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Digital Gene Expression Analysis Provides Insight into the Transcript Profile of the Genes Involved in Aporphine Alkaloid Biosynthesis in Lotus (Nelumbo nucifera)

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The predominant alkaloids in lotus leaves are aporphine alkaloids. These are the most important active components and have many pharmacological properties, but little is known about their biosynthesis. We used digital gene expression (DGE) technology to identify differentially-expressed genes (DEGs) between two lotus cultivars with different alkaloid contents at four leaf development stages. We also predicted potential genes involved in aporphine alkaloid biosynthesis by weighted gene co-expression network analysis (WGCNA). Approximately 335 billion nucleotides were generated; and 94% of which were aligned against the reference genome. Of 22 thousand expressed genes, 19,000 were differentially expressed between the two cultivars at the four stages. Gene Ontology (GO) enrichment analysis revealed that catalytic activity and oxidoreductase activity were enriched significantly in most pairwise comparisons. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, dozens of DEGs were assigned to the categories of biosynthesis of secondary metabolites, isoquinoline alkaloid biosynthesis, and flavonoid biosynthesis. The genes encoding norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), N-methylcoclaurine 3′-hydroxylase (NMCH), and 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) in the common pathways of benzylisoquinoline alkaloid biosynthesis and the ones encoding corytuberine synthase (CTS) in aporphine alkaloid biosynthetic pathway, which have been characterized in other plants, were identified in lotus. These genes had positive effects on alkaloid content, albeit with phenotypic lag. The WGCNA of DEGs revealed that one network module was associated with the dynamic change of alkaloid content. Eleven genes encoding proteins with methyltransferase, oxidoreductase and CYP450 activities were identified. These were surmised to be genes involved in aporphine alkaloid biosynthesis. This transcriptomic database provides new directions for future studies on clarifying the aporphine alkaloid pathway.
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ORIGINAL RESEARCH
published: 31 January 2017
doi: 10.3389/fpls.2017.00080
Frontiers in Plant Science | www.frontiersin.org 1January 2017 | Volume 8 | Article 80
Edited by:
Michael Deyholos,
University of British Columbia, Canada
Reviewed by:
Jianfei Zhao,
University of Pennsylvania, USA
Shengchao Yang,
Yunnan Agricultural University, China
*Correspondence:
Yanling Liu
liuyanling@wbgcas.cn
These authors have contributed
equally to this work.
Specialty section:
This article was submitted to
Plant Genetics and Genomics,
a section of the journal
Frontiers in Plant Science
Received: 23 September 2016
Accepted: 13 January 2017
Published: 31 January 2017
Citation:
Yang M, Zhu L, Li L, Li J, Xu L, Feng J
and Liu Y (2017) Digital Gene
Expression Analysis Provides Insight
into the Transcript Profile of the Genes
Involved in Aporphine Alkaloid
Biosynthesis in Lotus (Nelumbo
nucifera). Front. Plant Sci. 8:80.
doi: 10.3389/fpls.2017.00080
Digital Gene Expression Analysis
Provides Insight into the Transcript
Profile of the Genes Involved in
Aporphine Alkaloid Biosynthesis in
Lotus (Nelumbo nucifera)
Mei Yang1 † , Lingping Zhu 1, 2 † , Ling Li 1, 3, Juanjuan Li 1, 3, Liming Xu 1, Ji Feng 4and
Yanling Liu 1*
1Key Laboratory of Aquatic Plant and Watershed Ecology, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan,
China, 2Department of Agricultural Sciences, Viikki Plant Science Center, University of Helsinki, Helsinki, Finland, 3College of
Life Science, University of Chinese Academy of Sciences, Beijing, China, 4Tobacco Research Institute of Hubei Province,
Wuhan, China
The predominant alkaloids in lotus leaves are aporphine alkaloids. These are the
most important active components and have many pharmacological properties,
but little is known about their biosynthesis. We used digital gene expression
(DGE) technology to identify differentially-expressed genes (DEGs) between two lotus
cultivars with different alkaloid contents at four leaf development stages. We also
predicted potential genes involved in aporphine alkaloid biosynthesis by weighted
gene co-expression network analysis (WGCNA). Approximately 335 billion nucleotides
were generated; and 94% of which were aligned against the reference genome.
Of 22 thousand expressed genes, 19,000 were differentially expressed between
the two cultivars at the four stages. Gene Ontology (GO) enrichment analysis
revealed that catalytic activity and oxidoreductase activity were enriched significantly
in most pairwise comparisons. In Kyoto Encyclopedia of Genes and Genomes
(KEGG) analysis, dozens of DEGs were assigned to the categories of biosynthesis of
secondary metabolites, isoquinoline alkaloid biosynthesis, and flavonoid biosynthesis.
The genes encoding norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase
(6OMT), coclaurine N-methyltransferase (CNMT), N-methylcoclaurine 3-hydroxylase
(NMCH), and 3-hydroxy-N-methylcoclaurine 4-O-methyltransferase (4OMT) in the
common pathways of benzylisoquinoline alkaloid biosynthesis and the ones encoding
corytuberine synthase (CTS) in aporphine alkaloid biosynthetic pathway, which have
been characterized in other plants, were identified in lotus. These genes had positive
effects on alkaloid content, albeit with phenotypic lag. The WGCNA of DEGs revealed
that one network module was associated with the dynamic change of alkaloid content.
Eleven genes encoding proteins with methyltransferase, oxidoreductase and CYP450
activities were identified. These were surmised to be genes involved in aporphine alkaloid
biosynthesis. This transcriptomic database provides new directions for future studies on
clarifying the aporphine alkaloid pathway.
Keywords: lotus, aporphine alkaloids, co-expression network, biosynthesis, putative genes
Yang et al. Alkaloid Transcriptomic Analysis in Lotus
INTRODUCTION
Lotus belongs to the genus Nelumbo, the sole member of the
Nelumbonaceae family. This genus consists of two species,
Nelumbo nucifera (Asia, Australia, Russia) and Nelumbo lutea
(North America) (Xue et al., 2012). It is an ornamental
plant and an important economical crop in Asian countries,
especially in China. Lotus is characterized by dainty flowers,
round leaves, ellipsoidal seeds and fleshy rhizomes, thus it
has been cultivated as ornamental or vegetable plant for
7000 years throughout Asia, for its beautiful flowers and its
edible rhizomes and seeds (Shen-Miller, 2002; Zhang et al.,
2011). In addition, lotus has religious significance in both
Buddhism and Hinduism throughout the history because of
its pure and sacred meaning. Virtually, as a source of herbal
medicine, every part of the lotus plant, including its leaves,
buds, flowers, anther, stamens, fruit, stalks, and roots, have been
used for treatment of various diseases, such as pectoralgia, liver
disease, heart disease, cancer, insomnia, diabetes, obesity, and
hypertension (Lacour et al., 1995; Kashiwada et al., 2005; Ono
et al., 2006; Huang et al., 2010; Nguyen et al., 2012; Zhang
et al., 2012; Poornima et al., 2014). Alkaloids are the most
important active components in lotus, more than 20 had been
identified to date (Zhu et al., 2011; Nakamura et al., 2013).
On the basis of their structures, alkaloids in lotus can be
divided into three categories: aporphines, bisbenzylisoquinolines,
and monobenzylisoquinolines. Aporphine alkaloids accumulate
mainly in leaves, and include nuciferine, O-nornuciferine, N-
nornuciferine, anonaine, and roemerine (Luo et al., 2005; Itoh
et al., 2011; Chen et al., 2013b; Nakamura et al., 2013). This
accumulation starts from the early developmental stages, peaks
when the leaves reaches its full size, and then decreases slightly
during senescence (Deng et al., 2016). The bisbenzylisoquinoline
alkaloids, including liensinine, isoliensinine, and neferineare,
accumulate predominantly in the seed embryo (Itoh et al., 2011;
Chen et al., 2013b; Deng et al., 2016). Monobenzylisoquinoline
alkaloids are intermediate products in the biosynthesis of
aporphine and bisbenzylisoquinoline alkaloids, and occur in
trace amounts in several lotus organs (Itoh et al., 2011; Nakamura
et al., 2013).
All of the aporphine, bisbenzylisoquinoline, and
monobenzylisoquinoline alkaloids belong to the
benzylisoquinoline alkaloids (BIAs). BIAs are a large and
diverse group of natural products found primarily in several
plant families, and include approximately 2500 defined
structures (Ziegler and Facchini, 2008; Chow and Sato, 2013;
Glenn et al., 2013). BIAs are not essential for normal growth
and development but appear to function in the defense of plants
against herbivores and pathogens. Many of the estimated BIAs
are pharmacologically active. The most prominent compounds
are the narcotic analgesic morphine, the vasodilator papaverine,
the potential anti-cancer drug noscapine, and the antimicrobial
agents sanguinarine and berberine (Liscombe and Facchini,
2008; Ziegler and Facchini, 2008; Chow and Sato, 2013; Glenn
et al., 2013). Alkaloids in lotus have pharmacological effects
such as anti-hypertensive, anti-obesity, anti-cancer, and anti-
human immunodeficiency virus (HIV) activities (Lacour et al.,
1995; Kashiwada et al., 2005; Ono et al., 2006; Huang et al.,
2010; Nguyen et al., 2012; Poornima et al., 2014). Owing to
these pharmacological properties, lotus has received increasing
attention in recent years.
Although BIAs show wide structural diversity, their
biosynthetic pathways are all initiated by the condensation
of dopamine and 4-hydroxyphenyl acetaldehyde, which is
catalyzed by norcoclaurine synthase (NCS) (Figure 1A). Then,
a series of enzymes, norcoclaurine 6-O-methyltransferase
(6OMT), coclaurine N-methyltransferase (CNMT),
(S)-N-methylcoclaurine-3-hydroxylase (NMCH), and 3-
hydroxy-N-methylcoclaurine 4-O-methyltransferase (4OMT),
exhibit catalytic activity to yield the central branch point
intermediate (S)-reticuline. (S)-Reticuline undergoes diverse
reactions resulting in the formation of a wide variety of
backbone structures, including morphinan (morphine),
benzophenanthridine (sanguinarine), protoberberine
(berberine), and aporphine alkaloid subclasses (Liscombe
and Facchini, 2008; Ziegler and Facchini, 2008; Chow and
Sato, 2013; Glenn et al., 2013; Hagel and Facchini, 2013).
