Flesh Color Diversity of Sweet Potato: An Overview of the Composition, Functions, Biosynthesis, and Gene Regulation of the Major Pigments

Sweet potato is a multifunctional root crop and a source of food with many essential nutrients and bioactive compounds. Variations in the flesh color of the diverse sweet potato varieties are attributed to the different phytochemicals and natural pigments they produce. Among them, carotenoids and anthocyanins are the main pigments known for their antioxidant properties which provide a host of health benefits, hence, regarded as a major component of the human diet. In this review, we provide an overview of the major pigments in sweet potato with much emphasis on their biosynthesis, functions, and regulatory control. Moreover, current findings on the molecular mechanisms underlying the biosynthesis and accumulation of carotenoids and anthocyanins in sweet potato are discussed. Insights into the composition, biosynthesis, and regulatory control of these major pigments will further advance the biofortification of sweet potato and provide a reference for breeding carotenoidand anthocyanin-rich varieties.

as the main pigments in sweet potato because of their high antioxidant properties and the many beneficial effects on human health. In summary, sweet potato is highly diversified due to its varied flesh and skin colors attributed to the different phytochemical components.

Orange-Fleshed Sweet Potato
Orange-fleshed sweet potato is a nutrient-rich crop with appreciable amounts of carotenoids which provides the characteristic orange color. It is a cheap source of dietary antioxidants with many physiological functions including anti-inflammation, anti-mutagenic, anti-cancer, anti-oxidation, antidiabetic, and cardiovascular disease prevention properties [14,32].
Carotenoids including αand β-carotenes have been identified in orange-fleshed sweet potato but the amount of β-carotene is relatively higher compared to other carotenoid-rich fruits and vegetables such as carrot, mango, and tomato [33][34][35][36]. For example, high β-carotene content of about 20-30 mg/100 g and 276.98 µg/g has been recorded in orange-fleshed sweet potato [37][38][39]. However, the amount of vitamin A correlates with the color intensity of sweet potato, thus, the darker the orange color, the higher the β-carotene content. β-carotene has potent pro-vitamin A activity which the body converts to vitamin A. Vitamin A promotes health by boosting the immune system, improving overall skin and eye health, and for good vision [40]. About 100-150 g of orange-fleshed sweet potato may provide the daily Vitamin A needs of children and prevent night blindness [41]. Studies have revealed that the consumption of a medium-size orange-fleshed sweet potato can double the required daily needs of vitamin A. The retention capacity of about 80% β-carotene in boiled orange-fleshed sweet potato remains unmatched [42], hence described as a "superfood" that promotes health [43,44].
As a food security crop, the orange-fleshed sweet potato could supplement as an alternative source of staple food in areas with increasing population and nutritional deficits and for resource-poor farmers [45]. Hence, this biofortified food crop with a good supply of vitamin A can serve as a beacon of hope to battle vitamin A deficiency in underdeveloped countries and also scuffle malnutrition in rural communities [46]. In consequence, the orange-fleshed sweet potato has been incorporated into the vitamin A deficiency prevention program in Africa [47] due to its cheap source of vitamin A.

Purple-Fleshed Sweet Potato
Purple-fleshed sweet potato is a nutritious crop with high levels of anthocyanin which provides its distinctive skin and flesh colors. Anthocyanin is a natural hydro-soluble pigment that provides the purple, red, and blue coloration of flowers, leaves, fruits, and other plant parts. The purple and red coloration of the leaves, stem, and storage roots of sweet potato results from the accumulation of acylated anthocyanins  [48]. Peonidin and cyanidin, acylated with either hydroxybenzoic, ferulic, or caffeic acids are the main anthocyanins among the 39 anthocyanins identified in purple-fleshed sweet potato [11,30]. In recent years, anthocyanins from purple-fleshed sweet potato have extensively been studied due to their potential beneficial health effects on humans. Purple-fleshed sweet potato anthocyanin has good bioactivity and scavenges free radicals [1] contributing to its diverse biological and antioxidant activities. The antioxidants also act as a good protective agent against inflammations, cancers, diabetes, tumors, and hypoglycemia [49][50][51]. Furthermore, the anthocyanin from purple-fleshed sweet potato has high heat and light stability owing to its acylated forms, hence used as natural food additives [52]. Purple-fleshed sweet potato anthocyanin reduced inflammations caused by oxidative stress and decreased oxidative stressors confirming its free radical scavenging ability, thus, the strong antioxidant ability of purple-fleshed sweet potato [16]. The robust anti-mutagenic properties of the purple-fleshed sweet potato anthocyanin attributed to its radical scavenging activity decreased the risk of hypertension and liver injury in rats [53]. Again, the purple-fleshed sweet potato anthocyanin was revealed to have resilient anti-microbial and antiinflammation properties in addition to its ability to protect against colorectal cancer, UV light, and reduce memory loss [49]. The daily intake of 400 mg beverage prepared from purple-fleshed sweet potato protected the liver from oxidative stresses [54]. Hence, the many physiological activities of purple-fleshed sweet potato make it a health-promoting functional food.

