Molecular Plant

Review Article

‘‘La Vie en Rose’’: Biosynthesis, Sources, and

Applications of Betalain Pigments
Guy Polturak and Asaph Aharoni*
Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot 76100, Israel
*Correspondence: Asaph Aharoni (asaph.aharoni@weizmann.ac.il)
https://doi.org/10.1016/j.molp.2017.10.008

ABSTRACT
Betalains are tyrosine-derived red-violet and yellow pigments found exclusively in plants of the Caryophyl-
lales order, which have drawn both scientific and economic interest. Nevertheless, research into betalain
chemistry, biochemistry, and function has been limited as comparison with other major classes of plant
pigments such as anthocyanins and carotenoids. The core biosynthetic pathway of this pigment class
has only been fully elucidated in the past few years, opening up the possibility for betalain
pigment engineering in plants and microbes. In this review, we discuss betalain metabolism in light of
recent advances in the field, with a current survey of characterized genes and enzymes that take part in be-
talain biosynthesis, catabolism, and transcriptional regulation, and an outlook of what is yet to be discov-
ered. A broad view of currently used and potential new sources for betalains, including utilization of natural
sources or metabolic engineering, is provided together with a summary of potential applications of beta-
lains in research and commercial use.
Key words: betalain biosynthesis, plant pigment, secondary metabolism, metabolic engineering, plant
biotechnology
Polturak G. and Aharoni A. (2018). ‘‘La Vie en Rose’’: Biosynthesis, Sources, and Applications of Betalain
Pigments. Mol. Plant. 11, 7–22.

INTRODUCTION betanin-, gomphrenin-, amaranthin-, and bougainvillein-type

Betalains are nitrogenous, water-soluble, red-violet and yellow
pigments that form one of the major pigment classes providing
striking colors to plant organs, alongside chlorophylls, anthocy-
anins (and other flavonoids), and carotenoids (Tanaka et al.,
2008). In addition to their attractive colors, betalains were also
found to have strong antioxidant activity (Escribano et al.,
1998; Kanner et al., 2001). They have thus been widely
studied with respect to their potential health-promoting proper-
ties, including anticancer, hypolipidemic, hepatoprotective, anti-
inflammatory, and antidiabetic activities, as was previously
reviewed (Stintzing and Carle, 2004; Clifford et al., 2015;
Esatbeyoglu et al., 2015; Gengatharan et al., 2015; Gandia-
Herrero et al., 2016; Khan, 2016). Their reported nutritional
values have led to commercialization of a variety of betalain-
based products in the dietary supplements industry, while
their stability in a wide range of pH has made them a pigment
of choice for the food industry, where they are widely used as
natural food colorants (Stintzing and Carle, 2004; Azeredo,
2009).
Based on their structural characteristics and light-absorption
properties, betalains are generally classified into two groups,
the red-violet betacyanins and the yellow betaxanthins. The
betacyanins can be further divided into subgroups that include
pigments (Figure 1) (Steglich and Strack, 1990). All betalains
are based on a common scaffold, betalamic acid, which
condensates with cyclo-DOPA derivatives, or with various
amino acids and other amines, to form betacyanins or
betaxanthins, respectively. The structural differences obtained
following the condensation of betalamic acid (a chromophore
itself) with various compounds determines the differential
appearance of the two betalain subgroups; betacyanins display
an absorption maximum at approximately 535–538 nm, while
betaxanthins typically show highest absorption at the 460–
480 nm range, depending on the molecular structure and
type of solvent (Stintzing and Carle, 2004; Khan and Giridhar,
2015). To date, structures of approximately 75 betalains
have been unambiguously identified from 17 different plant
families (Khan and Giridhar, 2015); however, many more
compounds have been detected to which structures have
only been putatively assigned. The number of reported
betalains is also continuously increasing due to the
introduction of new analytical technologies and development of
better methodologies in liquid chromatography and mass
spectrometry (Sekiguchi et al., 2013).
Published by the Molecular Plant Shanghai Editorial Office in association with
Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

Molecular Plant 11, 7–22, January 2018 ª The Author 2017. 7

Molecular Plant Betalain Biosynthesis, Sources, and Applications

Figure 1. Representative Betalain Structures, Exemplifying Betalain Glucosylation, Acylation, Decarboxylation, and Isomerization.
Compounds are categorized into one betaxanthin and four betacyanin groups: betanin (betanidin 5-O-b-glucoside), gomphrenin (betanidin 6-O-
b-glucoside), amaranthin (5-O-b-glucuronosylglucoside), and bougainvillein (betanidin 5-O-b-sophoroside or betanidin 6-O-b-sophoroside). Betalains
are frequently named after the species from which they were first isolated.

Evolution and Distribution of Betalains Roles of Betalains in Plants

Unlike the ubiquitous anthocyanin and carotenoid classes of
pigments, betalains are fairly rare in nature and are restricted
to a single plant order, the Caryophyllales. Within the Caryophyl-
lales, betalains occur in a mutually exclusive fashion with
anthocyanins, as no plant was found to naturally produce
both types of pigments. Species in all families belonging
to the core Caryophyllales clade have been thought to
produce merely betalains, excluding the Molluginaceae and
Caryophyllaceae families in which plants exclusively produce
anthocyanins (Clement and Mabry, 1996). However, more
recent phylogenetic analyses show that additional lineages
within the core Caryophyllales are anthocyanic, including
the Kewaceae (previously included in Hypertelis) and
Macarthuriaceae (Brockington et al., 2011, 2015). Interestingly,
betalains are also produced in fungi of the genus Amanita in
what seems to be a converging evolution of the pathways to
produce similar products (Gill and Steglich, 1987). The
molecular basis behind the mutual exclusion of betalains and
anthocyanins in plants is not entirely understood, and several
hypotheses have been proposed to try and explain this
phenomenon, as previously reviewed (Brockington et al., 2011).
A recent study applying phylogenetic analyses proposed a
single origin of betalain pigmentation early in the evolutionary
history of the Caryophyllales, followed by the subsequent
loss of betalains in anthocyanic lineages (Brockington et al.,
2015).
8 Molecular Plant 11, 7–22, January 2018 ª The Author 2017.
Betalains occur in a wide range of plant tissues including leaves,
stem, fruits, flowers, roots, and seeds (Gandia-Herrero and
Garcia-Carmona, 2013). Like other plant pigments prevalent in
flowers and fruits, they likely play an important role in attraction
of pollinators and frugivores for fertilization and seed dispersal.
Additionally, betalains are likely to participate in plant defense
against various biotic and abiotic stress cues, although they
have been considerably less studied in this aspect compared
with their anthocyanin counterparts (Jain and Gould, 2015a).
Involvement of betalains in plant photoprotection has been
established based on experiments showing reduced damage to
photosynthetic capacity in red-pigmented versus green leaves
following exposure to excess light (Nakashima et al., 2011; Jain
and Gould, 2015b; Jain et al., 2015) and the fact that betalain
production is upregulated when plants are exposed to light or
UV radiation (Kishima et al., 1995; Vogt et al., 1999b; Ibdah
et al., 2002). Similarly, increased betalain accumulation under
drought and salt stress conditions (Hayakawa and Agarie,
2010; Nakashima et al., 2011; Jain and Gould, 2015b; Jain
et al., 2015) coupled with induced expression of betalain-
related genes (Casique-Arroyo et al., 2014) indicate a role in
protection against these stress factors. The role of betalains in
protection from abiotic stress coincides with their occurrence
in the Caryophyllales; one of the most prominent features of
this order is its dominance in arid and semi-arid regions, and
habitation of saline and alkaline soils. Members of several families
Betalain Biosynthesis, Sources, and Applications
Figure 2. The Betalain Biosynthetic Pathway.
Several betacyanins are given as examples for betanin acylation or glycosylation reactions. Dashed lines designate reactions of an alternative pathway, in
which cyclo-DOPA is first glucosylated and then condensates with betalamic acid to form betanin. Additional decoration reactions (e.g., glucuronidation)
may occur on cyclo-DOPA-glucoside instead of betanin (not shown in the scheme). Betanidin can also be glucosylated at the 60 O position to form
gomphrenin (not shown in scheme). Spon., spontaneous condensation reactions.
are particularly adapted to arid or saline regions, including the
Aizoaceae (ice-plant family), Portulacaceae (purslane family)
and, most notably, the Cactaceae (cacti family). Betalain activity
in defense against abiotic stress is most likely mediated through
their strong antioxidant capacity, as is the case for anthocyanins
(Jain and Gould, 2015a).
The involvement of betalains in plant protection against biotic
stress is less characterized. A presumed role for betalains in de-
fense against pathogenic fungi has been suggested as a major
factor driving their evolution (Brockington et al., 2011), although
experimental evidence for their antifungal activity is exceedingly
scarce in scientific literature. In a recent study, transgenic
betalain-producing tobacco plants exhibited increased resis-
tance to leaf infection by gray mold (Botrytis cinerea), possibly
due to their radical scavenging activity, causing delayed cell death
and proliferation of the necrotrophic fungus (Polturak et al., 2017).
Scavenging of reactive oxygen species was also suggested as the
reason for which betalain synthesis is induced in red beet leaves
following bacterial infiltration with Agrobacterium tumefaciens or
Pseudomonas syringae (Sepulveda-Jimenez et al., 2004).
BETALAIN BIOSYNTHESIS
Betalains are synthesized from tyrosine, an aromatic amino acid
that is mainly produced in plants via the shikimate pathway
Molecular Plant
(Figure 2) (Herrmann, 1995; Tzin and Galili, 2010). Tyrosine
is initially hydroxylated to form 3,4-dihydroxy-L-phenyalanine
(L-DOPA) (Steglich and Strack, 1990). L-DOPA is subsequently
converted to betalamic acid, the core backbone of all betalain
compounds, in a two-step reaction initiated by the DOPA
4,5-dioxygenase enzyme (Girod and Zryd, 1991; Christinet
et al., 2004). Alternatively, L-DOPA is oxidized and cyclized to
cyclo-DOPA, which spontaneously condenses with betalamic
acid forming the betacyanin precursor betanidin (Schliemann
et al., 1999). Betanidin is next glucosylated at the 50 O or 60 O
position to form betanin or gomphrenin, respectively, which in
turn may go through additional glycosylation and acylation
reactions, leading to synthesis of a wide assortment of
betacyanin compounds (Strack et al., 2003). A variation of the
core pathway toward betanin formation occurs in some
Caryophyllales species (e.g., Mirabilis jalapa, Celosia cristata),
where cyclo-DOPA is first glycosylated at the 50 O position,
followed by condensation of the cyclo-DOPA-5-O-glucoside
with betalamic acid to directly produce betanin (Sasaki et al.,
2004, 2005b). Formation of the yellow betaxanthins occurs via
spontaneous condensation of betalamic acid with amino acids
or other amines, rather than with cyclo-DOPA or cyclo-DOPA
derivatives (Schliemann et al., 1999). Betalains are thought to
be synthesized in the cytoplasm and endoplasmic reticulum,
based on subcellular localization of their key biosynthetic
enzymes (Christinet et al., 2004; DeLoache et al., 2015; Chen
Molecular Plant 11, 7–22, January 2018 ª The Author 2017. 9
Molecular Plant
et al., 2017). Just as for many other plant secondary metabolites,
they are eventually stored in the vacuole as glycosides
(Grotewold, 2006). In this section we review the current
knowledge with respect to enzyme-catalyzed steps and tran-
scriptional regulation of the biosynthetic pathway as well as beta-
lain catabolism.
Tyrosine Hydroxylation and L-DOPA Oxidation
The first step in betalain biosynthesis, namely the 3-hydroxylation
of tyrosine to L-DOPA, as well as the subsequent conversion
of L-DOPA to cyclo-DOPA, has commonly been attributed to
catalysis by a tyrosinase (polyphenol oxidase [PPO]) enzyme
(Strack et al., 2003; Gandia-Herrero and Garcia-Carmona,
2013). Transcripts encoding two copper-binding PPOs with
expression patterns correlating with betalain accumulation were
identified in Phytolacca americana. cDNA clones of the two genes
were isolated from cell culture, but enzyme activity assays
were not performed (Joy et al., 1995). Partial purification of
tyrosinase proteins showing tyrosine hydroxylation activity were
reported in plants and cell cultures of red beet and Portulaca
grandiflora (Steiner et al., 1996, 1999; Yamamoto et al., 2001).
