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Article J. Braz. Chem. Soc., Vol. 00, No. 00, 1-10, 2018.
Printed in Brazil - ©2018 Sociedade Brasileira de Química
http://dx.doi.org/10.21577/0103-5053.20170238
*e-mail: andrluca@yahoo.com.br; maysaf@iq.unesp.br
Biosynthetic Insights into p-Hydroxybenzoic Acid-Derived Benzopyrans in
Piper gaudichaudianum
Andrea N. L. Batista,*,a João M. Batista Jr.,b Tatiana M. Souza‑Moreira,c Sandro R. Valentini,c
Massuo J. Kato,d Cleslei F. Zanellic and Maysa Furlan*,a
aInstituto de Química, Universidade Estadual Paulista (UNESP), 14800-060 Araraquara-SP, Brazil
bDepartamento de Química, Universidade Federal de São Carlos (UFSCar), 13565-905 São Carlos-SP, Brazil
cFaculdade de Ciências Farmacêuticas, Universidade Estadual Paulista (UNESP),
14801-902 Araraquara-SP, Brazil
dInstituto de Química, Universidade de São Paulo (USP), 05508-000 São Paulo-SP, Brazil
Piper gaudichaudianum Kunth (Piperaceae) accumulates gaudichaudianic acid, a prenylated
benzopyran, as its major component. Interestingly, this trypanocidal compound occurs as a
racemic mixture. Herein, transcriptomic investigations of Piper gaudichaudianum using the RNA-
seq approach are reported, and from the analysis of the transcripts expressed it was possible to
propose a complete biosynthetic pathway for the production of gaudichaudianic acid, including
the steps that originate its precursor, p-hydroxybenzoic acid. Peperomia obtusifolia (L.) A. Dietr.
(Piperaceae) also accumulates racemic benzopyrans, however, its chromanes originate from the
polyketide pathway, while the chromenes from Piper derives from the shikimate pathway. Recent
transcriptomic and proteomic studies of the former species did not identify polyketide synthases
involved in the production of the benzopyran moiety, but revealed the expression of tocopherol
cyclase, which may be responsible for the cyclization of the 3,4-dihydro-2H-pyran ring. The
analysis of the enzymes involved in the secondary metabolism of Piper gaudichaudianum and the
comparison with the data previously obtained from Peperomia obtusifolia can provide valuable
information on how these compounds are biosynthesized.
Keywords: Piperaceae, chromene, transcriptome, RNA-seq, de novo assembly, biosynthesis
Introduction
Chromanes and chromenes, which are secondary
metabolites commonly found in some species from
Piper and Peperomia genera (Piperaceae), are characterized
by the presence of a benzopyran ring with various levels
of oxidation.1-3 This nucleus is considered a privileged
structure that is very common in bioactive natural products,
such as coumarins, flavones, tocopherols (vitamin E) and
tetrahydrocannabinoids.4,5
The benzopyran moieties, 2H-chromenes and
chromanes, isolated from Piper gaudichaudianum and
Peperomia obtusifolia, respectively, have been demonstrated
as potent trypanocidal compounds.6,7 Curiously, both
classes of compounds occur as racemic mixtures in these
species, though their formation follows two distinct
biosynthetic routes.6,8,9 In Piper gaudichaudianum,
chromenes originate from the shikimate pathway and
use p-hydroxybenzoic acid (p-HBA) as a precursor.10 In
the case of Peperomia obtusifolia, chromanes are formed
through the polyketide pathway and use orsellinic acid as
a precursor.1,11 Additionally, the formation of both classes
of metabolites involves the condensation of an aromatic
unit (p-HBA or orsellinic acid) and an isoprene unit
(dimethylallyl pyrophosphate, isopentenyl pyrophosphate
or geranyl pyrophosphate), followed by cyclization that
gives rise to the benzopyran ring. During cyclization, the
carbon atom at C-2 becomes a stereogenic center and,
different from other benzopyrans such as vitamin E, both
enantiomers are biosynthesized (Figure 1). Thus, the study
of the proteins and genes possibly responsible for the
biosynthesis of benzopyrans in these species, as well as
the comparison between them, may provide new insights
into how these compounds are produced.
