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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

Khan BMC Genomics (2022) 23:740
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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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
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
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.
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.
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.
Not applicable.
Availability of data and materials
Not applicable.
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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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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Background Elucidating the candidate genes and key metabolites responsible for pulp and peel coloration is essential for breeding pitaya fruit with new and improved appeal and high nutritional value. Here, we used transcriptome (RNA-Seq) and metabolome analysis (UPLC-MS/MS) to identify structural and regulatory genes and key metabolites associated with peel and pulp colors in three pitaya fruit types belonging to two different Hylocereus species. Result Our combined transcriptome and metabolome analyses suggest that the main strategy for obtaining red color is to increase tyrosine content for downstream steps in the betalain pathway. The upregulation of CYP76ADs is proposed as the color-breaking step leading to red or colorless pulp under the regulation by WRKY44 transcription factor. Supported by the differential accumulation of anthocyanin metabolites in red pulped pitaya fruit, our results showed the regulation of anthocyanin biosynthesis pathway in addition to betalain biosynthesis. However, no color-breaking step for the development of anthocyanins in red pulp was observed and no biosynthesis of anthocyanins in white pulp was found. Together, we propose that red pitaya pulp color is under the strict regulation of CYP76ADs by WRKYs and the anthocyanin coexistence with betalains is unneglectable. We ruled out the possibility of yellow peel color formation due to anthocyanins because of no differential regulation of chalcone synthase genes between yellow and green and no detection of naringenin chalcone in the metabolome. Similarly, the no differential regulation of key genes in the carotenoid pathway controlling yellow pigments proposed that the carotenoid pathway is not involved in yellow peel color formation. Conclusions Together, our results propose several candidate genes and metabolites controlling a single horticultural attribute i.e. color formation for further functional characterization. This study presents useful genomic resources and information for breeding pitaya fruit with commercially attractive peel and pulp colors. These findings will greatly complement the existing knowledge on the biosynthesis of natural pigments for their applications in food and health industry.
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Within the angiosperm order Caryophyllales, an unusual class of pigments known as betalains can replace the otherwise ubiquitous anthocyanins. In contrast to the phenylalanine-derived anthocyanins, betalains are tyrosine-derived pigments which contain the chromophore betalamic acid. The origin of betalain pigments within Caryophyllales and their mutual exclusion with anthocyanin pigments, has been the subject of considerable research. In recent years, numerous discoveries, accelerated by -omic scale data, phylogenetics and synthetic biology, have shed light on the evolution of the betalain biosynthetic pathway in Caryophyllales. These advances include: the elucidation of the biosynthetic steps in the betalain pathway, identification of transcriptional regulators of betalain synthesis, resolution of the phylogenetic history of key genes, and insight into a role for modulation of primary metabolism in betalain synthesis. Here we review how molecular genetics have advanced our understanding of the betalain biosynthetic pathway, and discuss the impact of phylogenetics in revealing its evolutionary history. In light of these insights we explore our new understanding of the origin of betalains, the mutual exclusion of betalains and anthocyanins, and the homoplastic distribution of betalain pigmentation within Caryophyllales. We conclude with a speculative conceptual model for the stepwise emergence of betalain pigmentation. This article is protected by copyright. All rights reserved.
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We previously developed red lettuce (Lactuca sativa L.) cultivars with high flavonoid and phenolic acid content and demonstrated their anti-diabetic effect. Here we report on developing three fertile and true-breeding lettuce lines enriched with flavonoids with reported beneficial health effects. These lines were identified in a segregating population of EMS-mutagenized red lettuce and characterized biochemically and genetically. Change in red coloration was used as a visual indicator of a mutation in a flavonoid pathway gene, leading to accumulation of flavonoid precursors of red anthocyanins. Pink-green kaempferol overproducing kfoA and kfoB mutants accumulated kaempferol to 0.6–1% of their dry weight, higher than in any vegetable reported. The yellow-green naringenin chalcone overproducing mutant (nco) accumulated naringenin chalcone, not previously reported in lettuce, to 1% dry weight, a level only observed in tomato peel. Kfo plants carried a mutation in the FLAVONOID-3′ HYDROXYLASE (F3′H) gene, nco in CHALCONE ISOMERASE (CHI). This work demonstrates how non-GMO approaches can transform a common crop plant into a functional food with possible health benefits.
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The genus Hylocereus (family Cactaceae) includes about 16 species. Now its reputation is spreading everywhere in the world due to its fruit (pitaya or pitahaya or dragon fruit), which is one of the most popular and widely used functional foods in the world. The fruit is a wealthy provenance of vitamins, minerals, antioxidants, and fiber. The ethno-pharmacological history of this genus indicated that it possesses antioxidant, anticancer, antimicrobial, hepato-protective, antihyperlipidemic, antidiabetic, and wound healing activities. Furthermore, it has been used for the treatment of cough, asthma, hyperactivity, tuberculosis, bronchitis, mumps, diabetes, and cervical lymph node tuberculosis. Different chemical constituents have been reported from this genus as betalains, flavonoids, phenolic acids, phenylpropanoids, triterpenes, sterols, fatty acids, etc. The current review focuses on the uses, botanical characterization, chemical constituents, nutritional importance, biological activities, and safety of Hylocereus species. Also, biosynthetic pathways of betalains have been discussed.