Tyrosinases exhibiting in vitro tyrosinase hydroxylase and/or
DOPA oxidase activity were subsequently purified from red
beet roots (Gandia-Herrero et al., 2004), Swiss chard leaves
(Gao et al., 2009), and Suaeda salsa seedlings (Chang-Quan
et al., 2007). However, conclusive evidence for tyrosinase
involvement in betalain biosynthesis has not been provided at
the gene level. Additionally, conceptual arguments against the
proposed role of tyrosinase have been raised, including (i) the
fact that plant PPOs are localized in plastids while betalain
biosynthesis is cytoplasmic and (ii) yellow mutants that produce
betaxanthins only, where tyrosine hydroxylase but not
DOPA oxidase activity would be functional, are prevalent in
Caryophyllales species. This phenomenon was expected to
be rare or non-existent if a single enzyme was involved in
the dual reaction (Hatlestad and Lloyd, 2015). Considering the
high prevalence of tyrosinases in the plant kingdom, it is
unsurprising that they also occur in the betalain-producing Car-
yophyllales, possibly playing other, previously suggested roles,
e.g., oxygen scavenging in the chloroplast (Vaughn et al., 1988)
and plant defense (Constabel et al., 1995; Sullivan, 2014). In
this case, in vitro tyrosine hydroxylase and DOPA oxidase
activities of tyrosinases purified from Caryophyllales plants
would still be expected, but do not necessarily indicate an
in planta function in betalain biosynthesis.
The role of tyrosinase in betalain biosynthesis was first chal-
lenged by the discovery of a cytochrome P450-type gene,
CYP76AD1, which was identified to be essential for conversion
of L-DOPA to cyclo-DOPA in red beet. This key finding was sup-
ported by gene-silencing assays, mutant complementation, and
recombinant expression in yeast. CYP76AD1 orthologs were
also identified based on sequence similarity in three additional
betalain-producing plant species (Hatlestad et al., 2012).
A comprehensive phylogenetic analysis in 100 Caryophyllales
plant species showed that multiple CYP76AD1-like genes are
found in each species, which could be categorized to three
clades within the CYP76AD1 group, named CYP76AD1-a,
CYP76AD1-b, and CYP76AD1-g (Brockington et al., 2015). The
CYP76AD1-a clade was suggested by the authors to be
10 Molecular Plant 11, 7–22, January 2018 ª The Author 2017.
Betalain Biosynthesis, Sources, and Applications
directly associated with betalain biosynthesis as it included the
B. vulgaris CYP76AD1 gene and its ortholog CYP76AD3, which
has also been implicated in playing a similar function in
M. jalapa (Suzuki et al., 2014). However, a functional role in
betalain biosynthesis could not be determined for genes
belonging to the CYP76AD1-b and CYP76AD1-g clades.
In a subsequent report, CYP76AD1 was shown to have an essen-
tial role in betalain biosynthesis not only in formation of cyclo-
DOPA but also in catalyzing tyrosine hydroxylation to L-DOPA.
Recombinant expression of CYP76AD1 in tyrosine-fed yeast cells
led to accumulation of L-DOPA when expressed alone and
formation of red-colored pigmentation when coexpressed with
M. jalapa DOPA dioxygenase (MjDOD) (DeLoache et al., 2015).
Transient expression in Nicotiana benthamiana and gene-
silencing assays in red beet provided in planta evidence for the
role of CYP76AD1 in tyrosine hydroxylation, in redundancy with
another newly identified, related gene, CYP76AD6 (Polturak
et al., 2016). Interestingly, while CYP76AD1 was shown to
catalyze both tyrosine hydroxylation to L-DOPA and its
subsequent conversion to cyclo-DOPA, CYP76AD6 exhibited
merely tyrosine hydroxylase activity. Sequence analysis of
the CYP76AD6 gene placed it in the CYP76AD1-b clade,
indicating that a similar phenomenon possibly occurs in other
Caryophyllales species, whereby enzymes belonging to the
CYP76AD1-a clade possess dual activity of L-DOPA and cyclo-
DOPA formation while CYP76AD1-b enzymes catalyze only
L-DOPA formation (Polturak et al., 2016). The occurrence of
two or more cytochrome P450 enzymes with single or dual
activity would also explain the high incidence of yellow mutants
in various Carophyllales plants. The involvement of CYP76AD1-
like genes in the first step of the pathway was further
supported by work of Sunnadeniya et al. (2016), who
demonstrated CYP76AD1 and CYP76AD6 activities in yeast
and plants together with an additional related B. vulgaris gene,
CYP76AD5, which exhibits activity similar to that of CYP76AD6.
A functional ortholog of CYP76AD6 was also identified in
M. jalapa, CYP76AD15, with its activity characterized by
recombinant expression in yeast (Sunnadeniya et al., 2016).
Formation of Betalamic Acid by DOPA Dioxygenase
DOPA dioxygenase (DOD), a key enzyme in betalain biosynthesis
that catalyzes formation of betalamic acid from L-DOPA, was
initially purified from the betalain-producing fungus Amanita mus-
caria (AmDOD) (Girod and Zryd, 1991; Terradas and Wyler, 1991).
The extradiol dioxygenase was found to perform both 2,3 and 4,5
cleavage of L-DOPA, thus forming either 2,3- or 4,5-secodopa,
which in turn spontaneously cyclize to give muscaflavin or
betalamic acid, respectively (Terradas and Wyler, 1991). The
gene encoding the fungal dioxygenase was later cloned from
Amanita cDNA libraries (Hinz et al., 1997) and expressed
in Escherichia coli (Mueller et al., 1997b). AmDOD was
also demonstrated to be functional in plants, where particle
bombardment-mediated expression enabled formation of red
and yellow cells in the background of white Portulaca grandiflora
petals (Mueller et al., 1997a). The first plant betalain-related DOD
was identified more than a decade after the fungal enzyme was
initially isolated, using an in silico analysis of differentially ex-
pressed cDNAs from differently colored P. grandiflora petals.
The Portulaca DOD was found to be entirely unrelated to
Betalain Biosynthesis, Sources, and Applications
AmDOD, showing no sequence homology and exhibiting strictly
4,5-dioxygenase activity (Christinet et al., 2004). DOD genes
have since been identified in several additional Caryophyllales
plants, including Mirabilis jalapa (Sasaki et al., 2009),
P. americana (Takahashi et al., 2009), Opuntia ficus (Felker
et al., 2008), Suaeda salsa (Zhao et al., 2011), B. vulgaris
(Gandia-Herrero and Garcia-Carmona, 2012; Hatlestad et al.,
2012) and Parakeelya mirabilis (Chung et al., 2015).
Glycosylation in Betalain Biosynthesis
Glycosylation reactions are frequent in betalain biosynthesis,
both as part of the core pathway that leads to formation
of ‘‘simple’’ betacyanins such as betanin (betanidin 5-O-
b-glucoside) and gomphrenin (betanidin 6-O-b-glucoside), as
well as in downstream decoration reactions that contribute to
the occurrence of a large variety of more complex betacyanins.
On the other hand, betaxanthin glycosylation is likely to be
exceedingly rare or absent in nature, as glycosylated betaxan-
thins have never been reported. However, a glycosylated betax-
anthin, i.e., dopaxanthin hexoside, was recently detected when
betaxanthins were produced in N. benthamiana following tran-
sient expression of CYP76AD6 and BvDODA1 (Polturak et al.,
2016). It is unclear whether the glycosylation reaction occurred
on L-DOPA prior to conjugation with betalamic acid, or on
dopaxanthin itself.
Uridine diphosphate (UDP)-glucose-dependent betanidin gluco-
syltransferases were first identified in cell-suspension cultures of
Livingstone daisy (Dorotheanthus bellidiformis). Betanidin 5-GT
and betanidin 6-GT enzymes were purified, which exhibit betani-
din glucosylation activity at either the 50 O or 60 O positions, thus
forming betanin or gomphrenin, respectively (Heuer and Strack,
1992; Heuer et al., 1996). An active site for the D. bellidiformis
betanidin 5-GT enzyme was later modeled, based on identifica-
tion of amino acids essential for its activity and crystallographic
structure of a bacterial UDP-glucose-dependent glucosyltrans-
ferase (Hans et al., 2004). The betanidin 5-GT was also cloned
and recombinantly expressed in E. coli. Interestingly, in addition
to 50 O glucosylation of betanidin, the enzyme also showed activ-
ity on ortho-dihydroxylated flavonols and flavones. This, together
with high homology of the betanidin 5-GT to flavonoid-related
glucosyltransferases, indicated that the betalain glycosylating
gene could have evolved from a gene involved in flavonoid glyco-
sylation (Vogt et al., 1999a). Moreover, this phenomenon
occurred more than once, as the D. bellidiformis 5-GT and
6-GT genes show very low sequence identity and have likely
evolved independently from ancestral flavonoid-related glucosyl-
transferases (Vogt, 2002). Flavonoid glucosyltransferases that
show betanidin 5-GT or 6-GT activity, albeit to a low extent,
were also cloned from B. vulgaris (Isayenkova et al., 2006). An
additional betanidin 5-GT that has high similarity to the D. bellidi-
formis betanidin 5-GT protein was identified in B. vulgaris. Stress
induction in leaves led to upregulation of betalain production and
betanidin 5-GT gene expression, while transient expression of
this gene in antisense orientation caused reduction of betalain
accumulation (Sepulveda-Jimenez et al., 2005).
An alternative route for betanin formation has been established, in
which glucosylation occurs on cyclo-DOPA and the resulting
cyclo-DOPA-5-O-glucoside conjugates with betalamic acid to
Molecular Plant
directly form betanin. Feeding experiments in Celosia plumosa
seedlings showed that amaranthin accumulation is much higher
following feeding with cyclo-DOPA and cyclo-DOPA glucoside
than with betanidin and betanin, suggesting that betanin and
amaranthin are formed by cyclo-DOPA glucosylation followed
by condensation with betalamic acid (Sciuto et al., 1974). Also
in support of this route was the occurrence of free cyclo-DOPA
glucoside in red beet (Wyler et al., 1984; Kujala et al., 2001).
Conclusive evidence for formation of betanin via the cyclo-
DOPA glucoside route were presented by Sasaki et al. (2004),
who identified an enzyme with cyclo-DOPA-5-O-glucosyltrans-
ferase (cDOPA5GT) activity in M. jalapa. The gene encoding
cDOPA5GT was later identified by the same group in M. jalapa,
as well as in another betalain producer, Celosia cristata
(Sasaki et al., 2005b). It is currently unclear which of the
two glucosylation routes toward formation of betanin is
more prevalent in the Caryophyllales, and to which extent
they may coexist in the same species. Notably, UDP-
glucuronosyltransferase activity on cyclo-DOPA glucoside was
detected in a C. cristata crude protein extract, suggesting that
the glucuronic acid moiety of amaranthin and its derivatives is
introduced at the cyclo-DOPA glucoside step rather than on be-
tanin (Sasaki et al., 2005a). It cannot be ruled out that additional
glycosylation or acylation reactions that lead to synthesis of
more complex betacyanins may also occur on the cyclo-DOPA
moiety, prior to conjugation with betalamic acid.
A large number of betacyanins, and particularly within the bou-
gainvillein group, have multiple glycosylations, including linkage
to glucose, apiose, rhamnose and, possibly, xylose sugars.
Such compounds have been described in several species,
most notably in Bougainvillea glabra, in which more than 140
different betacyanins were detected (Piattelli and Imperato,
1970; Heuer et al., 1994; Wybraniec et al., 2010). This large
variety of metabolites could hypothetically be obtained by the
occurrence of numerous betalain-related glycosyltransferases,
or alternatively by few glycosyltransferases with low substrate
specificity. Clarification of this issue will require the identification
of novel glycosyltransferases involved in downstream decoration
of betanin or other betacyanins. No such genes, however, have
been functionally characterized to date.
