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Phytochemistry 153 (2018) 11–27

Contents lists available at ScienceDirect

Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Review

Understanding the genetic regulation of anthocyanin biosynthesis in plants – T


Tools for breeding purple varieties of fruits and vegetables
Samuel Chaves-Silvaa,b,1, Adolfo Luís dos Santosa,b,1, Antonio Chalfun-Júniorb, Jian Zhaoc,
Lázaro E.P. Peresd, Vagner Augusto Beneditoa,∗
a
Division of Plant and Soil Sciences, West Virginia University, 3425 New Agricultural Sciences Building, 6108, Morgantown, WV 26506-6108, USA
b
Biology Department, Universidade Federal de Lavras (UFLA), Lavras, MG, 37200-000, Brazil
c
State Key Laboratory of Tea Plant Biology and Utilization, College of Tea and Food Science & Technology, Anhui Agricultural University, Hefei, 230036, China
d
Department of Biological Sciences, Escola Superior de Agricultura “Luiz de Queiroz” (ESALQ), University of São Paulo (USP), Piracicaba, SP, 13418-900, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Anthocyanins are naturally occurring flavonoids derived from the phenylpropanoid pathway. There is increasing
Anthocyanin evidence of the preventative and protective roles of anthocyanins against a broad range of pathologies, including
Biosynthesis different cancer types and metabolic diseases. However, most of the fresh produce available to consumers ty-
Genetic regulation pically contains only small amounts of anthocyanins, mostly limited to the epidermis of plant organs. Therefore,
Biofortification
transgenic and non-transgenic approaches have been proposed to enhance the levels of this phytonutrient in
Functional food
vegetables, fruits, and cereals. Here, were review the current literature on the anthocyanin biosynthesis pathway
Nutraceutical
Plant breeding in model and crop species, including the structural and regulatory genes involved in the differential pigmen-
tation patterns of plant structures. Furthermore, we explore the genetic regulation of anthocyanin biosynthesis
and the reasons why it is strongly repressed in specific cell types, in order to create more efficient breeding
strategies to boost the biosynthesis and accumulation of anthocyanins in fresh fruits and vegetables.

1. Introduction et al., 2017; Toufektsian et al., 2008). Most importantly, these benefits
are expected to be achieved only when a considerable amount of an-
Anthocyanins are specialized metabolites of the phenylpropanoid thocyanins is regularly consumed in the diet (Butelli et al., 2008;
pathway that are widely distributed in the plant kingdom. These gly- Habanova et al., 2016; Petroni et al., 2014). However, most of the
cosylated flavonoids are one of the most important water-soluble pig- vegetables available in the market contain only small quantities of
ments in plants and provide the shades of blue, purple, red and pink to anthocyanins in their edible parts, with the pigment often restricted to
plant organs, including leaves, petals, fruits, and seeds (Mazza and the epidermal layers (peel/skin), such as leaf and petal epidermis, the
Miniati, 2018; Onslow, 2014). Anthocyanins present in the epidermis of seed coat (testa), and the epidermal cells of the fruit (exocarp or peel)
flowers and fruits provide visual cues to attract pollinators and seed (Butelli et al., 2008). Since the peel usually accounts for less than 5% of
dispersers (Rieseberg and Blackman, 2010; Tanaka et al., 2008). The the total mass of edible parts of the plant (Sestari et al., 2014), the lack
fact that anthocyanins are induced under diverse biotic and abiotic of anthocyanin accumulation in parenchymal cells of cortical tissues
stresses suggests a role in cell stress coping mechanisms (Ferreyra et al., considerably limits the total amount of these compounds in most fresh
2012; Landi et al., 2015; Zhang et al., 2014a). Moreover, it is quite foods available to the consumer. Even blueberry, which is popularly
puzzling that different stresses induce distinct anthocyanin profiles regarded as a “superfood” due to their anthocyanin content (Reque
(Kovinich et al., 2014), which further suggest that individual types et al., 2014), the biosynthesis and accumulation of these pigments are
might play distinct roles in the physiology of the plants. exclusively restricted to the fruit skin, as seen in the white flesh of
Despite being a minor component of the human diet, many studies commercial varieties.
have highlighted the health benefits of anthocyanins and other phenolic This differential pigmentation pattern of plant organs showing
compounds, thus potentially helping in the prevention of different cyanic epidermis (colored skin/exocarp) and acyanic cortex (e.g., white
chronic pathologies (Amiot et al., 2016; Hooper et al., 2008; Khoo flesh made of non-pigmented parenchymal cells) is commonly found in


Corresponding author.
E-mail address: vagner.benedito@mail.wvu.edu (V.A. Benedito).
1
Both authors contributed equally to this work.

https://doi.org/10.1016/j.phytochem.2018.05.013
Received 24 October 2017; Received in revised form 15 May 2018; Accepted 17 May 2018
Available online 24 May 2018
0031-9422/ © 2018 Elsevier Ltd. All rights reserved.
S. Chaves-Silva et al. Phytochemistry 153 (2018) 11–27

Fig. 1. Dietary sources of anthocyanins. (A) Purple cabbage. (B) Onion bulb. (C) Apple. (D) Grape. (E) Eggplant. (F) Blueberry. (G) Potato. (H) Purple tomato (Aft/
atv/hp2 triple mutant, cv. Micro-Tom). (I) Purple potato. (J) Red cherry. Except for H-J, all other examples accumulate anthocyanin in the epidermal tissue, while
parenchymal cells remain acyanic. Scale: 20 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this
article.)

eggplant, grape, plum, apple, radish, as well as purple varieties of to- species derived from conventional breeding, such the cauliflower (Chiu
mato, onion, and cabbage (Fig. 1). Nonetheless, this is not a universal and Li, 2012; Chiu et al., 2010), potato (Liu et al., 2015b), and carrot
fact: there are remarkable examples of plant varieties bearing edible (Xu et al., 2017) attest to the demand for cyanic varieties with poten-
organs with cyanic parenchyma, such as purple potatoes, blood or- tially higher nutritional value. On the other hand, whereas it is quite
anges, red-flesh apples, teinturier grapes, purple carrots, as well as dark- straightforward to use biotechnology to boost anthocyanin content in
flesh stone fruits (e.g., cherries, peaches, and plums). Genetic en- crops, the transgenic approach can severely limit product marketing.
gineering, as well as molecular and conventional breeding, have been The reasons for that are due to legislation that forbids the commer-
employed to generate purple versions of some horticultural crops. The cialization of transgenic foods and the public perception and market-
public interest in the transgenic purple tomato (Butelli et al., 2008) as ability, since the consumer niche most concerned with food nutrition
well as the commercial success of many purple varieties of horticultural tends to reject transgenic foods, even in countries where transgenic

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S. Chaves-Silva et al. Phytochemistry 153 (2018) 11–27

foods are allowed (Dalla Costa et al., 2017). Therefore, a better un- (4CL). At this point, the phenylpropanoid pathway branches off to
derstanding of the mechanisms underlying the anthocyanin repression biosynthesis of coumarins, monolignols (lignans and lignin, aromatic
in parenchymal cells (e.g., genetic regulation of the immediate bio- volatiles –and in some species, salicylic acid) or polyketides (e.g., fla-
synthesis regulators) will facilitate breeding using non-transgenic vonoids, isoflavonoids, stilbenes and pyrones). The flavonoid pathway
methods. advances with the condensation of p-coumaroyl-CoA with three mole-
Here, we present basic information on the enzymology, subcellular cules of malonyl-CoA to produce chalcone (naringenin chalcone or
localization and gene expression regulation of the anthocyanin bio- tetrahydroxychalcone) by chalcone synthase (CHS) (Ferreyra et al.,
synthesis pathway in plant models as well as crop species. We then 2012). Chalcone is converted by chalcone isomerase (CHI) to the fla-
focus our discussion on the differential patterns of anthocyanin accu- vanone naringenin, a central flavonoid intermediate. Thereon, flava-
mulation in tissues and the cell types that comprise the fleshy fruit, and none 3-hydroxylase (F3H) converts naringenin into the flavononol di-
present breeding strategies that can be used to boost anthocyanin hydrokaempferol (DHK or aromadendrin), which can be used by
content in edible structures of the plant. Overall, a better understanding flavonoid 3′-hydroxylase (F3′H) to produce dihydroquercetin (taxi-
of the genetic regulation of anthocyanin biosynthesis and the reasons folin), or alternatively by flavonoid 3′,5′-hydroxylase (F3′5′H) to form
why the pathway is repressed in parenchymal cells will lead to efficient dihydromyricetin (ampelopsin). Next, a set of enzymes with broad
ways to breed food for high nutrient-density in vegetables (Jiang et al., substrate specificity accepts flavononols to move the pathway forward.
2016), fruits (Qi et al., 2014; Sestari et al., 2014) and cereals (Himi and Dihydroflavonol 4-reductase (DFR) converts dihydrokaempferol (or the
Taketa, 2015), potentially with a transformative impact on the health of direct products of F3′H or F3′5′H enzymes) into leucoanthocyanidins,
the consumer population. which are then converted into colored anthocyanidins (e.g., cyanidin,
pelargonidin, delphinidin) by anthocyanidin synthase (ANS, same as
2. The anthocyanin biosynthesis pathway leucocyanidin oxygenase: LDOX). These anthocyanidins can be further
decorated by transferases, such as methyltransferases (OMT) and
Anthocyanins are flavonoid pigments synthesized via the phenyl- acetylases (Sasaki et al., 2014), and further processed by 3-O-glyco-
propanoid pathway (Fig. 2). The entry metabolite is the aromatic amino syltransferases (3GT, same as UDP-glucose:flavonoid-3-O-glycosyl-
acid, phenylalanine (which is produced via the shikimate pathway). transferase: UFGT) to produce anthocyanidin-3-O-glucosides, which are
This precursor is first deaminated by phenylalanine ammonia lyase chemically stable, water-soluble pigments.
(PAL) in the cytoplasm, at the outer surface of the endoplasmic re- Finally, anthocyanins associate with glutathione S-transferase (GST)
ticulum (ER) membrane (Park et al., 2015a; Rasmussen and Dixon, for efficient sequestration into the vacuole (Mueller et al., 2000) with
1999) to produce trans-cinnamic acid, which is then converted into p- the assistance of ABC and MATE transporters localized at the tonoplast
coumaric acid (4-coumaric acid or trans-p-hydroxycinnamic acid, (Zhao and Dixon, 2009). Anthocyanins can also be enclosed in mem-
pHCA) by cinnamate 4-hydroxylase (C4H, CYP73A cytochrome P450 brane-bound bodies (anthocyanin-rich vesicles, often called anthocya-
monooxygenase). Alternatively, in some plant species, tyrosine can be noplasts), which are possibly prevacuolar compartments en route to the
converted to p-coumaric acid by tyrosine ammonia lyase (TAL) or even vacuole (Kallam et al., 2017) and are engulfed by the organelle in a
be used as a minor substrate by PAL (Manela et al., 2015; Yoo et al., process that resembles microautophagy (Chanoca et al., 2015). Fur-
2013). Thereon, p-coumaric acid, usually the most limiting inter- thermore, in some species, the accumulation at high levels of ar-
mediate in this pathway (Nishiyama et al., 2010), is conjugated with omatically-acylated anthocyanins in the vacuole leads to the formation
coenzyme A to produce p-coumaroyl-CoA by 4-coumarate-CoA ligase of aggregates called anthocyanin vacuolar inclusions (AVI) (Markham

