Review

pubs.acs.org/JAFC

The State of the Art in Biosynthesis of Anthocyanins and Its

Regulation in Pigmented Sweet Oranges [(Citrus sinensis) L. Osbeck]
Angela Roberta Lo Piero*
Department of Agriculture, Food and Environment (Di3A), University of Catania, Via Santa Sofia 98, 95123 Catania, Italy

ABSTRACT: Anthocyanins are water-soluble pigments belonging to the flavonoid compound family involved in nature in
several aspects of plant development and defense. By bestowing much of the color and flavor on fruits and vegetables, they are
components of the human diet and, thanks to their radical-scavenging properties, are not considered exclusively as food products
but also as therapeutic agents. Several cultivars of red (or blood) oranges [Citrus sinensis (L.) Osbeck], such as Tarocco, Moro,
and Sanguinello, are characterized by the presence of anthocyanins in both the rind and fruit juice vesicles. The amount and
composition of anthocyanins in the pigmented orange cultivar vary greatly depending on variety, maturity, region of cultivation,
and many other environmental conditions. Most of the blood orange varieties require a wide day−night thermal range to
maximize color formation. Therefore, the production of red oranges characterized by high anthocyanin levels is limited to a few
regions and in particular to the Sicilian area around Mount Etna in Italy, where the characteristic climate conditions yield fruits of
unique color intensity and quality. In this review, both the basic information and the most recent advances in red orange
anthocyanins are reported, with intense attention given to their biosynthesis and regulation.
KEYWORDS: Citrus sinensis, anthocyanin, blood orange, biosynthesis, transcriptional regulation

■ INTRODUCTION
Anthocyanins are water-soluble pigments belonging to the
flavonoid compound family involved in nature in several
aspects of plant development and defense. They color flowers
and fruits, favoring seed and pollen dispersal and contributing
to plant adaptation to unfavorable environmental conditions
such as UV light damage, cold, and pathogen attacks.1 As
components of the human diet, they are not considered
exclusively as food products but also as therapeutic agents; in
fact, anthocyanins have been suggested as a protection against
under the same conditions, from very high pigmentation
(OTA9), high (Moro), medium and low pigmentation
(Tarocco) to no pigmentation (Navelina) (Figure 1). In the
mid 19th century, it was believed that blood oranges arose by a
bud mutation occurring in the Mediterranean region following
the insertion of the sweet orange (C. sinensis) in the 16th
century.12 More recently, an authoritative text suggests three
independent derivations: one Italian/Sicilian from Doppio
Sanguigno/Maltaise Sanguine, a second in Spain from
Doblefina, and a third from Shamouti orange referred to as
Shamouti Blood or Palestinian Blood Jaffa orange.13,10

oxidative stress, coronary heart diseases, certain cancers, and
other age-related diseases, although some researchers have cast
doubt on their bioavailability.2 At least part of these presumed
health-promoting features can be attributed to the antioxidant
properties of these compounds, the chemical structures of
which appear to be ideal for free radical scavenging.3 Several
cultivars of red (or blood) orange [Citrus sinensis (L.) Osbeck],
such as Tarocco, Moro, and Sanguinello, are characterized by
the presence of anthocyanins in both the rind and fruit juice
vesicles.4 The amount and composition of anthocyanins present
in the pigmented orange cultivar vary greatly depending on
variety, maturity, region of cultivation, and many other
environmental factors.5−9 Most of the blood orange varieties
require a wide day−night thermal range to maximize color
formation.10 Therefore, the production of red oranges
characterized of high anthocyanin levels is limited to a few
countries such as Brazil and Florida, and in particular to the
Sicilian area around Mount Etna, where the climate conditions
give fruits of unique color intensity and quality.11 As Butelli and
co-workers report,10 most modern varieties of blood orange
have been derived from old Italian varieties, such as Doppio
Sanguigno, and include more recently derived varieties, such as
Tarocco and Moro. The different orange accessions and the
hybrids display a range of pigmentation levels when grown
■
STRUCTURAL DIVERSITY OF ANTHOCYANINS IN
RED ORANGES
Anthocyanins are glycosides and acylglycosides of anthocyani-
dins, and the aglycones, flavyliums (2-phenylbenzopyrilium),
differ in the different hydroxyl or methoxyl substitutions in their
basic structures.14,15 The core of the anthocyanidin, the
flavylium, has the typical C6−C3−C6 flavonoid skeleton,
which contains one heterocyclic benzopyran ring (as the C
ring), one fused aromatic ring (as the A ring), and one phenyl
constituent (as the B ring) (Figure 2). In the cation form,
anthocyanidins have two double bonds in the C ring and hence
carry a positive charge.14,16 Both the hue and the color stability
are directly affected by the hydroxylation and methylation
pattern of the B ring of the anthocyanidins. Blueness is
enhanced with the increase of free hydroxyl groups, whereas
redness intensifies with the rise of the methylation of the
hydroxyl groups. The adjacent hydroxyl groups of o-diphenols
Received: March 4, 2015
Revised: April 10, 2015
Accepted: April 14, 2015

© XXXX American Chemical Society A DOI: 10.1021/acs.jafc.5b01123

J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Figure 1. Phenotypes and genotypes of orange varieties and hybrids: Navelina (A); Doppio Sanguigno (B); Tarocco (CRA) (C); Moro (CRA) (D);
OTA3 (E); OTA7 (F); OTA9 (G); OTA17 (H); Jingxian (I). Independent Blood Orange Accession, Jingxian, China. Reproduced with permission
from ref 10. Copyright 2012 American Society of Plant Biologists.