Major Pigments in Sweet Potato
Pigments are mainly produced by plants and are responsible for the color variations observed in many plant tissues. Natural pigments accumulate in different plant parts including flowers, fruits, leaves, and stems. Generally, these natural pigments provide several physiological activities with many beneficial health effects [55]. The relative quantity of pigments accumulated mainly determines the colors produced by the various plant tissues. However, the concentration of pigments correlates directly to the color intensity [56]. The storage root of sweet potato is the main repository organ of natural pigments that provide the different flesh colors (white, yellow, orange, and purple) compared to other crops [57].
Carotenoids and anthocyanins are the major sweet potato pigments which provide the yellow, orange, and purple colors. These pigments are synthesized through different metabolic pathways with different structural and regulatory genes regulating their biosynthesis and accumulation. They also accumulate in different sweet potato genotypes, the orange-and purple-fleshed respectively. As a result, it may be likely to observe no form of interaction between their biosynthesis and accumulation. However, there is evidence of transgenic sweet potato accumulating both color pigments in a single storage root. For instance, Park et al. [58] produced a dual-pigmented transgenic sweet potato through Agrobacteriummediated transformation. The transgenic plants expressing the IbMYB1 gene (a key regulator of anthocyanin biosynthesis in sweet potato), accumulated high levels of both anthocyanins and carotenoids in a single storage root. Overexpression of the gene slightly increased most carotenoid biosynthetic genes, such as phytoene desaturase, lycopene ε-cyclase, zeta-carotene desaturase, and lycopene β-cyclase in transgenic plants than in control plants, suggesting that IbMYB1 expression might affect carotenoid biosynthesis-related gene expression. The above result indicates a possible interaction in the regulation of carotenoid and anthocyanin biosynthesis but needs to be clarified through further research.

Carotenoids
Carotenoids are lipid-soluble natural pigments responsible for the yellow, orange, and red colors of flowers, vegetables, seeds, fruits, and roots [59]. They are synthesized only in photosynthetic organisms including plants, some bacteria, fungi, and algae. Carotenoids are the most abundant of all plant pigments [60]. The storage root of sweet potato has an excellent supply of carotenoids including β-cryptoxanthin, violaxanthin, β-carotene, lycopene, and zeaxanthin [26,61] among which β-carotene is the main carotenoid with the highest pro-vitamin A activity both in terms of widespread distribution and bioavailability [47,57]. Sweet potato leaves have an adequate supply of carotenoids including lutein, neoxanthin, β-carotene, and violaxanthin [19] with the composition equivalent to other plant chloroplasts [62]. Research evidence revealed that orange-fleshed sweet potato has high β-carotene content, thus about 80% of the carotenoids being trans-β-carotenes [63,64]. However, other studies also reported the presence of lutein and zeaxanthin in the orange-fleshed sweet potato which provides the orange color [65]. Inclusion of orange-fleshed sweet potato in the diet provides dietary pro-vitamin A due to the high β-carotene content [19], hence used as a model crop for many small-scale research to increase vitamin A status [45,66]. The carotenoids identified in orange-fleshed sweet potato with their concentrations reported by different authors have been presented in Tab. 1.

Functions of Carotenoids
Carotenoids are naturally occurring pigments prevalent in plants with their accumulation in flowers, fruits, and other plant parts, giving those parts the yellow, orange, and red colors [59]. Carotenoids are mostly found in the chloroplast and chromoplast with several functions in both plants and animals. In plants, these molecules serve as accessory pigments for harvesting light in photosynthetic reaction sites and inhibit photo-oxidative damage to cells and tissues. Mostly, they combine with chlorophyll to absorb blue-green wavelengths, thereby protecting cells from superfluous light, increase tolerance to herbicide and salt stress and also safeguard photosynthetic apparatus from photo-oxidative damage [18].