Recent studies in Peperomia obtusifolia using a
combination of shotgun proteomics and transcriptome
analysis did not identify an orsellinic acid synthase.
However, the transcriptome analysis revealed the
Biosynthetic Insights into p-Hydroxybenzoic Acid-Derived Benzopyrans in Piper gaudichaudianum J. Braz. Chem. Soc.
2
expression of tocopherol cyclase that may be responsible
for the cyclization of the prenylated orsellinic acid precursor
that yields the 3,4-dihydro-2H-pyran ring. Because
orsellinic acid is commonly found in sordariomycetes
and eurotiomycetes, these results suggest that orsellinic
acid-derived benzopyrans may be formed by the
combination of biosynthetic efforts from the host plant
and endophytic fungi.12
Some studies have already been performed in
Piper gaudichaudianum aimed at elucidating the
biosynthesis of gaudichaudianic acid (1) (Figure 2),10
which is the major prenylated chromene isolated from this
species and described as a potent trypanocidal compound
against the Y-strain of Trypanosoma cruzi.13 Interestingly,
trypanocidal assays indicated that the (+)-enantiomer was
more active than its antipode and that the enantiomer
mixtures showed a synergistic effect, with the racemic
mixture being the most active.6 Regarding its biosynthesis,
the origin of the terpene moieties was shown to involve
both the mevalonate and methylerythritol phosphate
pathways.10
Thus, to complement the data in the literature and
elucidate the complete biosynthesis of chromenes from
Piper gaudichaudianum, a transcriptome study was
performed on leaves from this plant using the RNA-seq
approach.
Results and Discussion
Transcriptome sequencing, de novo assembly and
annotation
The racemic prenylated chromene gaudichaudianic
acid (1) is the major constituent in all organs in
Piper gaudichaudianum adult plants, though it is found
exclusively in the roots of seedlings.6 The occurrence
of this compound has been reported in leaves over
12 months of age, and it becomes the major compound
by the 15th month of growth.14 Thus, to obtain the
Piper gaudichaudianum transcriptome, three RNA-seq
libraries were constructed from a pool of leaves from
young plants (older than 15 months) and sequenced using
the HiSeq 2500 Illumina paired-end sequencing system.
RNA-seq is an approach to transcriptome profiling that uses
deep-sequencing technologies in which RNA is sequenced
via a high-throughput manner enabling robust assessments
of eukaryotic transcriptomes.15,16 Furthermore, paired-end
sequencing produces twice the number of reads at the
same time, and sequences aligned as read pairs enable
more accurate read alignment.17 This approach has been
particularly useful in non-model species.16
In this study, an average of 34,846,653 raw reads
with a length of 100 bp each and a percentage of bases
with Q (Phred quality score) ≥ 30 over 87.5% were
generated from the three samples submitted to Illumina
sequencing. These values are adequate to guarantee
high-quality data. When the sequencing quality reaches
Q30, virtually all the reads will be perfect and have zero
errors and ambiguities. After removing the adaptors and
low-quality reads, 30,449,535 clean reads were retained,
which reflected a loss of ca. 12.6%. These clean data were
de novo assembled into a total of 216,786 transcripts with
Figure 1. The proposed biosynthetic pathway of benzopyrans from Peperomia obtusifolia and Piper gaudichaudianum.
Figure 2. Chemical structure of the gaudichaudianic acid.
Batista et al. 3Vol. 00, No. 00, 2018
average length of 770 bases and an N50 length (weighted
metric that represents the minimum assembly size in which
≥ 50% of the assembled bases are found)16 of 1,362 bases
using the Trinity software,18 which generates transcript
contigs based on overlapping information. The transcript
size distribution revealed that there were 165,192 contigs
(76.2%) ranging from 100 to 1,000 bases, 32,880 contigs
(15.2%) ranging from 1,001 to 2,000 bases, and 18,701
contigs (8.6%) longer than 2,000 bases (Figure S1,
Supplementary Information). The details of the transcript
assembly are summarized in Table 1. The present results
are in accordance with those recently published for
transcriptomes for the Piperaceae species Piper nigrum
and Peperomia obtusifolia, which were assembled using
Illumina HiSeq 2000 and 2500 platforms, respectively.12,19
Sequence homology searches were conducted for the
transcript functional annotation. Therefore, all transcripts
were submitted to BLASTX20 searches against the NCBI
non-redundant (Nr) protein database, and 148,113 (68%)
transcripts had significant similarity with an E (expect) value
less than 1e−5 (Table 2). The sequences were also searched
against a custom plant protein database, resulting in 113,374
(52%) annotated transcripts (E values lower than 1e−5) (Table
2). Even after these two different searches, a significant
number of transcripts (68,401, 32%) remained unannotated,
which can be a great resource for the discovery of novel
genes. This is an expected situation given the absence of
genomic information for Piper gaudichaudianum.