Figure 2. Structure of 3,5,7,4′-tetrahydroxylflavilium ion and of the six
basic anthocyanins. Reproduced from ref 20.
are more sensitive to oxidation to produce o-diquinones, or
even o-diphenol dimers.17 Therefore, cyanidin, delphinidin, and
petunidin, which contain the o-diphenol structure on the B ring,
are more sensitive to oxidation. On the contrary, neither
malvidin nor peonidin possesses the ortho-positioned hydroxyl
groups, which results in their comparatively higher resistance to
oxidation.17 In most plants only O-glycosylation occurs for
anthocyanins in which the sugar moiety is typically glucose, but
it can be substituted by other sugars such as galactose,
rhamnose, arabinose, or xylose.18 In Citrus pigmented varieties,
the glucose molecule can be linked to anthocyanidins through
the glycosidic bonds at the C3 position to form the 3-O-
monoglycoside anthocyanins. The junctions can also occur at
both C3 and C5 positions to produce 3,5-O-diglycoside
anthocyanins,19 which seem to be more stable than their
monoglucosidic counterparts. Blood orange [C. sinensis (L.)
B DOI: 10.1021/acs.jafc.5b01123
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Osbeck] juices contain anthocyanins in differing concentrations
depending on the variety, each showing a characteristic seasonal
variation of the pigment content. 20 The two major
anthocyanins of blood oranges (ca. 90%) have been identified
as cyanidin 3-glucoside19,21−23 and cyanidin 3-(6″-malonylglu-
coside)23 in comparable concentrations, these data being
confirmed in later studies.20,24,25 The pioneering work of
Maccarone et al.,19,23 which was carried out by HPLC
separation of pigments associated with their spectrophoto-
metric detection, also led to the identification of minor
anthocyanin components of blood orange juice [delphinidin 3-
glucoside, petunidin 3-glucoside, pelargonidin 3-glucoside,
cyanidin 3-(acetyl)glucoside], some of them found as
hydroxycinnamic acids derivatives of cyanidin 3-glucoside
[cyanidin 3-(ferulyl)glucoside, cyanidin 3- (coumarylferulyl)-
glucoside, cyanidin 3-(sinapyl)glucoside, peonidin 3-
(coumaryl)glucoside] as well as the diglucosylated forms
(3,5) of delphinidin, cyanidin, petunidin, and pelargonidin.
Dugo and co-workers20 identified for the first time peonidin 3-
glucoside, cyanidin 3-rutinoside, and the 6″-malonylglucose
esters of delphinidin, peonidin, and petunidin as minor
additional anthocyanins of blood orange juice by using
microhigh-performance liquid chromatography (HPLC)−elec-
trospray ionization mass spectrometry (ESI-MS) (Figure 3).
Further investigations revealed the occurrence of cyanidin 3-
(6″-dioxalylglucoside), as well as four other anthocyanin-
derived pigments, which are formed through a direct reaction
between anthocyanins and hydroxycinnamic acids only during
prolonged storage of the juice.25 These novel pyranoanthocya-
nins (i.e., anthocyanins that contain an additional pyran ring
between the C-4 and the hydroxyl group attached to C-5) were
identified as the 4-vinylphenol, 4-vinylcatechol, 4-vinylguaiacol,
and 4-vinylsyringol adducts of cyanidin 3-glucoside.25
■
BIOSYNTHESIS OF ANTHOCYANINS IN RED
ORANGES
Free anthocyanins are synthesized via the flavonoid pathway,
which is a ubiquitous and well-described plant secondary
metabolite pathway.16 It has been found that at least two
groups of genes are required for anthocyanin biosynthesis: the
structural genes encoding the enzymes directly implicated in all
of the metabolic reactions and the regulatory genes encoding
the transcription factors that control the expression of the
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Journal of Agricultural and Food Chemistry
Figure 3. Structures of the anthocyanins from blood orange juice:
cyanidin 3,5-diglucoside (1), delphinidin 3-glucoside (2), cyanidin 3-
sophoroside (3), cyanidin 3-glucoside (4), delphinidin 3-(6″-
malonylglucoside) (5), cyanidin 3-(6″-malonylglucoside) (6), cyanidin
3-(6″-dioxalylglucoside) (7), and peonidin 3-(6″-malonylglucoside)
(8). Reproduced from ref 25.
above-mentioned structural genes.26−28 Most genes encoding
the enzymes that biosynthesize the core structure have been
cloned and characterized in various species.1 Previous studies
have revealed that the genes acting earlier in the flavonoid
pathway are usually encoded by larger gene families, whereas
the enzymes directly involved in later steps are encoded by
single active genes.29 This rule, probably resulting from the fact
that the earlier acting genes are involved in diverse metabolic
functions that require a specific control of gene expression,
seems to be applicable to C. sinensis anthocyanin biosynthesis.