Generally, exposure to stress leads to the production of free radicals by reactive oxygen species (ROS) causing oxidative damage which is mostly inhibited by antioxidants [81,82]. Carotenoids exhibit antioxidant properties that inhibit the harmful effects of various environmental stresses such as high temperature, resilient light, ultra-violet radiation, and drought in plants [83,84]. This ability was reported in genes that code for carotenoid. For instance, IbPSY1, a carotenoid gene was reported to be crucial in plants' resistance to abiotic stress in vivo. In some plant species (daffodil, maize, potato), the upregulation of the PSY gene was reported to highly increase carotenoid levels [11], this then explains the ability of the IbPSY1 gene to increase tolerance to environmental stress including drought, salinity, and high/low temperature which may negatively affect growth and yield of sweet potato. Another gene observed to improve sweet potato resistance to environmental stress is IbOr, a gene involved in carotenoid accumulation, and its overexpression improves resistance to heat stress and oxidative damage [19,85]. These genes can therefore be beneficial for engineering plants with improved tolerance to abiotic stress and can be proposed to be essential in the defense response mechanism of plants to abiotic stresses. Similarly, the upregulation of the IbOr gene in transgenic potato and alfalfa increased tolerance to certain abiotic stress such as salinity, drought and heat stress [86,87].
The brightly colored parts of plants resulting from carotenoid accumulation, aids in pollination and seed dispersal by attracting pollinators and other agents of dispersal [19]. Similar antioxidant properties of carotenoids exhibited in plants have also been observed in animals that feed on plants with carotenoids. Carotenoids have positive impacts on human health as they are sources of dietary antioxidants which reduces the risk of many age-related illnesses including macular degeneration, cancer, and cardiovascular diseases [2,18]. Recently, orange-fleshed sweet potato has been on the research spotlight due to the high carotenoid content particularly β-carotene.
Orange-fleshed sweet potato is considered among the main sources of vitamin A to animals and humans that cannot synthesize vitamin A but can only obtain them through their diet [88,89]. Owing to that, several studies recommend the daily intake of orange-fleshed sweet potato which helps increase the levels of vitamin A and improve general well-being [90,91]. For instance, the daily intake of orange-fleshed sweet potato significantly elevated the vitamin A status of men, women, and children in some developing nations including Bangladeshi, Kenya, and Mozambique respectively [45,66,90]. Orange-fleshed sweet potato has also been revealed to be used to prevent blindness and maternal mortality resulting from vitamin A deficiency in most developing countries [92]. This then explains the incorporation of sweet potato as an excellent food source to fight vitamin A deficiency especially in underdeveloped countries, hence, its integration in the vitamin A deficiency prevention program [93].
Carotenoids are also used as additives to improve pigmentation and to prevent UV radiation damage. In ornamental fishes, dietary astaxanthin, a carotenoid is used on commercial bases as a natural colorant [94] and also used to improve the pigmentation of egg yolks [95]. Humans have also benefited from the photo-protective properties of carotenoids through their inclusion in cosmetic products to impede damages from UV radiation.
The lycopene cyclase genes, LCY-β, and LCY-ε are involved in the biosynthesis of the branch components of carotenoids in diverse plant species. Regulating the expression levels of these genes (LCY-ε and LCY-β) may affect the relative activity and production of cyclic carotenoid genes associated with lutein synthesis in some plants including rice, Arabidopsis, and tomato [103,104]. Both genes are interrelated in producing α and β branch carotenoids in that, the overexpression of one can suppress the other. LCY-β is reported as a vital enzyme associated with the synthesis of both αand β-branch carotenoids, like α-carotene and β-carotene.
According to Haskell et al. [26], the LCY-β gene functions to increase carotenoid content, oxidative ability, and resistance to abiotic stress in sweet potato. It also increases the production of β-branch carotenoids including zeaxanthin, β-carotene, violaxanthin, and β-cryptoxanthin. In sweet potato, the downregulation of IbLCY-ε in non-embryogenic calli of light orange-fleshed sweet potato cv. Yulmi increased the content of β-branch carotenoids resulting in an orange coloration of the ensuing transgenic calli [25].
Other research has also reported that silencing IbLCY-ε or IbCHY-β during carotenoid metabolic engineering can result in increased β-carotene and total carotenoid content in sweet potato. According to Kim et al. [18], the suppression of IbCHY-β in the catalytic hydroxylation of β-carotene to yield β-cryptoxanthin which is further converted to zeaxanthin in sweet potato increased the β-carotene and the overall total carotenoids content. Based on these results, it can be established that CHY-β and LCY-β are the primary regulatory enzymes involved in carotenoid biosynthesis in sweet potato making β-carotene the main cellular carotenoid in sweet potato. Though the pathway for carotenoid biosynthesis is well elucidated in all higher plants, it is slightly implicit in sweet potato. Therefore, the pathway for the biosynthetic of carotenoids needs to be further characterized in sweet potato.