Bisbenzylisoquinoline alkaloids are not produced via (S)-
reticuline, but are intermediates from the (S)-reticuline
precursor N-methylcoclaurine, which is catalyzed by the
P450 enzyme CYP80A1 (Kraus and Kutchan, 1995; Ziegler
and Facchini, 2008). The common pathway (from the initial
step to the produce of (S)-reticuline) and the five branches
of morphinan, benzophenanthridine, protoberberine, and
bisbenzylisoquinoline have been characterized clearly in
Opium poppy (Papaver somniferum), Eschscholzia californica,
Japanese goldthread (Coptis japonica) and yellow meadow rue
(Thalictrum flavum). Most of the genes encoding the enzymes in
these pathways have been isolated (Liscombe and Facchini, 2008;
Ziegler and Facchini, 2008; Chow and Sato, 2013; Glenn et al.,
2013; Hagel and Facchini, 2013). However, little is known about
aporphine alkaloid biosynthesis.
The first committed step in the aporphine synthetic pathway
begins with the conversion of (S)-reticuline to corytuberine,
which is catalyzed by corytuberine synthase (CTS) (Liscombe and
Facchini, 2008; Chow and Sato, 2013). CTS has been isolated
and characterized in C. japonica (Ikezawa et al., 2008). Then,
reticuline N-methyltransferase (RNMT) catalyzes corytuberine
to yield magnoflorine by C8-C2 coupling, and has been cloned
in P. somniferum (Morris and Facchini, 2016). However, in lotus,
it remains unclear how the five aporphine alkaloids: nuciferine,
O-nornuciferine, N-nornuciferine, anonaine, and roemerine
are yielded from corytuberine and magnoflorine. Structural
analysis of these five aporphine alkaloids suggests that they
are produced from corytuberine and/or magnoflorine through
oxidation, dehydroxylation, and demethylation (Figure 1B).
These processes require oxidoreductases, methyltransferases, and
demethylase. Dissecting these late steps is a key to understand the
biosynthetic pathway of aporphine alkaloids in lotus.
Besides the enzymatic genes in the BIA biosynthetic pathway,
two transcription factors regulate the BIA metabolism. A
WRKY (CjWRKY1) is the first discovered transcription factor
involved in the coordinate regulation of BIA biosynthesis. Its
expression caused a substantial increase in the expression of
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
several berberine biosynthetic genes in opium poppy (Kato et al.,
2007). The ectopic expression of Arabidopsis WRKY1 increase
morphinan alkaloid content in California poppy cells (Apuya
et al., 2008). Transient RNA interference and overexpression of
CjbHLH1, a novel basic helix–loop–helix protein in C. japonica,
revealed the regulation of CjbHLH in transcription of berberine
biosynthetic genes. CjbHLH1 had similar activity to CjWRKY1
(Yamada et al., 2011).
RNA-seq is a valuable tool for transcriptomic profiling, such as
digital gene expression (DGE) technology (Glaser, 2009; Wang
et al., 2009; Asmann et al., 2012). Recently, DGE has been used
widely to detect differentially expressed genes (DEGs) in crops,
such as rice (Ding et al., 2016), maize (Eveland et al., 2010; Liu
et al., 2015), cotton (Wei et al., 2013) and Brassica (Jiang et al.,
2015); in woody plants, such as peach (Li et al., 2015), orange
(Yang et al., 2013; Sun et al., 2014), pear (Zhang et al., 2015), white
spruce (Albouyeh et al., 2010), and Metasequoia glyptostroboides
(Zhao et al., 2015); and in orchids, such as cymbidium (Yang
and Zhu, 2015), Rosa chinensis (Yan et al., 2015), and Tagetes
erecta (Ai et al., 2016). DGE promotes our understanding of the
major changes in metabolic processes and enables us to detect
DEGs. For example, DGE analysis have been used successfully to
predict biosynthetic pathways of the alkaloids, rhynchophylline
and isorhynchophylline, in Uncaria rhynchophylla, a non-model
plant with potent anti-Alzheimer’s properties (Guo et al., 2014),
which suggests that DGE analysis can be used to understand
alkaloid biosynthesis in lotus.
We sequenced the genome of an ancient lotus cultivar, “China
Antique” (Ming et al., 2013), and performed transcriptome
profiling analysis to investigate the expression of genes related
to floral transition and rhizome development in lotus (Yang
et al., 2014, 2015). These provide a solid foundation for the
use of DGE analysis to understand the transcript profiling of
the genes involved in alkaloid biosynthesis in lotus. Building
on this previous work, here, we performed DGE analysis of
two lotus cultivars, “10–48” (a low-leaf -alkaloid cultivar) and
“Luming Lian” (a high-leaf-alkaloid cultivar). We analyzed four
leaf development stages: leafbud (S1), curly leaf underwater (S2,
the day before leaf emergence from water), unfolded leaf (S3,
day 4 after leaf emergence from water, the beginning of leaf
expansion), and mature leaf (S4, day 20 after leaf emergence
from water), with three biological repeats. By examining the
levels of gene expression digitally, we identified the DEGs among
the cultivars and stages. Using weighted gene co-expression
network analysis (WGCNA), the co-expression networks were
constructed and the potential genes responsible for the late steps
in aporphine alkaloid biosynthesis were predicted. These results
lay a foundation for further characterization of the aporphine
alkaloid pathway in lotus.
MATERIALS AND METHODS
Plant Materials
Two cultivars of N. nucifera, “10–48” and “Luming Lian,” were
used for DGE analysis. “10–48” is a low-alkaloid cultivar, and
“Luming Lian” is a high-alkaloid cultivar. They were planted
in a trial plot at Wuhan Botanical Garden, Chinese Academy
of Sciences, Hubei Province, China on 13 April, 2012. Each
cultivar was planted in a separate cement pool of 25 m2(5 ×
5 m) and supplemented with 20-cm-deep paddy soil and 1 kg
organic fertilizer (mature soybean seedcake) before planting. A
total of 200 g chemical fertilizer was top dressed at both the
early developmental stage of standing leaves and the initial
stage of flowering. Throughout the growth season, the depth of
water in the pool was maintained at 20–25 cm, with an average
temperature of 30C during the day and 20C at night.
Leaves from “10–48” and “Luming Lian” were collected at
six stages: leafbud (T1), curly leaf underwater (T2, the day
before leaf emergence from water), curly leaf emergence from
water (T3, day 2 after leaf emergence from water), unfolded
leaf (T4, day 4 after leaf emergence from water, the beginning
of leaf expansion), semi-mature leaf (T5, day 10 after leaf
emergence from water, leaf area does not increase) and mature
leaf (T6, day 20 after leaf emergence from water), throughout
the growth season (Figure 2A). Analysis of leaf development
and alkaloid content for the two cultivars identified four stages,
T1, T2, T4, and T6, that would be particularly appropriate for
transcriptome sampling, which were renamed S1, S2, S3, and S4,
respectively. The leaves from each stage were collected from three
comparable plants used as three biological replicates, transferred
immediately to liquid nitrogen, and then stored at 80C until
RNA extraction.
Alkaloid content of the leaves at each developmental stage
was determined using the HPLC-MS, as described by Chen
et al. (2013b). Alkaloid was extracted from approximately
600 mg of fresh leaves with 30 mL methanol–1.0% hydrochloric
acid aqueous solution (1:1, v/v) by ultrasonication for 30 min,
followed by centrifugation at 15,000 ×g for 5 min. The
supernatant was then collected and diluted to 50 mL in a
volumetric flask. 2 mL of each solution was passed through a
0.22-µm filter and 10 µL of the filtered solution was used for
HPLC analysis. A-eluent was Milli-Q water containing 1‰;
(v/v) triethylamine, and B-eluent was acetonitrile. The gradient
profile started with 40–80% B at 0–15 min, followed by 80% B
at 18 min, 80–40% B at 19 min, and then equilibration of the
column at 40% B for 3 min; all at a flow rate of 0.8 mL/min
with a column temperature at 30C. The chromatograms were
acquired at 272 nm. The UV-vis and photodiode array spectra
were recorded from 180 to 400 nm.
RNA Extraction and Sequencing
Total RNA was extracted using the Easyspin RNA reagent
(RN38; Aidlab Biotechnologies, Beijing, China), and treated with
RNase-free DNase I (Takara, Dalian, China) to remove genomic
DNA contamination. The RNA integrity was evaluated with a
1.0% agarose gel stained with ethidium bromide. The quality
and quantity of RNA were assessed using a NanoPhotometer R
spectrophotometer (Implen, Westlake Village, CA, USA) and an
Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA,
USA). The RNA integrity number was >8.0 for all samples.
The RNA samples were later used to construct cDNA
libraries and Illumina sequences which were completed by
Beijing Novogene Bioinformatics Technology Co. Ltd (Beijing,
China). Poly(A) mRNA was prepared and sequences from
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
FIGURE 1 | The biosynthesis of benzylisoquinoline alkaloids (BIAs) in plant (A) and the structures of five prominent aporphine alkaloids in lotus (B). The
biosynthesis of BIAs includes five branch pathways: aporphine, benzophenanthridine, morphinan, protoberberine, and bisbenzylisoquinoline alkaloids. These branch
pathways are synthesized through a common pathway derived from L-tyrosine. The pathways are color-coded: white, common pathway; pink, aporphine alkaloids;
green, benzophenanthridine alkaloids; blue, morphinan alkaloids; purple, protoberberine alkaloids; and orange, bisbenzylisoquinoline alkaloids. Abbreviations of the
enzymes for these pathways: BBE, berberine bridge enzyme; BBS, berbamunine synthase; CAS, canadine synthase; CHS, cheilanthifoline synthase; CNMT,
coclaurine N-methyltransferase; CODM, codeine 3-O-demethylase; COR, codeinone reductase; CTS, corytuberine synthase; DBOX, dihydrobenzophenanthridine
oxidase; DRR, 1,2-dehydroreticuline reductase; DRS, 1,2-dehydroreticuline synthase; MSH, N-methylstylopine 14-hydroxylase; NCS, norcoclaurine synthase; NMCH,
N-methylcoclaurine 3-hydroxylase; 4OMT, 3-hydroxy-N-methylcoclaurine 4-O-methyltransferase; 6OMT, norcoclaurine 6-O-methyltransferase; PRH, protopine
6-hydroxylase; SalAT, salutaridinol-7-O-acetyltransferase; SalR, salutaridine:NADPH 7-oxidoreductase; SalSyn, salutaridine synthase; SOMT,
scoulerine-9-O-methyltransferase; STOX, (S)-tetrahydroprotoberberine oxidase; STS, stylopine synthase; THS, thebaine synthase; TNMT, tetrahydroprotoberberine
cis-N-methyltransferase; T6ODM, thebaine 6-O-demethylase.
each of the four developmental stages were indexed with
unique nucleic acid identifiers. Library quality was assessed
on the Agilent Bioanalyzer 2100 system. The libraries were
sequenced on an Illumina Hiseq 2000 platform and 100-
bp paired-end reads were generated. All raw-sequence reads
data were deposited in NCBI Sequence Read Archive (SRA,
http://www.ncbi.nlm.nih.gov/Traces/sra) with accession number
SRP095042.