Betalain Acylation
In addition to structural complexity attained through glycosylation
reactions, betacyanins can undergo various acylation reactions.
Detection of acylated betacyanins has been reported in members
of at least four different families within the Caryophyllales, with
linkage to various substitutes including salicyl, malonyl,
3-hydroxy-3-methyl glutaryl and, most commonly, hydroxycin-
namoyl groups (Strack et al., 2003). Betacyanin acylation
would most probably be catalyzed by either acyl-coenzyme
A-dependent acyltransferases from the BAHD superfamily or by
serine-carboxy-peptidase-like (SCPL) acyltransferases (Tanaka
et al., 2008). Acylation with a hydroxycinnamoyl group by an
SCPL-like acyltransferase would require the presence of glucose
esters such as hydroxycinnamoyl glucoses (HCG) as acyl donors
(Milkowski and Strack, 2004). HCGs were previously shown
to serve as acyl donors for acylation of betalains in cell cultures
of Chenopodium rubrum and petals of Lampranthus sociorum
(Bokern et al., 1992; Bokern and Strack, 1988). They are
Molecular Plant 11, 7–22, January 2018 ª The Author 2017. 11
Molecular Plant
synthesized by transfer of the glucose group from UDP-glucose
to a hydroxycinnamic acid, in a reaction catalyzed by hydroxycin-
namate glucosyltransferases (HCGT) (Fleuriet et al., 1980; Strack,
1980). A sinapate glucosyltransferase, GgSGT, was previously
isolated from the betalain-producing plant Gomphrena globosa.
Recombinant expression of GgSGT was utilized for the pro-
duction of HCGs as substrates to determine acyltransferase ac-
tivity in protein extracts prepared from cultured cells of the
anthocyanin-producers Daucus carota and Glehnia littoralis.
However, the relation of GgSGT to betalain acylation was not
explored (Matsuba et al., 2008).
Decarboxylation of Betalains
Another group of modified betacyanins comprises the
2-descarboxy-betacyanins. Compounds in this group contain a
leuko-dopamine-chrome moiety instead of leuko-dopa-chrome
present in the more common compounds (Gandia-Herrero and
Garcia-Carmona, 2013). Occurrence of these compounds, as
well as the occurrence of dopamine- and tyramine-derived
betaxanthins, would require tyramine and dopamine precursors
and implies activity of an enzyme involved in betalain
biosynthesis that catalyzes the decarboxylation of either
tyrosine or L-DOPA (Strack et al., 2003). Alternative
pathways for the biosynthesis of dopamine-derived betacyanins
have been proposed, including condensation between
2-descarboxy-cyclo-DOPA and betalamic acid leading to forma-
tion of 2-descarboxybetanidin (Kobayashi et al., 2001) or
conversion of tyramine/dopamine betaxanthins to 2-descarboxy-
betanidin via oxidation by tyrosinase (Gandia-Herrero et al.,
2005). Decisive evidence for the occurrence of either of these
alternative routes in vivo has not been provided. Decarboxylation
is also considered with relation to betalain degradation rather
than biosynthesis, as mono-, bi-, and tridecarboxylated
betacyanins have been detected as degradation products in
betalain extracts derived from red beet and pitaya (Wybraniec,
2005; Wybraniec and Mizrahi, 2005).
Transcriptional Regulation of the Betalain Biosynthetic
Pathway
Regulation of betalain biosynthesis remains a poorly understood
area. Early studies described changes in betalain production in
seedlings and cell cultures, in response to exogenous feeding
of abscisic acid, gibberellic acid, or cytokinin hormones
(Kinsman et al., 1975; Biancocolomas, 1984; Hirano et al.,
1996). Response to changes in environmental conditions, in
particular to light and UV radiation, is also well documented
(Kishima et al., 1991, 1995; Vogt et al., 1999b; Ibdah et al., 2002).
Preliminary indication of the factors involved in transcriptional
regulation was found through examination of the promoter re-
gions of two DOD genes in P. americana. Several putative binding
sites of MYB, basic helix-loop-helix (bHLH), and environmental
stress-responsive transcription factors were detected in the up-
stream sequences of the two DOD genes (Takahashi et al.,
2009). Indeed, two R2R3-MYB-type transcription factors were
later putatively identified as betalain regulators from an in silico
analysis of the B. vulgaris (sugar beet) genome, based on
homology to known anthocyanin-related MYB transcription fac-
tors, located in the vicinity of the R locus (CYP76AD1) on chromo-
some 2 (Stracke et al., 2014). Hatlestad et al. (2015) concurrently
12 Molecular Plant 11, 7–22, January 2018 ª The Author 2017.
Betalain Biosynthesis, Sources, and Applications
identified the MYB transcription factor BvMYB1 to correspond to
the Y locus, a major determinant of color in B. vulgaris that had
been known to beet breeders for decades. BvMYB1 was
shown to have an essential role as a positive regulator of
betalain biosynthesis through activation of the CYP76AD1 and
BvDODA1 genes, acting by direct binding of their respective
promoter regions. The high sequence similarity of BvMYB1 to
anthocyanin-related MYBs and the strikingly similar spatiotem-
poral pigmentation patterns of anthocyanins and betalains indi-
cate that BvMYB1 most likely evolved from an ancestral
anthocyanin-regulating MYB transcription factor. Interestingly,
the BvMYB1 protein does not include a bHLH-binding consensus
motif and is therefore not likely to be part of an MYB-bHLH-WD40
complex as in the case of anthocyanins (Hatlestad et al., 2015).
While BvMYB1 is the only currently known betalain-related tran-
scription factor, it is to be expected that additional genetic and
epigenetic factors take part in the regulation of betalain biosyn-
thesis in B. vulgaris and other species. This is particularly relevant
considering the complex betalain pigmentation patterns found in
plants, and the involvement of several such factors in the case of
anthocyanins. In M. jalapa, for example, flower color in varieties
that display red-yellow variegated flowers was found to be deter-
mined by activity of a transposon, dTmj1. The class II DNA trans-
posable element of the En/Spm superfamily resides in an intron of
the cytochrome P450 gene CYP76AD3, which regains function
upon excision and transposition of dTmj1, leading to formation
of red spots on the yellow background (Suzuki et al., 2014).
Catabolism of Betalains
Very little is currently known with respect to genes involved in
betalain catabolism. Understanding the betalain degradation
process is crucial for retaining strong coloration in betalain-
containing food products (Azeredo, 2009). In addition to various
non-enzymatic factors contributing to betalain degradation that
include among others light, oxygen, pH, and high temperature,
evidence for involvement of PPOs and peroxidases was also pro-
vided (Zakharova et al., 1995; Zakharova and Petrova, 1997;
Martinez-Parra and Munoz, 2001; Gandia-Herrero et al., 2007;
Wybraniec and Michalowski, 2011). PPO oxidizing activities
require monophenolic or diphenolic structures that are rarely
found in betaxanthins and would be found in betacyanins only
following removal of the sugar moiety (Stintzing and Carle,
2008). Additionally, peroxidase enzymes purified from red beet
exhibited stronger activity toward the aglycone betanidin as
compared with betanin (Martinez-Parra and Munoz, 2001).
Enzymatic betalain degradation would therefore require a
concerted action of PPOs, peroxidases, and glucoside-cleaving
enzymes such as b-glucosidases (Stintzing and Carle, 2008).
Indeed, b-glucosidase activity toward betacyanins was shown
to occur in B. vulgaris leaves and roots (Zakharova and
Petrova, 2000). No PPOs, peroxidases, or b-glucosidases
related to betalain catabolism have been identified at the gene
level to date.
Uncharacterized Reactions and Enzymes
While there has been major progress in our understanding of the
biochemical reactions that take place in the betalain biosynthetic
pathway and enzymes catalyzing these reactions, much still re-
mains unknown with respect to betalain metabolism, regulation
Betalain Biosynthesis, Sources, and Applications
of the pathway, and subcellular transport. The increasing avail-
ability of whole-genome sequence data for Caryophyllales spe-
cies and complementary gene expression information will
certainly facilitate the identification of additional betalain-related
genes in the near future. Indeed, several recent studies largely
based on ‘‘omics’’ data report on unknown genes with putative
betalain-related functions (Casique-Arroyo et al., 2014; Qingzhu
et al., 2015; Xu et al., 2016; Zheng et al., 2016; Jarvis et al., 2017).
SOURCES AND APPLICATIONS OF
BETALAINS
Betalains, as are other natural pigments, are highly valued by
man. They provide vibrant colors to many cherished ornamental
plants (e.g., bougainvillea, four o’clocks, cockscomb, moss
rose, and globe amaranth) and several food crops (e.g., beetroot,
Swiss chard, prickly pear, and dragon-fruit). They serve as a basis
for numerous dietary supplement products, and have also
been explored for potential use in dye-sensitized solar cells
(Zhang et al., 2008; Calogero et al., 2012) and as textile dyes
(Sivakumar et al., 2009; Guesmi et al., 2012; Ganesan and
Karthik, 2017). In science, their color properties can be utilized
as chemical biosensors (DeLoache et al., 2015), protein-
labeling fluorophores (Cabanes et al., 2016), and markers for
genetic transformation (Harris et al., 2012; Polturak et al., 2016).
Perhaps the most prominent use of betalains is their application
as natural food colorants, although their use is limited to certain
food products due to their tendency to degrade upon exposure
to high temperatures and light (Herbach et al., 2006). However,
betalains hold several advantages for use as food colorants in
comparison with other widely used natural water-soluble red pig-
ments, the anthocyanins. Betalains have higher solubility in water
than anthocyanins, exhibit significantly higher tinctorial strength,
and are generally stable in the pH range of 3–7, making them
more suitable for application in low-acid and neutral foods
(Stintzing and Carle, 2007). Indeed, betalains already have a
long history of use as food colorants, with pokeberry juice
being in use as early as the 19th century to enhance the color
of red wine, but it has since been banned due to its reported
toxicity (Petit-Paly et al., 1994). Today, red beet extract is the
sole commercially used source of betalains as food colorants
(Rodriguez-Amaya, 2016).
The wide variety of possible uses and applications for betalains,
together with the limited occurrence of betalain-producing spe-
cies in nature, and particularly the small number of edible plants
that contain betalains, have prompted research toward improve-
ment and development of new sources of betalains. The various
efforts may be categorized into three different approaches:
(i) improving betalain yield in beet, (ii) exploring alternative plant
species and cell cultures for betalain extraction, and (iii) devel-
oping new betalain sources through metabolic engineering of
plants and microbes. In this section we describe developments
in these three approaches, with a focus on metabolic engineering
of betalains and its potential biotechnological applications.
Improving Yields in Beet
Beet was domesticated many centuries ago and is widely culti-
vated today in parts of Europe and North America, yet only a small
Molecular Plant
fraction of the land used to grow red beet is destined for colorant
production. The use of beet-derived betalains as food colorants
is likely very old, but breeding programs with specific objectives
for increasing and modifying pigment composition were only
initiated in the 1970s. Breeding efforts have been mainly
directed toward increasing pigment levels while simultaneously
decreasing sucrose concentration, which can be 100-fold higher
than pigment concentration in typical beet cultivars (Baranksi
et al., 2016). Betalain accumulation in beet acts as a
quantitative trait and has been increased through recurrent
selection (Wolyn and Gabelman, 1990). Several loci associated
with the quantities and types of betalains produced must be
present in order for selection to be possible. Most notably,
the R and Y loci are essential for betalain production and
determine beet color to be red, yellow, or white. While the
R and Y loci have been known to breeders for many decades,
they were only recently identified as the cytochrome P450 gene
CYP76AD1 and the MYB-type transcription factor BvMYB1,
respectively (Hatlestad et al., 2012, 2015).