Fig. 2. Anthocyanin biosynthesis pathway. Phenylalanine (and occasionally, tyrosine) enters the general phenylpropanoid pathway with PAL (or TAL) activity. CHS
directs p-coumaroyl-CoA to the flavonoid pathway, and the route becomes committed to anthocyanin by the activity of F3H. The broad substrate specificity of DFR
converts flavononols into leucoanthocyanidins, which are oxidized by ANS/LDOX, yielding anthocyanin pigments. Anthocyanins are further decorated with sugars
and other moieties, increasing their stability and solubility. At last, they are conjugated with glutathione by GST and transported for storage into the vacuole via
transporters in the tonoplast, or by the formation of autophagic vacuolar inclusions. PAL: phenylalanine ammonia lyase; TAL: tyrosine ammonia lyase; C4H:
cinnamate 4-hydroxylase; 4CL: 4-coumarate-CoA ligase; CHS: chalcone synthase; CHI: chalcone isomerase; F3H: flavanone 3-hydroxylase; F3′H: flavonoid 3′-hy-
droxylase; F3′5′H: flavonoid 3′,5′-hydroxylase; DFR: dihydroflavonol 4-reductase; ANS: anthocyanidin synthase; LDOX: leucocyanidin oxygenase; UFGT: UDP-glu-
cose:flavonoid-3-O-glycosyltransferase; GST: glutathione S-transferase; MATE: multi-antimicrobial extrusion protein; ABC: ATP-binding cassette transporter; EBGs:
early biosynthesis genes; LBGs: late biosynthesis genes.

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S. Chaves-Silva et al. Phytochemistry 153 (2018) 11–27

Fig. 3. Chemical structures of anthocyanins. (A) The flavylyum cation is the basic structure of anthocyanins. The ring names, atom numbering, and positions of
common substitutions are indicated. (B) Nomenclature key of the most common anthocyanidins. Glycosylation generally occurs in the R3 position to form antho-
cyanins. Anthocyanidins without the hydroxyl group in R3, which precludes glycosylation in this position, are called 3-deoxyanthocyanidins. Commonly, the R4
position consistently contains a hydroxyl group. (C) Cyanidin-3-O-glucoside (chrysanthemin) is a very common anthocyanin in plant tissues.

et al., 2000). diversity found even in closely related species. The expression of the
The general structure of most common anthocyanins is character- early flavonoid biosynthesis genes (EBGs: CHS, CHI, F3H, F3′H, and
ized as a flavylium cation (2-phenylbenzopyrylium or 2-phenylchro- FLS) is modulated by subgroup 7 R2R3-MYB transcription factors
menylium) with 3,5,7-trihydroxylations (Fig. 3). Currently, at least 29 (MYB11, MYB12, MYB111) in addition to MYB75/PAP1 (Liu et al.,
different forms of anthocyanins have been identified only in Arabi- 2015a). The late biosynthesis genes (LBGs: DFR, ANS/LDOX, UFGT)
dopsis, including cis and trans isomers. Cyanidins are the predominant and transporters embedded in the tonoplast (MATE, ABC) are regulated
structure, while all other types are considered cyanidin derivatives by a trio of transcription factors known as the MBW ternary complex
carrying different decorations, such as glycosylation, acylation, and (R2R3-MYB, bHLH, and WD40) (Dubos et al., 2010; Stracke et al.,
methylation (Shi and Xie, 2014). Furthermore, anthocyanin structures 2007). Importantly, in other systems like maize, the expression of MYBs
are cataloged numerically (A1, A2, … A19 and so forth), indicating alone do not suffice, and the MBW complex is also necessary to active
different properties of solubility, light absorption, and distribution EBGs (Petroni and Tonelli, 2011).
pattern in various parts of the plant (Luo et al., 2007). For example, A11 The main R2R3-MYB transcription factors associated with the for-
molecules {i.e., the cyanidin 3-O-[2-(2-(sinapoyl)xylosyl)-6-O-(4(glu- mation of the MBW complex and induction of late anthocyanin bio-
cosyl)-p-coumaroyl)glucoside]5-[6-O-(malonyl)glucoside]} are most synthesis in Arabidopsis belong to subgroup 6: PAP1/MYB75, PAP2/
abundant in leaves (Rowan et al., 2009), whereas A5 anthocyanins MYB90, MYB113, and MYB114 (Shi and Xie, 2014). Transgenic plants
{cyanidin 3-O-[2-xylosyl-6-O-(4(glucosyl)-p-coumaroyl)]5-[6-O-(mal- overexpressing PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENTA-
onyl)glucoside]} are most frequently found in roots (Tohge et al., TION 1) accumulate high amounts of anthocyanins in roots, stems,
2005). Interestingly, the composition profile of anthocyanins can leaves and flowers (Borevitz et al., 2000; Rowan et al., 2009; Tohge
change under different stresses, and the biological reasons for this fact et al., 2005). Consistently, pap1 mutants are acyanic, confirming that
remain highly speculative (Kovinich et al., 2014, 2015). To date, more this transcription factor is an essential positive regulator of the pathway
than 650 different anthocyanin structures derived from 35 monomeric in Arabidopsis. Studies have shown that in particular environmental
anthocyanidins have been identified in nature, over 90% of which de- conditions or specific developmental stages of the plant, PAP2,
rived from the six most common anthocyanidins (cyanidin, delphinidin, MYB113, and MYB114 can also activate the expression of structural
pelargonidin, malvidin, peonidin, and petunidin) (Andersen and genes, such as DFR and ANS (Shi and Xie, 2014), although to a lesser
Jordheim, 2010). extent when compared to PAP1 (Petroni and Tonelli, 2011). The role of
inhibitory MYBs on anthocyanin biosynthesis is discussed in a sub-
sequent section.
3. Transcriptional regulation of anthocyanin biosynthesis in the Regarding bHLH factors of the MBW complex, GLABRA3 (GL3),
Arabidopsis model ENHANCER OF GLABRA 3 (EGL3) and TRANSPARENT TESTA 8 (TT8)
play redundant functions in modulating the proanthocyanidin and an-
Although some branches of the phenolic pathway are restricted to thocyanin biosynthesis (Baudry et al., 2004; Zhao et al., 2008). Al-
certain species, such as phlobaphenes (in maize, kola nut, and Sequoia though both GL3 and EGL3 can stimulate F3′H expression, EGL3 is the
redwood) and isoflavones (in legumes), other compounds are broadly predominant factor responsible for activating the LBGs DFR and ANS/
distributed in the plant kingdom, such as flavonols, proanthocyanidins, LDOX (Petroni and Tonelli, 2011). For a long time, EGL3 was suggested
and anthocyanins (Grotewold, 2005; Hernández et al., 2009; Mackova to be the most prominent regulator during anthocyanin biosynthesis.
et al., 2006). However, a study conducted under low nitrogen conditions demon-
A major regulatory mechanism of the flavonoid pathway is through strated that it is GL3 the most critical regulatory factor in leaves under
the transcriptional coordination of structural genes, such as biosyn- this condition (Feyissa et al., 2009). These observations suggest that
thetic enzymes (Petroni and Tonelli, 2011). The enzymatic genes of the EGL3 and GL3 have physiological specificities during development or
flavonoid metabolism are usually conserved in structure and function, stress. Finally, the Arabidopsis TT8 protein is required in siliques and
although minimal differences can lead to the myriad of chemical