Analysis of RNA-Seq Data
After removing those reads with only adaptor and unknown
nucleotides >5%, or those that were of low quality (Q<
20), the clean reads were filtered from the raw reads, then
aligned to the reference genome sequence (Ming et al., 2013)
using the program Tophat version 2.0.9 (Trapnell et al., 2012).
The tolerance parameters were the default settings, allowing
mismatches of no more than two bases. For inclusion in the
calculation of Reads Per Kilobases per Millionreads (RPKM)
values (Mortazavi et al., 2008), cut-offs were set such that 50%
of a read in contiguous nucleotides must be aligned to the
reference transcript with 98% identity. When reads could
be mapped to multiple reference locations, they were assigned
to reference transcripts that were based proportionally on the
relative number of unique reads.
The DESeq package was used to detect DEGs between the
two samples (Anders and Huber, 2010). The false discovery
rate (FDR) was used to determine the P-value threshold in
multiple tests. FDR 0.005 and absolute value of the log2(fold
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
change) with RPKM 1 were used as the thresholds to
determine significant differences in gene expression (Benjamini
and Yekutieli, 2001). The heatmap of DEGs was produced with
Cluster 3.0 (de Hoon et al., 2004). Before cluster formation,
RPKM expression values for each transcript were normalized to
between 2.0 and 2.0 by log-transformation. The heatmap was
clustered using the complete linkage hierarchical analysis based
on Euclidean distance.
Functional enrichment analyses, including Gene Ontology
(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
were performed to identify which GO terms or metabolic
pathways were significantly associated with the DEGs. The
GO enrichment analysis of DEGs was implemented using the
GOseq R package, in which gene length bias was corrected.
GO terms with a corrected P0.05 were considered
significantly enriched among the DEGs. The GO annotations
were classified functionally using WEGO software for gene
function distributions. KOBAS software was used to test the
statistical enrichment of DEGs in KEGG pathways. The pathways
with an FDR 0.05 were defined as those with genes that show
significant levels of differential expression.
Phylogenetic Analysis of the Genes
Protein sequences of the genes that are known to be involved in
BIA biosynthesis were used for phylogenetic analysis. Sequence
alignment was performed using ClustalW in MEGA5 (Tamura
et al., 2011) and adjusted manually, as necessary. The resulting
data were analyzed using the neighbor-joining method. The
bootstrap values were calculated from 1000 replicates.
Gene Co-Expression Network Analysis
The highly co-expressed gene modules were inferred from the
DEGs using WGCNA, an R package (Langfelder and Horvath,
2008). WGCNA network construction and module detection
were conducted using an unsigned type of topological overlap
matrix (TOM), a power βof 10, a minimal module size of
30, and a branch merge cut-off height of 0.25. The most
significant correlated genes with WGCNA edge weight >0.15
were visualized using Cytoscape 3.3 (Kohl et al., 2011).
Quantitative Real-Time PCR Validation of
RNA-Seq Data
Seven genes involved in the pathways of BIAs biosynthesis
and 11 candidate genes were selected for validation using
quantitative real-time PCR (RT-qPCR). Primers for RT-
qPCR, which were designed with the Primer 3.0 software
(http://biotools.umassmed.edu/bioapps/primer3_www.cgi), are
listed in Supplementary Table 1. RT-qPCRs were analyzed in
the ABI StepOneTM Plus Real-Time PCR System with the SYBR
Green PCR Master Mix (Takara), and amplified with 1 µL cDNA
template, 5 µL 2×SYBR Green Master Mix, and 0.2 µL each
primer (10 µmol/µL), to a final volume of 10 µL by adding
water. The amplification program consisted of one cycle of 95C
for 10 s, followed by 40 cycles of 95C for 15 s and 60C for 60
s. Fluorescent products were detected in the last step of each
cycle. Melting curve analysis was performed at the end of 40
cycles to ensure proper amplification of target fragments. All
RT-qPCR procedures for each gene were performed in three
biological replicates, with three technical repeats per experiment.
Relative gene expression was normalized by comparison with
the expression of lotus β-actin (NNU24864), and analyzed using
the 211CT method (Livak and Schmittgen, 2001). The data are
presented as means ±SE (n=9). Statistical analysis of RT-qPCR
data was conducted using the ANOVA procedure of SAS 8.1
(SAS Institute, Cary, NC, USA).
RESULTS
Alkaloid Content in Leaves at Different
Developmental Stages
Leaves from “10–48” and “Luming Lian” were sampled at six
stages (T1–T6, Figure 2A). Five profiles of alkaloids: roemerine,
anonaine, nuciferine, O-nornuciferine, and N-nornuciferine,
were tested for the two cultivars at the six stages (Figures 2C,D).
Nuciferine and N-nornuciferine were the dominant compounds
for “10–48,” and nuciferine and O-nornuciferine were the
dominant compounds for “Luming Lian.” Total alkaloid content
of “10–48” increased from 430.0 to 2060.7 mg kg1in stages
T1–T5, and decreased to 1346.1 mg kg1at T6. Total alkaloid
content of “Luming Lian” increased rapidly throughout the
period, ranging from 308.6 to 6203.0 mg kg1. In the first two
stages, there was no significant difference in total alkaloid content
between the two cultivars, and from stages T3 to T6, total alkaloid
content of “Luming Lian” was higher than that of “10–48.”
Therefore, total alkaloid content of “Luming Lian” at T6 was
4.6 times higher than that of “10–48” (Figures 2C,D). Given our
interest in the changes in expression of genes involved in alkaloid
metabolic pathways, based on these data, we chose the stages T1,
T2, T4, and T6 for DEG analysis. Leaves from these four stages
of “10–48” and “Luming Lian” were sampled to construct eight
cDNA libraries: LS1–4 and HS1–4.
HiSeq mRNA Sequencing and Number of
Expressed Genes
Three biological repeats were set for each stage of
the two cultivars, and 24 samples were sequenced
(Supplementary Table 2). Every sample generated 6 million
nucleotides of sequence data. Therefore, 335 billion nucleotides
were obtained from these samples. Ninety-four percent of
the short clean reads were aligned against the lotus “China
Antique” reference genome (Ming et al., 2013). To verify the
reproducibility of the sequencing data, Person’s correlation
coefficients for three biological replicates at each stage were
calculated by log10RPKM. All biological replicates had a
strong correlation (R20.97) except for the library HS1R3
(Supplementary Table 3).
Among the 26,685 annotated genes, 22,893 and 22,782 genes
were expressed in the low-alkaloid cultivar “10–48” and high-
alkaloid cultivar “Luming Lian,” respectively. Of these expressed
genes, 22,225 were detected in both cultivars. Only 668 and
557 genes were specifically expressed in “10–48” and “Luming
Lian,” respectively (Supplementary Figure 1A). Approximately
22,000 genes were expressed in each of the samples, and 21,000
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
FIGURE 2 | Leaf development and alkaloid content of “10–48” (low-alkaloid cv.) and “Luming Lian” (high-alkaloid cv.). (A) Six leaf developmental stages,
leafbud (T1), curly leaf underwater (T2, the day before leaf emergence from water), curly leaf emergence from water (T3, day 2 after leaf emergence from water),
unfolded leaf (T4, day 4 after leaf emergence from water, the beginning of leaf expansion), semi-mature leaf (T5, day 10 after leaf emergence from water, leaf area does
not increase) and mature leaf (T6, day 20 after leaf emergence from water). The leaves at the stages T1, T2, T4, and T6 are selected for DGE analysis and are
indicated with an asterisk, being renamed as S1, S2, S3, and S4, respectively. (B) Leaf area at different developmental stages for “10–48” and “Luming Lian.” (C) Five
profiles of alkaloids, roemerine, anonaine, nuciferine, O-nornuciferine, and N-nornuciferine at different leaf developmental stages for “10–48.” (D) Five profiles of
alkaloids, roemerine, anonaine, nuciferine, O-nornuciferine, and N-nornuciferine at different developmental stages of leaf for “Luming Lian.”
genes were co-expressed in three biological repeats for each
stage of the two cultivars (Supplementary Table 2). Of these
co-expressed genes, there were approximately 18,000 genes that
were commonly expressed in the four stages for each cultivar
(Supplementary Figures 1B,C). The following analyses were
based on the co-expressed genes in three biological repeats for
each stage of the two cultivars.
DEGs during Leaf Development
Differences in gene expression at the four leaf development stages
in the two cultivars were examined, and DEGs were identified
by pairwise comparisons of these libraries (Figure 3). The total
number of DEGs across the four stages was higher in “10–48”
than in “Luming Lian” at each stage. Comparisons of the four
stages of “10–48” revealed 3009, 10,998, 8269, and 15,127 DEGs
in the pairs LS2 vs. LS1, LS3 vs. LS2, LS4 vs. LS3, and LS4 vs. LS1,
respectively. Comparisons of the four stages of “Luming Lian”
revealed 415, 5434, 7982, and 7141 DEGs in the pairs of HS2 vs.
HS1, HS3 vs. HS2, HS4 vs. HS3, and HS4 vs. HS1, respectively
(Figures 3A,B). The number of DEGs detected in same-stage
comparisons between the two lotus cultivars was generally lower
than that detected from same-cultivar comparisons at different
stages. Comparisons of the same stage between the two lotus
cultivars identified 149, 1555, 1327, and 3882 DEGs in the
pairs HS1 vs. LS1, HS2 vs. LS2, HS3 vs. LS3, and HS4 vs.
LS4, respectively (Figure 3C). The number of the up-regulated
DEGs was larger than that of the down-regulated ones in the
pairs HS1 vs. LS1 and HS2 vs. LS2, but lower than that of the
down-regulated DEGs in HS4 vs. LS4 (Figure 3D).