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The tribe Hylocereeae are represented by mainly Central American-Mexican epiphytic, hemi-epiphytic and climbing cacti. They are popular due to their spectacular nocturnal flowers and have some importance as crops grown for their edible fruits. We present the first comprehensive phylogenetic study of the Hylocereeae sampling 60 out of the 63 currently accepted species and 17 out of 19 infraspecific taxa. Based on four plastid regions (trnK/matK, the rpl16 intron, rps3-rpl16, and trnL-F) we find a highly supported core Hylocereeae clade that also includes Acanthocereus and Peniocereus p.p., while Strophocactus is depicted as polyphyletic and is resolved outside of the Hylocereeae tribe. The clades found within Hylocereeae agree, in general terms, with the currently accepted genera but none of the genera are entirely monophyletic in their current circumscription. A new concept for the Hylocereeae is presented to include the genera Acanthocereus (incl. Peniocereus p.p.), Aporocactus, Disocactus, Epiphyllum, Selenicereus (incl. Hylocereus and Weberocereus p.p.), Pseudorhipsalis, Kimnachia gen. nov., and Weberocereus. New nomenclatural combinations are provided to make these genera monophyletic. The genus Deamia is reinstated for Strophocactus testudo and S. chontalensis, while Strophocactus is newly circumscribed to include S. wittii, Pseudoacanthocereus brasiliensis, and P. sicariguensis. Both genera are excluded from Hylocereeae. A taxonomic synopsis of Hylocereeae is provided.
Anthocyanins, the color compounds of plants, are known for their wide applications in food, nutraceuticals and cosmetic industry. The biosynthetic pathway of anthocyanins is well established with the identification of potential key regulatory genes, which make it possible to modulated its production by biotechnological means. Various biotechnological systems, including use of in vitro plant cell or tissue cultures as well as microorganisms have been used for the production of anthocyanins under controlled conditions, however, a wide range of factors affects their production. In addition, metabolic engineering technologies have also used the heterologous production of anthocyanins in recombinant plants and microorganism. However, these approaches have mostly been tested at the lab- and pilot-scales, while very few up-scaling studies have been undertaken. Various challenges and ways of investigation are proposed here to improve anthocyanin production by using the in vitro plant cell or tissue culture and metabolic engineering of plants and microbial culture systems. All these methods are capable of modulating the producing anthocyanins, which can be further utilized for pharmaceutical, cosmetics and food applications.
Pitaya (Hylocereus spp.) is the only commercial cultivation of fruit containing abundant betalains for consumer. Betalains are water-soluble nitrogen-containing pigments with high nutritional value and bioactivities. In this study, contents of betaxanthins and betacyanins were compared between ‘Guanhuabai’ (H. undatus) and ‘Huanglong’ (H. megalanthus) pitayas and key genes involved in betalain biosynthesis were screened from ‘Huanglong’ pitaya by RNA-Seq technology. Twenty-nine candidate genes related to betalain biosynthesis were obtained from the transcriptome data. Based on expression characteristics and sequence analyses, HmB5GT1 and HmHCGT2 were further analyzed. HmB5GT1 and HmHCGT2 were both conserved in ‘PSPG-box’ and localized in nucleus. Silencing of HmB5GT1 and HmHCGT2 resulted in a significant reduction in betacyanin and betaxanthin contents. Those results suggested that HmB5GT1 and HmHCGT2 are possibly involved in betalain biosynthesis in H. megalanthus. The present work provides new information on betalain biosynthesis in Hylocereus at the transcriptional level.
Betalains are tyrosine-derived pigments that occur solely in one plant order, the Caryophyllales, where they largely replace the anthocyanins in a mutually exclusive fashion. In this study, we conducted multi-species transcriptome and metabolic profiling in Mirabilis jalapa and additional betalain-producing species to identify candidate genes possibly involved with betalain biosynthesis. Among the candidates identified, betalain-related cytochrome P450 and glucosyltransferase-type genes, which catalyze tyrosine hydroxylation or (hydroxy)cinnamoyl-glucose formation, respectively, were further functionally characterized. We detected the expression of genes in the flavonoid/anthocyanin biosynthetic pathways as well as their metabolite intermediates in betalain-accumulating M. jalapa flowers, and found that the anthocyanin-related gene ANTHOCYANIDIN SYNTHASE (MjANS) is highly expressed in the betalain-accumulating petals. However, it appears that MjANS contains a significant deletion in a region spanning the corresponding enzyme active site. These findings provide novel insights into betalain biosynthesis and a possible explanation for how anthocyanins have been lost in this plant species. Our study also implies a complex, non-uniform history for the loss of anthocyanin production across betalain producers, previously assumed to be strictly due to diminished expression of anthocyanin-related genes.
Metabolites are building blocks of cellular function. These species are involved in enzyme-catalyzed chemical reactions and are essential for cellular function. Upstream biological disruptions result in a series of metabolomic changes and, as such, the metabolome holds a wealth of information that is thought to be most predictive of phenotype. Uncovering this knowledge is a work in progress. The field of metabolomics is still maturing; the community has leveraged proteomics experience when applicable and developed a range of sample preparation and instrument methodology along with myriad data processing and analysis approaches. Research focuses have now shifted toward a fundamental understanding of the biology responsible for metabolomic changes. There are several types of metabolomics experiments including both targeted and untargeted analyses. While untargeted, hypothesis generating workflows exhibit many valuable attributes, challenges inherent to the approach remain. This Critical Insight comments on these challenges, focusing on the identification process of LC-MS-based untargeted metabolomics studies—specifically in mammalian systems. Biological interpretation of metabolomics data hinges on the ability to accurately identify metabolites. The range of confidence associated with identifications that is often overlooked is reviewed, and opportunities for advancing the metabolomics field are described.