAs), SPLs, JAZ and NAC transcription factors (Gou et al. 2011; Cui et al. 2014; Yang et al. 2019; LaFountain and Yuan 2021). Recent research has also identified numerous genes involved in light-regulated anthocyanin production, making use of callus cultures or transient expression/silencing systems (Ni et al. 2019; Li et al. 2020; Hu et al. 2021; Ma et al. 2021). Functional characterisation of these genes using stable mutants is required to see which components are amenable to mutagenesis, allowing a decoupling of environmental inhibition and flavonoid production, without deleterious effects on other quality attributes.

Concluding comments

New genetic technologies are providing tools for asking deep questions into flavonoid biology and ways to translate fundamental research from model plants into crops and plant products. We can anticipate gene-editing technologies will be widely adopted for altering flavonoid content in plants. This will likely include ornamentals with new flower colours and patterns, fruits and vegetables fortified with health-promoting phytochemicals, and high-PA forages that help mitigate methane emissions from agriculture. These technologies will also provide new ways to perform robust health studies, helping to understand the interactions and synergistic effects that particular flavonoids and other phytochemicals confer. Just as flavonoids have played important roles understanding genetics and genetic improvement, it is likely that this fascinating group of metabolites will continue to inspire new discoveries and understanding within plant biology.

Funding Statement

NWA and DJL are supported by the New Zealand Ministry of Business, Innovation, and Employment contract C11X1704 ‘Filling the Void: boosting the nutritional content of New Zealand fruit.’ KMD is supported by the Marsden Fund of New Zealand /Te Pūtea Rangahau a Marsden [contract PAF2002] and a James Cook Research Fellowship [Contract JCF-PAF2001].

Disclosure statement

No potential conflict of interest was reported by the author(s).