There were 19,000 genes which were expressed differentially
for “10–48” and “Luming Lian” at the four stages. We
performed hierarchical clustering of these DEGs using the
Euclidean distance method associated with complete-linkage.
The expression patterns of six clusters, K1–K6, were plotted
(Figure 3E). The K1 cluster included 2613 genes that showed
down-regulation in both cultivars as the stages progressed, and
the expression levels of these genes were low at the S3 and S4
stages. Most of the genes in the K2 cluster, despite being up-
regulated as development progressed, had low expression levels.
Most of the genes in K3 and K4 were up-regulated as the stages
progressed in both cultivars, and the levels of expression of the
genes in K4 were higher than those in K3. The clusters K5 and
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
FIGURE 3 | Venn diagram and cluster analysis of differentially expressed genes (DEGs) identified by pairwise comparisons of the four stages in the
two cultivars. (A) Venn diagram of DEGs in “10–48” (low-alkaloid cv.) by pairwise comparisons of the four stages. (B) Venn diagram of DEGs in “Luming Lian”
(high-alkaloid cv.) by pairwise comparisons of the four stages. (C) Number of DEGs between “10–48” and “Luming Lian” at the particular stage. The separate and
overlapping areas in Venn diagrams represent the numbers of specifically expressed and co-expressed genes between different stages, respectively. (D) Numbers of
up-regulated and down-regulated DEGs between “10–48” and “Luming Lian” at the particular stage. (E) Heatmap of DEGs across the four developmental stages in
the lotus cultivars, “10–48” and “Luming Lian.” Expression values are presented as RPKM normalized log2transformed counts. Red and blue colors indicate up- and
down- regulated transcripts, respectively.
K6 possessed 7828 DEGs, that were significantly down-regulated
as the stages progressed in both cultivars, and the expression
of genes in K5 was higher than those in K6. Overall, the levels
of expression of genes in K5 and K6 were higher than those in
K1–K4 (Figure 3E).
Functional Classification of DEGs during
Leaf Development
To evaluate the potential functions of DEGs, GO assignments
were used to classify the functions of DEGs in pairwise
comparisons of the library between two cultivars and between
four developmental stages. The overrepresented GO terms of
DEGs in the three GO categories (cellular component, molecular
function, and biological process) are listed in Figure 4. In
the cellular component category, no GO terms were enriched
significantly in HS1 vs. LS1 and HS3 vs. LS3. The other pairwise
comparisons had high proportions of transcripts associated
with photosystem and thylakoid. The enriched GO terms in
the comparisons of two cultivars at each stage were fewer
than those across the stages for each cultivar (Figure 4A).
In the molecular function category, no GO terms were
enriched significantly in all pairwise comparisons, but catalytic
activity and oxidoreductase activity were enriched significantly
in most pairwise comparisons (Figure 4B). In the biological
process category, three GO terms, single-organism metabolic
process, photosynthesis, and oxidation–reduction process, were
enriched significantly in most comparisons. There were fewer
enriched GO terms in same-stage comparisons between the two
lotus cultivars than those across the stages for each cultivar
(Figure 4C).
KEGG analysis was performed to explore the pathways in
which DEGs were involved (Figure 5). Dozens of DEGs in
all pairwise comparisons were assigned to the biosynthesis of
secondary metabolites, isoquinoline alkaloid biosynthesis, and
flavonoid biosynthesis. BIA biosynthesis is part of isoquinoline
alkaloid biosynthesis, and belongs to the category of biosynthesis
of secondary metabolites. Therefore, the transcriptomic
profiling analysis could provide new information to characterize
aporphine alkaloid biosynthesis.
Genes in the BIAs Biosynthetic Pathway
The biosynthesis of BIAs includes the common pathway and five
branch pathways: aporphine, benzophenanthridine, morphinan,
protoberberine, and bisbenzylisoquinoline alkaloids, which are
synthesized through a common pathway derived from L-tyrosine
(Figure 1). We identified only 23 genes involved in the common
pathway, aporphine, morphinan and protoberberine pathways.
There are four homologs for NCS, four for 6OMT, one for CNMT,
two for NMCH, four for 4’OMT, and two for CTS, three for
scoulerine-9-O-methyltransferase (SOMT), one for codeine 3-O-
demethylase (CODM), and two for thebaine 6-O-demethylase
(T6ODM).
NCS is the first committed enzyme in BIA biosynthesis,
which catalyzes the condensation of dopamine and 4-
hydroxyphenylacetaldehyde (Figure 1). The four identified
NCSs (NNU14334, NNU21730, NNU21731, and NNU21732)
were classified into two groups (Figure 6A). NNU21730
is closely related to Populus euphratica,Theobroma cacao,
Phoenix dactylifera, and Elaeis guineensis. Meanwhile, the
other three NCSs are closely related to T. flavum,C. Japonica,
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
FIGURE 4 | GO-term function enrichment analysis of pairwise comparisons: (A) cellular component, (B) molecular function, and (C) biological process. The
significance of the most represented GO Slims in each comparison pair is indicated using log-transformed P-value (red); the dark gray areas represent missing values.
Corydalis saxicola,P. somniferum, and Argemone mexicana. The
expression of NNU21730 for the four stages in two cultivars
differed from those of the other three genes (Figures 6B–E).
NNU21730 was up-regulated as the stages progressed, and was
expressed more highly in “10–48” than in “Luming Lian.” The
expression of NNU14334 and NNU21731 increased at stages
S1 and S2, and then decreased from stage S3. NNU21732 had
a low level of expression, which may have had a little effect on
alkaloids. Considering the dynamic change of alkaloid content
(Figure 2), NNU21730 was not specific for BIA biosynthesis, and
NNU14334 exhibited phenotypic lag, wherein its expression was
prior to the dynamic phenotypic changes of alkaloid content.
There are two O-methyltransferases (6OMT and 4OMT) and
an N-methyltransferase (CNMT) in the common pathway of the
BIA biosynthesis (Figure 1). The genes encoding these enzymes
were identified (Figure 7A). The four homologs encoding 6OMT
(NNU03165, NNU03166, NNU19035, and NNU23168) had
the highest expression at stage S2, and their expression in
“Luming Lian” was significantly higher than that in “10–48”
(Figure 7B). Of the four homologs encoding 4OMT, NNU24728
had low expression, although it was up-regulated as the stages
progressed. The other three homologs, NNU15801, NNU15809,
and NNU25948, had the highest expression at stage S2, and
their expression in “10–48” was significantly higher than that in
“Luming Lian” in the first three stages (Figure 7C). The homolog
encoding CNMT, NNU11880, was up-regulated as the stages
progressed, and its expression in “Luming Lian” was significantly
higher than that in “10–48” (Figure 7D). Considering the
dynamic change in alkaloid content (Figure 2), the three
homologs of 6OMT (NNU03165, NNU03166, and NNU23168)
exhibited phenotypic lag, wherein their expression were prior to
the dynamic phenotypic changes of alkaloid content. CNMT is
positively correlated with alkaloid accumulation.
CYP80B and CYP80G encode NMCH and CTS, the
committed enzymes for the common pathway and aporphine
alkaloids branch, respectively (Figure 1). Two homologs for
CYP80B (NNU03539 and NNU08355) and two homologs
for CYP80G (NNU21372 and NNU21373) were identified
(Figure 8A). The expression of NNU03539 was low in the two
cultivars, so it may have had little effect on alkaloids (Figure 8E).
The expression of NNU08355 peaked at stage S3 in the two
cultivars, which was significantly higher in “Luming Lian” than
in “10–48” (Figure 8D). In addition, the expression pattern of
NNU21372 differed between the two cultivars. In “10–48” it was
down-regulated as the stages progressed, while in “Luming Lian,
it peaked at stage S3. The expression level of NNU21372 in
“Luming Lian” was higher than that in “10–48” at stages S2 and
S3 (Figure 8B). The expression of NNU21373 peaked at stage S2
in the two cultivars, and was significantly higher in “10–48” than
in “Luming Lian” (Figure 8C). Combining these findings with
the dynamic change of alkaloid content (Figure 2), we predicted
that CYP80B (NNU08355) and CYP80G (NNU21372) would
have positive effects on alkaloid content, albeit with phenotypic
lag.
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
FIGURE 5 | KEGG pathways that were significantly enriched in pairwise comparisons. The significance of the most strongly represented pathway in each
comparison pair is indicated using log-transformed P-value (red); the dark gray areas represent missing values.
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
FIGURE 6 | Phylogenetic relationship of norcoclaurine synthase (NCS) for different species based on protein sequence (A) and the expression of four
lotus NCS genes at four developmental stages in two lotus cultivars (B–E). In (A), the value at each node is the bootstrap support value from 1000 replicates. In
(B–E), RPKM value for each gene used three biological replicates; the error bars indicate standard error (n=3).
The homologs encoding SOMT, CODM, and T6ODM
were identified (Supplementary Figure 2). There were
three homologs (NNU20253, NNU20903, and NNU24927)
encoding SOMT, which is involved in the protoberberine
alkaloid pathway. The expression of NNU20253 in “Luming
Lian” were significantly higher than that in “10–48” as the
developmental stages progressed. NNU20903 only showed
significant difference between the two cultivars at stage
S1. The expression of NNU24927 increased as the stages
progressed, and was significantly higher in “Luming Lian”
than in “10–48” (Supplementary Figure 2A). One gene
encoding CODM (NNU26506) and two genes encoding
T6ODM (NNU14369 and NNU16999) were identified that
are involved in the morphinan alkaloid pathway. NNU26506
was significantly up-regulated as the stages progressed, but
showed no significant difference between the two cultivars
(Supplementary Figure 2B). NNU14369 only showed a
significant difference between the two cultivars at stage S1. The
expression of NNU16999 increased as the developmental stages
progressed, and was significantly higher in “Luming Lian” than
in “10–48” (Supplementary Figure 2C).
Genes Potentially Involved in the Late
Steps of Aporphine Alkaloid Biosynthesis
Predicted by WGCNA
The five predominant alkaloids in lotus are aporphine alkaloids.
The first two steps in aporphine alkaloid biosynthesis are to
yield corytuberine and magnoflorine. Little is known about
the late steps how corytuberine and magnoflorine produce
these five aporphine alkaloids of lotus. Given the structural
analysis of the five aporphine alkaloids, we conjectured
that oxidoreductases and methyltransferases are involved in
the late steps. Therefore, we used WGCNA to predict
the genes involved in the late steps of aporphine alkaloid
biosynthesis.
WGCNA was performed with all DEGs, which enabled
the identification of seven WGCNA modules (Figure 9A).