The characterization of genes corresponding to the R and Y loci,
together with the rise of precise genome-editing techniques, will
likely allow the implementation of new approaches for increasing
pigment content in beet. For example, introduction of point
mutation in the F309 position in CYP76AD1 is likely to reduce
significantly its capacity to catalyze L-DOPA oxidation to cyclo-
DOPA (DeLoache et al., 2015), and consequently result
in the production of yellow beet varieties with boosted
betaxanthin production (Wang et al., 2017). The increasing
knowledge regarding the betalain biosynthetic pathway will also
facilitate the identification of its limiting factors. In a recent
study, comparative analyses of betacyanins, betaxanthins, and
tyrosine content were conducted in various beet genotypes.
Red beet varieties were found to have higher betalain
concentration than yellow beets but lower tyrosine levels.
Additionally, increased tyrosine levels correlated with higher
betalain accumulation in red but not yellow genotypes. This
indicated that red and yellow genotypes have different
bottlenecks in betalain production, which are limited by the
supply or utilization of tyrosine, respectively (Wang et al., 2017).
Alternative Plant Sources
While red beet extract remains the only source of betalains in
commercial use today, it holds several disadvantages and draw-
backs. Its betacyanin profile is mostly composed of betanin, thus
offering limited color range. It moreover carries adverse flavors
due to the presence of geosmin and other pyrazines and holds
the risk of carry-over of soil-bound bacteria (Stintzing and
Carle, 2007). Therefore, alternative plant species have
been explored as potential sources of betalains. Amaranth
(Amaranthus spp.), a member of the same family as B. vulgaris,
the Amaranthaceae, was also proposed as an alternative
betalain source to red beet. A study evaluating color, spectral
characteristics, and stability of betacyanins from seven
Amaranth species found that the extracted pigments exhibit
stability and color characteristics similar to red radish
anthocyanins, indicating their potential use as commercial food
colorants (Cai et al., 1998). An additional genus in the amaranth
family, Celosia, has been suggested as a possible source for
betacycanins and betaxanthins (Schliemann et al., 2001).
Molecular Plant 11, 7–22, January 2018 ª The Author 2017. 13
Molecular Plant
However, high saponin content in Amaranthus spp. and high
dopamine content in Celosia spp. might a pose a toxicological
hurdle that will restrict their global commercialization as
sources of food colorants (Stintzing and Carle, 2004). Other
than plants of the Amaranthaceae, the cacti family (Cactaceae)
seems to be the most promising source for edible betalains
(Azeredo, 2009), with prickly pear (Opuntia spp.) and dragon-
fruit (Hylocereus spp.) being the most cultivated and best-
suited cacti fruit crops for this purpose (Mizrahi et al., 1997).
Betalain profiles and concentrations were investigated in
several dragon-fruit (Wybraniec et al., 2001; Stintzing et al.,
2002a; Wybraniec and Mizrahi, 2002) and prickly pear cultivars
(Stintzing et al., 2002b, 2003, 2005; Castellanos-Santiago and
Yahia, 2008). Opuntia betalains have also been studied with
respect to their antioxidant capacity and biological activities
(Butera et al., 2002; Tesoriere et al., 2004; Stintzing et al.,
2005), and stability (Castellar et al., 2003; Mosshammer et al.,
2005, 2007).
Various other plant materials derived from Caryophyllales plants
have been suggested as betalain sources, the most promising of
which include the red and yellow tubers of the Andean tuberous
crop ulluco (Ullucus tuberosus) (Svenson et al., 2008), red-
fruited malabar spinach (Basella alba) (Lin et al., 2010), grains of
djulis (Chenopodium formosanum), a cereal native to Taiwan
(Tsai et al., 2010), ripe berries of the pigeonberry plant (Rivina
humilis) (Khan et al., 2012), grains of quinoa (Chenopodium
quinoa) (Escribano et al., 2017), and the leaf vegetable
waterleaf (Talinum triangulare) (Swarna et al., 2013). Several of
these plants could provide added value in replacing red beet as
a commercial source of betalains, offering a wide spectrum of
colors, higher-quality pigments devoid of adverse flavors, and
the advantage of containing additional bioactive and nutritional
phytochemicals (Khan and Giridhar, 2015).
In addition to extraction from plant material, plant hairy root cul-
tures and cell cultures provide promising alternative sources for
production of betalains. Such sources possess advantages in
terms of rapidity and reproducibility of production quality and
yield, together with efficient downstream recovery of the product
(Rao and Ravishankar, 2002). Red beet has been the most widely
studied source for in vitro production of betalains, including both
hairy root and cell culture systems, as previously reviewed
(Georgiev et al., 2008; Neelwarne, 2012a, 2012b). In addition
to numerous studies focused on developing B. vulgaris cell
or hairy root culture for betalain production, several other
Caryophyllales plants were explored for the same purpose,
including C. rubrum (Berlin et al., 1986), Amaranthus tricolor
(Biancocolomas and Hugues, 1990), Celosia argentea
(Guadarrama-Flores et al., 2015), P. americana (Schliemann
et al., 1996), P. grandiflora (Bohm et al., 1991) and Mammillaria
candida (Santos-Diaz et al., 2005).
Metabolic Engineering of Betalains in Plants and
Microbes
The possibility to metabolically engineer betalain production in
plants and microbes became feasible with the increasing number
of genetic components associated with their production uncov-
ered. In vitro biosynthesis of the fluorescent yellow betalamic
acid, the backbone of all betalain structures, was first
14 Molecular Plant 11, 7–22, January 2018 ª The Author 2017.
Betalain Biosynthesis, Sources, and Applications
enabled by recombinant expression of the M. jalapa DOPA
4,5-dioxygenase gene (MjDOD) in E. coli and reacting it with its
substrate L-DOPA (Sasaki et al., 2009). The same group later
reported the in vitro production of 24 different betaxanthins by
allowing the spontaneous condensation of the recombinantly
produced betalamic acid together with amino acids or other
amines (Sekiguchi et al., 2010). Similarly, in vitro production of
betalamic acid and methionine-betaxanthin was also achieved
by recombinant expression of a B. vulgaris DOD in E. coli
(Gandia-Herrero and Garcia-Carmona, 2012).
A first example for in vivo production of betaxanthins in microbes
was enabled by expression of B. vulgaris DOD (BvDODA1) in
baker’s yeast (Saccharomyces cerevisiae), and feeding with
L-DOPA as substrate. Additionally, betacyanins were produced
when BvDODA1 was expressed together with CYP76AD1 and
yeast were fed with L-DOPA (Hatlestad et al., 2012). Early work
showing heterologous production of betalains in plants also
relied on feeding of substrates. Betaxanthins were produced in
the non-Caryophyllales plants broad bean (Vicia faba) and
pea (Pisum sativum) by feeding betalamic acid directly into plant
seedlings, thus providing evidence for the spontaneous character
of the betalamic acid condensation reaction with amines
(Schliemann et al., 1999). A major advance in heterologous
production in plants was achieved when a DOD gene derived
from the betalain-producing fungus AmDOD was stably trans-
formed into Arabidopsis plants. L-DOPA-fed transformant seed-
lings showed deep orange coloration due to betaxanthin accu-
mulation. Concurrently, transient expression of AmDOD or the
P. grandiflora DOD led to the formation of betaxanthins (and
remarkably, also glycosylated betacyanins betanin and isobeta-
nin) in L-DOPA-fed potato cell cultures and snapdragon petal
tissues (Harris et al., 2012).
Genetic engineering could potentially provide new sources for
commercial applications of betalains, such as high-yield crop
plants or microbial fermentation. However, to be commercially
viable, betalain production would have to be achieved without
the need for substrate feeding. Progress toward this objective
was delayed primarily due to the lack of characterization of the
first step of the pathway, namely the hydroxylation of tyrosine
to form L-DOPA (Schwinn, 2016). Attempted stable production
of betalains without substrate feeding was carried out in
suspension-cultured Arabidopsis T87 and tobacco BY2 cells,
by expression of a tyrosinase originating from shiitake mush-
room, together with the M. jalapa MjDOD gene. Expression of
the tyrosine-hydroxylating mushroom tyrosinase obviated the
need for L-DOPA feeding. However, the cultured cells turned
brown and stunted within several weeks, most probably due to
accumulation of the toxic dopaquinone and its derivatives, as a
result of tyrosinase activity (Nakatsuka et al., 2013).
The recently discovered involvement of cytochrome P450-type
enzymes in the tyrosine hydroxylase step in B. vulgaris enabled
betalain production in yeast without L-DOPA supplementation.
Red-violet-colored yeast cultures that mainly produce betacya-
nins were obtained via expression of MjDOD together with
CYP76AD1 (DeLoache et al., 2015; Polturak et al., 2016;
Sunnadeniya et al., 2016). Betaxanthin-producing cultures were
generated by expression of DOD together with a mutated
CYP76AD1 gene, which holds tyrosine hydroxylase but not
Betalain Biosynthesis, Sources, and Applications
L-DOPA oxidase activity (DeLoache et al., 2015). Moreover,
betaxanthin production in yeast was achieved by expressing
the native beet genes CYP76AD6, CYP76AD5, or CYP76AD15
(Polturak et al., 2016; Sunnadeniya et al., 2016). Elucidation of
the tyrosine hydroxylation step enabled the first examples of
stable heterologous betalain production in plants, which were
initially achieved in the model plants tobacco (Polturak et al.,
2016) and Arabidopsis (Sunnadeniya et al., 2016).
Metabolic engineering of betalains was also subsequently
demonstrated in a number of solanaceous plant species,
including the ornamental plant petunia and three food crops,
namely tomato, potato, and eggplant (Polturak et al., 2017). The
engineered plants exhibited red-violet pigmentation throughout
plant development, following the introduction of a single
binary vector driving constitutive expression of the B. vulgaris
CYP76AD1 and DODA1, together with M. jalapa cDOPA5GT. Me-
tabolomics analysis of red-pigmented tissues revealed that in
addition to the expected production of betanin, additional beta-
lains were being uniquely produced in the different species,
some of which have not been previously reported in literature
and are possibly new to nature (Polturak et al., 2017). Betalain-
producing tobacco plants with different flower colors were
obtained through expression of different combinations of
betalain-related genes. Specifically, expression of CYP76AD1,
CYP76AD6, or both genes led to formation of plants that respec-
tively accumulate predominantly betacyanins, betaxanthins, or
both types of pigments. Similarly, introduction of the same
vectors into tobacco BY2 cells also enabled the generation of
intensely yellow- or red-pigmented cell-suspension cultures
(Figure 3) (Polturak et al., 2017).
Heterologous production of betalains in plants and microbes offers
a number of potential biotechnological applications. For example,
Molecular Plant
Figure 3. Metabolic Engineering of Beta-
lains in Plants and Microbes.
(A) Flowers of wild-type (bottom right) and three
betalain-producing tobacco lines.
(B) Cell-suspension cultures of wild-type (left) and
betalain-producing BY2 tobacco cell lines.
(C) Ripe and unripe fruit of a betalain-producing
tomato line.
(D) Betalains accumulate in both roots and shoots
of transgenic tobacco plants.
production of betalains in plant cell-suspen-
sion culture using cell lines such as tobacco
BY2 or Arabidopsis T87 cells may be advan-
tageous over culture of cells derived from
naturally producing plants; methods for their
culture are well established in comparison
with other plant cell lines. Optimization of be-
talain yields, which typically relies on exter-
nally provided elicitors in the case of naturally
producing cells (Georgiev et al., 2008), can
instead be manipulated by precise control
of expression levels of the heterologous
proteins. Importantly, in a heterologous
system the production of specific betalain
compounds can be targeted, such as
several acylated betacyanins, which were shown to have
increased stability compared with betanin (Reynoso et al., 1997;
Schliemann and Strack, 1998; Herbach et al., 2005). However,
this will depend on future identification of currently unknown
enzymes performing decoration activities including betacyanin
glycosylation and acylation. Considering the economic aspects
of production in comparison to alternative heterologous systems,
betalain production will likely be significantly more cost-effective
in fermentation-derived production in engineered microbes
such as E. coli and S. cerevisiae, due to their shorter doubling
times, inexpensive carbon sources, ease of genetic modification,
and well-established methods for scale-up (Wilson and Roberts,
2012).