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S. Chaves-Silva et al. Phytochemistry 153 (2018) 11–27

endothelial cells of the seed for regular expression of DFR as well as the structural genes (Cui et al., 2014; Lei et al., 2015). Additionally, LAT-
anthocyanidin reductase (ANR) BANYULS (BAN) (Nesi et al., 2000), ERAL ORGAN BOUNDARY DOMAIN (LBD) factors (LBD37, LBD38,
which expression reduces anthocyanin biosynthesis by competing for LBD39) have been implicated in suppressing anthocyanin biosynthesis
substrates to produce flavanols and condensed tannins (Kitamura et al., under nitrogen-sufficient conditions, and particularly by nitrate (Zerche
2016; Xie et al., 2003). Moreover, the transcriptome profile of pap1-d et al., 2016) via the transcriptional repression of PAP1 and PAP2 (Rubin
mutant leaves overexpressing PAP1-D revealed that TT8 is strongly et al., 2009; Zhou et al., 2012). Furthermore, in opposition to other
induced in seedlings by PAP1-D (Shi and Xie, 2011). NAC factors found to induce anthocyanin biosynthesis, strong abiotic
Among the 269 genes in the Arabidopsis genome coding for proteins stresses trigger the expression of ANAC032, which induces the expres-
containing at least one WD-40 motif (van Nocker and Ludwig, 2003), to sion of anthocyanin repressors (e.g., MYBL2 and SPL9) (Mahmood
date, only TRANSPARENT TESTA GLABRA 1 (TTG1) has been char- et al., 2016). If these networks are evolutionarily conserved beyond the
acterized as a regulator of anthocyanin biosynthesis. TTG1 is expressed Brassicaceae, this knowledge can significantly impact crop molecular
constitutively in all tissues and throughout plant development, with breeding.
little response to changes in environmental conditions at the tran-
scriptional level (Cominelli et al., 2008; Olsen et al., 2009). Mutations 5. Post-transcriptional and post-translational regulation of
in TTG1 resulted in numerous pleiotropic effects, including defective anthocyanin metabolism in Arabidopsis
trichome development, and deficient biosynthesis of proanthocyanidin
and anthocyanins in vegetative tissues and the seed coat (Cominelli Whereas we know in great details about the anthocyanin bio-
et al., 2008; Walker et al., 1999). synthesis regulation at the transcriptional level, the post-transcriptional
Although the transcription factors referred above are central reg- and post-translational mechanisms controlling this pathway have only
ulators of anthocyanin production in Arabidopsis (Petroni and Tonelli, recently started to unveil. Anthocyanin accumulation induced by high
2011) and other species (Albert et al., 2014; Goodman et al., 2004), the light intensities in Arabidopsis requires phosphorylation of MYB75/
precise identification of which bHLH and MYB orthologs in other spe- PAP1 by MAP KINASE 4 (MPK4) (Li et al., 2016). On the other hand,
cies form functional MBW complexes to activate the anthocyanin bio- dark induces the expression of CONSTITUTIVE PHOTOMORPHOGENIC
synthesis during the various stages of plant development and environ- 1/SUPPRESSOR OF PHYTOCHROME A (COP1/SPA) E3 ubiquitin li-
mental conditions remains quite limited. Different (positive and gase, which targets MYB75 and MYB90/PAP2 for degradation (Maier
negative) feedback mechanisms control the responses to environmental and Hoecker, 2015). The utilization of these regulatory mechanisms in
changes (Rowan et al., 2009). Such responses must occur in a fast and other species remains to be determined.
precise way and are dependent on the coordinated action between ac- In a mechanism well conserved in dicots, PAP1, PAP2, and MYB113
tivators and repressors. Cominelli et al. (2008) demonstrated that under transcripts are targeted by the trans-acting siRNA TAS4-siRNA81(-) in
high light intensity situations, PAP1 is the first factor to be tran- response to sugar exposure. Under non-inductive conditions, TAS4
scriptionally activated. Thus, together with EGL3 and TTG1, PAP1 is transcripts are targeted by miR828 in a negative feedback regulatory
thought to compose the initial MBW complex that induces the pathway. loop fashion (Luo et al., 2012). Additionally, miR156 targets transcripts
This original complex possibly activates the expression of TT8, which in of SPL repressors, thus boosting anthocyanin biosynthesis (Cui et al.,
turn competes with EGL3 for the complex. Importantly, TT8 expression 2014; Gou et al., 2011; Zhao et al., 2017). However, miR156 also
is transcriptionally self-regulated by a positive feedback loop me- participates in the regulation of other developmental processes, which
chanism, leading to a robust activation of LBGs (Baudry et al., 2006). makes it an unlikely candidate for molecular crop breeding due to
potential pleiotropic effects.
4. Repression of anthocyanin biosynthesis in Arabidopsis
6. Environmental control of anthocyanin biosynthesis in
Several inhibitory transcription factors are involved in the tran- Arabidopsis
scriptional repression of the anthocyanin pathway. In Arabidopsis, the
MYB repressors first discovered were MYB4 and MYB32, which induce Several environmental factors induced anthocyanin biosynthesis. In
lignin biosynthesis (Preston et al., 2004). Later, MYB7 and MYB4 were general, stresses cause the expression of MYB and bHLH transcription
found to repress the flavonoid biosynthesis by negatively regulating factors of the MBW complex via inhibiting the expression of their
DFR and UGT gene expression (Fornalé et al., 2014). These factors form transcriptional repressors. For example, high-light conditions rapidly
a distinct phylogenetic clade of R2R3-MYB repressors (subgroup 4) that inhibit MYBL2 expression, resulting in the activation of PAP1, PAP2,
prevent the complete formation of the MBW complex in addition to and GL3, thus favoring the accumulation of anthocyanin at high levels
having the active C-terminal EAR transcription repressor motif (Hiratsu (Dubos et al., 2008). In turn, low temperatures coupled with a high
et al., 2003; Kranz et al., 1998). incidence of light increases MYBL2 transcription, thus inhibiting PAP1,
Furthermore, single-repeat R3-MYB factors, such as CAPRICE (CPC) TT8, TTG1, and EGL3. The MYBL2 expression can reduce anthocyanin
and MYBL2, are negative regulators of the anthocyanin pathway by biosynthesis in leaves, even in transgenic plants constitutively expres-
acting via competitive inhibition of the MBW complex formation sing PAP1 (Rowan et al., 2009).
(Dubos et al., 2008; Nemie-Feyissa et al., 2014; Rubin et al., 2009; Shi Nitrogen and phosphate deficiencies activate PAP1, PAP2, GL3, and
and Xie, 2010; Zhu et al., 2009) (Fig. 4b). In addition to directly reg- MYB12 transcription, leading to anthocyanin accumulation (Lea et al.,
ulating the MBW complex by competing with positive MYB factors, 2007). This mechanism involves the E3 ubiquitin ligase NITROGEN
MYBL2 contains a C2 repressor motif that acts indirectly by preventing LIMITATION ADAPTATION (NLA) (Peng et al., 2007, 2008), which
the expression of positive regulators, such as TT8, PAP1, and PAP2 recruits the E2 conjugase PHOSPHATE 2 (PHO2/UBC24) to target the
(Matsui et al., 2008). Moreover, although TT8 self-promotes its own phosphate transporter PT2 for degradation by the proteasome (Park
expression, it also activates the expression of its repressor, MYBL2, thus et al., 2014). The molecular connection between phosphate and ni-
counterbalancing the activation mechanism (Fig. 4a). trogen deficiency signaling pathways through NLA have yet to be fully
SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcrip- established, especially regarding the involvement of anthocyanins in
tion factors negatively regulate anthocyanin biosynthesis in Arabidopsis the process.
by interacting with MYB factors and preventing the MBW complex as- Exogenous application of sugars is another potent inducer of an-
sembly (Gou et al., 2011). However, under low phosphate, a condition thocyanin biosynthesis in Arabidopsis (Teng et al., 2005). In response to
known to stimulate anthocyanin biosynthesis, various SPL transcripts high sucrose, expression of NAC factors activate anthocyanin bio-
(e.g., SPL3, SPL9) are targeted by miR156 to relieve the repression of synthesis, such as ANAC078 and JUNGBRUNNEN1 (ANAC042/JUB1),

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S. Chaves-Silva et al. Phytochemistry 153 (2018) 11–27

Fig. 4. The MBW ternary complex can be formed by


distinct orthologous transcription factors to regulate
the late biosynthesis genes (LBGs) of the anthocyanin
pathway. (A) The complete ternary complex can act
as an activator of anthocyanin biosynthesis genes,
but the joining of repressors can hinder protein-
protein interactions, thus precluding proteins to as-
semble into the complex, as well as by actively in-
hibiting activation of gene expression through re-
pressive domains. (B) For the formation of the MBW
complex, MYB proteins play a pivotal role in dic-
tating the transcriptional control. Moreover, in
Arabidopsis, TT8 shows positive feedback self-acti-
vation, which has not been reported for alternate
bHLH orthologs (EGL3 and GL3). The alternative
MYB repressor, CPC, has not been shown to possess
such inhibitory activity on the expression of a bHLH
factor. The expression of the WD40 factor is fre-
quently not limiting in most species. The stoichio-
metry of the complex is not represented in the figure.

(Morishita et al., 2009; Wu et al., 2012). Additionally, the histone biosynthesis has been expanding in cultivated species through nu-
chaperone FACT, which is an elongation factor associated with RNA merous studies. The evolutionary conservation of this pathway
Polymerase II, is required for the induction of anthocyanin biosynthesis throughout land plant evolution makes much of what we have learned
under stress conditions, such as high light (Pfab et al., 2018). The FACT in Arabidopsis and other model species also applicable to crops.
factor is a heterodimer formed by SSRP1 and SPT6 subunits and seems Although not food crops, petunia (Petunia x hybrida, Solanales) and
to act directly on the anthocyanin structural genes, possibly by opening snapdragon (Antirrhinum majus, Lamiales) are well-established models
the chromatin of promoter regions of these genes to facilitate tran- for anthocyanin biosynthesis in the Asterids clade. For example, R2R3-
scription of both, early as well as late enzymes, but not on transcrip- type MYB27 and the R3-MYBx are anthocyanin repressors in petunia
tional regulators. Overall, the picture of the molecular pathways from (Albert et al., 2014). The identification of specific functional orthologs
the stimuli to the expression of structural genes of the biosynthesis in crops playing comparable roles to those characterized in model
pathway via signal transduction, transcription factors, chromatin re- species is necessary, as well as the characterization of possible reg-
modelers, and post-transcriptional control remains incomplete, but it is ulatory nuances in each species of interest. Furthermore, advancing our
slowly being revealed in model species. comprehension on how the anthocyanin pathway is regulated in spe-
cific tissues of edible plant structures will better guide our efforts to-
7. Genetic regulation of anthocyanin biosynthesis in crops wards nutrient-dense breeding.