The module “Greenyellow” had expression patterns that
were associated with the dynamic change of alkaloid
content. Ninety genes with WGCNA edge weight >0.15
were selected in this module, which indicated that these
genes were highly connected among them. As a result, 329
pairs of co-expression edges were linked among these genes
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
FIGURE 7 | Phylogenetic relationships of the genes encoding three methyltransferases in the BIA pathways for different species based on protein
sequence (A) and the expression of these genes at four developmental stages in two lotus cultivars (B–D). The three methyltransferases are 6-O-methyltransferase
(6OMT), 3-hydroxy-N-methylcoclaurine 4-Omethyltransferase (4OMT), and coclaurine N-methyltransferase (CNMT). In (A), the value at each node is the bootstrap
support value from 1000 replicates. In (B–D), RPKM value for each gene used three biological replicates; the error bars indicate standard error (n=3).
(Figure 9B). Among the 90 genes, 13 were methyltransferases,
11 were CYP450 family genes, eight were oxidoreductases, 30
encoded the enzymes with other catalytic activity, 19 encoded
uncharacterized proteins, and the others encoded transporters
or transcription factors. For the 13 methyltransferases, six
were 6OMT and 4OMT. For the 11 genes for CYP450,
two were CYP80G, which encodes CTS (Figures 9B–D and
Supplementary Table 4).
DGE analysis showed that the seven methyltransferase
genes had the highest expression at stage S2 or S3, which
was similar to the expression patterns of 6OMT and 4’OMT
(Figure 9C). For the CYP450s, six and three genes had
patterns similar to the CYP80Gs, NNU21372, and NU21373,
respectively (Figure 9D). The eight oxidoreductase genes
were expressed most highly at stage S2, and “Luming Lian”
had higher expression than “10–48” in the last three stages
(Figure 9E). Eleven genes were found to cluster closely with
6OMT, 4OMT, and CYP80G (correlation >0.96), including five
methyltransferase genes (NNU04690, NNU07912, NNU09848,
NNU23605, and NNU24169), one CYP450 gene (NNU21317),
and five oxidoreductase genes (NNU04336, NNU04520,
NNU05340, NNU08935, and NNU10680). These 11 genes were
conjectured to be involved in the late steps of aporphine alkaloid
biosynthesis.
Experimental Confirmation of Gene
Expression by RT-qPCR
To confirm the results obtained through DGE analysis, 18 genes
involved in the pathways of BIA biosynthesis were selected to
analyze their expression in a biologically independent experiment
by RT-qPCR, including three genes encoding NCS, two encoding
NMCH, two genes encoding CTS, and the 11 candidate genes
(Figure 10). The RT-qPCR results for all of the genes were
tested statistically, and each gene showed significantly different
expression among the treatments (P=0.05). Moreover, 16
genes, except for NNU09848 and NNU23605, showed significant
correlations (P=0.05) between the RT-qPCR data and the
RNA-seq results, which indicated that the expression of the
genes studied was consistent between the RT-qPCR and DGE
analyses, despite some quantitative differences in expression level
(Figure 10).
DISCUSSION
The Importance of Alkaloid in Nelumbo
nucifera
Alkaloids are the most important active components in lotus
leaves, playing important roles in treating various diseases
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
FIGURE 8 | Phylogenetic relationships of NMCH (CYP80B) and CTS (CYP80G) for different species based on protein sequence (A) and the expression of
these genes at four stages in two lotus cultivars (B–E). In (A), the value at each node is the bootstrap support value from 1000 replicates. In (B–E), RPKM value for
each gene used three biological replicates; the error bars indicate standard error (n=3).
(Lacour et al., 1995; Kashiwada et al., 2005; Ono et al.,
2006; Huang et al., 2010; Nguyen et al., 2012; Poornima
et al., 2014). Alkaloids were present in each organ of lotus,
such as leaf, petiole, petal, seed, plumule and rhizome (Chen
et al., 2013b; Zhou et al., 2013; Deng et al., 2016). Lotus
leaves and plumules are rich in BIAs, whereas petioles and
rhizomes contain trace amounts of alkaloids. In leaves, five
aporphine-type alkaloids, N-nornuciferine, O-nornuciferine,
anonaine, nuciferine, and roemerine are the most dominant
component. Alkaloid composition and content ranged from
different lotus genotypes (Chen et al., 2013b; Deng et al.,
2016). These five aporphine alkaloids were identified at the six
developmental stages of leaves in two lotus cultivars. Nuciferine
was the dominant alkaloid in both cultivars, followed by N-
nornuciferine in “10–48” and O-nornuciferine in “Luming
Lian,” respectively (Figure 2). This result was consistent with
a previous study (Deng et al., 2016). Total content and the
dynamic change of alkaloids in two cultivars was different.
Alkaloid content in “Luming Lian” increased rapidly throughout
the period, and was 4.6 times higher than that of “10–48”
at last stage (Figures 2C,D), which was same as the result
tested by Deng et al. (2016). Hence, “10–48” and “Luming
Lian” are the ideal materials to study aporphine alkaloids in
lotus.
Because of the pharmacological significance of lotus, more
and more researches on lotus alkaloid have been reported,
including the pharmacological function of alkaloid, the
qualitative and quantitative analysis of alkaloid composition,
the alkaloid variation among lotus genotypes, and so on
(Ono et al., 2006; Huang et al., 2010; Nguyen et al., 2012;
Chen et al., 2013b; Poornima et al., 2014; Deng et al.,
2016). Lotus NCS genes were isolated and characterized
(Vimolmangkang et al., 2016), however, less report is
on aporphine alkaloid biosynthesis in lotus. Among the
different BIAs, only the details of the aporphine alkaloid
biosynthetic pathway remain largely unclear, while the
biosynthetic pathways for the other alkaloids have been
well elucidated (Liscombe and Facchini, 2008; Ziegler and
Facchini, 2008; Chow and Sato, 2013; Glenn et al., 2013; Hagel
and Facchini, 2013). In order to characterize the aporphine
alkaloid pathway in lotus, four stages of leaf development that
significantly differ between “10–48” and “Luming Lian” were
selected to detect genes expression using DGE technology
(Figure 2).
Ninety-four percent of clean pair-end reads were aligned
against the lotus “China Antique” reference genome, and 86% of
the predicted genes in the lotus genome were identified (Ming
et al., 2013), which is consistent with previous estimates of the
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
FIGURE 9 | Weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs) identified from the two lotus cultivars at
four developmental stages. (A) Hierarchical cluster tree showing seven modules of co-expressed genes. Each of the DEGs is represented by a leaf in the tree.
Seven modules were identified, which are shown in designated colors: “Black,” “Blue,” “Brown,” “Cyan,” “Greeenyellow,” “Red,” and “Tan.” (B) Cytoscape
representation of co-expressed genes with edge weight 0.15 in module “Greenyellow.” Gene IDs in red color refer to 6OMT, 4OMT, and CYP80G, the ones in blue
refer to methyltransferases, the ones in green refer to CYP450, and the ones in yellow refer to oxidoreductases. The other genes are in white. (C) Heatmap of
methyltransferases across the four developmental stages in the lotus cultivars, “10–48” and “Luming Lian.” (D) Heatmap of CYP450 across the four developmental
stages in the lotus cultivars, “10–48” and “Luming Lian.” (E) Heatmap of oxidoreductases across the four developmental stages in the lotus cultivars “10–48” and
“Luming Lian.” Expression values are presented as RPKM normalized log2transformed counts. Red and blue colors indicate up- and down- regulated transcripts,
respectively.
flower and rhizome transcriptome generated in lotus by RNA-seq
studies (Yang et al., 2014, 2015). Dozens of DEGs were assigned
to the categories of biosynthesis of secondary metabolites,
isoquinoline alkaloid biosynthesis, and flavonoid biosynthesis
by KEGG analysis (Figure 5). This provided a meaningful
framework for the specific biological activities. The secondary
metabolisms vary considerably depending on the enzyme(s)
associated with the pathways. Lotus contain abundant alkaloids,
steroids, flavonoids, triterpenoids, glycosides and polyphenols
(Chen et al., 2013a,b; Deng et al., 2013, 2016). The expressions
of the genes in biosynthesis of secondary metabolites were
changed with the development of the lotus leaf, and regulated
the content of these metabolites. Lotus aporphine alkaloids are
isoquinoline alkaloid, and the genes involved in isoquinoline
alkaloid pathway should differentially express. The KEGG result
confirmed the accuracy of the data and provided new clues that
deepen the understanding of aporphine alkaloids metabolism in
lotus.
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
FIGURE 10 | Quantitative real-time RT-qPCR confirmation of seven genes at the four stages, S1, S2, S3, and S4, between the two cultivars, “10–84”
and “Luming Lian.” The left y axis indicates relative gene expression levels determined by RT-qPCR. Relative gene expression was normalized by comparison with
the expression of lotus βactin (NNU24864). The expression values were adjusted by setting the expression of LS1 to 1 for each gene. All RT-qPCRs for each gene
used three biological replicates, with three technical replicates per experiment; the error bars indicate standard error. Different lower-case letters (a–e) indicate a
significant difference among eight treatments at P=0.05. Correlation coefficient (R) shown for each gene between the RT-qPCR and DGE data are those that
reached significance at P<0.05.
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Yang et al. Alkaloid Transcriptomic Analysis in Lotus
Genes Related to BIA Biosynthesis in Lotus
BIAs constitute the group of the most abundant alkaloids in
the plant. They are derived biosynthetically from the amino
acid tyrosine generating the fundamental intermediate dopamine
and 4-hydroxyphenyl acetaldehyde, and yielded through the
branch pathways, including morphinan, benzophenanthridine,
protoberberine and aporphine alkaloid pathways (Liscombe
and Facchini, 2008; Ziegler and Facchini, 2008; Chow and
Sato, 2013; Glenn et al., 2013; Hagel and Facchini, 2013). The
genes for the common pathway and the branches (morphinan,
benzophenanthridine and protoberberine) have been cloned in
the opium poppy, Japanese goldthread, and yellow meadow
rue. The two enzymes in aporphine alkaloid pathway, CTS and
RNMT, were characterized in Japanese goldthread and opium
poppy, respectively (Kraus and Kutchan, 1995; Ikezawa et al.,
2008; Liscombe and Facchini, 2008; Ziegler and Facchini, 2008;
Chow and Sato, 2013; Glenn et al., 2013; Morris and Facchini,
2016). In lotus, the genes involved in the common pathway and
aporphine alkaloid pathway were identified, which were NCS,
6OMT, CNMT, NMCH, 4OMT and CTS (Figures 68).