Gene Regulation of Carotenoid Biosynthesis
A key determining factor of carotenoid content is the regulation of essential biosynthetic genes [106]. Regulation of these genes and the allelic variation of genes in the biosynthetic pathway may influence the different accumulation levels of carotenoids [107,108]. Fluctuations in the levels of these genes have been associated with the development of some crops with increased carotenoid content. Most of the genes have been reported to be involved in the regulation of the three key processes (biosynthesis, degeneration, and storage) in carotenoid accumulation at different stages of plant growth [109]. Genes coding for practically all enzymes involved in the biosynthesis of carotenoids have been isolated from bacteria, fungi, and plants ( Fig. 3) [92]. Carotenoid biosynthetic genes in sweet potato including PSY, GGPS, CrtISO, PDS, LCY-ε, ZDS, ZEP, CHY-β, and LCY-β have been cloned and characterized [18,[25][26][27].
Research evidence on the specific gene that regulates the accumulation of carotenoids in plants is limited. However, the Or (orange) gene is a gene of interest and has been researched in several plant species including sorghum, cauliflower, melon, alfalfa, potato, Arabidopsis, and sweet potato. Orange denotes an extraordinary group of regulatory genes that facilitate the accumulation of carotenoids which are highly conserved in diverse species and exhibit functions like preserving homeostasis of carotenoids, maintaining photosynthesis, and regulating carotenoid biosynthesis [110,111].
The overexpression of the Or gene increased carotenoid accumulation in plants including Brassica oleracea var. botrytis [112], Cucumis melo [113], Solanum tuberosum and Arabidopsis thaliana [114]. In sweet potato, the Orange gene (IbOr) originally cloned from the sweet potato cv. Sinhwangmi (orangefleshed) based on the BoOr sequence, induced the accumulation of carotenoids in various tissues (leaves, stem, and storage root) [25]. However, the gene expression levels vary in different parts of the various sweet potato varieties. It is highly expressed in the storage root of the orange-fleshed varieties whilst in other colored varieties (white, orange, and purple), its expression is highly observed in the leaves [110]. The IbOr functions to aid the buildup of carotenoids and regulate carotenoid homeostasis in sweet potato. The orange gene has also been reported to interact with PSY and carotenoid cleavage dioxygenases (CCDs). In sweet potato, IbOr interrelates with IbPSY to enable higher stability of IbPSY through the holdase chaperone activity of IbOr [85] which offers a substitute and complement strategy for increased carotenoid levels, chromoplast differentiation and PSY stabilization [115]. Apparently, carotenoid catabolism negatively regulates accumulation. In potato, the accumulation of carotenoid was controlled negatively by CCD1 and CCD4 [116]. However, the purple-fleshed sweet potato expressing higher levels of IbOr also contained increased levels of CCD1, CCD4, and NCED transcripts [77] which proposes the ability of carotenoid catabolism genes (IbCCD1 and/or IbCCD4) to increase carotenoid accumulation in sweet potato. This is however inconclusive and exposes us to the complex mechanism involved in carotenoid accumulation, regulated by the molecular function of the IbOr gene. The interrelation between carotenoid accumulation and catabolism needs to be further elucidated.

Anthocyanins
Anthocyanins are a subclass of flavonoid compounds and an essential water-soluble natural pigment in vascular plants, responsible for the wide-ranging colors in several plant species [117]. They are existent in diverse plant tissues including flowers, fruits, and storage organs like the root and stem. Anthocyanins occur naturally as glycosides of anthocyanidins attached to different sugar moieties [118] and are highly appreciated for their anti-oxidant activities which provide several health benefits such as anti-cancer, antiinflammatory, anti-diabetic, anti-mutagenic, and cardiovascular diseases prevention properties [119,120].
Purple-fleshed sweet potato mounts up high levels of anthocyanins in their storage roots, with anthocyanin 3-O-sophoroside and its derivatives as the major compounds [121]. Anthocyanins from purple-fleshed sweet potato are non-toxic, resource-rich, and unscented bioactive compounds with stable physicochemical properties compared to anthocyanins from other plant sources including cherry, strawberry, and grapes [122]. Because these anthocyanins are acylated, they have high stability against heat and UV radiations, hence used as natural food additives [52]. The various anthocyanins identified by different authors in purple-fleshed sweet potato are summarized in Tab. 2.

