Article
pubs.acs.org/jpr
Carnivorous Nutrition in Pitcher Plants (Nepenthes spp.) via an
Unusual Complement of Endogenous Enzymes
Linda Lee,† Ye Zhang,† Brittany Ozar,† Christoph W. Sensen,‡ and David C. Schriemer*,†
†
Department of Biochemistry and Molecular Biology and the Southern Alberta Cancer Research Institute, University of Calgary,
Calgary, Alberta T2N 4N1, Canada
‡
Institute of Molecular Biotechnology, Graz University of Technology, Graz 8010, Austria
S Supporting Information
*
ABSTRACT: Plants belonging to the genus Nepenthes are carnivorous, using
specialized pitfall traps called “pitchers” that attract, capture, and digest insects as
a primary source of nutrients. We have used RNA sequencing to generate a cDNA
library from the Nepenthes pitchers and applied it to mass spectrometry-based
identification of the enzymes secreted into the pitcher fluid using a nonspecific
digestion strategy superior to trypsin in this application. This first complete
catalog of the pitcher fluid subproteome includes enzymes across a variety of
functional classes. The most abundant proteins present in the secreted fluid are
proteases, nucleases, peroxidases, chitinases, a phosphatase, and a glucanase.
Nitrogen recovery involves a particularly rich complement of proteases. In
addition to the two expected aspartic proteases, we discovered three novel
nepenthensins, two prolyl endopeptidases that we name neprosins, and a putative serine carboxypeptidase. Additional proteins
identified are relevant to pathogen-defense and secretion mechanisms. The full complement of acid-stable enzymes discovered in
this study suggests that carnivory in the genus Nepenthes can be sustained by plant-based mechanisms alone and does not
absolutely require bacterial symbiosis.
KEYWORDS: Nepenthes, transcriptomics, carnivory, mass spectrometry, fluid, enzymes
■
INTRODUCTION
Carnivorous plants use specialized trapping structures to
attract, capture, digest, and absorb nutrients from insect prey
and other sources of nitrogen, phosphorus, and minerals.1−3
Trapping mechanisms include snap traps (e.g., Dionaea, also
known as Venus flytrap), flypaper or adhesive traps (Drosera,
Pinguicula), sucking bladder traps (e.g., Utricularia), and pitfall
traps (e.g., Nepenthes). Plants belonging to the genus Nepenthes,
also known as pitcher plants or monkey cups, are particularly
intriguing. The genus consists of over 100 species found mostly
in Southeast Asia and other tropical regions.4 The Nepenthes
pitcher buds from the end of the leaf and progressively forms a
pitfall trap consisting of three sections: a slippery upper rim
called the peristome that is involved in attracting and trapping
prey; a slippery, waxy inner wall to trap and prevent escape; and
a bottom pit filled with an acidic viscoelastic fluid used to digest
the trapped prey. Several structural and chemical variations of
the trap seem to be adapted for a variety of noninvertebrate
prey, including plant detritus and even tree-shrew feces.5,6 The
traps contribute a remarkably high percentage of the total
nutrient requirement for the plant7 and raise interesting
questions regarding the processes involved, particularly those
related to prey digestion.
A common feature of all traps is the containment and
presentation of the digestive fluid. Glands line the bottom
section of the pitcher and perform numerous functions,
including the detection of stimuli, absorption of nutrients,
© 2016 American Chemical Society
and secretion of digestive enzymes and acid into the pitcher
fluid. Attempts in recent years to identify and characterize the
protein composition of the Nepenthes pitcher fluid yielded a
surprisingly low number of enzymes. On the basis of this body
of work, it appears that the most abundant proteins in the
Nepenthes digestive fluid are Nepenthesins 1 and 2 (Nep1/2).8,9
These are noncanonical aspartic proteases that exhibit broad
cleavage specificity, high thermal stability, and optimal activities
at pH as low as 2.5.9−12 Aside from these two aspartic
proteases, a handful of other proteins have been identified in
pitcher fluid, including a β-1,3-glucanase, a β-D-xylosidase,
chitinases, a peroxidase, pathogenesis-related protein PR-1, a
thaumatin-like protein, and a ribonuclease.13 Even this minimal
composition begins to suggest a concerted mechanism for
nutrient acquisition and inhibition of microbial pathogens.13−20
Prior to modern proteomics methods, other enzymatic
activities were either detected or suspected. Activities arising
from cysteine proteases, phosphatases, esterases, oxidoreductases, and lipases have been attributed to the Nepenthes
fluid.8,19−25 Nevertheless, the identity and source of most of
these enzymes are not known. The Nepenthes fluid harbors a
diverse microbial community, some of which may contribute
enzymatic capacity to the fluid and form a symbiotic
relationship with the plant, although their relative contribution
Received: March 14, 2016
Published: July 20, 2016
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J. Proteome Res. 2016, 15, 3108−3117
Article
Journal of Proteome Research
to prey digestion is still under investigation.26−28 However,
studies have questioned whether the bacterial load in the trap
has a role to play in the processing of insect prey, prompting a
closer inspection of fluid composition.
