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The reported colour formation mechanism in pitaya fruit through co-accumulation of anthocyanins and betalains is inconsistent and fails to establish the co-accumulation

Authors:
Khan BMC Genomics (2022) 23:740
https://doi.org/10.1186/s12864-022-08957-z
CORRESPONDENCE
The reported colour formation mechanism
inpitaya fruit throughco-accumulation
ofanthocyanins andbetalains isinconsistent
andfails toestablish theco-accumulation
Mohammad Imtiyaj Khan*
Keywords: Anthocyanins, Betalains, Gene expression, Amaranthin, Gomphrenin-I
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Background
e premise of the paper authored by Zhou et al. [1]
published in BMC Genomics is that, in pitayas Hylocer-
eus undatus (red peel-red pulp or RR; green peel-white
pulp or GW) and H. megalanthus (yellow peel-white
pulp or YW, also called Selenicereus megalanthus, http://
legacy. tropi cos. org/ Name/ 50251 405? tab= accep tedna
mes, accessed on 14/11/2020) [2, 3]), anthocyanins and
betalains co-accumulate, and hence both contribute to
peel and pulp colour formation. Transcriptome sequenc-
ing, metabolome analysis, and qPCR were carried out.
Despite inconsistencies, incomplete data, and inaccurate
interpretation of data in the paper, the authors concluded
that anthocyanins and betalains might co-accumulate in
the same plant. Recently, a similar claim of co-accumula-
tion of anthocyanins and betalains in Hylocereus spp. was
systematically refuted [4]. In nature, anthocyanins and
betalains have been found to be mutually exclusive [5,
6]. However, it is possible that some plants may accumu-
late both the pigments. Herein, I systematically point out
the inconsistencies and misinterpretations of data in the
paper by Zhou etal. [1], to demonstrate that this study
does not disprove the mutual exclusiveness of anthocya-
nins and betalains.
Main text
Proling betacyanins andamaranthin donotmeet
established standards
White pulp of H. undatus fruits have been reported to
contain no betacyanins and betaxanthins [710]. Since
betalains were detected in white pulp of H. undatus by
Zhou etal. [1], the analysis they carried out should be
confirmed in accordance with the available standard
practices [11]. Nevertheless, they did not report any con-
firmatory data, except referring to two previous studies
among which only one reported data on white and red
species of Hylocereus [9], which was contrary to the find-
ings of Zhou etal. [1]. is indicates that the analytical
data presented by Zhou et al. [1] was unreliable. What
furthers this assumption is that no Hylocereus sp. has
been reported to accumulate amaranthin and/or con-
tain more gomphrenin-I than betanin and hylocerenin
[1215], unlike what Zhou et al. reported [1]. In fact,
in none of the references that Zhou etal. [1] depended
upon for secondary metabolite identification by declaring
…….metabolites with similar fragment ions were sug-
gested to be the same compounds. (page 15, column 2,
lines 5–6)”, there was detection of either amaranthin or
gomphrenin-I in pitaya samples. However, Zhou et al.
[1] reported the contrary without thorough chemical/
spectral characterisation. Such characterisation steps
include profiling authentic reference compounds, frag-
mentation or neutral loss patterns, and matching of ten-
tative structures’ precursor m/z to relevant databases.
Open Access
*Correspondence: imtiyaj@gauhati.ac.in
Biochemistry and Molecular Biology Lab, Department of Biotechnology,
Gauhati University, Assam 781014 Guwahati, India
Page 2 of 5
Khan BMC Genomics (2022) 23:740
Accurate mass determination based on isotopic abun-
dance and various charged and adduct ion forms must
be performed to rule out multiple candidate structures
of a single molecular formula [11, 16]. Moreover, multi-
ple analytical steps/techniques for confirmation would
be absolutely required, because the only difference in
the structures of gomphrenin-I and betanin is the posi-
tion of glucose moiety attachment, and hence MS spectra
of these two compounds are the same. erefore, there
is need for additional spectroscopic characterisation to
confirm the identity of gomphrenin-I. However, quite
questionably, on page 11, column 2, it is reasoned with
regard to the presence of high gomphrenin-I that “……
this is possibly due to the conversion of betanin into
gomphrenin-I as the latter was the significantly enriched
metabolite mapped on the betalain biosynthesis path-
way.” is explanation has no scientific basis because the
literature cited to support the explanation is a review
paper focussed on betalain evolution in which there is no
mention of gomphrenin-I and the (bio)chemistry of its
conversion into betanin. With regard to betanin content,
on page 11, column 2, the authors mention that “…[beta-
nin] was present in low quantities in RR-peel as com-
pared to GW and YW-peels…the quantity [of betanin] in