In the Citrus genus the early studies on the genes encoding the
key enzymes of anthocyanin biosynthesis and their expression
analysis occurred more than 10 years ago,30−32 and new
information has since been released.6,33−35 As shown below,
anthocyanins are synthesized by an extremely complex network
of all the structural enzymes in the pathway. Therefore, for an
efficient production of anthocyanins at the different steps, it is
speculated that all of the key enzymes involved in the
anthocyanin biosynthesis are associated with each other to
form a multienzyme complex.36 Published evidence indicates
the existence of such a complex in some model plants, such as
Arabidopsis thaliana.36 Phenylalanine is a direct precursor for
the synthesis of anthocyanidins. The conversion of phenyl-
alanine to anthocyanins requires a series of reactions: the first
of them is represented by the transformation of phenylalanine
to trans-cinnamic acid through the elimination of ammonia
catalyzed by phenylalanine ammonia-lyase (PAL). PAL has
been extensively studied because of its importance in plant
stress responses and tissue wounding;37 protection against UV
radiation,38 low temperature,6,30 and levels of nitrogen,
phosphate, and iron;38 and pathogenic attack and ethylene
response.39 Two full-length cDNA clones (FPAL1 and FPAL2)
were isolated from the flavedo tissue of the Fortune cultivar
(Citrus clementina Hort. ex Tanaka × Citrus reticulata Blanco)
library, and the deduced amino acid sequences showed a 75−
85% similarity with PAL genes from other plant species.30 A
partial cDNA clone putatively coding for PAL was isolated from
C DOI: 10.1021/acs.jafc.5b01123
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the Tarocco fruit juice vesicles [C. sinensis (L.) Osbeck] by RT-
PCR. The partial nucleotide sequence (DQ088064.1) showed
98.0% identity with the available C. clementina sequence.6 By
browsing the C. sinensis genome using the BLASTP tool at
Phytozome (http://www.phytozome.net/), four sequences
matching the translated partial sequence of the Tarocco PAL
have been found, and all of them were recognized as PAL
(scaffold00201, position 117,512−120,607, e value 6.8e‑117;
scaffold00201, position 95,337−97,004, e value 2.8e‑115;
scaffold00349, position 92,401−96,606, e value 3.1e‑111;
scaffold08490, position 1,157−2,599, e value 2.4e‑109), thus
suggesting that the gene is present in four polymorphic forms
in the C. sinensis genome as also emerged by the in silico
analysis of CitEST database performed by Lucheta and co-
workers.40
In the following steps, 4-hydroxylation of cinnamic acid by
cinnamate 4-hydroxylase (C4H) generates p-coumaric acid,
which is activated by the 4-coumarate:CoA ligase (4CL) to the
respective CoA ester. In Valencia sweet orange (C. sinensis) two
C4H genes were described coding a constitutively expressed
C4H2 (AF255014) enzyme that plays an ordinary role in the
phenylpropanoid pathway, in contrast to a wound-induced
C4H1 (AF255013) isoform.40,41 In silico studies revealed that
the 4-coumarate:CoA ligase (4CL), which converts 4-
coumarate (or p-coumaric acid) to 4-coumaroyl-CoA, is
encoded by a multigene family in the Citrus genus.40
The first specific enzyme of the anthocyanin biosynthetic
pathway is chalcone synthase (CHS) (Figure 4), which
condenses three malonyl-CoA molecules and one p-coumaro-
yl-CoA to produce the naringenin chalcone, which is implicated
not only in the anthocyanin biosynthesis but also in the
creation of other phenolic compounds.42 As a consequence,
different CHS genes may act in different pathways producing
distinct secondary metabolites. Two cDNA clones encoding
CHS were isolated (CitCHS1, AB009350; and CitCHS2,
AB009351) from the Valencia orange (C. sinensis) seed library.
Southern blot analysis indicated that CitCHS1 (1481 bp long
encoding a 389 amino acid protein) is present in a single copy,
whereas some sequences related to CitCHS2 (1412 bp long
encoding a 391 amino acid protein) are in the C. sinensis
genome.31 Furthermore, because hybridization patterns of
CitCHS1 and CitCHS2 are different from each other, these
cDNA clones have been likely originated from different loci.
The expression analysis showed that CitCHS2 mRNA is
notably induced by embryogenesis but CitCHS1 mRNA is not,
thus indicating that two CHS genes are differentially expressed
during citrus somatic embryogenesis and that CitCHS2 may
regulate the accumulation of flavonoids in citrus cell cultures.31
The naringenin chalcone is isomerized by chalcone isomerase
(CHI) to the flavanone naringenin, which is subsequently
converted to dihydrokaempferol by flavanone 3′-hydroxylase
(F3H). The gene active in the aforementioned stereospecific
isomerization of naringenin chalcone into naringenin flavanone
(CHI) and the one involved in the following naringen
flavanone hydroxylation into dihydrokaempferol (F3H) were
later isolated from a Citrus unshiu cDNA library, and their
expression patterns were analyzed during citrus (Marc.) fruit
development in relation to their flavonoid contents. The
respective cDNA clones from chalcone isomerase (CHI,
CitCHI, AB011794) and flavanone 3′-hydroxylase (F3H,
CitF3H, AB011795) are 931 and 1360 bp long, encoding
proteins of 222 and 362 amino acid residues, respectively.
CitCHI seems to be a single-copy gene, as also confirmed by
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Journal of Agricultural and Food Chemistry Review

Figure 4. Outline of the biosynthetic pathway leading to the synthesis of anthocyanins. Enzyme names are abbreviated as follows: chalcone synthase
(CHS); chalcone isomerize (CHI); flavanone 3′-hydroxylase (F3H), flavanoid 3′-hydroxylase (F3′H); flavanoid 3′,5′-hydroxylase (F3′5′H); flavonol
synthase (FLS); dihydroflavonol 4-reductase (DFR); anthocyanidin synthase (ANS); UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT).
Reproduced with permission from ref 7. Copyright 2006 American Society for Horticultural Science.

browsing the C. sinensis genome using the BLASTP tool at
Phytozome (http://www.phytozome.net/) (scaffold00024, po-
sition 1,113,233−1,115,603, e value 2.9e‑125), whereas a few
CitF3H-related sequences are in the C. unshiu genome32 as also
found by the in silico analysis of the CitEST database.40
Messenger RNA levels of the flavonoid biosynthetic genes
CitCHI and CitF3H and two previously isolated chalcone
synthases (CHS, CitCHS1, and CitCHS2) were analyzed in
various tissues during Satsuma mandarin fruit development.