The wide spectrum of hues that can possibly be obtained by
means of combining red and yellow betalains makes them an
excellent target for development of new ornamental varieties
with an array of flower colors. However, as stable metabolic en-
gineering of betalains in plants has been delayed until recently
due to the lack of a fully characterized pathway, efforts in genetic
engineering of flower color have thus far mostly concentrated
on inducing or modifying the production of flavonoid/anthocyanin
or carotenoid pigments (Nishihara and Nakatsuka, 2010;
Chandler and Sanchez, 2012). Similarly, engineered production
of carotenoids and anthocyanins in means of nutritional
enhancement (‘‘biofortification’’) of fruits and vegetables has
been extensively explored (Dixon et al., 2013; Martin, 2013;
Giuliano, 2017), while first examples for the exploitation of
betalains for the same purpose have only recently been
described with betalains being produced in tomato, potato, and
eggplant (Polturak et al., 2017). Nevertheless, the simple
biosynthetic pathway and ubiquitous occurrence of tyrosine
imply that heterologous betalain production may hypothetically
be achieved in many additional species.
Molecular Plant 11, 7–22, January 2018 ª The Author 2017. 15
Molecular Plant Betalain Biosynthesis, Sources, and Applications

Figure 4. L-DOPA-Derived Metabolites.

L-DOPA is a precursor in biosynthetic pathways leading to formation of betalains (e.g., betanin), benzylisoquinoline alkaloids (e.g., morphine, codeine,
berberine), ipecac alkaloids (e.g., emetine), and phenetylisoquinoline alkaloids (e.g., colchicine). Dashed lines designate multiple reactions.

The antioxidant activities and potential health-promoting prop-
erties of betalains have been studied in edible plants such as
red beet (Georgiev et al., 2010), dragon-fruit (Tenore et al.,
2012), and prickly pear (Butera et al., 2002). However, it is
difficult to differentiate the suggested biological activities
of betalains from the activity of other phytochemicals found in
these plant extracts (e.g., flavonoids and other polyphenols).
Similarly, pinpointing the specific contribution of betalains in
resistance to abiotic and biotic stress factors when they are
16 Molecular Plant 11, 7–22, January 2018 ª The Author 2017.
studied in their natural hosts and habitats is complicated
by the possible involvement of other non-related factors.
Engineering betalain production in naturally non-producing
food crops and other plants enables to distinguish between
betalain activity and the one of other factors. Thus, such
plants serve as an exceptional platform for studying the nutri-
tional value of betalains in the human diet, as well as the central
roles of these pigments in plant development and stress
response.
Betalain Biosynthesis, Sources, and Applications
Finally, the betalain-related cytochrome P450s displaying solely
tyrosine hydroxylase activity may be utilized for efficient produc-
tion of L-DOPA, an economically important compound that has
been used for decades as the primary treatment for Parkinson’s
disease (Nagatsu and Sawada, 2009). L-DOPA is also a
precursor for dopamine-mediated biosynthesis of additional
high-value metabolites (Figure 4), which include among
others benzylisoquinoline alkaloids (e.g., morphine and other
opiates) (Beaudoin and Facchini, 2014), ipecac alkaloids (e.g.,
emetine, cephaeline) (Nomura and Kutchan, 2010), colchicine
(Ehrenworth and Peralta-Yahya, 2017), and catecholamines,
which are mostly known as neurotransmitters in animals but
have also been reported to occur in a large number of plants
species (Kuklin and Conger, 1995; Kulma and Szopa, 2007).
The use, for example, of CYP76AD6 or F309L-mutated
CYP76AD1 for this purpose would likely be advantageous to
employing other enzymes used for tyrosine-derived production
of L-DOPA, due to their inherent limitations such as diphenolase
activity on L-DOPA (tyrosinase), high cofactor requirements
(tyrosine hydroxylase), or broad substrate range (hydroxyphenyl-
acetic acid hydroxylase) (DeLoache et al., 2015; Trenchard et al.,
2015; Narcross et al., 2016).
CONCLUDING REMARKS
The study of betalain biochemistry has seen major advances in
recent years, eventually leading to the full elucidation of the
core betalain biosynthetic pathway in plants. This has already
led to several examples of metabolic engineering of betalains,
and may thus provide new platforms to facilitate the study of
the roles betalains play in plants, as well as in human nutrition
and health. Additionally, heterologous production of betalains in
plants or microbes may be considered a viable new opportunity
for commercial production of betalains, to be further developed
alongside other ongoing efforts that include increasing yield in
beet through breeding, exploring untapped plant sources, and
improving methods of hairy root and cell culture from betalain-
producing species.
Advancements in betalain pathway elucidation could be largely
attributed to high-throughput RNA-sequencing analyses, e.g.,
the identification of CYP76AD1, CYP76AD6, BvDODA1, and
BvMYB1, highlighting the value of this approach for the study
of metabolic pathways in non-model plants. The increasing
availability of transcriptomics and genomic data from Caryo-
phyllales plants is expected to enable the identification of
additional genes and enzymes that take part in betalain biosyn-
thesis, most notably those involved in glycosylation and acyla-
tion reactions that lead to the wide variety of betacyanins found
in nature. Identification of such genes will in turn permit prog-
ress in metabolic engineering possibilities, such as the produc-
tion of betalains with varying hues or compounds with increased
chemical stability.
ACKNOWLEDGMENTS
No conflict of interest declared.
Received: July 27, 2017
Revised: October 11, 2017
Accepted: October 19, 2017
Published: October 25, 2017
Molecular Plant
REFERENCES
Azeredo, H.M.C. (2009). Betalains: properties, sources, applications, and
stability—a review. Int. J. Food Sci. Technol. 44:2365–2376.
Baranksi, R., Goldman, I., Nothnagel, T., and Scott, J. (2016).
Improving color sources by plant breeding and cultivation. In
Handbook on Natural Pigments in Food and Beverages: Industrial
Applications for Improving Food Color, R. Carle and R. Schweiggert,
eds. (New York: Springer), pp. 429–472.
Beaudoin, G.A.W., and Facchini, P.J. (2014). Benzylisoquinoline alkaloid
biosynthesis in opium poppy. Planta 240:19–32.
Berlin, J., Sieg, S., Strack, D., Bokern, M., and Harms, H. (1986).
Production of betalains by suspension-cultures of Chenopodium
rubrum L. Plant Cell Tissue Organ Cult. 5:163–174.
Biancocolomas, J. (1984). Effect of a cytokinin antagonist on cytokinin
and light-dependent amaranthin synthesis in Amaranthus tricolor
seedlings. J. Plant Growth Regul. 2:281–287.
Biancocolomas, J., and Hugues, M. (1990). Establishment and
characterization of a betacyanin producing cell-line of Amaranthus
tricolor—inductive effects of light and cytokinin. J. Plant Physiol.
136:734–739.
Bohm, H., Bohm, L., and Rink, E. (1991). Establishment and
characterization of a betaxanthin-producing cell-culture from
Portulaca grandiflora. Plant Cell Tissue Organ Cult. 26:75–82.
Bokern, M., Heuer, S., and Strack, D. (1992). Hydroxycinnamic acid
transferases in the biosynthesis of acylated betacyanins—purification
and characterization from cell-cultures of Chenopodium rubrum and
occurrence in some other members of the caryophyllales. Bot. Acta
105:146–151.
Bokern, M., and Strack, D. (1988). Synthesis of hydroxycinnamic acid-
esters of betacyanins via 1-0-acylglucosides of hydroxycinnamic
acids by protein preparations from cell-suspension cultures of
Chenopodium rubrum and petals of Lampranthus sociorum. Planta
174:101–105.
Brockington, S.F., Walker, R.H., Glover, B.J., Soltis, P.S., and Soltis,
D.E. (2011). Complex pigment evolution in the Caryophyllales. New
Phytol. 190:854–864.
Brockington, S.F., Yang, Y., Gandia-Herrero, F., Covshoff, S.,
Hibberd, J.M., Sage, R.F., Wong, G.K., Moore, M.J., and Smith,
S.A. (2015). Lineage-specific gene radiations underlie the evolution
of novel betalain pigmentation in Caryophyllales. New Phytol.
207:1170–1180.
Butera, D., Tesoriere, L., Di Gaudio, F., Bongiorno, A., Allegra, M.,
Pintaudi, A.M., Kohen, R., and Livrea, M.A. (2002). Antioxidant
activities of Sicilian prickly pear (Opuntia ficus indica) fruit extracts
and reducing properties of its betalains: betanin and indicaxanthin.
J. Agric. Food Chem. 50:6895–6901.
Cabanes, J., Gandia-Herrero, F., Escribano, J., Garcia-Carmona, F.,
and Jimenez-Atienzar, M. (2016). Fluorescent bioinspired protein
labeling with betalamic acid. Derivatization and characterization of
novel protein-betaxanthins. Dyes Pigm. 133:458–466.
Cai, Y.Z., Sun, M., and Corke, H. (1998). Colorant properties and stability
of Amaranthus betacyanin pigments. J. Agric. Food Chem. 46:4491–
4495.
Calogero, G., Yum, J.H., Sinopoli, A., Di Marco, G., Gratzel, M., and
Nazeeruddin, M.K. (2012). Anthocyanins and betalains as light-
harvesting pigments for dye-sensitized solar cells. Solar Energy
86:1563–1575.
Casique-Arroyo, G., Martinez-Gallardo, N., de la Vara, L.G., and
Delano-Frier, J.P. (2014). Betacyanin biosynthetic genes and
enzymes are differentially induced by (a)biotic stress in Amaranthus
hypochondriacus. PLoS One 9:e99012.
Molecular Plant 11, 7–22, January 2018 ª The Author 2017. 17
Molecular Plant
Castellanos-Santiago, E., and Yahia, E.A. (2008). Identification and
quantification of betalains from the fruits of 10 Mexican prickly
pear cultivars by high-performance liquid chromatography and
electrospray ionization mass spectrometry. J. Agric. Food Chem.
56:5758–5764.
Castellar, R., Obon, J.M., Alacid, M., and Fernandez-Lopez, J.A.
(2003). Color properties and stability of betacyanins from Opuntia
fruits. J. Agric. Food Chem. 51:2772–2776.
Chandler, S.F., and Sanchez, C. (2012). Genetic modification;
the development of transgenic ornamental plant varieties. Plant
Biotechnol. J. 10:891–903.
Chang-Quan, W., Heng, S., Xiang-Zhong, G., Qin-Guang, H., Feng, L.,
and Bao-Shan, W. (2007). Correlation of tyrosinase activity and
betacyanin biosynthesis induced by dark in C-3 halophyte Suaeda
salsa seedlings. Plant Sci. 173:487–494.
Chen, N., Yu, Z.H., and Xiao, X.G. (2017). Cytosolic and nuclear co-
localization of betalain biosynthetic enzymes in tobacco suggests
that betalains are synthesized in the cytoplasm and/or nucleus of
betalainic plant cells. Front. Plant Sci. 8:831.
Christinet, L., Burdet, F.R.X., Zaiko, M., Hinz, U., and Zryd, J.P. (2004).
Characterization and functional identification of a novel plant 4,5-
extradiol dioxygenase involved in betalain pigment biosynthesis in
Portulaca grandiflora. Plant Physiol. 134:265–274.
Chung, H.H., Schwinn, K.E., Ngo, H.M., Lewis, D.H., Massey, B.,
Calcottt, K.E., Crowhurst, R., Joyce, D.C., Gould, K.S., Davies,
K.M., et al. (2015). Characterisation of betalain biosynthesis in
Parakeelya flowers identifies the key biosynthetic gene DOD as
belonging to an expanded LigB gene family that is conserved in
betalain-producing species. Front. Plant Sci. 6:499.
Clement, J.S., and Mabry, T.J. (1996). Pigment evolution in the
Caryophyllales: a systematic overview. Bot. Acta 109:360–367.
Clifford, T., Howatson, G., West, D.J., and Stevenson, E.J. (2015). The
potential benefits of red beetroot supplementation in health and
disease. Nutrients 7:2801–2822.