To classify the functions of the predicted transcripts,
functional annotations were also created by using the
Blast2GO21 software with the Gene Ontology (GO)
database, which is an internationally standardized
gene functional classification system. A total of 65,579
transcripts were assigned at least one GO term distributed
among the three main ontologies comprising biological
process, molecular function, and cellular component. The
biological process ontology distribution (level 2 of detail)
contained mainly proteins involved in metabolic and
cellular processes (Figure 3). In the same level of detail,
catalytic activity and binding in the molecular function
Table 1. Overview of the sequencing and de novo assembly of
Piper gaudichaudianum transcriptome
Description Piper gaudichaudianum (leaves)
Total raw readsa34,846,653
Total clean readsa30,449,535
Bases ≥ Q30a / % 87.5
Number of trinity transcripts 216,786
Number of trinity ‘genes’ 148,419
GC / % 46.04
N50 value 1,362
Median contig length 407
Average contig length 771
Total assembled bases 167,094,492
aAverage of the triplicate. Q30: Phred quality score of 30; GC: guanine-
cytosine content; N50: minimum contig length needed to cover 50% of
assembled bases.
Table 2. Summary of sequence annotation for Piper gaudichaudianum
Database Hit number (percentage)
NCBI Nr 148,113 (68%)
Custom plant 113,374 (52%)
Figure 3. Distribution of GO classifications (level 2). Annotated transcripts were classified into 3 major categories (biological processes (BP), cellular
components (CC), and molecular function (MF)).
Biosynthetic Insights into p-Hydroxybenzoic Acid-Derived Benzopyrans in Piper gaudichaudianum J. Braz. Chem. Soc.
4
represented the major subcategories (Figure 3). For cellular
components, the assignments were mostly given to cell
and cell parts (Figure 3). This pattern of GO annotation
distribution was similar to those of other species and is
typically seen in the transcriptome of samples undergoing
development processes.19,22
KEGG pathway mapping assignment
The annotated transcripts were also analyzed by searching
them against the KEGG (Kyoto Encyclopedia of Genes and
Genomes) database for KEGG (K) number assignments
and subsequent reconstruction of the biosynthetic pathways
active in Piper gaudichaudianum leaves. KEGG is an
integrated database resource for the biological interpretation
of genome sequences and other high-throughput data.23 As
a result, 35,192 transcripts were identified with a K number
corresponding to a total of 3,594 distinct genes. Among them,
961 (26.7%) are involved in general metabolic pathways
that include energy metabolism, carbohydrate and lipid
metabolism, nucleotide and amino acid metabolism, and
secondary metabolism subcategories.
Within the secondary metabolism subcategory,
423 genes were identified. However, only the transcripts
with an FPKM (fragments per kilobase of transcript
per million fragments mapped) ≥ 1 were considered,
resulting in 393 genes encoding enzymes involved
in some manner in secondary metabolism (Table S1,
Supplementary Information). This result allowed for
the identification of the active secondary metabolism
pathways in Piper gaudichaudianum; terpenoid and
steroid biosynthesis (61 genes) represented the largest
group, followed by phenylpropanoid biosynthesis
(18 genes), flavonoid biosynthesis (12 genes), and
isoquinoline alkaloid biosynthesis (7 genes). The
terpenoid and steroid group includes terpenoid backbone,
monoterpenoid, sesquiterpenoid, diterpenoid, triterpenoid,
steroid, and carotenoid biosynthesis. These results are
in accordance with the data in the literature because
terpenoids, mainly those present in the essential oil, and
steroids have already been described in this species.24-26
Although there are reports of phenylpropanoids only in
the leaves of seedlings,14,27 the transcriptome indicates
their occurrence in the young plant. These data may
suggest that these compounds are produced and then
immediately used as precursors of lignins, since the
complete biosynthesis pathways of p-hydroxyphenyl,
guaiacyl, 5-hydroxyguaiacyl, and syringyl lignins have
been identified (Figure S2, Supplementary Information).