Composition of Anthocyanin
Anthocyanin, a major plant secondary metabolite is a subclass of flavonoid compounds made of monoor di-glycosylated aglycones of anthocyanidins attached to a sugar moiety [123]. The molecular structure of anthocyanin mainly exists as glycosides of poly-hydroxyl or poly-methoxyl derivative of the flavylium (2-phenylbenzopyrylium) cation which consist of a double aromatic ring [A and B], divided by a heterocyclic ring [C] (Fig. 4) [124]. The presence of a positive charge on the C-ring distinguishes anthocyanins from other flavonoids. Anthocyanins are natural plant pigments with varied and complex structures. The structural variation of the various anthocyanins is attributed to the number of the sugar moiety, type of functional group, and the natural acyl group present [49].    The basic anthocyanin structure consists of aglycone base (anthocyanidin), two (2) or three (3) chemical units, sugars, and organic acids as in acylated anthocyanins [125]. Among the over twenty-six (26) anthocyanidins discovered in nature, only six (6) main types; petunidin, cyanidin, malvidin, pelargonidin, delphinidin, and peonidin are found in plants [126,127]. The six (6) main types are mostly responsible for the diverse color variations in plants (Tab. 3). These anthocyanidins usually work together with genes and enzymes to regulate the various colors of anthocyanins. According to Tanaka and Brugliera [128], the enzymes F3′H and F3′5′H which determines the hydroxylation pattern of the B-ring by different substitution patterns at R1, R2, and R3, influences the diversity and color variations of anthocyanins ( Fig. 4; Tab. 3). At present, there are over 600 kinds of anthocyanins identified in plants [127].

Functions of Anthocyanins
Anthocyanins are among the major secondary metabolites, responsible for distinctive colors in plants [134,135]. As a water-soluble natural pigment, anthocyanin plays a significant role in both plants and animals, especially in human health. In plants, anthocyanins aid reproduction by alluring insect pollinators. The brightly colored parts of plants resulting from the accumulation of anthocyanin attract insect pollinators which aid in pollination and seed dispersal [17,136]. Although some of these insects are essential in influencing the reproductive ability of the plant, others act as pathogens that infest plants with diseases. Anthocyanins are effective in reducing the infestations from these pathogenic insects. For example, tomato fruits enriched in anthocyanin exhibited tolerance to gray mold [137]. Also, large numbers of African bollworm died and pupation delayed in tropical armyworm when fed with anthocyanin-rich leaves relative to those fed with green leaves [138].
Anthocyanins also safeguard plants against some biotic and abiotic stress which may offer them better adaptation to climatic changes [139]. Although much has been reported on anthocyanin-related stress response in diverse plant species, little information is available in sweet potato. Dihydroflavonol-4-reductase (DFR), a gene involved in the biosynthesis of several flavonoids including anthocyanins was reported to influence sweet potato tolerance to cold stress [140] with the increase attributed to the enhanced antioxidant ability. Research has reported that the enhanced antioxidant activity of purple-fleshed sweet potato was due to its resilient ability to scavenge free radicals [141]. These findings then propose the role of anthocyanins in the maintenance of ROS homeostasis as the sweet potato grows and develops.
As photo-protective agents, anthocyanins protect the photosynthetic tissues by absorbing excess visible ultraviolet radiation and also act as scavengers of free radicals [142]. Furthermore, anthocyanins accumulate in immature non-reproductive tissues and light-exposed parts of fruits to offer protection against photoinhibition and photo-bleaching under light stress without considerably affecting the process of photosynthesis [143,144].
Despite its countless roles in plants, anthocyanins have beneficial health effects on mammals owing to their antioxidant properties. Purple-fleshed sweet potato has high anthocyanin content and subsequently high antioxidant properties which influences its health-promoting functions [49]. These antioxidant properties enable the scavenging of free radicals associated with aging and degenerative diseases [145]. Typically, anthocyanins are administered to animals through their diets with dietary anthocyanins offering protection against cardiovascular diseases, cancer, and other chronic disorders [146]. Some studies have attributed the shielding effects of dietary anthocyanins to their antioxidative and anticancer properties [147,148]. In an experiment on rats, it was observed that feeding rats with purple-fleshed sweet potato anthocyanin reduced hepatoxin-induced liver injury [53]. This was reported to be due to the enhanced expression of some antioxidant enzymes like glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT) in the liver.
Also, the anti-aging and anti-oxidative properties of anthocyanins make it safe for the manufacturing of natural skin-care products in the cosmetic industry [135]. This inhibits the impact of UV radiation on the skin, hence, reducing inflammations and diseases. Furthermore, anthocyanins from purple-fleshed sweet potato are used as a replacement for some synthetic pigments in cosmetic products like shampoos, rouge, creams, and lipsticks among others. In the pharmaceutical industry, purple-fleshed sweet potato anthocyanins are used as potential components for the production of pharmaceuticals such as anti-neoplastic and anti-inflammatory agents due to their antioxidant properties [57]. As a non-toxic natural pigment, purple-fleshed sweet potato anthocyanin can be used to substitute synthetic pigments in the production of colored medicines as the long-term effect of these synthetic pigments could be detrimental to the human body [149].