Constabel, C.P., Bergey, D.R., and Ryan, C.A. (1995). Systemin
activates synthesis of wound-inducible tomato leaf polyphenol
oxidase via the octadecanoid defense signaling pathway. Proc. Natl.
Acad. Sci. USA 92:407–411.
DeLoache, W.C., Russ, Z.N., Narcross, L., Gonzales, A.M., Martin,
V.J., and Dueber, J.E. (2015). An enzyme-coupled biosensor
enables (S)-reticuline production in yeast from glucose. Nat. Chem.
Biol. 11:465–471.
Dixon, R.A., Liu, C.G., and Jun, J.H. (2013). Metabolic engineering
of anthocyanins and condensed tannins in plants. Curr. Opin.
Biotechnol. 24:329–335.
Ehrenworth, A.M., and Peralta-Yahya, P. (2017). Accelerating
the semisynthesis of alkaloid-based drugs through metabolic
engineering. Nat. Chem. Biol. 13:249–258.
Esatbeyoglu, T., Wagner, A.E., Schini-Kerth, V.B., and Rimbach, G.
(2015). Betanin—a food colorant with biological activity. Mol. Nutr.
Food Res. 59:36–47.
Escribano, J., Pedreno, M.A., Garcia-Carmona, F., and Munoz, R.
(1998). Characterization of the antiradical activity of betalains from
Beta vulgaris L. roots. Phytochem. Anal. 9:124–127.
Escribano, J., Cabanes, J., Jimenez-Atienzar, M., Ibanez-Tremolada,
M., Gomez-Pando, L.R., Garcia-Carmona, F., and Gandia-Herrero,
F. (2017). Characterization of betalains, saponins and antioxidant
power in differently colored quinoa (Chenopodium quinoa) varieties.
Food Chem. 234:285–294.
Felker, P., Stintzing, F.C., Muessig, E., Leitenberger, M., Carle, R.,
Vogt, T., and Bunch, R. (2008). Colour inheritance in cactus pear
(Opuntia ficus-indica) fruits. Ann. Appl. Biol. 152:307–318.
18 Molecular Plant 11, 7–22, January 2018 ª The Author 2017.
Betalain Biosynthesis, Sources, and Applications
Fleuriet, A., Macheix, J.J., Suen, R., and Ibrahim, R.K. (1980).
Partial-purification and some properties of a hydroxycinnamoyl
glucosyltransferase from tomato fruits. Z. Naturforsch.C 35:967–972.
Gandia-Herrero, F., Garcia-Carmona, F., and Escribano, J. (2004).
Purification and characterization of a latent polyphenol oxidase from
beet root (Beta vulgaris L.). J. Agric. Food Chem. 52:609–615.
Gandia-Herrero, F., Escribano, J., and Garcia-Carmona, F. (2005).
Characterization of the monophenolase activity of tyrosinase on
betaxanthins: the tyramine-betaxanthin/dopamine-betaxanthin pair.
Planta 222:307–318.
Gandia-Herrero, F., Escribano, J., and Garcia-Carmona, F. (2007).
Characterization of the activity of tyrosinase on betanidin. J. Agric.
Food Chem. 55:1546–1551.
Gandia-Herrero, F., Escribano, J., and Garcia-Carmona, F. (2016).
Biological activities of plant pigments betalains. Crit. Rev. Food Sci.
Nutr. 56:937–945.
Gandia-Herrero, F., and Garcia-Carmona, F. (2012). Characterization of
recombinant Beta vulgaris 4,5-DOPA-extradiol-dioxygenase active in
the biosynthesis of betalains. Planta 236:91–100.
Gandia-Herrero, F., and Garcia-Carmona, F. (2013). Biosynthesis of
betalains: yellow and violet plant pigments. Trends Plant Sci.
18:334–343.
Ganesan, P., and Karthik, T. (2017). Analysis of colour strength, colour
fastness and antimicrobial properties of silk fabric dyed with natural
dye from red prickly pear fruit. J. Textile Inst. 108:1173–1179.
Gao, Z.J., Han, X.H., and Xiao, X.G. (2009). Purification and
characterisation of polyphenol oxidase from red Swiss chard (Beta
vulgaris subspecies cicla) leaves. Food Chem. 117:342–348.
Gengatharan, A., Dykes, G.A., and Choo, W.S. (2015). Betalains: natural
plant pigments with potential application in functional foods. Lwt-Food
Sci. Technol. 64:645–649.
Georgiev, V., Ilieva, M., Bley, T., and Pavlov, A. (2008). Betalain
production in plant in vitro systems. Acta Physiol. Plant. 30:581–593.
Georgiev, V.G., Weber, J., Kneschke, E.M., Denev, P.N., Bley, T., and
Pavlov, A.I. (2010). Antioxidant activity and phenolic content of
betalain extracts from intact plants and hairy root cultures of the red
beetroot Beta vulgaris cv. Detroit dark red. Plant Foods Hum. Nutr.
65:105–111.
Gill, M., and Steglich, W. (1987). Pigments of fungi (Macromycetes). In
Fortschritte der Chemie Organischer Naturstoffe/Progress in the
Chemistry of Organic Natural Products, Vol. 51, W. Herz, H.
Grisebach, G.W. Kirby, and C. Tamm, eds. (New York: Springer),
pp. 1–297.
Girod, P.A., and Zryd, J.P. (1991). Biogenesis of betalains—purification
and partial characterization of dopa 4,5-dioxygenase from amanita-
muscaria. Phytochemistry 30:169–174.
Giuliano, G. (2017). Provitamin A biofortification of crop plants: a gold
rush with many miners. Curr. Opin. Biotechnol. 44:169–180.
Grotewold, E. (2006). The genetics and biochemistry of floral pigments.
Annu. Rev. Plant Biol. 57:761–780.
Guadarrama-Flores, B., Rodriguez-Monroy, M., Cruz-Sosa, F.,
Garcia-Carmona, F., and Gandia-Herrero, F. (2015). Production of
dihydroxylated betalains and dopamine in cell suspension cultures of
Celosia argentea var. plumosa. J. Agric. Food Chem. 63:2741–2749.
Guesmi, A., Ladhari, N., Ben Hamadi, N., and Sakli, F. (2012). Isolation,
identification and dyeing studies of betanin on modified acrylic fabrics.
Ind. Crops Prod. 37:342–346.
Hans, J., Brandt, W., and Vogt, T. (2004). Site-directed mutagenesis
and protein 3D-homology modelling suggest a catalytic mechanism
for UDP-glucose-dependent betanidin 5-O-glucosyltransferase from
Dorotheanthus bellidiformis. Plant J. 39:319–333.
Betalain Biosynthesis, Sources, and Applications
Harris, N.N., Javellana, J., Davies, K.M., Lewis, D.H., Jameson, P.E.,
Deroles, S.C., Calcott, K.E., Gould, K.S., and Schwinn, K.E.
(2012). Betalain production is possible in anthocyanin-producing
plant species given the presence of DOPA-dioxygenase and L-
DOPA. BMC Plant Biol. 12:34.
Hatlestad, G.J., Sunnadeniya, R.M., Akhavan, N.A., Gonzalez, A.,
Goldman, I.L., McGrath, J.M., and Lloyd, A.M. (2012). The beet R
locus encodes a new cytochrome P450 required for red betalain
production. Nat. Genet. 44:816–820.
Hatlestad, G.J., Akhavan, N.A., Sunnadeniya, R.M., Elam, L., Cargile,
S., Hembd, A., Gonzalez, A., McGrath, J.M., and Lloyd, A.M. (2015).
The beet Y locus encodes an anthocyanin MYB-like protein that
activates the betalain red pigment pathway. Nat. Genet. 47:92–96.
Hatlestad, G.J., and Lloyd, A. (2015). The betalain secondary metabolic
network. In Pigments in Fruits and Vegetables, C. Chen, ed. (New York:
Springer), pp. 127–140.
Hayakawa, K., and Agarie, S. (2010). Physiological roles of betacyanin in
a halophyte, Suaeda japonica Makino. Plant Prod. Sci. 13:351–359.
Herbach, K.M., Stintzing, F.C., and Carle, R. (2005). Identification of heat-
induced degradation products from purified betanin, phyllocactin and
hylocerenin by high-performance liquid chromatography/electrospray
ionization mass spectrometry. Rapid Commun. Mass Spectrom.
19:2603–2616.
Herbach, K.M., Stintzing, F.C., and Carle, R. (2006). Betalain stability
and degradation—structural and chromatic aspects. J. Food Sci.
71:R41–R50.
Herrmann, K.M. (1995). The shikimate pathway—early steps in the
biosynthesis of aromatic-compounds. Plant Cell 7:907–919.
Heuer, S., Richter, S., Metzger, J.W., Wray, V., Nimtz, M., and Strack,
D. (1994). Betacyanins from bracts of Bougainvillea glabra.
Phytochemistry 37:761–767.
Heuer, S., and Strack, D. (1992). Synthesis of betanin from betanidin and
UDP-glucose by a protein preparation from cell-suspension cultures of
Dorotheanthus bellidiformis (Burm. f.) N. E. Br. Planta 186:626–628.
Heuer, S., Vogt, T., Bohm, H., and Strack, D. (1996). Partial purification
and characterization of UDP-glucose: betanidin 5-O- and 6-O-
glucosyltransferases from cell suspension cultures of Dorotheanthus
bellidiformis (Burm f) NEBr. Planta 199:244–250.
Hinz, U.G., Fivaz, J., Girod, P.A., and Zyrd, J.P. (1997). The gene coding
for the DOPA dioxygenase involved in betalain biosynthesis in Amanita
muscaria and its regulation. Mol. Gen. Genet. 256:1–6.
Hirano, H., Sakuta, M., and Komamine, A. (1996). Inhibition of
betacyanin accumulation by abscisic acid in suspension cultures of
Phytolacca americana. Z. Naturforsch.C 51:818–822.
Ibdah, M., Krins, A., Seidlitz, H.K., Heller, W., Strack, D., and Vogt, T.
(2002). Spectral dependence of flavonol and betacyanin accumulation
in Mesembryanthemum crystallinum under enhanced ultraviolet
radiation. Plant Cell Environ. 25:1145–1154.
Isayenkova, J., Wray, V., Nimtz, M., Strack, D., and Vogt, T. (2006).
Cloning and functional characterisation of two regioselective
flavonoid glucosyltransferases from Beta vulgaris. Phytochemistry
67:1598–1612.
Jain, G., and Gould, K.S. (2015a). Are betalain pigments the functional
homologues of anthocyanins in plants? Environ. Exp. Bot. 119:48–53.
Jain, G., and Gould, K.S. (2015b). Functional significance of betalain
biosynthesis in leaves of Disphyma australe under salinity stress.
Environ. Exp. Bot. 109:131–140.
Jain, G., Schwinn, K.E., and Gould, K.S. (2015). Betalain induction
by l-DOPA application confers photoprotection to saline-exposed
leaves of Disphyma australe. New Phytol. 207:1075–1083.
Molecular Plant
Jarvis, D.E., Ho, Y.S., Lightfoot, D.J., Schmockel, S.M., Li, B., Borm,
T.J.A., Ohyanagi, H., Mineta, K., Michell, C.T., Saber, N., et al.
(2017). The genome of Chenopodium quinoa. Nature 542:307–312.
Joy, R.W., Sugiyama, M., Fukuda, H., and Komamine, A. (1995).
Cloning and characterization of polyphenol oxidase cDNAs of
Phytolacca americana. Plant Physiol. 107:1083–1089.
Kanner, J., Harel, S., and Granit, R. (2001). Betalains—a new class of
dietary cationized antioxidants. J. Agric. Food Chem. 49:5178–5185.
Khan, M.I. (2016). Plant betalains: safety, antioxidant activity, clinical
efficacy, and bioavailability. Compr. Rev. Food Sci. Food Saf.
15:316–330.
Khan, M.I., and Giridhar, P. (2015). Plant betalains: chemistry and
biochemistry. Phytochemistry 117:267–295.
Khan, M.I., Harsha, P., Giridhar, P., and Ravishankar, G.A. (2012).