Transcript levels for CitCHS1, CitCHS2, CitCHI, and CitF3H
are generally high in young tissues that correspond to high
accumulation of flavonoids.32 However, the crucial role of both
CitCHS and CitF3H in the development of anthocyanin
pigmentation was confirmed by a later study regarding the
analysis of the proteomic profile of two orange cultivars with
different pigmentation (the blood orange Moro and the blond
or common orange Cadenera), at ripening time.43 Chalcone
synthase as well as flavanone hydroxylase were identified as
differentially expressed proteins among merely 55 proteins,
suggesting that the activation of anthocyanin biosynthetic
pathway in blood oranges is one of the major differences
distinguishing blood and blond oranges.43
The dihydroflavonols, dihydroquercetin and dihydromyrice-
tin, are synthesized from dihydrokaempferol by hydroxylation
D DOI: 10.1021/acs.jafc.5b01123
reactions catalyzed by flavonoid 3′-hydroxylase (F3′H)44 and
flavonoid 3′,5′-hydroxylase, respectively. Dihydroflavonol 4-
reductase (DFR) can reduce the dihydroflavonols to their
corresponding leucoanthocyanidins. The gene encoding
dihydroflavonol 4-reductase was isolated from both a cDNA
library (AY519363.1) and genomic DNA (DQ084722.1)
(Tarocco and Navel varieties). The data revealed that the
cDNA sequences of orange DFR belonging to the red and
blond varieties were 100% homologous and contained a 1017
bp open reading frame that encodes a protein of 338 amino
acid residues, corresponding to a molecular mass of 38010.76
Da, with a theoretical pI of 5.96. Southern blot analysis of
genomic DNA indicated that df r is a single-copy gene (as
confirmed by browsing the C. sinensis genome using the
BLASTP tool at Phytozome, http://www.phytozome.net/,
scaffold00002, position 3,110,202−3,111,942) in both pig-
mented and nonpigmented orange varieties. The successful in
vitro expression of orange DFR led to an active DFR enzyme
that converts dihydroquercetin to leucoanthocyanidin, thus
confirming the involvement of the isolated genes in the
biosynthesis of anthocyanins.34 Furthermore, the promoter of
the DFR gene was cloned and analyzed.34 The examination of
the sweet orange dfr promoter sequence revealed a number of
motifs that may be involved in the transcriptional activation of
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Journal of Agricultural and Food Chemistry
the DFR genes: a putative unit I, which includes the
homologous binding site for the G-box factor, and the
downstream MYB homologous binding site were found.45
Unit I was initially identified in the chs promoter and shown to
be involved in light induction of the gene expression; it was
shown to be necessary and sufficient to impart photoregulation
through the phytochromes and the UV-A/blue and UV-B
photoreceptors.46 Two putative GA-myb (gibberellic acid
activated myb) homologous binding sites are also located
upstream of unit I. Several core elements for Dof3 (one zinc
finger), which have been reported to be involved in tissue
specificity and light regulation,47 were observed in the df r
promoter. A sequence with homology to the TACpyAT box of
the chs promoter,48 which is responsible for organ-specific
expression, was also identified immediately upstream of the
TATA box. Moreover, three homologous SBF-1 transcription
factor binding sites were also detected in the df r promoter.49
Finally, it was also found that there are no differences in the df r
coding and promoter sequences between red and blond
oranges. Therefore, as the DFR transcripts are normally
detected only in red oranges, its expression should be under
extremely strict control of the transcriptional regulators.34
Anthocyanidin synthase (ANS), an oxoglutarate-dependent
Fe2+/Fe3+ dioxygenase, converts the colorless leucoanthocyani-
dins into the colored anthocyanidins, that, once formed, must
be immediately modified as they are inherently unstable under
physiological conditions (Figure 4). Therefore, the UDP-
glucose-flavonoid glucosyl transferase (UFGT) catalyzes the
addition of one glucose moiety in the 3-OH positions of
anthocyanidins, increasing their hydrophilicity and stability.
The sweet orange [(C. sinensis) Osbeck, Tarocco] ANS and
UFGT genes have been cloned by Lo Piero and co-workers
(unpublished data), and their sequences are available at http://
www.ncbi.nlm.nih.gov/ (ANS, AY581048; UFGT, AY519364).
Both genes are likely in single copy in the C. sinensis genome as
resulted by the BLASTP search tool at Phytozome (http://
www.phytozome.net/) (ANS scaffold00074, position 275,559−
279,543; UFGT scaffold00204, position 195,714−197,012).
The presence of diglycosylated anthocyanins in blood orange
juice19−23 indicates that other glucosylating enzymes might be
involved in anthocyanin modification as occurs in grape,17
although a homologous gene has not yet been identified in
Citrus. Acylation is one of the most common modifications of
red orange anthocyanins, resulting in greatly increased
structural diversity of anthocyanins from the addition of
aromatic and/or aliphatic constituents linked to the C6″
positions of the glucosyl groups.23 Anthocyanins, without the
protection of acylation, can be easily and quickly decolorized in
neutral or weakly acidic aqueous solutions. The acylation of
anthocyanins in plants is catalyzed by the action of anthocyanin
acyltransferases (ACT, also known as AAT), which have a really
high substrate specificity for both the anthocyanin acceptors
and the acyl group donors. In plants, there are mainly two types
of ACTs that are classified on the basis of the acyl group
donors: the BAHD family using acyl-CoA and the serine
carboxypeptidase-like (SCPL) group using acyl-activated
sugars.50 Although in some important C. sinensis pigmented
varieties the acylated anthocyanins can account for >40% of the
total anthocyanin content (cyanidin 3-(6″-malonylglucoside),23
until now there has been no report about the exact genes
required for sweet orange anthocyanin acylation by ACTs.