Our knowledge of the regulatory mechanisms of anthocyanin

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S. Chaves-Silva et al. Phytochemistry 153 (2018) 11–27

7.1. Fleshy fruits: absence of anthocyanin biosynthesis in parenchymal cells expression occurs during fruit ripening, where it balances pigment ac-
of the mesocarp cumulation during the late stages of fruit maturation (Aharoni et al.,
2001). Also, FaMYB1 is possibly responsible for the fruit white core
Regarding the transcriptional regulation of anthocyanin synthesis, present in many cultivars. It would be interesting to assess whether the
apple, strawberry, and grape are the best-studied cases in fruit crops - FaMYB1 expression is negatively regulated by light, hindering the more
per their horticultural classifications as fruit, since botanically, apple internal tissues to accumulate pelargonidin-3-O-glucosides, the primary
and strawberry are accessory fruits (Lin-Wang et al., 2010; Matus et al., anthocyanin type in strawberry (Giampieri et al., 2012). Interestingly,
2010; Takos et al., 2006). Besides the pigmented epidermis, the failure RNAi knock-down of bHLH33, thought to be a potential partner of
of parenchymal cells of the fleshy mesocarp to synthesize anthocyanins MdMYB10, did not have any significant impact on anthocyanin content
can be witnessed by the pigmented vascular tissues commonly found (Lin-Wang et al., 2014).
embedded in the mesocarp, as occasionally seen in some white-fleshed In peach, a study with the red-fleshed variety ‘Dahongpao’ points to
apple varieties. This fact also underscores the non-essentiality of direct the participation of NAC and SPL transcription factors operating in
light incidence over the cells to induce the pathway. internal tissues of the fruit. In this variety, the red flesh develops due to
In apple, besides the typical varieties with non-pigmented flesh, two the expression of PpMYB10.1, which is activated during fruit matura-
different groups of red-fleshed fruit variants have been identified. In tion by the NAC transcription factors BLOOD (BL) and PpNAC1, in
type-I apples, the pigment occurs not only in the fruit (peel and flesh) addition to the repression of the PpSPL1 repressor (Zhou et al., 2015).
but also in vegetative organs of the whole plant. On the other hand, the This study leads to a possible breeding strategy to created blood-fleshed
red pigmentation of type-II apples occurs exclusively in the fruit (peel peaches by simply inactivating the PpSPL1 gene.
and flesh) tissues. Besides the genetic difference responsible for pig- Given that anthocyanins are the pigments responsible for the color
mentation types of red-fleshed apples, they also show a different pat- of red wines, there is an immense interest from this industry to better
tern of anthocyanin accumulation during fruit development. Type-I understand the control of the pathway in grape berries. MYB factors
cultivars show intense pigmentation in the early stages of fruit devel- have been confirmed to play crucial roles in the phenylpropanoid
opment, reducing the intensity of the fruit color during ripening. pathway in this species (Zhang et al., 2016). Anthocyanin synthesis
Meanwhile, type-II varieties bear fruits that are acyanic in the early during berry ripening in dark cultivars is induced by at least three MYB
stages of development, and pigmentation is gained during maturation factors (VvMYBA1, VvMYBA2, VvMYB5b), which specifically regulate
(Hamada et al., 2015). the expression of the LBGs, such as UFGT, GST, OMT, and anthoMATE
The type-I apple phenotype is caused by the expression of a MYB (Chiu et al., 2010; Deluc et al., 2008). VvMYBA1 has also been im-
activator (the allelic versions MdMYBA, MdMYB10 and MdMYB1), plicated in inducing the expression of an anthocyanin acyltransferase
which induces anthocyanin biosynthesis (Espley et al., 2009; Lin-Wang (Vv3AT) (Rinaldo et al., 2015). Congruently, some studies comparing
et al., 2010). Rearrangements of the R1 cis-element in the MdMYB10 cyanic and white grape cultivars showed that UFGT is the critical en-
promoter, such as the tandem addition of five identical 23-bp repeats zyme limiting the accumulation of anthocyanin in several white grape
(making the R6 element), were sufficient to promote its transcriptional varieties (Boss et al., 1996; Kobayashi et al., 2001).
self-activation and subsequently cause constitutive anthocyanin accu- The WD40 (VvWDR1 and VvWDR2) and bHLH (VvMYC1 and
mulation, including in the parenchymal cells of the mesocarp VvMYCA1) transcription factors were identified as potential compo-
(Brendolise et al., 2017; Espley et al., 2009). When overexpressed in nents of the MBW complex in grapevine (Cutanda-Perez et al., 2009).
Arabidopsis, MdMYB10 strongly induced anthocyanin biosynthesis in Their expression patterns are correlated with the VvMYBA1 and UF3GT
virtually all tissues of the plant (Espley et al., 2009). On the other hand, transcriptional profiles as well as with the anthocyanin accumulation in
the type-II accumulation pattern is independent of MdMYB10. In vari- the berry. Overexpression of VvWDR1 in Arabidopsis activated antho-
eties such as 'Pink Pearl' and 'JPP35′, the red color of the fruit flesh is cyanin biosynthesis, demonstrating the functional conservation of this
rather regulated by MdMYB110a (Chagné et al., 2013), which is a genetic network since their last common ancestor that existed circa 115
dominant allele genetically linked to the S3-RNase locus that is re- million years ago (Matus et al., 2010; Schneider et al., 2004; Wikström
sponsible for self-incompatibility (Sekido et al., 2010; Umemura et al., et al., 2001). Similarly to TT8 in Arabidopsis, VvMYC1 also self-reg-
2013). ulates its expression through a positive feedback mechanism. Studies
bHLH transcription factors (MdbHLH3 and MdbHLH33) control the showed that VvMYC1 can interact with all MYB factors analyzed
expression of anthocyanin biosynthesis genes in cyanic cells of apple (VvMYB5a/5b, VvMYBA1/A2, VvMYBPA1) and that it also induces the
(Espley et al., 2007, 2009). Furthermore, despite the fact that the WD40 anthocyanin and proanthocyanidins biosynthetic genes in grapevine
factor MdTTG1 (Brueggemann et al., 2010) is considered as an essential (Hichri et al., 2010).
factor for the induction of polyphenol biosynthesis in apple, its ability Whereas most dark grapes develop non-pigmented mesocarp, some
to interact with MYB and bHLH proteins to form the MBW complex has few genotypes (called teinturier varieties, such as ‘Alicante Bouchet’,
not been fully confirmed. Apparently, MdTTG1 associates with the ‘Salvador’, ‘Rubired’) do develop cyanic flesh. Despite their generally
bHLH, but not directly with MYB proteins to regulate anthocyanin ac- poor vinification quality, teinturier berries are used in blends of deep-
cumulation (An et al., 2012). colored wines (Guan et al., 2016; He et al., 2010). Regarding the
MdMYB10 orthologs have also been identified in other Rosaceae structural genes in grape, Xie et al. (2015) compared the expression of
fruit species, such as pear, plum, cherry, peach, raspberry, and straw- anthocyanin biosynthetic genes in the berry skin and pulp of teinturier
berry (An et al., 2012; Kadomura-Ishikawa et al., 2015; Shen et al., and common grape varieties. The results showed that OMT, AM3, GST,
2013; Zhou et al., 2015). In strawberry, FaMYB10 expression only F3′5′H, ANS/LDOX and MYBA1 were highly expressed in the pulp of
correlates with ripening after the fleshy receptacle reaches its maximum teinturier grapes while hardly detectable in the other group. These re-
size (Kadomura-Ishikawa et al., 2015; Lin-Wang et al., 2010). Fur- sults reveal the primary genes responsible for pigment accumulation in
thermore, FaMYB10 overexpression increased anthocyanin accumula- teinturier varieties (Xie et al., 2015). Several R2R3-MYB repressors (C2
tion by 70% compared to non-transgenic wild strawberries (Lin-Wang repressor motif clade) expressed in the grape berry showed to repress
et al., 2014). Conversely, knockout mutants of this transcription factor either the general phenylpropanoid biosynthesis (VvMYB4a and
failed to express anthocyanin structural genes (both, EBGs as LBGs), VvMYB4b) or more specifically the anthocyanin and proanthocyanidin
supporting the central role of FaMYB10 in stimulating anthocyanin pathways (VvMYBC2-L1 and VvMYBC2-L3) in heterologous models
biosynthesis in strawberry (Medina-Puche et al., 2014). On the other (petunia and Nicotiana benthamiana) (Cavallini et al., 2015).
hand, FaMYB1 was the first anthocyanin repressor characterized in a In Asian pear (Pyrus pyrifolia), a comparison between the red peel
fruit species (Aharoni et al., 2001; Lin-Wang et al., 2010). High FaMYB1 (‘Aoguan’) and the acyanic cultivar (‘Mantianhong’) showed that