These phenylpropanoids may also act as precursors for
the chromenes, flavokawains and flavonoids reported in
this species.26,28,29 With respect to isoquinolinic alkaloids,
the genes identified are involved in the early steps
of their biosynthesis involving dopamine production
(Figure S3, Supplementary Information). Although there
are no reports of the occurrence of these compounds in
Piper gaudichaudianum, two aporphinoid alkaloids,
cepharadione A and piperolactam E, were isolated from
Piper caninum and Piper taiwanense, respectively.30
Candidate genes involved in benzopyran biosynthesis
In Piper gaudichaudianum, chromenes originate from
the precursor p-HBA via the shikimate pathway. Despite the
simple structure of p-HBA and its widespread distribution
in plants, the enzymatic steps for its biosynthesis are not
clearly understood.31 Hydroxybenzoates have been reported
to originate directly from shikimate via chorismic acid or
from phenylalanine (Figure 4).31,32
In the former case, chorismate-pyruvate lyase (CPL)
converts chorismic acid into p-HBA.32 Although some reports
mention that this enzyme is restricted to bacterial lineages,33,34
a chorismate pyruvate lyase-catalyzed reaction, similar
to that observed in Escherichia coli, seems to also occur
in eukaryotic microorganisms.32,35 However, as expected,
CPL was not identified in the Piper gaudichaudianum leaf
transcriptome, suggesting that p-HBA is not biosynthesized
by this pathway. This result is in agreement with literature
data on microorganisms being the only known organisms to
transform chorismic acid into p-HBA.
In the latter case, the biosynthesis of p-HBA from
phenylalanine may follow two different routes, either
by means of a β-oxidative or non-β-oxidative pathway.36
The first route proceeds via p-coumaroyl-CoA and
p-hydroxybenzoyl-CoA for the formation of p-HBA
from p-coumaric acid. This pathway involves the
activation of p-coumaric acid by the action of the
enzyme 4-coumarate-CoA ligase (4CL), leading to
its thioester with subsequent chain-shortening into
p-hydroxybenzoyl-CoA in a reaction mechanism
analogous to that of NAD-dependent β-oxidation of fatty
acids.37,38 The operation of this pathway is supported by
in vitro enzymatic activity assays using cell-free extracts
from Lithospermum erythrorhizon cell cultures.37 However,
this conversion requires steps not fully elucidated yet, i.e.,
those corresponding to the hydration, dehydrogenation,
and thiolation of the β-oxidative cycle followed by the
final hydrolysis of the 4-hydroxybenzoyl-CoA thioester.33
A Petunia gene encoding the bifunctional peroxisomal
enzyme cinnamoyl-CoA hydratase-dehydrogenase
(CHD), which is responsible for the initial two-step
conversion of cinnamoyl-CoA into benzoic acid, was
Batista et al. 5Vol. 00, No. 00, 2018
identified by a functional genomics approach and has been
shown to be active with p-coumaroyl-CoA (Figure S4,
Supplementary Information).39 The next steps are expected
to be catalyzed by thiolases and CoA thioesterases. A
3-ketoacyl-CoA thiolase (PhKAT1) was confirmed to
be involved in the production of benzoyl-CoA from
cinnamoyl-CoA in Petunia hybrid.36 The specific
4-hydroxybenzoyl-CoA thioesterase (4HBT), which is
part of the bacterial 2,4-dichlorobenzoate degradation
pathways, appears to occur only in microorganisms.