Pigment identification, nutritional composition, bioactivity, and
in vitro cancer cell cytotoxicity of Rivina humilis L. berries, potential
source of betalains. Lwt-Food Sci. Technol. 47:315–323.
Kinsman, L.T., Pinfield, N.J., and Stobart, A.K. (1975). The hormonal
control of amaranthin synthesis in Amaranthus caudatus seedlings.
Planta 127:207–212.
Kishima, Y., Nozaki, K., Akashi, R., and Adachi, T. (1991). Light-
inducible pigmentation in Portulaca callus—selection of a high
betalain producing cell-line. Plant Cell Rep. 10:304–307.
Kishima, Y., Shimaya, A., and Adachi, T. (1995). Evidence that blue light
induces betalain pigmentation in Portulaca callas. Plant Cell Tissue
Organ Cult. 43:67–70.
Kobayashi, N., Schmidt, J., Wray, V., and Schliemann, W. (2001).
Formation and occurrence of dopamine-derived betacyanins.
Phytochemistry 56:429–436.
Kujala, T., Loponen, J., and Pihlaja, K. (2001). Betalains and phenolics
in red beetroot (Beta vulgaris) peel extracts: extraction and
characterisation. Z. Naturforsch.C 56:343–348.
Kuklin, A.I., and Conger, B.V. (1995). Catecholamines in plants. J. Plant
Growth Regul. 14:91–97.
Kulma, A., and Szopa, J. (2007). Catecholamines are active compounds
in plants. Plant Sci. 172:433–440.
Lin, S.M., Lin, B.H., Hsei, W.M., Ko, H.J., Liu, C.D., Chen, L.G., and
Chiou, R.Y.Y. (2010). Structural identification and bioactivities of red-
violet pigments present in Basella alba fruits. J. Agric. Food Chem.
58:10364–10372.
Martin, C. (2013). The interface between plant metabolic engineering and
human health. Curr. Opin. Biotechnol. 24:344–353.
Martinez-Parra, J., and Munoz, R. (2001). Characterization of
betacyanin oxidation catalyzed by a peroxidase from Beta vulgaris
L. roots. J. Agric. Food Chem. 49:4064–4068.
Matsuba, Y., Okuda, Y., Abe, Y., Kitamura, Y., Terasaka, K., Mizukami,
H., Kamakura, H., Kawahara, N., Goda, Y., Sasaki, N., et al. (2008).
Enzymatic preparation of 1-O-hydroxycinnamoyl-beta-D-glucoses
and their application to the study of 1-O-hydroxycinnamoyl-beta-
D-glucose-dependent acyltransferase in anthocyanin-producing
cultured cells of Daucus carota and Glehnia littoralis. Plant
Biotechnol. 25:369–375.
Milkowski, C., and Strack, D. (2004). Serine carboxypeptidase-like
acyltransferases. Phytochemistry 65:517–524.
Mizrahi, Y., Nerd, A., and Nobel, P.S. (1997). Cacti as crops. Hort. Rev.
18:291–319.
Mosshammer, M.R., Stintzing, F.C., and Carle, R. (2005). Colour
studies on fruit juice blends from Opuntia and Hylocereus cacti and
betalain-containing model solutions derived therefrom. Food Res. Int.
38:975–981.
Molecular Plant 11, 7–22, January 2018 ª The Author 2017. 19
Molecular Plant
Mosshammer, M.R., Rohe, M., Stintzing, F.C., and Carle, R. (2007).
Stability of yellow-orange cactus pear (Opuntia ficus-indica L. Mill.
cv. ’Gialla’) betalains as affected by the juice matrix and selected
food additives. Eur. Food Res. Technol. 225:21–32.
Mueller, L.A., Hinz, U., Uze, M., Sautter, C., and Zryd, J.P. (1997a).
Biochemical complementation of the betalain biosynthetic pathway
in Portulaca grandiflora by a fungal 3,4-dihydroxyphenylalanine
dioxygenase. Planta 203:260–263.
Mueller, L.A., Hinz, U., and Zryd, J.P. (1997b). The formation of
betalamic acid and muscaflavin by recombinant dopa-dioxygenase
from Amanita. Phytochemistry 44:567–569.
Nagatsu, T., and Sawada, M. (2009). L-dopa therapy for Parkinson’s
disease: past, present, and future. Parkinsonism Relat. Disord.
15:S3–S8.
Nakashima, T., Araki, T., and Ueno, O. (2011). Photoprotective function
of betacyanin in leaves of Amaranthus cruentus L. under water stress.
Photosynthetica 49:497–506.
Nakatsuka, T., Yamada, E., Takahashi, H., Imamura, T., Suzuki, M.,
Ozeki, Y., Tsujimura, I., Saito, M., Sakamoto, Y., Sasaki, N., et al.
(2013). Genetic engineering of yellow betalain pigments beyond the
species barrier. Sci. Rep. 3:1970.
Narcross, L., Fossati, E., Bourgeois, L., Dueber, J.E., and Martin, V.J.J.
(2016). Microbial factories for the production of benzylisoquinoline
alkaloids. Trends Biotechnol. 34:228–241.
Neelwarne, B. (2012a). Cell and tissue culture studies in Beta vulgaris L. In
Red Beet Biotechnology: Food and Pharmaceutical Applications, B.
Neelwarne, ed. (Boston, MA: Springer), pp. 175–198.
Neelwarne, B. (2012b). Red beet hairy root cultures. In Red Beet
Biotechnology: Food and Pharmaceutical Applications, B. Neelwarne,
ed. (Boston, MA: Springer), pp. 199–249.
Nishihara, M., and Nakatsuka, T. (2010). Genetic engineering of novel
flower colors in floricultural plants: recent advances via transgenic
approaches. Methods Mol. Biol. 589:325–347.
Nomura, T., and Kutchan, T.M. (2010). Three new O-Methyltransferases
are sufficient for all O-methylation reactions of ipecac alkaloid
biosynthesis in root culture of Psychotria ipecacuanha. J. Biol. Chem.
285:7722–7738.
Petit-Paly, G., Andreu, F., Chénieux, J.C., and Rideau, M. (1994).
Phytolacca americana L. (Pokeweed): in vitro production of
betacyanins and medicinal compounds. In Medicinal and Aromatic
Plants VII, Y.P.S. Bajaj, ed. (Berlin Heidelberg: Springer), pp. 366–385.
Piattelli, M., and Imperato, F. (1970). Pigments of Bougainvillea glabra.
Phytochemistry 9:2557–2560.
Polturak, G., Breitel, D., Grossman, N., Sarrion-Perdigones, A.,
Weithorn, E., Pliner, M., Orzaez, D., Granell, A., Rogachev, I., and
Aharoni, A. (2016). Elucidation of the first committed step in betalain
biosynthesis enables the heterologous engineering of betalain
pigments in plants. New Phytol. 210:269–283.
Polturak, G., Grossman, N., Vela-Corcia, D., Dong, Y., Nudel, A.,
Pliner, M., Levy, M., Rogachev, I., and Aharoni, A. (2017).
Engineered gray mold resistance, antioxidant capacity and
pigmentation in betalain-producing crops and ornamentals. Proc.
Natl. Acad. Sci. USA 114:9062–9067.
Qingzhu, H., Chengjie, C., Zhe, C., Pengkun, C., Yuewen, M., Jingyu,
W., Jian, Z., Guibing, H., Jietang, Z., and Yonghua, Q. (2015).
Transcriptomic analysis reveals key genes related to betalain
biosynthesis in pulp coloration of Hylocereus polyrhizus. Front. Plant
Sci. 6:1179.
Rao, S.R., and Ravishankar, G.A. (2002). Plant cell cultures: chemical
factories of secondary metabolites. Biotechnol. Adv. 20:101–153.
20 Molecular Plant 11, 7–22, January 2018 ª The Author 2017.
Betalain Biosynthesis, Sources, and Applications
Reynoso, R., Garcia, F.A., Morales, D., and deMejia, E.G. (1997).
Stability of betalain pigments from a cactacea fruit. J. Agric. Food
Chem. 45:2884–2889.
Rodriguez-Amaya, D.B. (2016). Natural food pigments and colorants.
Curr. Opin. Food Sci. 7:20–26.
Santos-Diaz, M.S., Velasquez-Garcia, Y., and Gonzalez-Chavez, M.M.
(2005). Pigment production by callus of Mammillaria candida
Scheidweiler(Cactaceae). Agrociencia 39:619–626.
Sasaki, N., Adachi, T., Koda, T., and Ozeki, Y. (2004). Detection of UDP-
glucose:cyclo-DOPA 5-O-glucosyltransferase activity in four o’clocks
(Mirabilis jalapa L.). FEBS Lett. 568:159–162.
Sasaki, N., Abe, Y., Wada, K., Koda, T., Goda, Y., Adachi, T., and
Ozeki, Y. (2005a). Amaranthin in feather cockscombs is synthesized
via glucuronylation at the cyclo-DOPA glucoside step in the
betacyanin biosynthetic pathway. J. Plant Res. 118:439–442.
Sasaki, N., Wada, K., Koda, T., Kasahara, K., Adachi, T., and Ozeki, Y.
(2005b). Isolation and characterization of cDNAs encoding an enzyme
with glucosyltransferase activity for cyclo-DOPA from four o’clocks
and feather cockscombs. Plant Cell Physiol. 46:666–670.
Sasaki, N., Abe, Y., Goda, Y., Adachi, T., Kasahara, K., and Ozeki, Y.
(2009). Detection of DOPA 4,5-dioxygenase (DOD) activity using
recombinant protein prepared from escherichia coli cells harboring
cDNA encoding DOD from Mirabilis jalapa. Plant Cell Physiol.
50:1012–1016.
Schliemann, W., Cai, Y.Z., Degenkolb, T., Schmidt, J., and Corke, H.
(2001). Betalains of Celosia argentea. Phytochemistry 58:159–165.
Schliemann, W., Joy, R.W., Komamine, A., Metzger, J.W., Nimtz, M.,
Wray, V., and Strack, D. (1996). Betacyanins from plants and cell
cultures of Phytolacca americana. Phytochemistry 42:1039–1046.
Schliemann, W., Kobayashi, N., and Strack, D. (1999). The decisive step
in betaxanthin biosynthesis is a spontaneous reaction. Plant Physiol.
119:1217–1232.
Schliemann, W., and Strack, D. (1998). Intramolecular stabilization of
acylated betacyanins. Phytochemistry 49:585–588.
Schwinn, K.E. (2016). The dope on L-DOPA formation for betalain
pigments. New Phytol. 210:6–9.
Sciuto, S., Oriente, G., Piattelli, M., Impellizzeri, G., and Amico, V.
(1974). Biosynthesis of amaranthin in Celosia plumosa.
Phytochemistry 13:947–951.
Sekiguchi, H., Ozeki, Y., and Sasaki, N. (2010). In vitro synthesis
of betaxanthins using recombinant DOPA 4,5-dioxygenase and
evaluation of their radical-scavenging activities. J. Agric. Food Chem.
58:12504–12509.
Sekiguchi, H., Ozeki, Y., and Sasaki, N. (2013). Biosynthesis and
regulation of betalains in red beet. In Red Beet Biotechnology, B.
Neelwarne, ed. (New York: Springer), pp. 45–54.
Sepulveda-Jimenez, G., Rueda-Benitez, P., Porta, H., and Rocha-
Sosa, M. (2004). Betacyanin synthesis in red beet (Beta vulgaris)
leaves induced by wounding and bacterial infiltration is preceded by
an oxidative burst. Physiol. Mol. Plant Pathol. 64:125–133.
Sepulveda-Jimenez, G., Rueda-Benitez, P., Porta, H., and Rocha-
Sosa, M. (2005). A red beet (Beta vulgaris) UDP-glucosyltransferase
gene induced by wounding, bacterial infiltration and oxidative stress.
J. Exp. Bot. 56:605–611.
Sivakumar, V., Anna, J.L., Vijayeeswarri, J., and Swaminathan, G.
(2009). Ultrasound assisted enhancement in natural dye extraction
from beetroot for industrial applications and natural dyeing of leather.
Ultrason. Sonochem. 16:782–789.