17
S. Chaves-Silva et al. Phytochemistry 153 (2018) 11–27

PyMYB10 and PyMYB10.1 are the primary MYB candidates to activate the course of evolution. Moreover, the miR156 expression during the
anthocyanin biosynthesis in the fruit peel (Feng et al., 2010). It was late stages of the fruit development was suggested as a potential
revealed later that both could form a functional complex with PybHLH strategy to enhance anthocyanin content in pomegranate (Saminathan
in yeast-two-hybrid as well as the BiFC system (in onion epidermis), et al., 2016). However, pleiotropic effects of miR156 overexpression
despite failing to show activation in the tobacco leaf infiltration system must be taken into account due to its many targets and involvement in
(Feng et al., 2015). A recent study showed that PpMYB10, PpbHLH, and the regulation of diverse biochemical and developmental mechanisms
PpWD40 are expressed in the peel and can form stable complexes (Qian in the plant (Guo et al., 2017; Xie et al., 2017; Xu et al., 2016).
et al., 2017). Moreover, a suite of nine SPL repressors was expressed in In the genus Solanum, many species bear fleshy fruits that are
this tissue, along with the SPL-targeting miR156 (Qian et al., 2017). On naturally pigmented with anthocyanins and other flavonoids, such as
the other hand, a genetic analysis of the Chinese pear (P. × bretsch- black nightshade (S. nigrum), eggplant (S. melongena) and tomato (S.
neideri) fruit identified PyMYB144 as essential to the development of lycopersicum). Non-transgenic varieties of purple tomato (e.g., 'Indigo
the red peel. Also, PyERF3 was found to interact with PyMYB144 and Rose' and 'Black Galaxy') were obtained by crosses of the cultivated
PybHLH3 to activate anthocyanin biosynthesis, and that the expression species (S. lycopersicum) with compatible wild species, such as S. lyco-
of PyMYB114 and PyMYB10 acted additively to boost the pathway (Yao persicoides, S. chilense, and S. cheesmaniae, leading to successful
et al., 2017). When assessing the epidermis of six different cultivars of breeding of plants producing fruits with higher anthocyanin content
European pear (P. communis), Yang et al. (2015) found no correlation (Jones et al., 2003). Moreover, the introgression of just three natural
between the expression of EBGs and anthocyanin content in fruits, re- allelic variants from wild species - Anthocyanin fruit (Aft), atroviolacium
vealing that these enzymes were not limiting. Moreover, the level of (atv) and high pigment 2 (hp2) – induced a dark purple color in the fruit
expression of the late enzymes ANS/LDOX and UFGT correlated with epicarp (Sestari et al., 2014). It is noteworthy that in addition to an-
anthocyanin content. Co-expression analysis revealed a strong asso- thocyanins, the triple allelic combination (Aft/atv/hp2) also induced
ciation between the expression of MYB10 and bHLH33 and anthocyanin high concentrations of ascorbate and lycopene, without neither loss in
in cyanic fruit epidermis (Yang et al., 2015). Surprisingly, however, was soluble solids content in the fruit nor yield compared to the near-iso-
the negative correlation between anthocyanin accumulation and the genic, red-fruit plants (Sestari et al., 2014). This finding demonstrates
expression of a WD40 factor, which usually is not a limiting element of that the trait ‘fruit with high anthocyanin content’ does not compete
the MBW complex in most species studied. If confirmed, this protein significantly for substrates (including photosynthates) with other re-
could be functioning as a decoy element that competes with positive levant metabolic pathways, or lower the synthesis of other nutrients.
components of the ternary complex to halt the expression of structural Therefore, this trait can rather be improved along with the increase of
genes in pear (Yang et al., 2015), making it a potential target for mo- other nutrients and without any loss in yield.
lecular breeding, perhaps via CRISPR-based genome editing. The identification of the exact MYB factor responsible for activation
In another species of the Rosaceae family, purple-leaf plum cultivar of the anthocyanin biosynthesis in tomato fruits is complex because the
(Ziyeli) accumulates anthocyanin throughout the plant, including in the locus associated with this trait on chromosome 10 (e.g., ant1, an2) is in
fruit flesh, much like type-I red-fleshed apples. Among the several MYB a genomic region containing four in-tandem duplications of the MYB
factors evaluated in this species, the primary gene affecting antho- gene. The introduction of an activation tag (4 × pro35S) upstream of
cyanin biosynthesis is PcMYB10.6, which was highly expressed in all the MYB ANT1 gene induced high and constitutive accumulation of
cyanic organs (Gu et al., 2015). In wild cherry (Prunus avium), anthocyanin in tomato (Mathews et al., 2003). This work reveals that
PaMYB10.1–1 correlated with anthocyanin levels in the fruit, although ANT1 is able to induce the pathway, but it does not confirm that it is the
PaMYB10.1–3, which is only moderately expressed in this organ, was key gene in natural conditions. Indeed, it has been recently shown that
capable of inducing anthocyanin biosynthesis in tobacco leaf infiltra- although functional when constitutively overexpressed, ANT1 is not
tion assay. PabHLH3 was shown to be a positive regulator, while expressed conspicuously in the plant, and that instead it is AN2 that
PabHLH33 was an effective inhibitor of the biosynthesis. Moreover, responds to light induction and low temperatures to activate antho-
much like in other systems, the PaWD40 expression is relatively con- cyanin biosynthesis (Cao et al., 2017). Overall, the actual MYB re-
stant and non-limiting, including in the fruit flesh (Starkevič et al., sponsible for purple skin in tomato has not been resolved. Moreover,
2015). the gene in the atv locus responsible for the anthocyanin phenotype has
The vast germplasm diversity available for pomegranate makes it recently been identified as SlMYBATV, an R3-MYB repressor. In S.
possible to find genotypes bearing fruits with black, red, pink, green cheesmaniae, the original source of the atv locus, a loss-of-function
and white peel (Rouholamin et al., 2015). In a comparative study of mutation leads to anthocyanin accumulation in epidermal cells of the
skin fruit pigmentation and expression of the transcription factors fruit (Cao et al., 2017).
PgAN1 (bHLH), PgAN2 (MYB), and PgWD40 (Rouholamin et al., 2015) The transgenic purple tomato expressing two heterologous tran-
demonstrated that PgWD40 and PgAN2 factors are the main factors scription factors from Antirrhinum majus (the MYB Rosea1 and the bHLH
responsible for cyanidin biosynthesis by activating the expression of Delila) reveals aspects that are highly significant for breeding (Butelli
PgDFR and PgANS/LDOX. The PgWD40 expression pattern was con- et al., 2008). The high levels of anthocyanins in both the peel and the
siderably different among the genotypes studied, being induced up to flesh of the fruit, especially delphinidin-3-O-(trans-coumaroyl)-rutino-
60-fold in varieties bearing black fruit compared with those with side-5-O-glucoside and petunidin-3-O-(p-coumaroyl)-rutinoside-5-O-
acyanic skin. The same trend was observed for PgDFR, which showed glucoside, demonstrate that substrates are not limiting in the flesh.
virtually no expression in green and white fruits. On the other hand, Therefore, the failure of parenchymal cells to accumulate anthocyanin
PgAN2 was continuously expressed in all four genotypes, but at a is fundamentally genetic due to lack of expression of the MYB and
higher level in fruit with darker peels (black and red). Therefore, in bHLH elements of the MBW ternary factor, since the WD40 component
pomegranate, the MBW complex could be formed by PgAN1, PgAN2, (W) is constitutively expressed in the tomato fruit (our data).
and PgWD40, and a positive correlations between PgAN2, PgWD40 and Eggplant (S. melongena) also displays genetic variation with fruits
PgDFR expression and anthocyanin accumulation were found in the showing peel that is entirely cyanic or acyanic, as well as bicolored in
fruit skin (Rouholamin et al., 2015). In addition, Ben-Simhon et al. gradient or striping patterns. Nevertheless, the fruit the flesh of the fruit
(2011) complemented Arabidopsis ttg1-9 mutants by overexpressing remains virtually acyanic in all commercial groups. By just expressing
PgWD40 and restored anthocyanin accumulation in vegetative tissues, SmMYB1 constitutively (Zhang et al., 2016), plants accumulated an-
restituted mucilage production in the seed coat, as well as the growth of thocyanins in leaves, petals, stamens, as well as fruit tissues, including
trichomes on stems and leaves. These results further confirm the high in internal tissues. Transcriptional analysis showed that most structural
conservation of the genetic network centered on the MBW complex over genes of the anthocyanin biosynthesis pathway were highly expressed