GW pulp was almost double than RR pulp.” is cannot
be reconciled with the betalain biosynthetic pathway, and
also not supported by the metabolite profile provided in
TableS9 in [1], i.e. RR pulp has more than 800 times total
betacyanins than GW pulp, and RR peel has more than
5 times than GW peel. As mentioned above, H. undatus
white pulp has been reported to contain no betaxanthins
or betacyanins, let alone betanin. erefore, it is contra-
dictory that green samples and yellow peels had more
betanin than red samples that had the highest betacya-
nin content among all studied samples. Contents of both
betacyanins (ca. 1.5mg/100g fresh peels) and betaxan-
thins (ca. 7mg/100g fresh peels) in H. megalanthus have
already been reported from China [17], whereas Colom-
bian Selenicereus megalanthus (or H. megalanthus) peels
were reported to contain ca. 2.5mg betaxanthins/100g
fresh weight [18]. e quantification of betacyanins, in
particular, and metabolites, in general, in Zhou etal. [1]
considers the area under the peak, whose relationship
with concentration could be established only through a
linear regression curve of the respective authentic refer-
ence compound. e cascading effect of the absence of
authentic reference compounds, and lack of proper iden-
tification of metabolites through spectral characteristics,
could be seen in the case of metabolite profile of differ-
ent samples, viz. pulp and peel of RR, GW and YW. For
example, green peel and white pulp samples of H. unda-
tus are expected to have the least betacyanin content,
as Israeli H. undatus (white pulp) has been reported to
contain no betacyanins and betaxanthins [10]. Surpris-
ingly, Zhou etal. [1] reported green peel and white pulp
to have total betacyanins content higher than yellow
pulp and yellow peel, when the areas under the curve of
all the major identified betacyanins in TableS9 [1] are
summedup. As for YW pulp, Ecuadorian S. megalanthus
(or H. megalanthus) pulp was earlier reported to contain
no betacyanins and betaxanthins [10]. In addition, there
are 433 metabolites listed in TableS9 [1], but, curiously,
some of the commonly reported amino acids or amines,
like L-DOPA, dopamine, and also ascorbic acid are not
among them, though their presence in H. megalanthus
[17, 19] and H. undatus [79, 19] has been established
beyond doubt.
Reported betalain biosynthetic gene expression
andbetalain‑ especially amaranthin‑ accumulation cannot
be reconciled
e expression patterns of unigenes of betalain biosyn-
thetic pathway presented in Fig.6 [1] do not corroborate
with betacyanin content presented in TableS9 [1]. Of the
four genes, viz. CYP76AD1-like (Cluster-864.132907),
Portulaca grandiflora DOD (Cluster-864.102567), Beta
vulgaris DOD (Cluster-864.111172) and Bougainvil-
lea spectabilis cD5GT (Cluster-864.24834), in RR peel
and pulp, only the expression of CYP76AD1-like seems
to correlate with their metabolite contents. at is, RR
pulp has higher betacyanin content and CYP76AD1-like
expression than RR peel. All the remaining genes were
either less expressed or not significantly different in RR
pulp than RR peel. Similarly, RR pulp had comparable
or lower betalain biosynthetic gene expression than GW
pulp, but total betacyanins content was much higher
in RR pulp. erefore, of all the four reported betalain
biosynthetic genes, only the expression of CYP76AD1-
like can be reconciled with the metabolite profile. How-
ever, based on Fig. 3 [1], CYP76AD1-like expression
should lead to L-DOPA formation, but L-DOPA was not
detected in any of the studied samples (TableS9 [1]).
Further, based on Fig.3 in [1], CYP76AD1-like gene was
not expressed in GW pulp and peel, and hence betalain
biosynthesis should not occur therein. Contrastingly,
TableS9 [1] shows that GW samples have higher betalain
content than corresponding YW samples. In addition,
all the 21 genes whose expressions are listed in Fig.3 [1]
have lower or similar expressions in YW pulp and GW
pulp compared to YW peel and GW peel. So, contrary
to what Fig.3 [1] suggests, i.e. betalain formation does
not occur in the absence of CYP76ADs, thereby result-
ing in white pulp and green peel, all the above-mentioned
observations do not support GW samples having higher
betacyanin content than YW samples.
Page 3 of 5
Khan BMC Genomics (2022) 23:740
In Fig.6 [1], none of the betalain biosynthetic genes
presented has higher expression in RR peel or pulp as
compared to GW pulp or peel. In fact, cluster-864.102567
(PgDOD-like) and cluster-864.111172 (uncharacterised
protein or BvDOD-like) are less expressed in both RR
peel and RR pulp, while the other genes remained not
significantly different from that of corresponding GW
samples. erefore, the gene expression pattern does not
support the metabolite profile, and it cannot be explained
by focussing only on betanin content in the samples, as
done on page 11, column 2, lines 8–13.