Purple-fleshed sweet potato anthocyanin is used as a functional ingredient in food processing industries as preservatives and sources of natural colorants with excellent color potency [150,151]. Purple-fleshed sweet potato anthocyanin exhibited high stability when added to beverages and prolonged the shelf life than pigments from grapes and blackberries [152]. Anthocyanins from purple-fleshed sweet potato can proliferate the growth of helpful bacteria especially those utilized in probiotics and as well inhibit the growth of harmful ones. According to Sun et al. [153], peonidin-based anthocyanins proliferate the Bifidobacterium spp. (bifidum, adolescentis, infantis) and Lactobacillus acidophilus whiles inhibiting the growth of Salmonella typhimurium and Staphylococcus aureus. Similarly, crude anthocyanin derived from purple-fleshed sweet potato inhibits the growth of Bacteroides, Prevotella, and Clostridium histolyticum [154] proposing the ability of anthocyanins to be involved in prebiotic-like activity by modulating intestinal microbiota. In effect, purple-fleshed sweet potato anthocyanins are essential due to their diversified functions.
Anthocyanins after their synthesis are transported from the cytosol to the vacuole for storage. Vacuolar sequestration is crucial to prevent anthocyanins from being oxidized [160] and to perform its function as bioactive pigments. Though anthocyanin biosynthesis and regulatory genes are well characterized, the mechanism involved in its translocation from the cytosol to the vacuole in plants is still debatable [161,162]. The Multidrug toxic compound extrusion (MATE) protein and ATP-binding cassette (ABC) transporters confined in the tonoplast help link anthocyanins to glutathione S-transferase (GSTF) for effective segregation into the vacuole and may also cling unto anthocyanoplasts, a pre-vacuolar segment proceeding to the vacuole [163,164]. Acylated anthocyanins accumulate at high levels inside the vacuole to form AVI (anthocyanic vacuolar inclusions) in some species [165].
DFR is an essential structural gene and its substrate specificity regulates the structure and color of anthocyanins. Characterization of IbDFR revealed its expression to be also associated with both biosynthesis and accumulation. According to Wang et al. [140], expression levels of IbDFR in the leaves, stem, and root correlated with anthocyanin accumulation in these plant parts. In purple-fleshed sweet potato, a decrease in the expression of IbDFR also impacted the flux dissemination of flavonoids like proanthocyanidins and flavonols. In Arabidopsis, the transparent testa (tt) loci encode several flavonoid (anthocyanin) biosynthetic enzymes such as CHI, DFR, and CHS at the tt5, tt3 and tt4 loci respectively. However, mutations in genes encoding these anthocyanin biosynthetic enzymes eliminate anthocyanin synthesis [166,167]. For example, Dong et al. [168] observed no pigment accumulation (anthocyanin and brown tannins in the hypocotyl and seed coat respectively) in Arabidopsis tt mutants compared to the seeds of the wild-type, suggesting the absence of biosynthetic enzyme (CHS, CHI, and DFR) activity. Introduction of IbDFR into the hypocotyls, cotyledons, and seed coat of Arabidopsis tt3 mutants gave the hypocotyls and cotyledons a purple color and restored the pigments in the seed coat, proposing the biosynthetic function of the IbDFR gene. Glutathione S-transferases (GSTs) function to detoxify xenobiotics (heterocyclic compounds) by connecting glutathione to a substrate to form a glutathione S-conjugate. According to Marrs et al. [169], these enzymes catalyze the conjugation of glutathione (GSH) to anthocyanins to form anthocyanin-GSH conjugates for onward sequestration into vacuoles by the glutathione pump, proposing that anthocyanin may be an endogenous substrate for the glutathione pump. GST plays an integral role in the intracellular transport of anthocyanin by coupling its synthesis and accumulation in the vacuole. GSTs also function as carrier proteins by physically binding to anthocyanins to facilitate the vacuolar sequestration of anthocyanin from the cytoplasm, though their functions remain indistinct [170]. For instance, Kitamura et al. [171] and Sun et al. [172] observed the localization of GSTs from other plants and Arabidopsis tt19 that accrue high proanthocyanidins than anthocyanins in the cytoplasm of undeveloped seed coats. Recently, Marrs et al. [160] and Alfenito et al. [173] reported the inability of Petunia hybrida (petunia) and Zea mays (maize) mutants to accumulate anthocyanins in their vacuoles due to the lack of GST. This suggests the function of GST as flavonoid binding protein, hence, confirming its involvement in anthocyanin accumulation. Again, GSTs are allied to high-anthocyanin producing membranes in the plant cell, possibly the vacuole and endoplasmic reticulum [172]. These results confirm the function of GSTs as carrier proteins and thus, the glutathionylation of flavonoids might not be catalyzed by GSTs due to their inability to conjugate GSH to anthocyanins [161] which therefore contrast with the report by Marrs et al. [169]. It is therefore noteworthy that, there is no evidence of anthocyanin-GSH conjugates in plants [170].