18
S. Chaves-Silva et al. Phytochemistry 153 (2018) 11–27

in transgenic lines compared to non-transformed controls. This reveals that accumulate anthocyanins in both, peel and flesh. The epidermis of
a pivotal role of SmMYB1 in the induction of the pathway (Zhang et al., purple potatoes still stores anthocyanins at higher levels than the purple
2016). flesh, where the expression of structural genes of the pathway is re-
A distinct group of Citrus sinensis cultivars is collectively called duced (Fogelman et al., 2015; Liu et al., 2016). The bHLH transcription
blood oranges. They accumulate anthocyanins predominantly in the factors StJAF13 and StAN1 were identified as responsible for hier-
endocarp of the fruit (hesperidium), which encompasses the edible archically controlling this trait, with StJAF13 first activating StAN1
portion (pulp), whereas the exocarp (the flavedo and albedo layers) expression, which then turns the structural genes on (Montefiori et al.,
remains lacking anthocyanin completely (orange and white, respec- 2015). Heterologous expression of StAN1 activated the expression of
tively). This phenotype is unusual given that in most species, antho- CHI, F3′H, F3H, and ANS/LDOX in tobacco flowers. Furthermore, RNAi
cyanins accumulate preferentially in the epidermis. Compared with silencing of the closest tobacco orthologs (NtAN1 and NtJAF13) pro-
blonde oranges, CHS, ANS/LDOX, and UFGT were differentially ex- duced plants with white and pale pink flowers, respectively, confirming
pressed and strongly correlated with anthocyanin levels in the blood the evolutionary conservation of this network in the Solanaceae family
pulp (Cotroneo et al., 2006). The first work published on anthocyanin (Montefiori et al., 2015).
genetic regulation in citrus described CsMYB8 and the bHLH CsMYC2 It is interesting to note the similarity of cyanic leafy structures as
as controlling the pathway, along with an R2R3-MYB factor (RUBY) evolutionarily divergent as onion and cabbage, each of which accu-
that was implicated in pulp anthocyanin levels (Cotroneo et al., 2006). mulates anthocyanins in epidermal cells while showing acyanic meso-
While RUBY expression was only found in the blood pulp, CsMYC2 phylls. The onion bulb is formed by a compact cluster of superimposed
expression was detected in both types and showed that the primary modified leaves (tunic) on a short basal stem (plate) (Slimestad et al.,
limitation to anthocyanin accumulation in the flesh of blonde oranges is 2007). Comparably, the cabbage head is formed by overlapping, com-
the absence of RUBY expression (Cultrone et al., 2010). Following, the pacted leaves (Yuan et al., 2009). This fact underlines that the tendency
sequencing of the RUBY promoter region in the cv. Moro (a blood of epidermal cells to synthesize anthocyanins compared to parenchymal
variety) compared to the Cadenera (a blonde variety) revealed a 500- cells is a highly conserved trait in plants. Furthermore, as demonstrated
nucleotide insertion in Moro. The sequence of this insert resembled long by some underground structures that can also be cyanic in some vari-
terminal repeats (LTRs) of Copia retrotransposons and activated RUBY eties (e.g., onion, potato, sweet potato, turnip, carrot), although light is
expression during cold stress. Indeed, low temperature is a major factor often a critical inducer of anthocyanin accumulation in aboveground
involved in pigment development in blood oranges during fruit ripening structures, in some cases the biosynthesis pathway can be induced in
(Butelli et al., 2017). the dark.
It is suggestive that in at least two independent instances (apple and Moreover, in onion, another study revealed that the R2R3-MYB
blood orange), insertions close to the transcription start site of a MYB factor MYB1 is the primary anthocyanin regulator a purple variety
gene associated with anthocyanin biosynthesis led to a ubiquitous ac- (‘California Red’). Anthocyanin biosynthesis was induced by the over-
cumulation of anthocyanin in internal organs of the fruit. As discussed expression of MYB1, whereas the opposite was observed in RNAi lines.
below for inflorescence, the same was found true for purple cauliflower Ectopic red pigmentation was also found in garlic plants expressing the
(Chiu et al., 2010). Therefore, a repressive cis-element potentially re- onion MYB1 together with a bHLH factor (Zm-Lc) but not when ex-
cognized by inhibitory transcription factors may hinder the activation pressed alone (Schwinn et al., 2016).
of anthocyanin activators. A closer analysis of putative conserved in- A comparative expression analysis of anthocyanin structural genes
hibitory cis-elements in promoter regions of a positive transcription between purple and green cabbage (Brassica oleracea var. capitata)
factor may unravel an efficient tool for breeding, especially with the showed that EBGs (CHS, F3H, and F3′H), as well as LBGs (DFR, ANS/
emerging genome-editing techniques. LDOX, and GST), are positively regulated in purple genotypes. Although
Color fading due to anthocyanin degradation has been observed in no drastic change was observed for PAL1 and CHI expression between
petals of ornamental flowers (e.g., petunia) as well as some fruit vari- the cultivar groups, the expression of the late structural gene, ANS/
eties, such as the ‘Red Bartlett’ pear (Wang et al., 2017) and lychee LDOX, was almost 100-fold higher in purple varieties (Yuan et al.,
(Fang et al., 2015). Enzymes potentially involved in this process include 2009). Regarding regulatory genes, both BoMYB2 and BoTT8 (bHLH)
laccase, polyphenol oxidases (PPO), class-III peroxidases (POD), and β- were highly expressed in the purple leaf epidermis, whereas BoMYB3
glucosidases (Oren-Shamir, 2009). A transcriptomic approach asso- was negatively correlated with anthocyanin content. Therefore, the
ciated the expression levels of laccase and POD with color fading of the respective positive and negative feedback mechanisms of BoMYB2 and
red pear (Wang et al., 2017). In lychee, although the red epicarp is not BoMYB3 that regulate the expression of one another are evolutionarily
edible, color fading induces browning, which affects consumer per- conserved in cabbage as described for Arabidopsis (Yuan et al., 2009).
ception and marketability. A laccase was also shown to induce color In Chinese cabbage (Brassica rapa ssp. pekinensis), purple varieties
fading through epicatechin-mediated anthocyanin degradation in ly- also accumulate anthocyanins, particularly between the first and third
chee (Fang et al., 2015). The identification of these enzymes can fa- outmost cell rows in epidermal and sub-epidermal tissues of the edible
cilitate breeding efforts to resolve the problem of color fading in these structure (head leaves) as well as reproductive organs. A regulatory
crops. mechanism very similar to that of cabbage was observed in this species,
in which the induction of structural genes (CHS, F3′H, DFR, ANS/
7.2. Beyond the fruit: anthocyanin accumulation in other edible plant LDOX, UFGTs, and GSTs) is regulated mainly by BrMYB2 (orthologous
structures to the Arabidopsis PAP2/MYB113) and BrTT8 (He et al., 2016). Like-
wise, in several other cruciferous vegetables, MYB2 and TT8 factors
In addition to fruit, other organs can often accumulate anthocya- were characterized as the primary activators of the anthocyanin
nins, such as leaves, stems, several floral structures and seeds, and even pathway, including purple varieties of bok choy (B. rapa var. chinensis)
roots and tubers. Most commonly, and much like in the fruit, the epi- (Zhang et al., 2014b), kale (B. oleracea var. acephala f. tricolor) (Zhang
dermis of other organs displays most of the anthocyanin accumulation, et al., 2012), and cauliflower (B. oleracea var. botrytis) (Chiu and Li,
while parenchymal cells of the cortex do not express the enzymes of the 2012). Remarkably, the purple phenotype in cauliflower is caused by a
pathway. Below, we describe instances which anthocyanin biosynthesis 695-bp Harbinger DNA transposon insertion in the BoMYB2 promoter
was studied at the molecular genetic level in vegetable species. (Chiu et al., 2010). This mutation activates the tissue-specific expres-
Among the many genotypes of potato (Solanum tuberosum), some sion of BoMYB2, triggering anthocyanin accumulation in the in-
develop entirely non-pigmented tubers, while some show red skin and florescence, seeds, and young vegetative tissues. Thereupon, the accu-
acyanic starchy parenchyma (flesh) and others have dark purple tubers mulation pattern of anthocyanins in purple cauliflower (cv. Graffiti) is

19
Table 1
Crop varieties with high-anthocyanin content in edible parts of the plant have been characterized genetically.
Crop species Genotypes studied Transcription factors involved Tissues analyzed Main findings References

Apple (Malus spp.) Red Field OP and other red-fleshed genotypes vs. MdMYB10 fruit - epicarp, mesocarp; leaf Insertion of 6 × 23-bp cis-element tandem repeat (R6 Espley et al., 2009
S. Chaves-Silva et al.

white-fleshed varieties element) in gene promoter 275-bp upstream the MdMYB10


start codon led to constitutive cyanic tissues (including red
flesh in fruit) by autoregulatory transactivation.
Asian pear (Pyrus pyrifolia) Aoguan (red peel) vs. Mantianhong (white peel) PyMYB10, PyMYB10.1, Fruit skin, young leaf and flower Overexpression of PyMYB10 in Arabidopsis induced Feng et al., 2015
PybHLH anthocyanin biosynthesis in immature seeds; expression of
PyMYB10 in fruit tissues correlates with anthocyanin
accumulation. PybHLH interacts in vivo with both MYB
factors.
Meirensu (red peel); Suli (white) PpbHLH3, PPbHLH33, Identification of factors that can form the MBW complex; Qian et al., 2017
PpWD40, PpSPL1-19, miR156 identification of a suite of nine SPL factors that repress the
anthocyanin pathway in white pears, whereas miR156
counteracts this repression by targeting SPL transcripts in
epidermal cells of red varieties.
Chinese pear (P. x Bayuehong x Dangshansuli segregating population; PyMYB114 A QTL study identifies a locus linked to the “red skin” trait Yao et al., 2017
bretschneideri) several red vs. green skin varieties PyERF3 on chromosome 5, which contains a MYB114 ortholog.
Transient expression in tobacco leaves of either PyMYB10
or PyMYB114 together with PybHLH3 and PyERF3
induced anthocyanin biosynthesis
Bilberry (Vaccinium myrtillus) Wildly grown in Finland's boreal forest VmTDR4 (MADS-box); Whole fruit Silencing of VmTDR4 resulted in a reduced anthocyanin Jaakola et al.,
Wildly grown in Slovenian forest near Ziri (blue vs. VmMYB2 Fruit skin and correlated with expression supression of CHS and 2010
albino fruits) VmMYBPA1; MYB2 compared to the wild type. Zorenc et al., 2017
VmMYBC2, VmMYBR3 VmMYBPA1 expression correlated with anthocyanin
biosynthesis. Fruit peel of albino berries expressed the
potential repressor VmMYBR3.

20
Blackberry (Rubus spp.) Arapaho RuMYB10 whole fruit – ripening stages RuMYB10 expression is upregulated during ripening, when Chen et al., 2012
anthocyanin accumulates
Cabbage (Brassica oleracea var. Red varieties (Royale, Cardinal, Cairo, Red Express) BrMYB2 and BrTT8 (bHLH) Seedling, leaf Expression of MYB and bHLH transcription factors Yuan et al., 2009
capitata) vs. green varieties correlated with expression of structural genes of the
anthocyanin pathway as well as anthocyanin accumulation
(in epidermal tissues); nutritional stresses induced
anthocyanin accumulation.
Carrot (Daucus carota) Purple varieties (Deep Purple, Purple 68, Tianzi2hao), DcMYB6 taproot The R2R3-MYB transcription factor DcMYB6 was identified Xu et al., 2017
orange varieties (Kuroda, Sanhongliucun, as a positive regulator of anthocyanin biosynthesis in
Junchuanhong) and yellow varieties (Bejo1719, carrot.
Qitouhuang, Baiyu)
Cauliflower (Brassica oleracea Graffiti (purple) vs. Stovepipe (white – progenitor BobHLH1, and BoMYB2 (Pr inflorescence (curd), and major Insertion of a Harbinger DNA transposon 373-bp upstream Chiu et al., 2010
var. botrytis) genotype) locus) organs of the plant of the semi-dominant Pr-D mutant allele in cv. Graffiti is
responsible for the purple phenotype; in young organs,
anthocyanin accumulates in more internal tissues rather
than the epidermis.
Eggplant – Gilo, bitter tomato Non-specified (material from the Chongqing Acad. of SmMYB1 epicarp, and major organs of the Overexpression of SmMYB1 gene induces anthocyanin Zhang et al., 2016
(Solanum aethiopicum Ag Sciences) plant accumulation.
group Gilo)
Grape (Vitis vinifera) Several contrasting varieties, including teinturier VvMYBA1-2, epicarp (berry skin), root, leaf The overexpression of VlmybA1-2 from V. labruscana was Cutanda-Perez
cultivars (Petit Rouge, Gamay Fréaux, Morrastel- VvMYB5b sufficient to induce constitutive accumulation of et al., 2009
Bouschet); anthocyanin by activating especially the final genes of the Deluc et al., 2008
Cabernet Sauvignon pathway (e.g., UFGT, OMT, GST and anthoMATE),
inclusive in inner tissues.
VvMYB5b is expressed in the Cabernet Sauvignon berry
skin during ripening, along with VvMYBA1 and VvMYBA2,
and induced anthocyanin and proanthocyanidin
biosynthesis in tobacco.
(continued on next page)
Phytochemistry 153 (2018) 11–27
Table 1 (continued)

Crop species Genotypes studied Transcription factors involved Tissues analyzed Main findings References

Kohlrabi (Brassica oleracea var. Azur-Star (purple peel) vs. Winner (green) BoPAP1, BoPAP2, BoMYB114; The acyanic epidermis is mainly due to the expression of Rahim et al., 2017
gongylodes) BoGL3, BoEGL3, BoTT8; the MYB repressor BoMYBL2.2.
S. Chaves-Silva et al.