E DOI: 10.1021/acs.jafc.5b01123
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■ ANTHOCYANIN TRANSPORTATION INTO THE
VACUOLES
Anthocyanins are synthesized by multienzyme complexes that
are localized at the cytoplasmic face of the endoplasmic
reticulum (ER)51,52 and then transported to the large vacuole,53
this localization being necessary to prevent oxidation and for
anthocyanins to function as pigments.54 In some plants,
anthocyanins are found in highly pigmented bodies located
inside the vacuole; these vacuole-accumulating anthocyanins
are also known as anthocyanic vacuolar inclusion (AVI) and
contain a dense complex mix of anthocyanins concentrated
above the levels that are possible in vacuolar solution.55 The
mechanism of anthocyanin transport to the vacuole has long
been debated, with numerous models proposed such as
membrane vesicle-mediated or membrane transporter-mediated
transport.56−58 To date, several molecular players involved in
the sequestration of anthocyanins have been identified. The
importance of glutathione-S-transferases (GSTs) for anthocya-
nin transport has been demonstrated in Zea mays (maize)
BZ2,54 petunia AN9,59 and Arabidopsis TT19,60 where
mutations in the encoding genes lead to a reduction in
anthocyanin accumulation and pigment mislocalization. A
mechanism similar to the detoxification process was then
proposed for anthocyanins, according to which they need to be
transferred into the vacuoles as glutathione S-conjugates.54
However, no anthocyanin−glutathione conjugate has been
observed in vitro, so it has been suggested that anthocyanins
could be transported via the noncovalent binding activity of
glutathione S-transferase functioning as ligandin.61,62 Two
major transporter families, the multidrug resistance-associated
protein (MRP) and the multidrug and toxic compound
extrusion transporter (MATE) families, would also be involved
in anthocyanin transport in maize63 and grape.64 However, the
involvement of these different proteins in anthocyanin
transport to the vacuole is still unclear. With regard to the
anthocyanin transfer into the vacuole in blood oranges, a GST
gene homologous to both the AN9 and BZ2 has been isolated
from red orange fruit flesh.35 Having considered gene
organization and homology data, it has been suggested that
the isolated GST gene is involved in the mechanism of vacuolar
import of anthocyanins as confirmed by in vitro expression of
orange GST, leading to an enzyme that is active against
cyanidin-3-O-glucoside.35 The comparison between colored
and uncolored varieties reveals that blood and blond orange
GST encoding genes share the same nucleotide sequences, but
the GST expression in the nonpigmented orange cultivars
(Navel and Ovale) was strongly reduced as compared to that of
the pigmented orange (Tarocco). Interestingly, in the crude
extracts of pigmented orange fruits, the GST activity is
reproducibly detected by providing either 1-chloro-2,4
dinitrobenzene (CDNB) or cyanidin-3-O-glucoside as sub-
strates; moreover, cyanidin-3-O-glucoside acts as a powerful
competitive inhibitor of 1-chloro-2,4-dinitrobenzene conjuga-
tion to reduced glutathione (GSH) in the pigmented oranges,
confirming that this molecule might easily bind to the active
site of the enzyme and functions as a putative substrate.35
Although the GST involved in anthocyanin conjugation to
GSH has been unequivocally identified, it is obvious that the
information to precisely understand the working mechanism of
the entire complex in vivo is still lacking. As a consequence,
more work is needed, and the just released C. sinensis genome
sequence65 will likely represent a valued source to recruit
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Journal of Agricultural and Food Chemistry
orthologous sequences to provide additional insight into this
metabolic pathway.
■ DEGRADATION OF RED ORANGE ANTHOCYANINS
In contrast to the detailed knowledge available on anthocyanin
synthesis, very little is known about pigment stability and
catabolism in plants. Transient anthocyanin accumulation and
disappearance during plant development or changes in
environmental conditions suggest that anthocyanin degradation
is controlled and induced when beneficial to the plant.66 Loss of
red pigmentation can occur in young foliage as it matures, in
developing flowers, or in response to changes in environmental
conditions.66 Anthocyanin degradation is also observed in
Sicilian sweet orange varieties (Tarocco, Moro, and Sangui-
nello): the pigments accumulated at the late stages of ripening
in both the rind and flesh may be degraded, leading to a
commercially undesirable partial loss of this color.67 Enzymatic
studies of anthocyanin degradation in postharvest fruits and
fruit juices have revealed several enzymes that degrade
anthocyanins in these systems and may be candidates for the
active in vivo degradation. Three enzyme groups that control
degradation rates of anthocyanins in fruit extracts and juices are
polyphenol oxidases, peroxidases, and β-glucosidases. Assuming
that in planta anthocyanin degradation occurs in the cell
vacuoles, the direct oxidation of anthocyanin with peroxidase,
which is present in cell vacuoles unlike the oxidizing polyphenol
oxidases, is more likely the candidate route for in planta
anthocyanin degradation. Nevertheless, the possibility that
anthocyanins are transferred out of the vacuoles before
degradation exists, and further studies are needed to determine
the site of degradation. Therefore, three alternative pathways
for enzymatic anthocyanin degradation are presented: (1)
direct oxidation of anthocyanin by peroxidase; (2) coupled
oxidation, that is, reduction of quinones of phenolic
compounds to the original phenolic compounds in parallel to
oxidation of anthocyanin to anthocyanin quinine; (3)
degradation in two steps, deglycosylation with anthocyanase
(β-glucosidase) and then oxidation with polyphenol oxidase or
peroxidase. The β-glucosidases may also be involved in the in
planta degradation process, because they increase the
degradation rate of anthocyanins in fruit extracts of both
lychee and eggplant.66 A specific β-glucosidase was isolated
from the fruit juice of Tarocco blood oranges, and the
anthocyanin degradation kinetic was followed physiochemically.