Gomphrenin-I is synthesised by a 6-O-GT in plants,
particularly betacyanin-accumulating ones (as reviewed
in [5]). erefore, if there were no 6-O-GTs expressed and
only 5-O-GTs were differentially expressed as shown in
Figs.3 and 6 [1], then gomphrenin-I cannot be the most
abundant betacyanin. However, gomphrenin-I has been
claimed to be the most abundant betacyanin (TableS9)
[1]. e following reasons make the claim unfeasible:
1) at least, in the case of H. megalanthus, there was no
6-O-GT expression observed by Xie etal. [17], 2) betan-
idin-5-O-glucosyltransferase (B5GT) and betanidin-6-O-
glucosyltransferase (B6GT) share only 19% amino acid
sequence identity suggesting that these enzymes are
paraphyletic evolutionarily even if they are present in
the same plant [20], and 3) betanin (betanidin-5-O-glu-
coside) has not been shown so far to convert into gom-
phrenin-I (betanidin-6-O-glucoside) via any enzymatic or
non-enzymatic step. Further, the presence of amaranthin
in pitaya has not been established so far through exten-
sive spectroscopic characterisation and quantification
[1215] though Zhou etal. [1] claimed to have detected
it. Amaranthin biosynthesis is completed only after glu-
curonylation at 2-OH of betanin. Since Zhou etal. [1]
did not report data on UDP-glucuronyltransferase and
Xie etal. [17] also could not find any upregulated glucu-
ronyltransferase gene, except for a down-regulated one in
H. megalanthus peel, it is very unlikely that amaranthin
was detected by Zhou etal. [1] in RR and GW samples,
especially when other researchers had not detected it
before in Hylocereus cacti [9, 12, 13].
Anthocyanins andANS proling fall shortofestablished
standards
Betalains are tyrosine-derived metabolites, whereas
anthocyanins are phenylalanine-derived. In plants,
anthocyanidin synthase (ANS) converts colourless leu-
coanthocyanidins into anthocyanidin pigments [21].
Only after this step does glucosylation and formation of
downstream compounds take place [21]. Consequently, it
is generally believed that ANS is the most crucial point
of separation between anthocyanin and betalain biosyn-
thesis in plants, the other two crucial points being the
convergence of one of the transcription factors involved
in betalain biosynthesis, and deregulation of arogenate
dehydrogenase to favour more tyrosine synthesis at the
cost of phenylalanine [6]. erefore, anthocyanins and
betalains are widely accepted to be mutually exclusive,
even though ANS is expressed in betalain-accumulating
plants. In Mirabilis jalapa, a 69 amino acid truncated and
catalytically inactive ANS is expressed, with the trunca-
tion involving a part of the active site [22]. Also, ANS is
present intact but not expressed in betalain-accumulat-
ing Spinacia oleracea and Phytolacca americana [23].
erefore, any finding contrary to the long-held and well-
supported concept of mutual exclusivity of anthocyanins
and betalains should be based on unquestionable evi-
dence. Zhou etal. [1] did not include any ANS in Fig.5
[1], but the expression of one ANS (cluster-864.105069)
was presented in Fig.6 [1]. However, the main concern
here is that the lone ANS whose differential expression
data is given in Fig.6 [1] is not a functionally validated
protein. Furthermore, its expression is not commensurate
with anthocyanin content presented in TableS9 [1]. For
example, RR peel has about ten times more total antho-
cyanins (all the differentially expressed anthocyanins
taken together) than RR pulp (TableS9 in [1]), however,
the ANS expression in both of them was not significantly
different (Fig.6 in [1]). Similarly, RR peel has about eight
times more anthocyanins than GW peel (TableS9 in [1]),
but the ANS expression was the same in both the sam-
ples (Fig.6 in [1]). It may not be even required to look
for downstream metabolite formation or corresponding
gene expression, if ANS expression itself is implausible,
because ANS acts as a catalyst that transforms colour-
less compounds/precursors into corresponding coloured
products that exhibit characteristic spectra which are dif-
ferent from that of its precursors or flavonoids derived
from the partially overlapping biosynthetic pathway [21,
24, 25]. erefore, it is questionable as to how this lone
ANS candidate whose expression does not correspond
to the anthocyanin content can support the premise of
anthocyanin accumulation in pitayas, let alone the co-
occurrence of anthocyanins and betalains.