NCS is the first committed enzyme in the common
pathway, and catalyzes the condensation of dopamine and 4-
hydroxyphenyl acetaldehyde to (S)-norcoclaurine. It plays an
important role in the regulation of BIA biosynthesis due to
its entry-point location in the pathway (Minami et al., 2007;
Liscombe and Facchini, 2008; Ziegler and Facchini, 2008;
Chow and Sato, 2013; Glenn et al., 2013). In plant, the NCS
genes are divided into two subfamilies. NCSI genes are only
identified in a limited number of dicotyledonous taxa that
produce BIAs, while NCSII genes are universal in plants (Lee
and Facchini, 2010). Seven NCS genes have been identified in
the sacred lotus genome. The high or low levels of alkaloids
may be inhibited or induced, respectively, by the expression of
NnNCS7, one of NCSI (Vimolmangkang et al., 2016). Four of
these seven NCS genes were also detected in our study. The
expression of NNU21730, belongs to NCSII, was not significantly
correlated with alkaloid content in leaf (Figures 6A,B). It is
not specific to BIA biosynthesis and its lack of expression
may not inhibit alkaloid accumulation (Vimolmangkang et al.,
2016). NNU21732, the NCSI gene, had a low expression level
(Figure 6D) and is a pseudogene because of the single-nucleotide
deletion adjacent to the GC dinucleotide at the 5border of the
intron (Vimolmangkang et al., 2016). The dynamic phenotypic
change of alkaloid content occurs later than the actual expression
change of NNU14334 and NNU21731, namely phenotypic lag
(Figures 6C,E). None of these four identified genes showed a
significant correlation with alkaloid content in leaves in this
study. It is likely that other NCS homologs, for example,
NnNCS7, play a prominent role in alkaloid accumulation
in leaves (Vimolmangkang et al., 2016). More studies are
needed to confirm the function of NCS in the biosynthesis
of BIAs.
The conversion of (S)-norcoclaurine to (S)-reticuline involves
a series of enzymes: 6OMT, CNMT, NMCH, and 4OMT
(Liscombe and Facchini, 2008; Ziegler and Facchini, 2008; Chow
and Sato, 2013; Glenn et al., 2013). Among them, 6OMT,
CNMT, and 4OMT are methyltransferases. Specifically, 6OMT
and 4OMT are both class II O-methyltransferases and display
strict regiospecificity (Ziegler et al., 2005). 6OMT is the rate-
limiting gene, and 4OMT is a surrogate for 6OMT that facilitates
BIA biosynthesis. Constitutive overexpression of 6OMT led
to increased alkaloid content. In contrast, overexpression of
4OMT was shown to have little effect on alkaloid content
(Inui et al., 2007; Desgagné-Penix and Facchini, 2012). Four
homologs for each of 6OMT and 4OMT were found in
lotus, and were clustered with taxa that produce BIAs, such
as P. somniferum,T. flavum, and C. japonica (Figure 7A).
6OMT showed differential expression pattern with 4OMT
in “Luming Lian” and “10–48” (Figures 7B,C). 6OMT had
positive regulation on alkaloid content, while 4OMT inhibited
alkaloid accumulation, which confirmed their distinct effects on
alkaloid regulation. Furthermore, their expressions were prior
to the dynamic changes of aporphine alkaloid content. This
phenomenon of phenotypic lag may be due to that 6OMT
and 4OMT are not specific to aporphine alkaloid biosynthesis.
NMCH is encoded by CYP80B, a gene in P450 superfamily and is
highly region- and stereo-specific (Liscombe and Facchini, 2008;
Ziegler and Facchini, 2008; Chow and Sato, 2013; Glenn et al.,
2013). Overexpression of CYP80B resulted in an increase in the
amount of total alkaloid in latex of opium poppy, but without
changing the ratio of the individual morphine alkaloids (Pauli
and Kutchan, 1998; Frick et al., 2007). VIGS-based silencing of
CYP80B did not affect papaverine levels (Desgagné-Penix and
Facchini, 2012). The expression of CYP80B, NNU08355, has
a positive effect on aporphine alkaloids content in lotus, but
exhibits phenotypic lag (Figures 8,10), which might be because
of NMCH which acts early in the BIA biosynthetic pathway
(Frick et al., 2007).
In the aporphine alkaloid pathway, CTS, the first committed
enzyme, has intramolecular C–C phenol-coupling activity to
produce corytuberine from (S)-reticuline. It is encoded by
CYP80G, and was firstly cloned and characterized in Japanese
goldthread (Ikezawa et al., 2008). CYP80G was shown to be
similar in respect of high amino acid sequence to CYP80A,
a berbamunine synthase involved in the bisbenzylisoquinoline
alkaloid pathway (Ikezawa et al., 2008; Ziegler and Facchini,
2008). Two homologs of CYP80G found in our study exhibited
different expression patterns (Figures 8,10). Combining their
expression pattern with the dynamic change of alkaloid content,
we predicted that NNU21372 has a positive effect on aporphine
alkaloid content, but NNU21373 has no specific effect on
this variable. The gene RNMT is responsible for the yield
of magnoflorine from corytuberine, and was isolated in P.
somniferum. Suppression of RNMT resulted in a significant
decrease in magnoflorine accumulation, and a concomitant
increase in corytuberine levels (Morris and Facchini, 2016).
RNMT is a member of NMTs, and forms a larger clade with
CNMTs from T. flavum,C. japonica, and P. somniferum, which
is in agreement with our analysis (Figure 7A). However, its
homolog was not found in lotus.
Transcription factors, WRKY1 and bHLH1, regulated the BIA
metabolism. The expression of the two transcription factors
Frontiers in Plant Science | www.frontiersin.org 15 January 2017 | Volume 8 | Article 80
Yang et al. Alkaloid Transcriptomic Analysis in Lotus
caused a substantial increase in the expression of several
berberine biosynthetic genes (Kato et al., 2007; Yamada et al.,
2011). Transcription factors may be used to improve the yields
of BIAs. To test whether these two transcription factors regulate
the BIAs biosynthesis in lotus, we selected WRKY and bHLH
which differentially expressed at the four developmental stages in
the two cultivars (Supplementary Figure 3). 35 WRKY and 45
bHLH1 were identified. Some had similar expression tendency
as the dynamic change of alkaloid content. However, no one
was predicted using WGCNA. Further studies are needed to
test the function of WRKY1 and bHLH1 in regulating the BIAs
biosynthesis.
As stated above, we concluded that these homologous
genes, NCS (NNU14334), 6OMT (NNU03165, NNU03166,
and NNU23168), CNMT (NNU11880), NMCH (NNU08355),
4OMT (NNU15801 and NNU25948), and CTS (NNU21372),
are involved in the common pathway and aporphine alkaloid
pathway in lotus. They are thus candidate genes for alkaloid
biosynthesis.
Candidate Genes for the Late Steps of the
Aporphine Alkaloid Pathway
WGCNA can capture the relationships of individual genes
comprehensively, and is a powerful tool for obtaining new
insights into both the function of genes and the mechanism
controlling complex traits (Fuller et al., 2011; Wisniewski et al.,
2013). WGCNA was developed to analyze more efficiently
microarray datasets and transcriptomic profiling experiments.
This method has been used successfully to dissect fruit
anthocyanin in apple (Malus ×domestica) (El-Sharkawy et al.,
2015), the tomato fruit metabolome (DiLeo et al., 2011; Gao et al.,
2013), and pollination in petunias (Broderick et al., 2014). In this
study, WGCNA was used to predict genes that may be involved
in the aporphine alkaloid pathway of lotus.
Upon performing structural comparisons corytuberine and
magnoflorine with five prominent aporphine alkaloids in lotus,
we predicted that these aporphine alkaloids were produced
through the chemical reactions of oxidation, dehydroxylation,
methyl transfer, and demethylation (Figure 1). The enzymes
involved in the late steps might be oxidoreductases and
methyltransferases. CYP450 plays an important role in
synthesizing plant secondary metabolites and has catalytic
oxidation function for carbon–carbon bond, alkyl hydroxylation,
and hydroxyl oxidation (Coon, 2005). Oxidoreductases involved
in aporphine alkaloid biosynthesis are likely to come from
the CYP450 family. WGCNA identify 90 genes associated
with alkaloid content, and 32 genes were methyltransferases,
CYP450 genes, and oxidoreductases (Figures 9A,B and
Supplementary Table 4). In the biosyntheses of BIAs, CYP450-
mediated hydroxylation, methylenedioxy bridge formation,
and phenolcoupling reactions have been reported. CYP80A,
CYP80B, and CYP80G catalyze hydroxylation and C–O
phenol-coupling, and CYP719A catalyze methylenedioxy bridge
formation. A series of reaction of BIA pathways were catalyzed
by methyltransferases and oxidoreductases (Chow and Sato,
2013; Glenn et al., 2013; Hagel and Facchini, 2013). Among the
32 predicted genes, eight were 6OMT, 4OMT, and CYP80G, and
the remaining genes are likely to be involved in the late steps of
aporphine alkaloid biosynthesis (Figures 9C–E).
6OMT, 4OMT, and CYP80G are key enzymes in the common
pathway of BIA synthesis and the aporphine alkaloid branch, and
their expression affects the metabolic activities of downstream
factors. Therefore, the expression profiles of the candidate gene
in the late steps of aporphine alkaloid synthesis should match the
expression profiles of 6OMT, 4OMT, and CYP80G. Eventually,
11 candidate genes were identified for the late steps of the
aporphine alkaloid pathway. The expression of these candidate
genes was confirmed by RT-qPCR (Figure 10). Since these genes
had not been characterized previously, this study provides not
only new insights into the aporphine alkaloid pathway, but also a
list of interesting candidate genes for more dedicated functional
studies in the future. Functional analysis of these candidate genes
might be useful for genetic engineering to regulate aporphine
alkaloid content in lotus.
AUTHOR CONTRIBUTIONS
MY participated in the design of the study, analyzed the data, and
drafted the manuscript. LZ collected the leaves samples, extracted
RNA, and sequenced the RNA libraries. LL aligned the sequenced
data to the reference genome and performed gene expression
analysis. JL conducted Gene Ontology enrichment and KEGG
analysis. LX cultivated and provided the plant materials. JF
constructed gene co-expression network and predicted the
candidate genes. YL conceived the study, participated in its
design and coordination, and helped to draft the manuscript. All
authors read and approved the final manuscript.