Although proteomics methods have been applied to the fluid,
the complement of proteins should not be considered
complete. A thorough identification of all the proteins present
in the Nepenthes digestive fluid is challenged by two major
problems: the unusual amino acid composition of the proteins
and the limited representation of carnivorous plants in the
genomic/protein databases. Many of the proteins identified in
the Nepenthes digestive fluid thus far have a low frequency of
Lys/Arg residues. These characteristics suggest that standard
methods based on tryptic digestion may not be fruitful. Indeed,
studies have resulted in low sequence coverage using
conventional proteomic analyses.9,13,14,29 Moreover, the identification of the proteins is complicated by the lack of a
complete sequence of the Nepenthes genome and transcriptome. Although some transcriptomics resources are
beginning to emerge, and used in a preliminary trypsin-based
proteomics workflow,29 an alternative strategy is required to
test for the completeness of the compositional analysis. To
acquire comprehensive insight into the enzymatic components
secreted by the plant, we sequenced the N. raf f lesiana
transcriptome and coupled it to proteomic analysis of the
fluid using a combination of approaches. In addition to trypsin,
we took advantage of the robust proteolytic capacity of the fluid
itself to increase the depth of coverage. Using these
complementary approaches, we were able to confirm many of
the known fluid proteins. We were also able to identify novel
proteins that likely function in prey digestion and defense.
■
Zero Plant reagents to remove the abundant cytoplasmic,
mitochondrial, and chloroplast rRNA. First strand cDNA was
reverse transcribed using random primers, followed by second
strand synthesis. Indexing adaptors were ligated to the dsDNA
ends to generate Illumina sequencing libraries. The libraries
were PCR amplified, normalized, and pooled. Paired-end 100
bp sequencing was performed on an Illumina HiSeq 2500
sequencer.
Transcriptome Assembly and Analysis
Cleaning filters were applied to the raw sequencing data files,
based on FastQC quality control statistics, and Illumina
sequencing adaptors were detected with Cutadapt. Reads
were clipped using TrimmomaticPE. Reads corresponding to
contaminant genomes such as all known bacteria, Tetrahymena
thermophila, Fusarium graminearum and Drosophila melanogaster
were removed from the transcriptome assembly. De novo
transcript assembly was performed with Trinity (v2.0.6) at the
default kmer size of 25 and a cutoff contig length of 200 bp.
Contigs that were expressed were considered for CDS
prediction in all six reading frames using Transdecoder
(v2.0.1). The translated database was filtered for polypeptide
≥150 amino acids in length, and initial annotation was based on
comparison with NCBI nt, NCBI nr, and SwissProt databases
using Tera-Blast with an e-value cutoff of e-15. The highest
scoring hit was used for the annotation.
Plant and Fluid Processing
N. ventrata (100 plantings in 8″ pots) were grown in a
dedicated greenhouse (Urban Bog, Langley, BC, Canada).
Upon pitcher maturity, the plants were fed with 1 or 2
Drosophila per pitcher, and the digestive fluid was harvested the
following week as previously described.31 In brief, the fluid
from approximately 1000 N. ventrata pitchers was collected,
pooled, clarified through a 0.2 μm filter to remove debris,
concentrated 10-fold using an Amicon Ultra 10 kDa molecular
weight cutoff centrifugal filter (Millipore), and washed with 100
mM glycine-HCl pH 2.5. The fluid was stored at 4 °C for shortterm or at −80 °C for long-term.