This enzyme, mainly bound to pectin chains, turns out to be
responsible for the degradation of anthocyanins in both the
juice and the ripening fruits.67 The yielded anthocyanidins may
be a good substrate for oxidases such as polyphenol oxidase
(PPO) in a successive step of the enzymatic degradation
process.66
Many physical and chemical factors affect the stability of
anthocyanins, including temperature, pH, the presence of
oxygen, enzymes, the presence of copigments, metal ions,
ascorbic acid, sulfur dioxide, sugars, and sugar degradation
products. Both low and high temperatures might induce
anthocyanin degradation. During cold storage (4 °C for 12
months), the anthocyanic pigments of blood orange juices
undergo modifications that lead to the transformation of
polycondensation compounds, the color of which changes from
brick-red to brown. The process of polymerization is
accompanied by the production of chemical indicators of
sugar and ascorbic acid degradation (furfural and hydrox-
ymethylfurfural).68 Reactions between anthocyanic pigments
F DOI: 10.1021/acs.jafc.5b01123
Review
and the aforementioned intermediates of degradation of sugar
and ascorbic acid in an acidic environment are the main causes
of the formation of these polymers.69 Moreover, blood orange
juice loses its color due to the anthocyanin degradation caused
by the standard thermal treatment directed at pectinesterase
inactivation, thus negatively influencing the commercial value of
blood orange juice.70 Studies on the thermal degradation of
cyanidin 3-(6″-malonyl) glucoside and cyanidin 3-glucoside,
which are the two major anthocyanins in blood orange
juice,19,23 as well as of cyanidin-3-glucoside-derived pyranoan-
́
thocyanins,25 indicate that the cyanidin-3-(6′-malonyl)-gluco-
side is the most stable. Conversely, cyanidin-3-glucoside-
derived pyranoanthocyanins show the highest degradation
rates, suggesting that acylated anthocyanins could be very
promising pigments contributing to the color of different
products as stable and intense color molecules.70 The major
intrinsic factors such as ascorbic acid and sugars significantly
accelerate the thermal degradation of blood orange anthocya-
nins, whereas flavonoids have a protective effect on the
degradation of anthocyanins; the protective effect of flavonoids
plays a more important role in the degradation of anthocyanins
compared to the negative effect of ascorbic acid or sugars.71
The effects of nonthermal preservation technologies including
high hydrostatic pressure, pulsed electric field, ultrasound,
irradiation, and ozone on the stability of anthocyanins in blood
orange juice are reviewed in Tiwari et al.72
■
REGULATION OF ANTHOCYANIN BIOSYNTHESIS
IN RED ORANGES
Sweet oranges can be easily divided into two groups based on
color, red (or blood) and blond. The red varieties can
synthesize and accumulate anthocyanins in both their rind and
flesh to express their color, whereas the blond cultivars
normally do not produce any anthocyanins in their fruits.
The expression profiles of six anthocyanin biosynthetic genes
(CHS, CHI, F3H, DFR, ANS, UFGT) were investigated in
both blood and blond oranges and revealed that genes involved
in the early steps of the anthocyanin biosynthetic pathway, such
as CHS, CHI, and F3H, are expressed in juice vesicles of both
uncolored and colored oranges. On the contrary, much lower
expression levels were observed in the cases of DFR, ANS, and
UFGT, the latter being barely detected or even totally
unexpressed in blond cultivars (Navel and Ovale).7,33 More-
over, in the pigmented varieties the anthocyanin content
increases during red orange fruit ripening;7 it is extremely
variable among the sweet orange pigmented varieties7,8 and also
may vary due to genetic factors and environmental conditions
such as temperature.5,6,9,73,74
Genetic Regulation of the Anthocyanin Biosynthesis
in Pigmented Orange Fruit. As the secondary metabolites of
the flavonoid pathway, anthocyanins are synthesized under the
complex regulation of multiple regulatory genes at the
transcriptional level. Generally, the spatial and temporal
expression of structural genes is under the control of several
different families of regulatory genes in plants, such as Myb,
Myc (encoding basic helix−loop−helix proteins, bHLH), and
WD40-like transcription factors, which also play crucial roles in
the regulation of other flavonoid products, such as flavonols
and proanthocyanidins.17 These transcription factors are
connected to form a ternary complex (MYB−bHLH−WD40,
MBW complex) in a hierarchical network to regulate the
expression of the structural genes involved in the flavonoid
synthesis, such as CHI, CHS, F3H, and DFR.17 As mentioned
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Review

Figure 5. RT real-time PCR detection of anthocyanin biosynthetic genes and total anthocyanin content in the Moro orange flesh during fruit
ripening. Fruits were collected at seven different stages, approximately every 3 weeks starting from October 19 and ending at February 21. CHS,
chalcone synthase; ANS, anthocyanidin synthase; UFGT, UDP-glucose flavonid 3-O-glucosyl transferase. Reproduced with permission from ref 7.
Copyright 2006 American Society for Horticultural Science.

Figure 6. RT real-time PCR detection of anthocyanin biosynthetic genes and total anthocyanin content in the Tarocco Moro orange flesh in 11
genotypes harvested at the end of winter. OTA9, C. reticulata ‘Oroval’ Clementine × C. sinensis Tarocco; 58-8D-1-Russo, nucellar line of C. sinensis
Moro; Stefano Fontanazza, old line of C. sinensis Tarocco; Tapi, nucellar line of C. sinensis Tarocco Arcimusa; C5787, nucellar line of C. sinensis
Tarocco Lempso; C5083, nucellar line of C. sinensis Tarocco Giarretta; C1882, nucellar line of C. sinensis Tarocco Sciara, D2071, nucellar line of C.
sinensis Tarocco Scirè; AMOA, poorly pigmented nucellar line of C. sinensis Moro; C1665, nucellar line of C. sinensis Tarocco Messina; Biondo
Cadenera, C. sinensis blond orange; CHS, chalcone synthase; ANS, anthocyanidin synthase; UFGT, UDP-glucose flavonid 3-O-glucosyl transferase.
Reproduced with permission from ref 7. Copyright 2006 American Society for Horticultural Science.