GST genes involved in anthocyanin accumulation have been identified in several plant species including strawberry, cyclamen, litchi, and grapevine [174][175][176][177]. For example, Hu et al. [175] reported the involvement of LcGST4 in the accumulation of anthocyanin in the fruit Litchi chinensis (litchi) and its overexpression in pigmented tissues. The results revealed that the expression of LcGST4 was regulated by the LcMYB1 gene. MYB gene family is a part of a larger family of transcriptional factors that regulate anthocyanin biosynthesis and accumulation in plants. For instance, the IbMYB1 gene regulates anthocyanin biosynthesis in sweet potato [58] whereas, in Arabidopsis, IbMYB1a increased anthocyanin accumulation in transgenic plants [178].
In sweet potato, a GST encoding gene, IbGSTF4 is reported to be involved in the accumulation of anthocyanin. For instance, the IbGSTF4 gene after characterization was found to be highly expressed in pigmented stems, leaves, and storage root with its expression correlating with the accumulation of anthocyanin. In the same study, the varied expression profiles of IbGSTF4 in the Arabidopsis tt19 (a knockout mutant of anthocyanin-related GST) gave the cotyledon and hypocotyl a purple color, suggesting IbGSTF4 participation in anthocyanin accumulation in sweet potato [22]. However, a dual luciferase assay pointed out that the IbMYB1 gene could not directly regulate the expression of IbGSTF4 which conflicts with the report by Hu et al. [175]. This then suggests that the regulation of anthocyanin biosynthesis and sequestration may involve other MYB regulatory factors [22], thus, proposing a complex regulatory mechanism of anthocyanin vacuolar sequestration and accumulation in sweet potato which needs to be elucidated through further research.

Gene Regulation of Anthocyanin Biosynthesis
The primary regulatory genes involved in anthocyanin biosynthesis have been studied extensively and sequestered in many plant species [124]. The transcriptional factors regulating the biosynthesis of anthocyanins are WD40-type co-regulators (WD40), R2R3-MYB protein, and a basic helix-loop-helix (bHLH, MYC) protein [179].
Structural and regulatory genes are the two main types of biosynthetic genes. The structural genes encode the enzymes which catalyze every reaction step while the regulatory genes encode transcriptional components that regulate structural gene expression [180,181]. Structural genes involved in anthocyanin biosynthesis are homogeneously expressed and their expression levels are dependent on the concentration [182]. There are two divisions of structural genes in dicot plants i.e., early (CHI, CHS, FLS, F3′H, and F3H,) and late (UFGT, ANS/LDOX, and DFR) biosynthetic genes [181]. These genes operate under the MYB-bHLH-WD40 (MBW) regulatory network made up of the MYB, basic helix-loop-helix (bHLH) and WD40 replicate families. For instance, the MYB domain C1 protein which regulates anthocyanin biosynthesis in maize requires a bHLH partner to activate the flavonoid structural genes and the dihydroflavonol reductase (DFR) promoter, although the MYB domain P protein which controls phlobaphene to stimulate the promoter lacks a bHLH partner [183]. These MYB proteins have a central responsibility of regulating the biosynthesis of secondary metabolites, signal transduction, resistance to diseases as well as growth and developmental fluctuations [181]. As reviewed in Amoanimaa-Dede et al. [20], the structurally conserved MYB genes comprise 100-160 bp DNA-binding regions with one or more replications. The R2R3 MYB genes with two repeats are the predominant group of MYB genes involved in the flavonoid pathway in plants. Therefore, the intensity of anthocyanin synthesis solely depends on the expression of structural genes that are related to a specific species [184].