EXPERIMENTAL METHODS
RNA Extraction
N. raff lesiana giant plants were purchased from Urban Bog
(Canada) and grown in a small terrarium in a 15:9 h light:dark
photoperiod. The plants were fed with 1 or 2 Drosophila sp. per
pitcher the day before the pitchers were to be harvested for
RNA extraction. Prior to harvesting the pitcher for RNA
extraction, the digestive fluid was decanted, and the pitcher was
washed with deionized water to remove partially digested
material and other debris. The bottom one-third of the pitcher
containing the secretory glands was excised, frozen in liquid
nitrogen, and ground to a fine powder in the presence of liquid
nitrogen. The total RNA was extracted using a modified CTAB
protocol described by Meisel et al.30 In brief, the ground
pitcher was lysed in warm CTAB extraction buffer (2% w/v
cetrimonium bromide, 100 mM Tris-HCl pH 8, 20 mM EDTA,
1.4 M NaCl, 1% w/v polyvinylpyrrolidone MW 40,000, 0.05%
w/v spermidine trihydrochloride, and β-mercaptoethanol) and
extracted several times with chloroform-isoamyl alcohol (24:1).
The RNA was then precipitated from the aqueous phase with
2.5 M LiCl, followed by chloroform extraction and precipitation
in ethanol. The quality and integrity of the RNA was assessed
on a 2200 TapeStation (Agilent) to have a RIN score >8.
In-Gel Fluid Proteomics Analyses
The N. ventrata digestive fluid was analyzed on an 8% SDSPAGE gel and silver stained with SilverSNAP stain for mass
spectrometry applications (Thermo Scientific, Rockford, USA)
following the manufacturer’s protocol. Visible protein bands
were excised, destained in 50 mM sodium thiosulfate and 15
mM potassium ferricyanide, and reduced with 10 mM DTT
followed by alkylation with 50 mM iodoacetamide in 100 mM
ammonium bicarbonate pH 8. The gel slices were then digested
with trypsin (Promega; enzyme to substrate ratio of 1:20) at 37
°C overnight followed by quenching with 0.1% trifluoroacetic
acid. The digests were desalted on a C18 ZipTip (Millipore)
prior to analysis by data-dependent LC−MS/MS on an Agilent
6550 iFunnel-QTOF mass spectrometer outfitted with a 1260
Infinity chip cube interface using a 25 min reversed-phase
gradient run (high-capacity chip: Agilent Zorbax 300SB-C18 5
μm particles, 150 μm × 75 μm, using 0.1% formic acid in 3%
acetonitrile for mobile phase A and 0.1% formic acid in 97%
acetonitrile for mobile phase B; flow rate of 300 nL/min and a
2% B/min linear gradient to 50% B). The MS/MS was
configured using CID for precursor ions in the m/z range
between 275 and 1700, and top-10 ion selection with a charge
of +2 and higher. The optimum collision energy was calculated
for each precursor based on its charge and mass. Ion exclusion
Transcriptome Sequencing
Illumina RNA sequencing of N. raf f lesiana transcriptomes was
performed by Omega Bioservices (Omega Biotek, Inc.,
Norcross, GA). RNA samples were processed using the
Illumina TruSeq Stranded Total RNA Sample Prep kit
(Illumina, San Diego, CA) following the manufacturer’s
protocol. Briefly, 1 μg of total RNA was treated with Ribo3109
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■
was released after 1 spectrum and 0.2 min to avoid reacquiring
MS/MS data for the same precursor. The data were searched
against the 6-frame translation of our N. raf f lesiana transcriptome databases using Mascot. The Mascot search
parameters for the tryptic digested samples were a maximum
of one missed cleavage, carbamidomethyl (C) as a fixed
modification, methionine oxidation as a variable modification, a
20 ppm peptide mass tolerance, and a 0.2 Da fragment mass
tolerance. Output was filtered for peptide hits with p < 0.05
(ion score ≥ 24).