above, in blood oranges, several anthocyanin biosynthetic genes
show increased expression compared with blond or-
anges.33−35,73−76 Variation in pigment content or tissue
specificity is largely governed by the activity of the R2R3Myb
transcription factor in the complex, which has recently been
isolated from blood orange and named Ruby.10 The analysis of
gene expression revealed that high levels of Ruby transcription
are in the flesh and rind of blood oranges, whereas no Ruby
expression is detectable in blond oranges, thus suggesting that a
different regulatory mechanism occurs during Ruby tran-
scription between blond and blood oranges.10 The isolation
and sequencing of the upstream regulatory regions of Ruby
from both Moro (blood) and Cadenera (blond) highlighted an
G DOI: 10.1021/acs.jafc.5b01123
insertion of 501 nucleotides in Moro, 254 bp upstream of the
initiating ATG in Ruby, compared with the otherwise identical
Ruby promoter from the blond Cadenera orange. This insertion
showed sequence similarity to the long terminal repeats
(LTRs) of the Copia family of retrotransposons and included
a 5 bp direct repeat, typical of LTR retroelement insertion
sites.77 The start of Ruby transcription mapped to an A, 551
nucleotides upstream of the initiating ATG of the Ruby gene.
The insertion generates a TATA box 32 bp upstream of the
start of transcription site within the LTR providing the
regulatory sequences for initiating expression of the Ruby
gene, whereas it is not possible to identify a TATA box in the
sequence upstream of the Ruby gene from blond oranges.10 The
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
finding that the CsMyc, gene encoding the bHLH partner in the
MWB complex78 is expressed at detectable levels in both blond
and blood oranges supports the hypothesis that the expression
of the Ruby gene limits anthocyanin biosynthesis in citrus
varieties.10 Recently, a large number of Citrus genes belonging
to a previously unidentified MYBA gene family were isolated,
molecularly characterized, and functionally evaluated.79 All of
these genes were recovered by PCR amplification using primers
corresponding to VvMybA1 of grapevine. Transient transgene
expression analysis using grapevine somatic embryos revealed
that many members of two Citrus MYBA subgroups (named b
and c) were fully functional and capable of activation
anthocyanin biosynthesis in nonanthocyanic explants.79 Notice-
ably, the null genes contained single-residue substitution
mutations within the R2R3 repeat corresponding to the DNA
binding domain. Most of these loss of function substitutions
were associated with positively charged amino acids. The
isolation of a large number of MYBA genes from Citrus suggests
the existence of highly homologous regulatory genes involved
in the anthocyanin pathway among taxonomically distant
genera such as Citrus and Vitis.79 Moreover, the identification
of many paralogue genes able to turn on the anthocyanin
biosynthesis needs to be further investigated to define their role
in anthocyanin production likely related with developmental
status, tissue specificity, or different color intensity.
Influence of Orange Fruit Ripening Stage and
Genotype on Anthocyanin Accumulation. The amount
of anthocyanins in blood oranges is strictly correlated with the
fruit ripening stage as well as with transcriptional levels of the
biosynthetic genes.7 Figure 5 shows the expression profile of
selected anthocyanin biosynthetic genes (CHS, ANS, and
UFGT) as well as the total anthocyanin content during the
Moro blood orange ripening period. The expression of CHS,
ANS, and UFGT increases during the entire period, and the
levels of all three transcripts appear to vary correspondingly.
Total anthocyanins are undetectable at the beginning, but their
levels rise from the third sampling (end of November) onward
and show a sharp increase between the VI (late January) and
VII samplings (mid-February). Further confirming results have
been obtained by microarray analysis performed to study gene
expression during blood orange ripening.76 The custom array
was hybridized using samples of the Moro (pigmented) and
Cadenera (nonpigmented) cultivars, at three different ripening
stages: immature phase, the halfway point of maturation
(corresponding to the start of Moro pigmentation), and full
ripe time. In Moro, the ripening process, which appears to be
closely related to the accumulation of anthocyanins in the fruit
flesh, is indeed associated with the induction of genes involved
in the anthocyanin pathway.76
As shown in Figure 6, the anthocyanin levels might also vary
depending on varieties or selections grown under the same
environmental conditions ranging from 34 mg/100 g (OTA 9)
to 0.25 mg/100g (Tarocco Messina). Generally, a relationship
between biosynthetic gene expression and fruit flesh
anthocyanin content is evident.7
Cold-Temperature Induction of Anthocyanin Content
in Blood Orange Fruit. Activation of anthocyanin biosyn-
thesis by cold has received more attention than other inducing
environmental factors. In fact, the productivity of most
commercially important Citrus varieties is severely affected by
low temperature due to the induction of several metabolic
alterations such as increased electrolyte leakage and decreased
photosynthetic capacity and respiration rate.80 The suscepti-
H DOI: 10.1021/acs.jafc.5b01123
Review
bility to cold stress varies widely in the Citrus genus, ranging
from very cold sensitive types, such as Citrus grandis
(pummelo), to the cold-hardy Citrus relative Poncirus trifoliata.