Steglich, W., and Strack, D. (1990). Betalains. The alkaloids: Chem.
Pharmacol. 39:1–62.
Betalain Biosynthesis, Sources, and Applications
Steiner, U., Schliemann, W., and Strack, D. (1996). Assay for tyrosine
hydroxylation activity of tyrosinase from betalain-forming plants and
cell cultures. Anal. Biochem. 238:72–75.
Steiner, U., Schliemann, W., Bohm, H., and Strack, D. (1999).
Tyrosinase involved in betalain biosynthesis of higher plants. Planta
208:114–124.
Stintzing, F.C., and Carle, R. (2004). Functional properties of
anthocyanins and betalains in plants, food, and in human nutrition.
Trends Food Sci. Technol. 15:19–38.
Stintzing, F., and Carle, R. (2007). Betalains—emerging prospects for
food scientists. Trends Food Sci. Technol. 18:514–525.
Stintzing, F.C., and Carle, R. (2008). N-heterocyclic pigments: betalains.
In Food Colorants: Chemical and Functional Properties, C. Socaciu,
ed. (Boca Raton: CRC Press), pp. 87–99.
Stintzing, F.C., Schieber, A., and Carle, R. (2002a). Betacyanins in fruits
from red-purple pitaya, Hylocereus polyrhizus (Weber) Britton & Rose.
Food Chem. 77:101–106.
Stintzing, F.C., Schieber, A., and Carle, R. (2002b). Identification of
betalains from yellow beet (Beta vulgaris L.) and cactus pear Opuntia
ficus-indica (L.) Mill. by high-performance liquid chromatography-
electrospray ionization mass spectrometry. J. Agric. Food Chem.
50:2302–2307.
Stintzing, F.C., Schieber, A., and Carle, R. (2003). Evaluation of colour
properties and chemical quality parameters of cactus juices. Eur.
Food Res. Technol. 216:303–311.
Stintzing, F.C., Herbach, K.M., Mosshammer, M.R., Carle, R., Yi, W.G.,
Sellappan, S., Akoh, C.C., Bunch, R., and Felker, P. (2005). Color,
betalain pattern, and antioxidant properties of cactus pear (Opuntia
spp.) clones. J. Agric. Food Chem. 53:442–451.
Strack, D. (1980). Enzymatic-synthesis of 1-sinapoylglucose from free
sinapic acid and UDP-glucose by a cell-free system from Raphanus
sativus seedlings. Z. Naturforsch.C 35:204–208.
Strack, D., Vogt, T., and Schliemann, W. (2003). Recent advances in
betalain research. Phytochemistry 62:247–269.
Stracke, R., Holtgrawe, D., Schneider, J., Pucker, B., Sorensen, T.R.,
and Weisshaar, B. (2014). Genome-wide identification and
characterisation of R2R3-MYB genes in sugar beet (Beta vulgaris).
BMC Plant Biol. 14:249.
Sullivan, M.L. (2014). Beyond brown: polyphenol oxidases as enzymes of
plant specialized metabolism. Front. Plant Sci. 5:783.
Sunnadeniya, R., Bean, A., Brown, M., Akhavan, N., Hatlestad, G.,
Gonzalez, A., Symonds, V.V., and Lloyd, A. (2016). Tyrosine
hydroxylation in betalain pigment biosynthesis is performed by
cytochrome P450 enzymes in beets (Beta vulgaris). PLoS One
11:e0149417.
Suzuki, M., Miyahara, T., Tokumoto, H., Hakamatsuka, T., Goda, Y.,
Ozeki, Y., and Sasaki, N. (2014). Transposon-mediated mutation of
CYP76AD3 affects betalain synthesis and produces variegated
flowers in four o’clock (Mirabilis jalapa). J. Plant Physiol. 171:1586–
1590.
Svenson, J., Smallfield, B.M., Joyce, N.I., Sanson, C.E., and Perry,
N.B. (2008). Betalains in red and yellow varieties of the Andean tuber
crop ulluco (Ullucus tuberosus). J. Agric. Food Chem. 56:7730–7737.
Swarna, J., Lokeswari, T.S., Smita, M., and Ravindhran, R. (2013).
Characterisation and determination of in vitro antioxidant potential
of betalains from Talinum triangulare (Jacq.) Willd. Food Chem.
141:4382–4390.
Takahashi, K., Takamura, E., and Sakuta, M. (2009). Isolation and
expression analysis of two DOPA dioxygenases in Phytolacca
americana. Z. Naturforsch.C 64:564–573.
Molecular Plant
Tanaka, Y., Sasaki, N., and Ohmiya, A. (2008). Biosynthesis of
plant pigments: anthocyanins, betalains and carotenoids. Plant J.
54:733–749.
Tenore, G.C., Novellino, E., and Basile, A. (2012). Nutraceutical potential
and antioxidant benefits of red pitaya (Hylocereus polyrhizus) extracts.
J. Funct. Foods 4:129–136.
Terradas, F., and Wyler, H. (1991). 2,3-SECODOPA AND 4,5-
SECODOPA, the biosynthetic intermediates generated from L-dopa
by an enzyme-system extracted from the fly agaric, Amanita
muscaria L., and their spontaneous conversion to muscaflavin and
betalamic acid, respectively, and betalains. Helvetica Chim. Acta
74:124–140.
Tesoriere, L., Butera, D., Pintaudi, A.M., Allegra, M., and Livrea, M.A.
(2004). Supplementation with cactus pear (Opuntia ficus-indica) fruit
decreases oxidative stress in healthy humans: a comparative study
with vitamin C. Am. J. Clin. Nutr. 80:391–395.
Trenchard, I.J., Siddiqui, M.S., Thodey, K., and Smolke, C.D. (2015). De
novo production of the key branch point benzylisoquinoline alkaloid
reticuline in yeast. Metab. Eng. 31:74–83.
Tsai, P.J., Sheu, C.H., Wu, P.H., and Sun, Y.F. (2010). Thermal and pH
stability of betacyanin pigment of Djulis (Chenopodium formosanum)
in Taiwan and their relation to antioxidant activity. J. Agric. Food
Chem. 58:1020–1025.
Tzin, V., and Galili, G. (2010). The biosynthetic pathways for shikimate
and aromatic amino acids in Arabidopsis thaliana. Arabidopsis Book
8:e0132.
Vaughn, K.C., Lax, A.R., and Duke, S.O. (1988). Polyphenol oxidase—
the chloroplast oxidase with no established function. Physiol. Plant.
72:659–665.
Vogt, T. (2002). Substrate specificity and sequence analysis define a
polyphyletic origin of betanidin 5- and 6-O-glucosyltransferase from
Dorotheanthus bellidiformis. Planta 214:492–495.
Vogt, T., Grimm, R., and Strack, D. (1999a). Cloning and expression of
a cDNA encoding betanidin 5-O-glucosyltransferase, a betanidin-
and flavonoid-specific enzyme with high homology to inducible
glucosyltransferases from the Solanaceae. Plant J. 19:509–519.
Vogt, T., Ibdah, M., Schmidt, J., Wray, V., Nimtz, M., and Strack, D.
(1999b). Light-induced betacyanin and flavonol accumulation in
bladder cells of Mesembryanthemum crystallinum. Phytochemistry
52:583–592.
Wang, M., Lopez-Nieves, S., Goldman, I.L., and Maeda, H.A. (2017).
Limited tyrosine utilization explains lower betalain contents in yellow
than in red table beet genotypes. J. Agric. Food Chem. 65:4305–4313.
Wilson, S.A., and Roberts, S.C. (2012). Recent advances towards
development and commercialization of plant cell culture processes
for the synthesis of biomolecules. Plant Biotechnol. J. 10:249–268.
Wolyn, D.J., and Gabelman, W.H. (1990). Selection for betalain pigment
concentrations and total dissolved solids in red table beets. J. Am. Soc.
Hortic. Sci. 115:165–169.
Wybraniec, S. (2005). Formation of decarboxylated betacyanins in heated
purified betacyanin fractions from red beet root (Beta vulgaris L.)
monitored by LC-MS/MS. J. Agric. Food Chem. 53:3483–3487.
Wybraniec, S., Jerz, G., Gebers, N., and Winterhalter, P. (2010).
Ion-pair high-speed countercurrent chromatography in fractionation
of a high-molecular weight variation of acyl-oligosaccharide linked
betacyanins from purple bracts of Bougainvillea glabra. J.
Chromatogr. B Analyt. Tech. Biomed. Life Sci. 878:538–550.
Wybraniec, S., and Michalowski, T. (2011). New pathways of betanidin
and betanin enzymatic oxidation. J. Agric. Food Chem. 59:9612–9622.
Wybraniec, S., and Mizrahi, Y. (2002). Fruit flesh betacyanin pigments in
Hylocereus cacti. J. Agric. Food Chem. 50:6086–6089.
Molecular Plant 11, 7–22, January 2018 ª The Author 2017. 21
Molecular Plant
Wybraniec, S., and Mizrahi, Y. (2005). Generation of decarboxylated and
dehydrogenated betacyanins in thermally treated purified fruit extract
from purple pitaya (Hylocereus polyrhizus) monitored by LC-MS/MS.
J. Agric. Food Chem. 53:6704–6712.
Wybraniec, S., Platzner, I., Geresh, S., Gottlieb, H.E., Haimberg, M.,
Mogilnitzki, M., and Mizrahi, Y. (2001). Betacyanins from vine
cactus Hylocereus polyrhizus. Phytochemistry 58:1209–1212.
Wyler, H., Meuer, U., Bauer, J., and Stravsmombelli, L. (1984). Cyclodopa
glucoside (=(2S)-5-(beta-D-glucopyranosyloxy)-6-hydroxyindoline-2-
carboxylic acid) and its occurrence in red beet (Beta vulgaris var.
rubra L.). Helvetica Chim. Acta 67:1348–1355.
Xu, S.X., Huang, Q.Y., Lin, C.S., Lin, L.X., Zhou, Q., Lin, F.C., and
He, E.M. (2016). Transcriptome comparison reveals candidate
genes responsible for the betalain-/anthocyanidin-production in
bougainvilleas. Funct. Plant Biol. 43:278–286.
Yamamoto, K., Kobayashi, N., Yoshitama, K., Teramoto, S., and
Komamine, A. (2001). Isolation and purification of tyrosine
hydroxylase from callus cultures of Portulaca grandiflora. Plant Cell
Physiol. 42:969–975.
22 Molecular Plant 11, 7–22, January 2018 ª The Author 2017.
Betalain Biosynthesis, Sources, and Applications
Zakharova, N.S., and Petrova, T.A. (1997). Investigation of betalains and
betalain oxidase of leaf beet. Appl. Biochem. Microbiol. 33:481–484.
Zakharova, N.S., and Petrova, T.A. (2000). Beta-glucosidases from
leaves and roots of the common beet, Beta vulgaris. Appl. Biochem.
Microbiol. 36:394–397.
Zakharova, N.S., Petrova, T.A., Shcherbukhin, V.D., and Gins, V.K.
(1995). Betacyanin and betalain oxidase in different Amaranthus
species. Appl. Biochem. Microbiol. 31:202–205.
Zhang, D., Lanier, S.M., Downing, J.A., Avent, J.L., Lum, J., and
McHale, J.L. (2008). Betalain pigments for dye-sensitized solar cells.
J. Photochem. Photobiol. A Chem. 195:72–80.
Zhao, S.Z., Sun, H.Z., Gao, Y., Sui, N., and Wang, B.S. (2011).
Growth regulator-induced betacyanin accumulation and dopa-4,5-
dioxygenase (DODA) gene expression in euhalophyte Suaeda salsa
calli. In Vitro Cell. Dev. Biol. Plant 47:391–398.
Zheng, X.L., Liu, S.C., Cheng, C.Z., Guo, R.F., Chen, Y.K., Xie, L.Y.,
Mao, Y.Y., Lin, Y.L., Zhang, Z.H., and Lai, Z.X. (2016). Cloning and
expression analysis of betalain biosynthesis genes in Amaranthus
tricolor. Biotechnol. Lett. 38:723–729.