However, CoA thioesterases members of the 4HBT
family (1,4-dihydroxy-2-naphthoyl-CoA thioesterase 1
and 2) capable of hydrolyzing aromatic acyl-CoA
substrates, including benzoyl-CoA, have been identified
in Arabidopsis.40 The second route involves a non-
oxidative pathway for the conversion of p-coumaric acid
or p-coumaroyl-CoA to p-hydroxybenzaldehyde in a
retro-aldol reaction with no co-factor requirement.37,38,41
A 4-hydroxybenzaldehyde synthase (HBS) and
4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL)
catalyzes the penultimate step of p-HBA biosynthesis by
performing the phenylpropanoid side-chain cleavage of
p-coumaric acid and p-coumaroyl-CoA, respectively.31,41
Then, the biosynthesis of p-HBA proceeds via
p-hydroxybenzaldehyde in a reaction catalyzed by the
enzyme 4-hydroxybenzaldehyde dehydrogenase.31 Studies
with Daucus carota and Vanilla planifolia support the
operation of this pathway.31,38
Analysis of the transcriptome of Piper gaudichaudianum
leaves against the NCBI non-redundant and custom
protein databases allowed for the identification of all the
enzymes involved in the biosynthesis of p-coumaric acid
and p-coumaroyl-CoA, starting from photosynthesis,
glycolysis, and pentose phosphate pathways (Table 3).
However, the next steps in the formation of p-HBA have not
been clearly determined. Many transcripts appear to encode
3-ketoacyl-CoA thiolases, and one transcript presented
homology with the gene encoding 4-hydroxybenzoyl-CoA
thioesterase from Sphingomonas sp., but the FPKM for
this transcript was < 1. Furthermore, the enzymes from
the non-β-oxidative pathway were not identified. Although
these data suggest that p-HBA biosynthesis proceeds via
the β-oxidative pathway, it is not possible to ensure which
route is actually operating in Piper gaudichaudianum.
Thus, to confirm the operant pathway in this species,
the transcripts were re-analyzed by comparing them with
a second custom database containing the sequences of
enzymes from the different pathways involved in benzoate
biosynthesis (from plants, fungi, and bacteria). By using
this more specific approach, 774 transcripts were annotated
(E value ≤ 1e−5). Of these transcripts, 34 showed significant
homology (E value ≤ 1e−50) and similarity over 80%
Figure 4. Schematic representation of the possible pathways for the biosynthesis of the p-hydroxybenzoic acid.
Biosynthetic Insights into p-Hydroxybenzoic Acid-Derived Benzopyrans in Piper gaudichaudianum J. Braz. Chem. Soc.
6
Table 3. Enzymes involved in gaudichaudianic acid biosynthesis based on the transcriptome data of Piper gaudichaudianum
Name K number EC number Pathway
E1 3-deoxy-7-phosphoheptulonate synthase K01626 EC:2.5.1.54 phenylalanine biosynthesis
E2 3-dehydroquinate synthase K01735 EC:4.2.3.4 phenylalanine biosynthesis
E3 3-dehydroquinate dehydratase / shikimate dehydrogenase K13832 EC:4.2.1.10 1.1.1.25 phenylalanine biosynthesis
E4 shikimate kinase K00891 EC:2.7.1.71 phenylalanine biosynthesis
E5 3-phosphoshikimate 1-carboxyvinyltransferase K00800 EC:2.5.1.19 phenylalanine biosynthesis
E6 chorismate synthase K01736 EC:4.2.3.5 phenylalanine biosynthesis
E7 chorismate mutase K01850 EC:5.4.99.5 phenylalanine biosynthesis
E8 arogenate/prephenate dehydratase K05359 EC:4.2.1.91 4.2.1.51 phenylalanine biosynthesis
E9 bifunctional aspartate aminotransferase and glutamate/aspartate-
prephenate aminotransferase
K15849 EC:2.6.1.1 2.6.1.78
2.6.1.79
phenylalanine biosynthesis
E10 histidinol-phosphate aminotransferase K00817 EC:2.6.1.9 phenylalanine biosynthesis