To complete the picture of the events taking place in cold-
exposed Citrus fruits also including the pigmented varieties, Lo
Piero and co-workers33,35,74 have shown that postharvest
exposure to cold induces an increase of the anthocyanin levels
in the blood orange flesh. This enhancement, which may
determine an 8-fold increase (from 0.5 to 7.46 mg/100g)
depending on storage time, is accomplished by the up-
regulation of the genes involved in the anthocyanin biosyn-
thesis (PAL, CHS, DFR, ANS, UFGT, GST). Further
transcriptome study based on the construction and analysis of
a subtractive cDNA library was performed to address the
overall activation of the gene expression in the blood orange
[(C. sinensis) L. Osbeck, Tarocco Sciara] after exposure to cold
stress conditions (4 °C for 77 days). Overall, the enhancement
of transcripts involved in the defense mechanisms against
oxidative damage, osmoregulating processes, and lipid desatu-
ration as well as many ESTs implicated in both the primary and
secondary metabolisms was observed. In particular, the results
show that cold stress induces transcriptome modifications
undoubtedly oriented toward the enhancement of the flavonoid
biosynthesis pathway in blood oranges. They include the
activation of genes directly involved in anthocyanin biosyn-
thesis (CHS and ANS) as well as genes intervening in the
metabolic pathways supplying it, such as the shikimate pathway,
which yields phenylalanine.9,74 Various functional genes and
regulator genes with their target proteins play important roles
in regulating low-temperature tolerance. Among low-temper-
ature-induced genes, the dehydration responsive element
binding protein (DREB)/C-repeat binding factor (CBF)
genes encode key transcription factors implicated in the
major transcriptional cascade that responds to cold.81 Many
abiotic-stress-inducible genes are controlled by abscisic acid
(ABA), but CBFs are not, thus indicating that both ABA-
dependent and ABA-independent regulatory systems are
involved in cold stress responsive gene expression.82 It is
worthwhile to mention that in the cited study,9 homologous
cDNAs to CBFs have not been retrieved among the specifically
cold-induced genes, suggesting that the blood orange response
to cold might involve an ABA-dependent pathway.9 Interest-
ingly, several transcription factors have been identified for the
first time as cold-responsive genes in plants.9 In particular, a
transcription factor belonging to the NAC transcription factor
family was found specifically induced in blood oranges, but not
in blond oranges, thus proposing it as a candidate gene involved
in the signal cascade triggered in response to cold.73 Recently, a
gene encoding a NAC domain transcription factor (TF) was
found to be highly up-regulated in blood-fleshed peaches when
compared with non-red-fleshed peaches. This NAC tran-
scription factor, designated BLOOD (BL), can activate the
transcription of PpMYB10.1, resulting in anthocyanin pigmen-
tation in transgenic tobacco.83 However, the cold dependency
of anthocyanin production in blood oranges might also depend
upon the cold induction of the retroelement controlling the
Ruby gene expression (see Genetic Regulation of the
Anthocyanin Biosynthesis in Pigmented Orange Fruit) as
elevated levels of these transcripts are observed in Tarocco and
Moro blood varieties following the storage of fruit in the cold.10
Finally, by comparing the blood orange response to cold stress
with those of other plant sources, such as grapefruit,84 it seems
to be similar to that of the chilling acclimated species.
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Therefore, it is likely that the rise of anthocyanin levels
occurring in the oranges subjected to cold stress might
contribute to the control of cell osmotic potential, thus coping
with the imposed stressful conditions.9
Other Anthocyanin Biosynthesis Induction Factors.
An assortment of additional intrinsic and environmental factors
has been linked to anthocyanin accumulation in several plant
species as reviewed by Chalker-Scott85 and more recently by de
Pascual-Teresa and Sanchez-Ballesta.86 Most research on
anthocyanins has focused on their photoinduction by wave-
lengths in the UV, visible, and far-red regions. Nutrient
deficiencies, especially of phosphorus (P) and nitrogen (N),
commonly induce the accumulation of anthocyanin in many
plant species. Also, exposure to lowered pH, methyl jasmonate,
wounding, pathogen infection, and fungal elicitors may trigger a
significant increase in the anthocyanin levels.85,86 Both auxins
and/or cytokinins have been shown to induce anthocyanins in
cell cultures or whole plant systems, and some authors have
linked gibberellins to anthocyanin production.85 Plant
hormones also affect anthocyanin biosynthesis: abscisic acid
(ABA) promotes biosynthesis of anthocyanin, and poor
coloration of fruit is improved by the application of ABA,
which increases the anthocyanin content in both grape skin and
cherry skin.86 Ethylene has been shown to influence the
biosynthesis of light-induced anthocyanin formation, improving
color development and increasing anthocyanin content in
apples.86 Probably, the above-mentioned factors might have the
same enhancing effect upon anthocyanin biosynthesis in
pigmented orange but, as far as is known, there is no published
experimental evidence confirming their role in activating the
anthocyanin biosynthetic pathway. Invaluable personal obser-
vations of several researchers report that fruits on the same tree
exposed to light in the top of the canopy produce more
pigment than those located in a shady position of the canopy
interior or that fruit that has fallen from the tree shows
increased levels of these pigments. All of these observations
leading to the consolidation of the physiological role of
anthocyanin as a “defending molecule” in red orange fruit
should be experimentally proven and lay the foundation for
future work. In this respect, the antifungal activity of salicylic
acid (SA) against the devastating pathogen Penicillium
digitatum, the casual agent of green mold disease of blood
oranges, has recently been investigated.87 The authors showed
that SA applications significantly decrease the postharvest decay
of fruit as well as positively influence fruit quality parameters
such as total soluble solid, titrable acidity, and anthocyanin
content. These findings demonstrate that an increase of
anthocyanin levels may be closely related to the antifungal
activity triggered by SA in blood oranges.87
■
CONCLUSIONS
The importance of anthocyanins is due both to their impact on
the organoleptic features of blood oranges, which may influence
their technological behavior during juice extraction and
processing, and to the beneficial effect on human health
correlated with their consumption. The experimental work of
several research groups led to greatly improved knowledge of
how anthocyanins are synthesized in blood oranges as well as
how this biosynthetic pathway is regulated at the transcriptional
level by genetic and environmental factors, this being achieved
with wide application of new biotechnological approaches such
as “omics”. However, there are still many aspects that need to
be clarified, and it would be worthwhile to focus the next efforts
I DOI: 10.1021/acs.jafc.5b01123
Review
on identifying the genes involved in anthocyanin modification,
elucidating the mechanism of vacuole compartmentation of
pigments, and defining the role of either phytohormones or
biotic and abiotic factors in inducing anthocyanin accumulation.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: rlopiero@unict.it. Phone: +39-95-7580238.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
Thanks are due to all of the researchers who contributed to the
enrichment of our knowledge on this topic.
■
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K DOI: 10.1021/acs.jafc.5b01123
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