ACKNOWLEDGMENTS
This research was financially supported by National Natural
Science Foundation of China (31272195 and 31471899).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.
00080/full#supplementary-material
Supplementary Figure 1 | Number of genes expressed at the four stages in
the two cultivars. (A) Venn diagram of genes identified in “10–48” (low-alkaloid
cv.) and “Luming Lian” (high-alkaloid cv.). (B) Venn diagram of genes in “10–48”
(low-alkaloid cv.) at four developmental stages. (C) Venn diagram of genes in
“Luming Lian” (high-alkaloid cv.) at four developmental stages. The individual and
overlapping areas in Venn diagrams represent the numbers of specifically
expressed and co-expressed genes between the different stages, respectively.
Supplementary Figure 2 | The expression of the genes encoding
scoulerine-9-O-methyltransferase (SOMT), codeine 3-O-demethylase
(CODM), and thebaine 6-O-demethylase (T6ODM) at four developmental
stages in the two cultivars. RPKM value for each gene used three biological
replicates; the error bars indicate standard error (n=3).
Supplementary Figure 3 | Heatmap of transcription factors, WRKY (A) and
bHLH1 (B) across the four developmental stages in the lotus cultivars “10–48”
and “Luming Lian.” Expression values are presented as RPKM normalized
log2transformed counts.
Frontiers in Plant Science | www.frontiersin.org 16 January 2017 | Volume 8 | Article 80
Yang et al. Alkaloid Transcriptomic Analysis in Lotus
Supplementary Table 1 | Primers used to assay gene expression by
RT-qPCR.
Supplementary Table 2 | Summary statistics of clean reads in the
transcriptomes of lotus.
Supplementary Table 3 | Pearson’s correlation coefficients for three
biological replicates at each stage in the two cultivars.
Supplementary Table 4 | Expression of the genes in the co-expression
network.
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Conflict of Interest Statement: The authors declare that the research was
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... IQAs are derived from the conversion of L-tyrosine to dopamine and 4-hydroxyphenylacetaldehyde and then the formation of various structures via intramolecular coupling, reduction, methylation, hydroxylation, and other reactions [6]. The biosynthetic pathways underlying several IQAs have been reported in some plants other than Stephania species, such as the biosynthesis of bisbenzylisoquinoline alkaloids in Berberis stolonifera and aporphine alkaloids in Nelumbo nucifera, and many enzymes related to IQA biosynthesis in these plants have been characterized [6,7]. ...
... IQAs are derived from the conversion of L-tyrosine to dopamine and 4-hydroxyphenylacetaldehyde and then the formation of various structures via intramolecular coupling, reduction, methylation, hydroxylation, and other reactions [6]. The biosynthetic pathways underlying several IQAs have been reported in some plants other than Stephania species, such as the biosynthesis of bisbenzylisoquinoline alkaloids in Berberis stolonifera and aporphine alkaloids in Nelumbo nucifera, and many enzymes related to IQA biosynthesis in these plants have been characterized [6,7]. ...
... Given the fact that plant secondary metabolites are often biosynthesized in a tissue-specific manner [8], transcriptomic comparisons between plant tissues combined with correlation analyses of chemical components represent an efficient approach to discover the key genes involved in secondary metabolism [9][10][11]. This method has already been successfully used to uncover candidate genes related to the biosynthesis of chemical constituents in some plant species [6,[12][13][14]. ...
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The roots of Stephania tetrandra are used as a traditional Chinese medicine. Isoquinoline alkaloids are considered to be the most important and effective components in this herb, but little is known about the molecular mechanism underlying their biosynthesis. In this context, this study aimed to reveal candidate genes related to isoquinoline alkaloid biosynthesis in S. tetrandra. Determination of tetrandrine and fangchinoline in the roots and leaves of S. tetrandra by HPLC showed that the roots had much higher contents of the two isoquinoline alkaloids than the leaves. Thus, a comparative transcriptome analysis of the two tissues was performed to uncover candidate genes involved in isoquinoline alkaloid biosynthesis. A total of 71 674 unigenes was obtained and 31 994 of these were assigned putative functions based on BLAST searches against 6 annotation databases. Among the 79 isoquinoline alkaloid-related unigenes, 51 were differentially expressed, with 42 and 9 genes upregulated and downregulated, respectively, when the roots were compared with the leaves. The upregulated differentially expressed genes were consistent with isoquinoline alkaloid accumulation in roots and thus were deemed key candidate genes for isoquinoline alkaloid biosynthesis in the roots. Moreover, the expression profiles of 10 isoquinoline alkaloid-related differentially expressed genes between roots and leaves were validated by quantitative real-time polymerase chain reaction, which indicated that our transcriptome and gene expression profiles were reliable. This study not only provides a valuable genomic resource for S. tetrandra but also proposes candidate genes involved in isoquinoline alkaloid biosynthesis and transcription factors related to the regulation of isoquinoline alkaloid biosynthesis. The results lay a foundation for further studies on isoquinoline alkaloid biosynthesis in this medicinal plant.
... The RNA-Seq has become a popular tool for uncovering the underlying molecular mechanisms of biological processes, including development, stress response, and metabolism processes in recent years (Fracasso et al., 2016;Goyal et al., 2016;Yang et al., 2017;Lanver et al., 2018;Xia et al., 2020;Sun et al., 2021). For example, the molecular mechanism of alkaloids biosynthesis has been clarified in numerous plants using RNA-seq (Guo et al., 2013;Cui et al., 2015;He et al., 2017;Deng et al., 2018). ...
... Most parts of the lotus plant have traditionally been used for various medicinal purposes due to its ability to accumulate abundant bioactive compounds, such as alkaloids and flavonoids (Mukherjee et al., 2009;Chen et al., 2012;Deng et al., 2016;Limwachiranon et al., 2018). To date, over 20 alkaloids categorized into aporphines, monobenzylisoquinolines, and bisbenzylisoquinolines have been identified in lotus (Deng et al., 2016;Yang et al., 2017). In this study, bis-BIAs, such as liensinine, isoliensinine, and neferine, were identified as the predominant alkaloids in lotus plumule, which is consistent with the results of previous studies (Deng et al., 2016;Menendez-Perdomo and Facchini, 2020). ...
... The biosynthesis pathway of aporphine-type BIAs in lotus leaf has previously been reported (Yang et al., 2017;Deng et al., 2018). However, the bis-BIAs biosynthesis pathway in plumule is yet to be characterized. ...
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Lotus plumule is a green tissue in the middle of seeds that predominantly accumulates bisbenzylisoquinoline alkaloids (bis-BIAs) and chlorophyll (Chl). However, the biosynthetic mechanisms of these two metabolites remain largely unknown in lotus. This study used physiological and RNA sequencing (RNA-Seq) approaches to characterize the development and molecular mechanisms of bis-BIAs and Chl biosynthesis in lotus plumule. Physiological analysis revealed that exponential plumule growth occurred between 9 and 15 days after pollination (DAP), which coincided with the onset of bis-BIAs biosynthesis and its subsequent rapid accumulation. Transcriptome analysis of lotus plumule identified a total of 8,725 differentially expressed genes (DEGs), representing ~27.7% of all transcripts in the lotus genome. Sixteen structural DEGs, potentially associated with bis-BIAs biosynthesis, were identified. Of these, 12 encoded O-methyltransferases (OMTs) are likely involved in the methylation and bis-BIAs diversity in lotus. In addition, functionally divergent paralogous and redundant homologous gene members of the BIAs biosynthesis pathway, as well as transcription factors co-expressed with bis-BIAs and Chl biosynthesis genes, were identified. Twenty-two genes encoding 16 conserved enzymes of the Chl biosynthesis pathway were identified, with the majority being significantly upregulated by Chl biosynthesis. Photosynthesis and Chl biosynthesis pathways were simultaneously activated during lotus plumule development. Moreover, our results showed that light-driven Pchlide reduction is essential for Chl biosynthesis in the lotus plumule. These results will be useful for enhancing our understanding of alkaloids and Chl biosynthesis in plants.
... Sacred lotus accumulates 1benzylisoquinolines, and derived aporphines (via C8-C2' intramolecular coupling) and bisbenzylisoquinolines (via C8-C3'/5' and C7-O-C3'/5' intermolecular coupling) (5), most of which are O-methylated at C6, C7, and/or C4' (13). The availability of a sacred lotus draft genome (30,31) greatly facilitates gene mining for candidate OMTs based on amino acid sequence similarity with respect to characterized O-methyltransferases (32)(33)(34). However, OMT candidates have so far been investigated only in terms of gene expression, with no functional characterization of the encoded proteins. ...
... In this regard, whereas reticuline constitutes a key branch-point intermediate in the biosynthesis of most BIAs in opium poppy (3), sacred lotus does not accumulate reticuline and other 1benzylisoquinolines containing a C3' functional group, or reticuline-derived alkaloids including phthalideisoquinolines, benzo[c]phenanthridines, protoberberines and morphinans, which occur in members of the Ranunculales (13). In contrast, aporphines and bisbenzylisoquinolines are the major BIAs in sacred lotus, although alkaloid profiles vary among nearly 600 known varieties, and across different plant organs and developmental stages (32)(33)(34)(35)(36)(37). In addition, sacred lotus aporphine alkaloids do not display substitutions in the benzyl moiety (13), in contrast with the reticuline-derived aporphines (e.g. ...
... Alkaloids containing 6-O-, 7-O-and/or 4'-Omethylations were detected in Pink and White varieties, supporting the search for specific Omethyltransferase activities in the plant. OMT gene candidates have been predicted in sacred lotus based on sequence similarity to functionally characterized OMTs from BIA-accumulating species (32)(33)(34), but the purported catalytic activities has not been assessed. We show that two of these candidates, NnOMT1 and NnOMT5, catalyze the O-methylation of 1benzylisoquinolines. ...