BoTTG1; BoMYBL2.2
Lychee (Litchi chinensis) Several varieties contrasting for skin color LcMYB1 Pericarp, aril, leaf, stem LcMYB1 expression correlates with anthocyanin Lai et al., 2014
biosynthesis.
Onion (Allium cepa) California Red MYB1 epidermis Knockdown expression of MYB1 by RNAi suppressed Schwinn et al.,
anthocyanin accumulation in epidermal tissues; MYB1 2016
overexpression in white petal of snapdragon mutant
(roseadorsea) complemented phenotype in epidermal cells,
as well as in white garlic.
Orange (Citrus x sinensis) Moro, Tarocco, Jingxian, and other blood varieties vs. RUBY (R2R3-MYB) Endocarp (flesh) Blood oranges present 500-bp LTR of Copia-like insertion in Butelli et al., 2012
blond oranges promoter region (Tc2 LTR) of a MYB gene (the dominant
Ruby locus) 254-bp upstream the start codon that leads to
its cold-induced expression in fruit tissues (flesh as well as
albedo); in the independent Jingxian blood orange variety,
a 5-kb insertion occurs instead at 450-bp upstream the start
codon.
Peach (Prunus persica) Redhaven, Roza, Fantasia MYB10.1, MYB10.3, bHLH3; epicarp, mesocarp These three genes are clustered on a single locus (Anther Rahim et al., 2014
Dahongpao (bloodflesh), Baifeng (white flesh) BLOOD (NAC) whole fruit (developmental time color, Ag) in the genome and may be responsible for Zhou et al., 2015
course) anthocyanin biosynthesis localized in the peel and flesh Zhou et al., 2015
inner cortex near the endocarp (stone).
BLOOD (BL) is a NAC transcription factor that forms a
hetero dimer with NAC1 to activate MYB10.1 in blood-
fleshed peach. In addition, SPL1 was shown as a repressor
of NAC1 expression during fruit maturation in white-
fleshed peach

21
Potato (Solanum tuberosum) Several, including acyanic, red skin/white flesh, and StAN1 (MYB), StMYBA1, tuber skin, tuber flesh, the Multiple MYB transcription factors can induce the Liu et al., 2016
the purple skin/purple flesh (cv. Hei Meiren) StMYB113; StbHLH1, StJAF13 vascular rings of a red-skin, anthocyanin pathway; expression of both bHLH gens
(bHLH), white flesh variety correlate with anthocyanin biosynthesis and are the
limiting factors for activating the pathway.
Radish (Raphanus sativus) Contrasting cultivars: acyanic (Seo Ho), green peel/ RsMYB root (skin and flesh) The study identified sequences of some regulatory and Park et al., 2011
pink flesh (Man Tang Hong), and red peel/white flesh structural genes of the anthocyanin pathway and
(Hong Feng #1) quantified anthocyanin in root tissues of contrasting
varieties; a MYB cDNA was identified, although its role in
the pathway was not demonstrated.
Strawberry (Fragaria x Sachinoka FaMYB10 whole fruit (developmental time Light and ABA promote anthocyanin biosynthesis by Kadomura-
ananassa) course) independently inducing FaMYB10; FaMYB10 Ishikawa et al.,
overexpression promoted while RNAi decreased 2015
anthocyanin content in the fruit.
Wild Strawberry (Fragaria Alpine FvMYB10 whole fruit FvMYB10 overexpression induced constitutive Lin-Wang et al.,
vesca ssp. vesca) anthocyanin accumulation, whereas RNAi promoted an 2014
acyanic phenotype.
Sweet cherry (Prunus avium) Hong-Deng (red flesh) PacMYBA whole fruit (developmental time PacMYBA overexpression in Arabidopsis led to Shen et al., 2014
Tieton (red fruit) vs. 13-33 (yellow fruit) course) development of pigmentation in seeds, and its knockdown Wei et al., 2015
whole fruit (developmental time via the virus-induced gene silencing (VIGS) inhibited
course) anthocyanin biosynthesis to a certain extent in the fruit;
ABA induced anthocyanin biosynthesis in the fruit by
activating the expression of PacMYBA, while cytokinin
inhibited the pathway.
Comparative transcriptome (RNA-Seq) of whole fruit in 4
different developmental stages led to identification of
genes potentially involved in the anthocyanin pathway in
the fruit, including a MYB10 (PacMYBA), as well as other
MYB, bHLH and WD40 transcription factors.
(continued on next page)
Phytochemistry 153 (2018) 11–27
S. Chaves-Silva et al. Phytochemistry 153 (2018) 11–27

quite distinctive because it is not restricted to the outer cell layers, but

Mano et al., 2007

Cao et al., 2017


it also occurs in the central region of the inflorescence meristem, near

Povero et al.,
the vascular bundles (Chiu and Li, 2012). Thus, the purple cauliflower
References

displays substantially high levels of anthocyanins although the details

2011
of the regulation mechanism underlying this surprising pattern remain
unknown.
Purple and “black” varieties of carrot (Daucus carota ssp. sativus)
anthocyanin ubiquitous accumulation in sweet potato calli

Genetic mapping of the locus atv identified the mutation on


Transcriptome analysis (Affymetrix microarray) correlated

a MYB repressor as the potential cause of the purple peel


as well as throughout the Arabidopsis plant; detection of
IbMYB1 is expressed primarily in the purple flesh of the

expression of SlMYB113 with anthocyanin biosynthesis


expressed in the purple flesh of the tuberous root, and

development or in stored tuberous root), thus possibly


have existed for over 3000 years, and are regarded as older than the
possible alternative splicing variants of IbMYB1, with
unknown consequences; the ortholog IbMYB2 is not

orange genotypes (Kammerer et al., 2003). Cyanic cultivars develop


slightly in special situations (during tuberous root
tuberous root; IbMYB1 overexpression induced

pigmented external root tissues (and occasionally, internal as well), in


which the anthocyanin pathway is expressed in a light-independent
fashion (Xu et al., 2014). A transcriptional analysis comparing purple
and orange carrots showed a high correlation between anthocyanin
and related genes in the fruit peel.

accumulation and expression of the structural genes PAL3/PAL4,


playing only a secondary role.

CA4H1 (C4H), 4CL1, CHS1, F3H1, F3′H1, DFR1, LDOX1/LDOX2 (Xu


et al., 2014). Moreover, the R2R3-MYB transcription factor DcMYB6
has recently been identified as a positive regulator of anthocyanin
biosynthesis in carrot (Xu et al., 2017).
Lastly, the pigmentation of the skin and flesh in tuberous roots of
Main findings

phenotype.

sweet potato (Ipomoea batatas) vary substantially. Purple, red, pink,


orange, yellow and white cultivars are available. Purple varieties can
develop entirely pigmented roots (Mano et al., 2007). The best-char-
acterized transcription factor involved in this trait is IbMYB1, which
Peel and flesh of tuberous roots,

induces anthocyanin biosynthesis exclusively in the tuber cortex, with


no activity in non-tuberous roots or other parts of the plant (Mano et al.,
2007). IbMYB1overexpression is sufficient to activate anthocyanin
biosynthesis even in orange cultivars (Park et al., 2015b). A compara-
Tissues analyzed

tive study between a purple cultivar (Ningzishu 1, N1) and its acyanic
other organs

alternate (Mutant of Ningzishu 1, MN1) showed that IbMYB1 tran-


fruit peel
fruit peel

scription was much higher in N1 and induced the expression of LBGs


(Ma et al., 2016). Several other transcription factors differentially ex-
pressed between the two genotypes remain to be characterized, in-
cluding 17 bHLH transcription factors (Ma et al., 2016).
Transcription factors involved

8. Metabolic engineering of anthocyanin content in horticultural


SlMYBATV (R3-MYB)

crops

The lessons learned directly from crop species are essential to


SlMYB113

creating effective breeding strategies aiming to enhance the antho-


IbMYB1

cyanin content in the fruit and other edible plant structures. The gen-
eral lack of anthocyanin accumulation in parenchymal cells is not due
to substrate limitation, given that when genetic inducers of structural
cultivars: Ayamurasak, Murasakimasari, Purple Sweet
7 contrasting genotypes, including the purple-fleshed

Anthocyanin fruit (Aft) and atroviolacia (atv) mutants,


Aft/atv double mutant (purple peel), cv. Ailsa Craig

genes are expressed, anthocyanin accumulates in these cells. Although


light is an inducer of anthocyanin biosynthesis, it is not an absolute
requirement, as shown in tissues that accumulate large amounts an-
thocyanins without direct light exposure (purple carrot, potato, sweet
potato, and blood oranges). The WD40 component is in most cases
ubiquitously available to form the MBW complex. It is often the lack of
MYB and/or bHLH inducers that precludes the expression of antho-
Indigo Rose vs. Heinz 1706

cyanin structural genes. In two instances (blood orange and red-fleshed


apple) characterized in fruit tissues, anthocyanin accumulated in the
Genotypes studied

mesocarp due to rearrangements of MYB promoters (Table 1).