In sweet potato, some structural and transcription factor genes have been characterized, with most of the genes functioning in both anthocyanin biosynthesis and accumulation. The IbMADS10 is a vital regulatory gene involved in anthocyanin biosynthesis of sweet potato [185]. Two MYB genes (IbMYB1 and IbMYB2) isolated from the storage root of purple-fleshed sweet potato cv. Ayamurasaki regulates anthocyanin biosynthesis in sweet potato [186]. According to Mano et al. [186], the IbMYB1 transcription factor from the MYB-family facilitates the accumulation of anthocyanins in sweet potato storage roots. Park et al. [58] reported that the overexpression of the IbMYB1 gene effectively caused the accumulation of anthocyanin in the storage root of an orange-fleshed sweet potato with high carotenoid content thereby increasing the radical scavenging activity.
Current research has identified some post-transcriptional modulators mostly miRNA including ib-miR164c, ib-miR160e-5p, ib-miR172e-3p, and ib-miR166m [187] to be involved in anthocyanin biosynthesis. These miRNA target genes are involved in auxin signaling. Auxin can inhibit the expression of the MBW complex which intends to regulate anthocyanin biosynthesis [36]. The ib-miR159, ib-miR319, ib-miR858, and ib-miR156 also regulated the MYB genes whereas the SPL gene was targeted by ib-miR156 and its upregulation reduced the expression of ibSPL in purple-fleshed sweet potato. The ib-miR156a-5p was also reported to cling unto ibSPL genes proposing that ib-miR156 may increase the biosynthesis of anthocyanin via structural gene regulation in the phenylpropanoid pathway.
Though there is post-transcriptional modulation of anthocyanin biosynthesis, the primary level at which anthocyanin biosynthesis is inducted or shut down in plants is controlled by the expression of biosynthetic genes [188]. From the above results, it can be deduced that the molecular regulation of anthocyanin biosynthesis and accumulation is complex both at the transcriptional and post-transcriptional levels. One keen observation made was that most molecular results were more tailored to individual research (transcriptome sequencing). This is due to the lack of a reference genome (the only one sequenced "Taizhong 6" is incomplete and inaccurate hence not representative of hexaploid sweet potato). The ability to sequence the reference genome will go a long way to improve molecular research in sweet potato.

Concluding Remarks and Perspectives
Sweet potato is a multifunctional food crop with rich nutritional composition and bioactive compounds. Several cultivated sweet potato varieties differ with flesh and skin colors (white, yellow, orange, red, and purple) of the storage root. Variations in phytochemicals and nutritional compositions, the pigments produced and the morphological traits may also distinguish the various sweet potato varieties. Carotenoid and anthocyanin are the major natural pigments in sweet potato known for their antioxidative properties which scavenge free radicals and protect both plants and animals from oxidative damage. The IbGGPS, IbLCY-ε, and IbCHY-β genes regulate carotenoid biosynthesis while IbCCD1, IbCCD4, and IbOr control its accumulation. Anthocyanin biosynthesis and accumulation are both regulated by the IbMYB, IbDFR, and IbGSTF3 genes. Besides, some post-transcriptional modulators basically miRNAs were revealed to be involved in anthocyanin biosynthesis. Further characterization of the biosynthesis and regulatory mechanism of carotenoids and anthocyanins will be beneficial to unravel the complex mechanism of carotenoid accumulation regulated by the molecular function of the IbOr gene. This will help elucidate the interrelation between carotenoid accumulation and catabolism. Although the molecular mechanism underlying the biosynthesis and regulatory control of carotenoids and anthocyanin is extensively studied in sweet potato, a lot is still unknown. Furthermore, the limited report on the role of anthocyanin in sweet potato stress response mechanism calls for further research.
Many innovative biotechniques such as CRISPR/Cas9 through synthetic transcription factor detection and gene activation, could modify the expression of targeted genes [189]. Although this technology has been used extensively for crop improvement, little is known about its application in sweet potato. For sweet potato pigmentation, transgenic technologies (genetic modification and metabolic engineering) have been used to further increase carotenoid and anthocyanin content by modifying the expression of single genes through Agrobacterium-mediated transformation. Also, the identification and development of synthetic transcription factors in sweet potato might increase the accumulation of carotenoids and anthocyanins. Therefore, CRISPR-Cas9-mediated genome editing technique may be significantly useful for the biofortification of sweet potato. The lack of a reference genome makes genetic and molecular studies very challenging, hence, whole-genome sequencing is suggested to improve molecular research in sweet potato. Overall, the complex molecular regulation of anthocyanin biosynthesis and accumulation both at the transcriptional and post-transcriptional levels due to the inconsistencies in previous reports should be addressed through further research. Understanding the biosynthesis and gene regulation of these major sweet potato pigments may provide appropriate resources and better schemes for breeding sweet potato varieties with high anthocyanin and carotenoid contents.