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Benzylisoquinoline alkaloids (BIAs) are a major class of plant metabolites with many pharmacological benefits. Sacred lotus (Nelumbo nucifera) is an ancient aquatic plant of medicinal value owing to antiviral and immunomodulatory activities linked to its constituent BIAs. Although more than 30 BIAs belonging to the 1-benzylisoquinoline, aporphine, and bisbenzylisoquinoline structural subclasses and displaying a predominant R enantiomeric conformation have been isolated from N. nucifera, its BIA biosynthetic genes and enzymes remain unknown. Herein, we report the isolation and biochemical characterization of two O-methyltransferases (OMTs) involved in BIA biosynthesis in sacred lotus. Five homologous genes, designated NnOMT1-5 and encoding polypeptides sharing > 40% amino acid sequence identity, were expressed in Escherichia coli Functional characterization of the purified recombinant proteins revealed that NnOMT1 is a regiospecific 1-benzylisoquinoline 6-O-methyltransferase (6OMT) accepting both R and S substrates, whereas NnOMT5 is mainly a 7-O-methyltransferase (7OMT), with relatively minor 6OMT activity and a strong stereospecific preference for S enantiomers. Available aporphines were not accepted as substrates by either enzyme, suggesting that O-methylation precedes BIA formation from 1-benzylisoquinoline intermediates. Km values for NnOMT1 and NnOMT5 were 20 μM and 13 μM for (R,S)-norcoclaurine and (S)-N-methylcoclaurine, respectively, similar to those for OMTs from other BIA-producing plants. Organ-based correlations of alkaloid content, OMT activity in crude extracts, and OMT gene expression supported physiological roles for NnOMT1 and NnOMT5 in BIA metabolism, occurring primarily in young leaves and embryos of sacred lotus. In summary, our work identifies two OMTs involved in BIA metabolism in the medicinal plant N. nucifera.
... The obtained DEGs were analyzed by weighted genes correlation network analysis (WGCNA) [14]. The steps of WGCNA mainly include gene co-expression similarity matrix calculation, adjacency function calculation, soft threshold selection, topological overlap matrix, heterogeneity matrix calculation, dynamic branch cutting calculation of gene module, and correlation analysis between gene modules and sample clinical information. ...
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Breast cancer is one of the most common malignant tumors in women worldwide. Early diagnosis, treatment, and prognosis of breast cancer are global challenges. Identification of valid predictive diagnosis and prognosis biomarkers and drug targets are crucial for breast cancer prevention. This study characterizes differentially expressed genes (DEGs) based on the TCGA database by using DESeq2, edgeR, and limma. A total of 2032 DEGs, including 1026 up-regulated genes and 1006 down-regulated genes were screened. Followed with WGCNA, PPI analysis, GEPIA 2, and HPA database verification, thirteen hub genes including CDK1, BUB1, BUB1B, CDC20, CCNB2, CCNB1, KIF2C, NDC80, CDCA8, CENPF, BIRC5, AURKB, PLK1, MAD2L1, and CENPE were obtained, and they may serve as potential therapeutic targets of breast cancer. Especially, overexpression of CCNB1 and PLK1 are strongly associated with the low survival rate of breast cancer patients, demonstrating their potentiality as prognostic markers. Moreover, CCNB1 and PLK1 are highly expressed in all breast cancer stages, suggesting that they could be further studied as potential drug targets. Taken together, our study highlights CCNB1 and PLK1 as potential anti-breast cancer drug targets and prognostic markers.
... ± 2.83 µg/g) in stage II, whereas N-nornuciferine content was the highest (4555.33 ± 26.40 µg/g) in stage III, and nuciferine was the highest (2534.67 ± 20.27 µg/g) in stage IV (Table S8, Supplementary Materials). Interestingly, the distributions and trends of alkaloids in lotus seed epicarp are consistent with those in lotus leaves, indicating that lotus seed epicarp may have comparable healthcare function to that of lotus leaves [47][48][49]. Figure 10. Contents of ten alkaloids in lotus seed epicarp at different growth stages. ...
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Lotus seed epicarp, a byproduct of lotus, is commonly discarded directly or burned in the cropland, resulting in waste of resources and environmental pollution. In this work, a green ultrasonic-assisted extraction method with ethyl lactate as the extraction solvent was established to extract alkaloids from lotus seed epicarp. The extraction conditions were optimized by response surface methodology. Under the optimal extraction conditions, the extraction of alkaloids from 1 g lotus seed epicarp was accomplished with only 10 mL of extraction solvent within 15 min. Combined with ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry with information-dependent acquisition mode, a total of 42 alkaloids were annotated in the lotus seed epicarp extracts. Among them, 39 alkaloids were reported in lotus seed epicarp for the first time. According to quantitative analysis, the distributions and trends of alkaloids in the lotus seed epicarp were found to be similar to those of lotus leaves. The five growth stages of lotus seed epicarp could be successfully distinguished based on the ten representative alkaloids. This study demonstrates that ultrasonic-assisted extraction with ethyl lactate as extractant solvent was efficient in the extraction of alkaloids from lotus seed epicarp, which is a potential renewable resource of bioactive ingredients.
... Till date, around 20 bisbenzylisoquinoline alkaloids have been identified from N. nucifera, each with its distinct chemical structure (Yang et al., 2017). Synthesis pathway of all 20 bisbenzylisoquinoline alkaloid compounds shares a common line up to a certain stage. ...
Article
Full-text available
Phytochemicals have recently received a lot of recognition for their pharmacological activities such as anticancer, chemopreventive, and cardioprotective properties. In traditional Indian and Chinese medicine, parts of lotus (Nelumbo nucifera) such as lotus seeds, fruits, stamens, and leaves are used for treating various diseases. Neferine is a bisbenzylisoquinoline alkaloid, a major component from the seed embryos of N. nucifera. Neferine is effective in the treatment of high fevers and hyposomnia, as well as arrhythmia, platelet aggregation, occlusion, and obesity. Neferine has been found to have a variety of therapeutic effects such as anti-inflammatory, anti-oxidant, anti-hypertensive, anti-arrhythmic, anti-platelet, anti-thrombotic, anti-amnesic, and negative inotropic. Neferine also exhibited anti-anxiety effects, anti-cancerous, and chemosensitize to other anticancer drugs like doxorubicin, cisplatin, and taxol. Induction of apoptosis, autophagy, and cell cycle arrest are the key pathways that underlying the anticancer activity of neferine. Therefore, the present review summarizes the neferine biosynthesis, pharmacokinetics, and its effects in myocardium, cancer, chemosensitizing to cancer drug, central nervous system, diabetes, inflammation, and kidney diseases. Practical applications Natural phytochemical is gaining medicinal importance for a variety of diseases like including cancer, neurodegenerative disorder, diabetes, and inflammation. Alkaloids and flavonoids, which are abundantly present in Nelumbo nucifera have many therapeutic applications. Neferine, a bisbenzylisoquinoline alkaloid from N. nucifera has many pharmacological properties. This present review was an attempt to compile an updated pharmacological action of neferine in different disease models in vitro and in vivo, as well as to summarize all the collective evidence on the therapeutic potential of neferine.
... Among these BIAs, most can be classified as protoberberines and aporphines according to their chemical structures 14 , but protopine-type and benzo[c]phenanthridine-type BIAs are also present in C. yanhusuo bulb extracts. Currently, thanks to the persistent efforts of biochemists and phytochemists, the labyrinthine BIA biosynthetic pathways have been well elucidated in the model species Papaver somniferum (opium poppy) 17,18 , but the clarification of the aporphine-type BIA biosynthetic pathway will require substantial additional efforts 19 . Additionally, a few amino acid sequences of key enzymes in protoberberine BIA pathways are still missing, which greatly impedes the molecular cloning and metabolic engineering of protoberberines. ...
Article
Full-text available
Corydalis yanhusuo W.T. Wang is a classic herb that is frequently used in traditional Chinese medicine and is efficacious in promoting blood circulation, enhancing energy, and relieving pain. Benzylisoquinoline alkaloids (BIAs) are the main bioactive ingredients in Corydalis yanhusuo. However, few studies have investigated the BIA biosynthetic pathway in C. yanhusuo, and the biosynthetic pathway of species-specific chemicals such as tetrahydropalmatine remains unclear. We performed full-length transcriptomic and metabolomic analyses to identify candidate genes that might be involved in BIA biosynthesis and identified a total of 101 full-length transcripts and 19 metabolites involved in the BIA biosynthetic pathway. Moreover, the contents of 19 representative BIAs in C. yanhusuo were quantified by classical targeted metabolomic approaches. Their accumulation in the tuber was consistent with the expression patterns of identified BIA biosynthetic genes in tubers and leaves, which reinforces the validity and reliability of the analyses. Full-length genes with similar expression or enrichment patterns were identified, and a complete BIA biosynthesis pathway in C. yanhusuo was constructed according to these findings. Phylogenetic analysis revealed a total of ten enzymes that may possess columbamine-O-methyltransferase activity, which is the final step for tetrahydropalmatine synthesis. Our results span the whole BIA biosynthetic pathway in C. yanhusuo. Our full-length transcriptomic data will enable further molecular cloning of enzymes and activity validation studies.
... WGCNA provides novel insights into the gene function and control mechanisms of complex traits by comprehensively capturing the relationship among several individual genes ( Garg et al., 2017). Determining the transcription modules can also help identify and control GRNs in the biosynthesis of secondary metabolites ( Yang et al., 2017). The overall analysis of different tissues in Arabidopsis could elucidate the network of genes that regulate flavonoid metabolism ( Saito et al., 2013). ...
Article
Different parts of lotus (Nelumbo nucifera Gaertn.) including the seeds, rhizomes, leaves, and flowers, are used for medicinal purposes with health promoting and illness preventing benefits. The presence of active chemicals such as alkaloids, phenolic acids, flavonoids, and terpenoids (particularly alkaloids) may account for this plant’s pharmacological effects. In this review, we provide a comprehensive overview and summarize up-to-date research on the biosynthesis, pharmacokinetics, and bioactivity of lotus alkaloids as well as their safety. Moreover, the potential uses of lotus alkaloids in the food, pharmaceutical, and cosmetic sectors are explored. Current evidence shows that alkaloids, mainly consisting of aporphines, 1-benzylisoquinolines, and bisbenzylisoquinolines, are present in different parts of lotus. The bioavailability of these alkaloids is relatively low in vivo but can be enhanced by technological modification using nanoliposomes, liposomes, microcapsules, and emulsions. Available data highlights their therapeutic and preventive effects on obesity, diabetes, neurodegeneration, cancer, cardiovascular disease, etc. Additionally, industrial applications of lotus alkaloids include their use as food, medical, and cosmetic ingredients in tea, other beverages, and healthcare products; as lipid-lowering, anticancer, and antipsychotic drugs; and in facial masks, toothpastes, and shower gels. However, their clinical efficacy and safety remains unclear; hence, larger and longer human trials are needed to achieve their safe and effective use with minimal side effects.