The transgenic approach used a decade ago to create the purple
tomato (Butelli et al., 2008) faced commercialization obstacles because
of transgenic food policies as well as public acceptance due to the
Lord

nature of the healthy, functional foods consumer basis, which is less


inclined to accept transgenic foods in their diet (Francis et al., 2017;
Sweet potato (Ipomoea batatas)

Saltzman et al., 2017). On the other hand, the success story of the
purple cauliflower as well as many other purple varieties of produce
(kale, lettuce, carrot), all derived from natural allelic variation, gained
Table 1 (continued)

shelf space in the market very quickly and demonstrates that the health-
Tomato (Solanum
lycopersicum)

inclined consumer market is open to biofortified produce.


Crop species

Currently, the availability of full genome sequences and gene an-


notations, coupled with the affordability and accessibility of RNA-Seq
analyses allow the identification of complete sets of regulators involved
with anthocyanin metabolism. The evolutionary conservation of

22
S. Chaves-Silva et al. Phytochemistry 153 (2018) 11–27

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Cavallini, E., Matus, J.T., Finezzo, L., Zenoni, S., Loyola, R., Guzzo, F., Schlechter, R.,
Funding Ageorges, A., Arce-Johnson, P., Tornielli, G.B., 2015. The phenylpropanoid pathway
is controlled at different branches by a set of R2R3-MYB C2 repressors in grapevine.
This work was supported by the Coordination for the Improvement Plant Physiol. 167, 1448–1470. https://doi.org/10.1104/pp.114.256172.
Chagné, D., Lin-Wang, K., Espley, R.V., Volz, R.K., How, N.M., Rouse, S., Brendolise, C.,
of Higher Education Personnel (CAPES) from the Brazilian Ministry of Carlisle, C.M., Kumar, S., De Silva, N., Micheletti, D., McGhie, T., Crowhurst, R.N.,
Education for the scholarship provided to A.L.S. and S.C.S. Storey, R.D., Valesco, R., Hellens, R.P., Gardiner, S.E., Allan, A.C., 2013. An ancient
duplication of apple MYB transcription factors is responsible for novel red fruit-flesh
phenotypes. Plant Physiol. 161, 225–239. https://doi.org/10.1104/pp.112.206771.
Declaration of interests Chanoca, A., Kovinich, N., Burkel, B., Stecha, S., Bohorquez-Restrepo, A., Ueda, T.,
Eliceiri, K.W., Grotewold, E., Otegui, M.S., 2015. Anthocyanin vacuolar inclusions
The authors declare that they have no competing interests. form by a microautophagy mechanism. Plant Cell 27, 2545–2559. https://doi.org/
10.1105/tpc.15.00589.
Chen, Q., Yu, H., Tang, H., Wng, X., 2012. Identification and expression analysis of genes
Acknowledgements involved in anthocyanin and proanthocyanin biosynthesis in the fruit of blackberry.
Sci. Hortic Amsterdam 141, 61–68. https://doi.org/10.1016/j.scienta.2012.04.025.
Chiu, L.-W., Li, L., 2012. Characterization of the regulatory network of BoMYB2 in con-
The authors would like to thank Luiz Carlos Rodrigues (UFES,
trolling anthocyanin biosynthesis in purple cauliflower. Planta 236, 1153–1164.
Federal University of Espírito Santo) for assisting with preparation of https://doi.org/10.1007/s00425-012-1665-3.
the figures. Chiu, L.-W., Zhou, X.J., Burke, S., Wu, X.L., Prior, R.L., Li, L., 2010. The purple cauli-
flower arises from activation of a MYB transcription factor. Plant Physiol. 154,
1470–1480. https://doi.org/10.1104/pp.110.164160.
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S. Chaves-Silva et al. Phytochemistry 153 (2018) 11–27

Zorenc, Z., Veberic, R., Slatnar, A., Koron, D., Miosic, S., Chen, M.-H., Haselmair-Gosch, regulation of flavonoids at the Samuel Roberts Noble Foundation (now, Noble Research
C., Halbwirth, H., Mikulic-Petkovsek, M., 2017. A wild ‘albino’bilberry (Vaccinium Institute) with Dr. Richard Dixon. He worked at Huazhong Agricultural University as a
myrtillus L.) from Slovenia shows three bottlenecks in the anthocyanin pathway and Professor of Plant Genetics and Molecular Biology between 2012 and 2017. His research
significant differences in the expression of several regulatory genes compared to the focused on primary and specialized metabolism in tea, soybean, and alfalfa to understand
common blue berry type. PLoS One 12, e0190246. https://doi.org/10.1371/journal. the biosynthesis, transport, and storage, as well as the regulation of health-promoting
pone.0190246. phytonutrients, such as flavonoids and lipids.

Dr. Samuel Chaves Silva graduate in Biological Sciences Dr. Lázaro Eustáquio Pereira Peres is an Associate
and in 2015 received his Ph.D. degree in Plant Professor of Plant Physiology at the University of São Paulo
Biotechnology from the Federal University of Lavras (UFLA, (USP, Brazil) from where he also received his Ph.D. His
Brazil). He was a visiting research scholar at West Virginia primary research interest is in Plant Development and its
University (WVU, United States) where he worked with Dr. interaction with the environment, with a focus on hormonal
Vagner Benedito on the characterization of the main tran- control of development and metabolism. Anthocyanins are
scription factors involved in anthocyanin biosynthesis in also of interest in Dr. Peres's Lab because they can be
purple tomatoes. He is currently a post-doctoral fellow at modulated by the environment, interact with plant hor-
the Federal University of Minas Gerais (UFMG, Brazil) mones and have an impact on plant development. Dr. Peres
working at Dr. Luzia Modolo's lab on the effects of novel has established and curates a large collection of induced
organic compounds on the urease activity in agricultural mutants, natural genetic variants and transgenic lines in-
soils. His research interests include plant molecular biology trogressed in the model tomato system cv. Micro-Tom,
and biochemistry, specialized plant metabolism and ni- which have been used worldwide for the study of physio-
trogen nutrition with a focus on the development of novel logical and genetic mechanisms involved in phenotypes of
urease inhibitors. agricultural relevance. Some of the genes studied at Dr. Peres's lab have alleles in-
trogressed from wild tomato species (natural genetic variation), representing a valuable
genetic resource for fundamental studies on plant adaptation, evolution, and domes-
Dr. Adolfo Luís dos Santos graduated in Agronomy from tication. The genotype collection studied is also endowed with wild-type alleles that were
José do Rosário Vellano University (UNIFENAS, Brazil) eliminated or modified during the domestication of tomato. Such alleles are essential
with expertise in Environment and Sanitary Engineering. resources for crop improvement aiming at the new consumer expectations, such as pig-
He holds an MSc in Ecology and Environmental Technology ment accumulation and nutritional enrichment.
from the Federal University of Alfenas (UNIFAL, Brazil) and
a Ph.D. in Plant Physiology from the Federal University of
Lavras-Brazil (UFLA, Brazil). Part of his doctoral research Dr. Vagner Augusto Benedito is an Associate Professor of
was carried out at West Virginia University (WVU, United Biochemical Genetics and Plant Physiology at West Virginia
States). He has experience in Molecular Plant Physiology University (WVU, United States. He graduated in Agronomy
and focused his research on the transcriptional regulation from the Federal University of Viçosa (UFV, Brazil). He
of metabolic pathways and transport of specialized meta- received his MSc degree in Horticulture from the University
bolites, with an emphasis on anthocyanins in tomato. of São Paulo (USP, Brazil) and his Ph.D. in Plant Sciences
from Wageningen University and Research Centre (WUR,
The Netherlands) in 2004. He was a post-doctoral fellow at
the Samuel Roberts Noble Foundation (now, Noble
Research Institute) with Dr. Michael Udvardi working on
Dr. Antonio Chalfun-Júnior is an Associate Professor of
genomics and transcriptomics of the model legume,
Plant Molecular Physiology at the Federal University of
Medicago truncatula. His research focuses on the genetic
Lavras (UFLA, Brazil). He graduated in Agronomy and ob-
control of plant traits of agricultural relevance, such as
tained his MSc degree in Horticulture from UFLA. His PhD
specialized metabolites and development of glandular tri-
in Plant Sciences was obtained in 2004 from Wageningen
chomes in tomato, and symbiotic nitrogen fixation in legumes. His focus on genetic
University and Research Centre (WUR, The Netherlands)
control of anthocyanin metabolism in edible structures of vegetables and fruits aims at
working on the molecular control of plant architecture.
providing molecular tools for high-nutrient density plant breeding.
Currently, his lab studies flowering mechanism in coffee,
sugarcane, tomato and other relevant crops using diverse
molecular and physiological tools to better understand this
important trait contributing to plant development.

Dr. Jian Zhao is a Professor in the College of Tea and Food


Science & Technology at Anhui Agricultural University in
Hefei, China. He received his M.S. degree from the College
of Life Science at Northeast Normal University, and his
Ph.D. of Pharmacognosy in 1999 from the Institute of
Materia Medica at the Chinese Academy of Medical
Sciences/Peking Union Medical College in Beijing, China.
He was then awarded a Foreigner Researcher Fellowship
from the Japan Society for the Promotion of Science to
research the biosynthesis of monoterpene derivatives at
Kyushu University in Fukuoka, Japan. He later moved to
the USA and worked on lipid metabolism at Kansas State
University with Dr. Xuemin Wang, cation nutrient transport
at the Baylor College of Medicine and Children's Nutrition
Research Centre (USDA/ARS) with Dr. Kendal Hirschi, and biosynthesis, transport and

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