E11 tyrosine aminotransferase K00815 EC:2.6.1.5 phenylalanine biosynthesis
E12 phenylalanine ammonia-lyase K10775 EC:4.3.1.24 phenylpropanoid
biosynthesis
E13 trans-cinnamate 4-monooxygenase K00487 EC:1.14.13.11 phenylpropanoid
biosynthesis
E14 4-coumarate-CoA ligase K01904 EC:6.2.1.12 phenylpropanoid
biosynthesis
E15 cinnamoyl-CoA hydratase-dehydrogenase – – β-oxidative pathway
E16 3-ketoacyl-CoA thiolase K07508/K07509/
K07513
EC:2.3.1.16 β-oxidative pathway
E17 4-hydroxybenzoyl-CoA thioesterase K01075 EC:3.1.2.23 ubiquinone biosynthesis
E18 glyceraldehyde 3-phosphate dehydrogenase K00134 EC:1.2.1.12 glycolysis / gluconeogenesis
E19 phosphoglycerate kinase K00927 EC:2.7.2.3 glycolysis / gluconeogenesis
E20 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase K01834 EC:5.4.2.11 glycolysis / gluconeogenesis
E21 2,3-bisphosphoglycerate-independent phosphoglycerate mutase K15633/K15634 EC:5.4.2.12 glycolysis / gluconeogenesis
E22 enolase K01689 EC:4.2.1.11 glycolysis / gluconeogenesis
E23 pyruvate kinase K00873 EC:2.7.1.40 glycolysis / gluconeogenesis
E24 pyruvate dehydrogenase E1 component alpha and beta subunits K00161/K00162 EC:1.2.4.1 glycolysis / gluconeogenesis
E25 pyruvate decarboxylase K01568 EC:4.1.1.1 glycolysis / gluconeogenesis
E26 pyruvate dehydrogenase E2 component K00627 EC:2.3.1.12 glycolysis / gluconeogenesis
E27 acetyl-CoA C-acetyltransferase K00626 EC:2.3.1.9 terpenoid backbone
biosynthesis
E28 hydroxymethylglutaryl-CoA synthase K01641 EC:2.3.3.10 terpenoid backbone
biosynthesis
E29 hydroxymethylglutaryl-CoA reductase (NADPH) K00021 EC:1.1.1.34 terpenoid backbone
biosynthesis
E30 mevalonate kinase K00869 EC:2.7.1.36 terpenoid backbone
biosynthesis
E31 phosphomevalonate kinase K00938 EC:2.7.4.2 terpenoid backbone
biosynthesis
E32 diphosphomevalonate decarboxylase K01597 EC:4.1.1.33 terpenoid backbone
biosynthesis
E33 1-deoxy-D-xylulose-5-phosphate synthase K01662 EC:2.2.1.7 terpenoid backbone
biosynthesis
E34 1-deoxy-D-xylulose-5-phosphate reductoisomerase K00099 EC:1.1.1.267 terpenoid backbone
biosynthesis
E35 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase K00991 EC:2.7.7.60 terpenoid backbone
biosynthesis
E36 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase K00919 EC:2.7.1.148 terpenoid backbone
biosynthesis
E37 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase K01770 EC:4.6.1.12 terpenoid backbone
biosynthesis
Batista et al. 7Vol. 00, No. 00, 2018
(Table S2, Supplementary Information). Interestingly, all
34 annotated transcripts are related to p-HBA biosynthesis
via the β-oxidative pathway, which corroborates this as
the main operant pathway in Piper gaudichaudianum for
p-HBA production.
With respect to the terpenoid portion, all the genes
codifying the enzymes from both the mevalonate and
methylerythritol phosphate pathways were identified in
this species, along with those genes encoding isopentenyl-
diphosphate isomerase and geranyl-diphosphate synthase
(Table 3). This result confirms the simultaneous operation
of these two pathways in Piper gaudichaudianum, which
is in accordance with previous studies.10 Many transcripts
were identified as genes encoding prenyltransferases.
Considering only E values ≤ 1e−50 and FPKM ≥ 1, 5
transcripts were initially aligned with 4-hydroxybenzoate
polyprenyltransferase genes as being involved in
ubiquinone biosynthesis. However, these transcripts also
present significant homology with 4-hydroxybenzoate
geranyltransferase 2 genes (Table S3, Supplementary
Information). These enzymes may be related to the
prenylation of p-HBA that yields the 2H-pyran moiety
after cyclization.