Yellow colour formation appears tobe notsupported
bytheprovided data
Zhou etal. [1] wrote on page 2, column 1, second para-
graph that “….the color of the peel and pulp [of pitayas]
which is contributed mainly by the pigment betalains
and other secondary metabolites such as anthocyanins
and carotenoids.” To support this statement, a reference
was cited, although it does not report the characterisa-
tion of the pigment contents of vine cacti, but reports
phenotypic and genomic characterisation. In the stud-
ied pitaya samples by Zhou et al. [1], YW peels were
Page 4 of 5
Khan BMC Genomics (2022) 23:740
supposed to accumulate betaxanthins to ascribe their
colour to, as contribution of anthocyanins (page 14,
column 1, second paragraph, lines 11–13) and carot-
enoids (page 14, column 1, third paragraph, lines 7–9)
in yellow colour formation was ruled out. However,
there was no betaxanthin detected in the metabolite
analysis data presented in TableS9 [1]. e presence of
dopamine has been explained by Zhou et al. [1] as an
indication of betaxanthin formation, however, the cor-
responding betaxanthin, miraxanthin V, which is yellow
in colour, is not reported in Table9 [1]. All the dopa-
mine that has been reduced in yellow samples com-
pared to green and red samples may not be completely
attributed to betaxanthin formation, as was hypoth-
esised by Zhou etal. [1]. On the other hand, betalamic
acid is also greenish yellow in colour. However, it is also
not listed in Table9 [1] though others have reported its
presence in Hylocereus spp. [9]. Any other amino acid,
such as phenylalanine, can form a yellow betaxanthin
(i.e.Phe-betaxanthin). However, such a yellow betaxan-
thin was also not identified in Table9 [1]. Additionally,
YW peels and pulps had betacyanins to be detected
unlike betaxanthins which were simply assumed to be
present but not detected by the same method of analy-
sis that could detect betacyanins. In a separate study,
Xie at al. [17], and Cejudo-Bastante etal. [18] reported
5–7mg, and ca. 2.5mg betaxanthins/100g fresh peels,
respectively, of H. megalanthus (or Selenicereus mega-
lanthus) after colour breaking stage. So, taking into
account all these inconsistencies, it is clear that the
metabolite analysis method used by Zhou etal. [1] was
not reliable enough to explain the colour formation in
the studied samples.
Abbreviations
5-O-GT: Betanidin-5-O-glucosyltransferase; 6-O-GT: Betanidin-6-O-glucosyl-
transferase; ANS: Anthocyanidin synthase; B5GT: Betanidin-5-O-glucosyltrans-
ferase; B6GT: Betanidin-6-O-glucosyltransferase; BvDOD: Beta vulgaris DOPA-
4,5-dioxygeanse; cD5GT: cyclo-DOPA-5-O-glucosyltransferase; CYP76AD1: A
cytochrome P450 protein with monooxygenase activity towards tyrosineand
diphenol oxidase activity towards L-DOPA; DOD: DOPA-4,5-dioxygenase; GW:
Green peel-white pulp; HPLC: High performance liquid chromatography;
L-DOPA: L-3,4-dihydroxyphenylalanine; m/z: Mass to charge ratio; MS: Mass
spectrometry; PgDOD: Portulaca grandiflora DOPA-4,5-dioxygenase; RR: Red
peel-red pulp; UDP: Uridyl diphosphate; YW: Yellow peel-white pulp.
Acknowledgements
MIK is grateful to the Department of Biotechnology (BT/PR16902/
NER/95/422/2015) and Science and Engineering Research Board
(ECR/2016/000952) of the Government of India for financial support to the
Biochemistry and Molecular Biology lab.
Authors’ contributions
Not applicable. The author(s) read and approved the final manuscript.
Funding
Not applicable.
Availability of data and materials
Not applicable.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
Not applicable.
Received: 3 November 2021 Accepted: 19 October 2022
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Article
Full-text available
Here we respond to the paper entitled “ Contribution of anthocyanin pathways to fruit flesh coloration in pitayas ” (Fan et al., BMC Plant Biol 20:361, 2020). In this paper Fan et al. 2020 propose that the anthocyanins can be detected in the betalain-pigmented genus Hylocereus , and suggest they are responsible for the colouration of the fruit flesh. We are open to the idea that, given the evolutionary maintenance of fully functional anthocyanin synthesis genes in betalain-pigmented species, anthocyanin pigmentation might co-occur with betalain pigments, as yet undetected, in some species. However, in absence of the LC-MS/MS spectra and co-elution/fragmentation of the authentic standard comparison, the findings of Fan et al. 2020 are not credible. Furthermore, our close examination of the paper, and re-analysis of datasets that have been made available, indicate numerous additional problems. Namely, the failure to detect betalains in an untargeted metabolite analysis, accumulation of reported anthocyanins that does not correlate with the colour of the fruit, absence of key anthocyanin synthesis genes from qPCR data, likely mis-identification of key anthocyanin genes, unreproducible patterns of correlated RNAseq data, lack of gene expression correlation with pigmentation accumulation, and putative transcription factors that are weak candidates for transcriptional up-regulation of the anthocyanin pathway.
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