Finally, similar to what was recently observed for
Peperomia obtusifolia, the transcriptome analysis of the
Piper gaudichaudianum leaves revealed the presence of
tocopherol cyclase.12 By using the BLASTN20 web tool
from NCBI, it was possible to verify that the transcripts
from both species present a high degree of similarity
(Figure S5, Supplementary Information). Thus, apart from
catalyzing the formation of (S)-tocopherols, these enzymes
may also be responsible for the non-stereoselective
cyclization that yields the racemic chromane and
chromene moieties. All the enzymes identified that appear
to participate in gaudichaudianic acid biosynthesis in
Piper gaudichaudianum are presented in Table 3, and a
proposed scheme for this pathway is shown in Figure 5.
Conclusions
This study presents for the first time the transcriptome
analysis of Piper gaudichaudianum using a next-generation
sequencing approach. Approximately 10% of the
transcribed genes identified are somehow involved in
secondary metabolism. This approach allowed for the
identification of the main active biosynthetic pathways
in this species, such as those involved in the formation of
terpenoids, phenylpropanoids, flavonoids, and isoquinoline
alkaloids. These data corroborate and complement previous
studies performed on Piper gaudichaudianum. However,
the main contribution of this work concerns the biosynthesis
of p-hydroxybenzoic acid-derived benzopyrans. Despite
the advent of sequencing technologies that facilitate the
elucidation of plant secondary metabolism, many genes
in the benzoic acid derivative biosynthetic network
remain to be discovered.42 With this study, it was possible
to propose that p-HBA is produced via the β-oxidative
pathway, providing the first insights into its biosynthesis
in Piper species. Moreover, the transcriptome analysis
revealed the presence of prenyltransferases and tocopherol
cyclase enzyme genes, which may be responsible for the
prenylation and cyclization that yield the 2H-pyran moiety.
These findings are in agreement with those found in other
Piperaceae species, such as Peperomia obtusifolia.
Experimental
General experimental procedures
Total RNA was extracted using the RNeasy® Plant Mini
Kit (Qiagen, Hilden, Germany). A NanoDrop 2000 UV-Vis
Spectrophotometer (Thermo Fisher Scientific, Wilmington,
DE, USA) was used to determine the concentration and
quality of each RNA sample. The quality of the isolated RNA
was checked on a 2100 Bioanalyzer (Agilent Technologies,
Name K number EC number Pathway
E38 (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase K03526 EC:1.17.7.1 1.17.7.3 terpenoid backbone
biosynthesis
E39 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase K03527 EC:1.17.7.4 terpenoid backbone
biosynthesis
E40 isopentenyl-diphosphate delta-isomerase K01823 EC:5.3.3.2 terpenoid backbone
biosynthesis
E41 geranyl diphosphate synthase K14066 EC:2.5.1.1 terpenoid backbone
biosynthesis
E42 4-hydroxybenzoate geranyltransferase 2 K13565 EC 2.5.1.93 ubiquinone biosynthesis
E43 tocopherol cyclase K09834 EC:5.5.1.24 ubiquinone biosynthesis
Table 3. Enzymes involved in gaudichaudianic acid biosynthesis based on the transcriptome data of Piper gaudichaudianum (cont.)
Biosynthetic Insights into p-Hydroxybenzoic Acid-Derived Benzopyrans in Piper gaudichaudianum J. Braz. Chem. Soc.
8
Santa Clara, CA, USA). The RNA libraries were prepared
using a TruSeq RNA Library Preparation Kit (Illumina, San
Diego, CA, USA) and were sequenced using a HiSeq2500
Sequencing System (Illumina, San Diego, CA, USA).
Plant materials
Leaves from young Piper gaudichaudianum Kunth
specimens, which were cultivated under greenhouse
conditions at the Instituto de Química (UNESP, Araraquara,
SP, Brazil), were collected. The specimens were identified
by Dr Elsie F. Guimarães, and a voucher specimen
(Kato-0093) was deposited at the Herbarium of the Instituto
de Botânica (São Paulo, SP, Brazil).
RNA extraction, library preparation, and RNA sequencing
Total RNA was extracted in triplicate from a pool of
Piper gaudichaudianum leaves using the RNeasy® Plant
Mini Kit following the manufacturer’s instructions. The
concentration and quality of the three isolated RNA samples
were checked by using the A260/280 and A260/230
ratios from a NanoDrop spectrophotometer. The quality
of the samples was also checked in a Bioanalyzer for the
presence of intact 28S and 18S bands. Paired-end libraries
were prepared using a TruSeq RNA Library Preparation
Kit according to the manufacturer’s protocol. After that,
the resulting libraries were sequenced using an Illumina
HiSeq 2500 device.
De novo transcriptome assembly and annotation
The raw reads obtained after sequencing were quality-
filtered using the Trimmomatic software (version 0.33)43
with default parameters to remove the Illumina adapters
and low-quality bases. The filtered reads were subjected to
a digital normalization algorithm to decrease the sampling
variation, discard the redundant data, and remove most of
the errors. For links to the digital normalization software,
see Supplementary Information section. De novo assembly
of the filtered clean reads was conducted with the Trinity
software,18 version r20140717, using default parameters (for
the assembled sequences, see Supplementary Information
section). Fragments per kilobase of transcript per million
fragments mapped (FPKM) values were calculated using
the Bowtie2 program.44
For annotation, all the assembled transcripts were
searched using the BLASTX20 tool with an E value
cut-off of 1e−5 against the following databases: (i) the
non-redundant NCBI protein database; (ii) a custom
protein database with a total of 948,000 sequences from
plant proteins, derived from the RefSeq and UniProt/
Swiss-Prot public banks; and (iii) a custom database
containing 13,675 enzymes from the different pathways
Figure 5. Biosynthesis of gaudichaudianic acid in Piper gaudichaudianum including all proteins identified from the transcriptomic study.
Batista et al. 9Vol. 00, No. 00, 2018
involved in benzoate biosynthesis (from plants, fungi, and
bacteria). This benzoate biosynthesis database included
the sequences for chorismate pyruvate lyase, cinnamoyl-
CoA hydrates dehydrogenase, 3-ketoacyl-CoA thiolase,
4-hydroxybenzoyl-CoA thioesterase, 1,4-dihydroxy-
2-naphthoyl-CoA thioesterase, hydroxycinnamoyl-CoA
hydratase lyase, and hydroxybenzaldehyde dehydrogenase,
which were derived from the RefSeq and UniProt/Swiss-Prot
public banks. The annotation assigned to each transcript
was based on the best hit (highest score). The Blast2GO21
program was used to assign Gene Ontology (GO) terms to
the annotated transcripts according to biological process,
molecular function, and cellular component ontologies.
The comparison of the nucleotide sequences to
verify their degree of similarity was performed using the
BLASTN20 webtool from NCBI.
KEGG pathway mapping assignment
Using the KEGG BlastKOALA23 annotation web tool,
the transcripts with significant hits against the custom
plant protein database were also searched against a non-
redundant set of KEGG genes from the Kyoto Encyclopedia
of Genes and Genomes (KEGG) database to assign K
numbers. The transcripts with assigned K numbers were
mapped using the Search&Color Pathway tool offered
by the KEGG database.45 This analysis was focused on
transcripts with functions that were assigned to a given
secondary metabolism biosynthetic pathway.
Supplementary Information
Supplementary information (transcript length
distribution, secondary metabolism enzymes (KEGG maps,
EC number, K number, FPKM), nucleotide alignment,
β-oxidative pathway scheme) is available free of charge
at http://jbcs.sbq.org.br as PDF file.
All links to the digital normalization software are
available electronically through http://ged.msu.edu/
papers/2012-diginorm/.
The assembled sequences file is available free of charge
at http://jbcs.sbq.org.br as txt file.
Acknowledgments
The authors are grateful to the São Paulo Research
Foundation (FAPESP, grant numbers 2013/07600-3
(CEPID-CIBFar), 2014/25222-9, 2014/50316-7 and
2015/07089-2) and to the National Council for Scientific
and Technological Development (CNPq) for the research
fellowships to M. F. and M. J. K.; A. N. L. B. thanks
FAPESP for the provision of a postdoctoral fellowship
(grant number 2011/01003-8).
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Submitted: October 20, 2017
Published online: December 20, 2017