Article
RESULTS
Transcriptome of the Stimulated Nepenthes Pitcher
To support the proteomics-based identification of the protein
composition of Nepenthes digestive fluid, we performed
Illumina RNA sequencing to generate a comprehensive
database of the Nepenthes transcriptome (Figure 1). We
Gel-Free Fluid Proteomic Analyses
For the proteome of the N. ventrata pitcher fluid to be analyzed,
the fluid was digested either with trypsin (specific digest) or N.
ventrata fluid (nonspecific digest) using a modified FASP
protocol.32 Briefly, 15 μg of 10× concentrated N. ventrata
digestive fluid was reduced with 100 mM DTT followed by
alkylation with 50 mM iodoacetamide under denaturing
condition (8 M Urea, 100 mM Tris-HCl pH 8) on a YM-10
microcon (Millipore). For digestion with trypsin, the reduced/
alkylated fluid was buffer exchanged to 50 mM ammonium
bicarbonate pH 8 and digested with trypsin (Promega; enzyme
to substrate ratio of 1:100) at 37 °C overnight followed by
quenching with 0.1% trifluoroacetic acid. For nonspecific
digestion, the reduced/alkylated fluid was washed with 100
mM Gly-HCl pH 2.5 and digested with N. ventrata fluid (1:30
enzyme:substrate) at room temperature for 40 min. The digests
were desalted on a C18 ZipTip (Millipore) prior to analysis by
data-dependent LC−MS/MS on an Orbitrap Velos ETD
(Thermo Scientific). The samples were analyzed twice using
90 min reversed-phase gradient runs configured for top-10 ion
selection using CID in a high/low configuration. One run
focused on peptides with a charge state of 1+, whereas the other
run focused on peptides with a charge state of 2+ and higher.
MS scans were acquired over the m/z 350−1800 range with a
resolution of 60,000 (m/z 400). The target value was 5.00 ×
105. For MS/MS, ions were fragmented in the ion trap with
normalized collision energy of 35%, activation q = 0.25,
activation time of 10 ms, and one microscan. The target value
was 1.00 × 104. Separation was achieved using a self-packed
pico-frit column (New Objective, packed with Phenomenex 5
μm Jupiter C18 beads, 100 mm × 75 μm), using 0.1% formic
acid in 3% acetonitrile for mobile phase A and 0.1% formic acid
in 97% acetonitrile for mobile phase B (flow rate of 300 nL/
min and a 0.5% B/min linear gradient to 45% B). The data
were searched against the 6-frame translation of our N.
raf flesiana transcriptome databases using Mascot. The search
parameters for the tryptic digested samples were one missed
cleavage allowed, carbamidomethyl (C) as a fixed modification,
methionine oxidation (M) as a variable modification, a 10 ppm
peptide mass tolerance, and a 0.6 Da fragment mass tolerance,
and the output was filtered with a false discovery rate of 1%
(peptide hits with p < 0.00045; ion score ≥ 42). The search
parameters for the fluid-digested samples were similar to those
for the tryptic sample, except the search was configured for “no
enzyme” specificity and the output set was filtered with a false
discovery rate of 1% (peptide hits with p < 0.009; ion score ≥
49). In cases where shorter partial ORF were contained within
or overlap with another transcript, the longer assembled
transcript was used for characterization.
Figure 1. Transcriptomics and proteomics workflow applied to
Nepenthes spp. Transcriptomics was applied to Nepenthes raf f lesiana
pitcher tissue, and proteomics was applied to Nepenthes ventrata
pitcher fluid. Photographs courtesy of Linda Lee. Copyright 2016.
extracted the RNA from the bottom third of a N. raf f lesiana
giant pitcher, where the secretory glands are located,4 which
had been stimulated by feeding with fruit flies to reduce fluid
pH and promote the digestive state.15,33 Because of the open
nature of the Nepenthes pitcher, bacteria and fungi as well as
fruit flies could be present in the pitcher. However, read pairs
corresponding to these contaminating genomes accounted for
less than 1% of the total read set (Figure S1 and Table S1) with
proteobacteria being the most dominant phylum (Figure 2).
After the raw data were cleaned, including the removal of the
contaminating genomes, the transcriptome was assembled from
154 million paired-end reads of 100 bp length. A total of 1.36
million contigs >200 bp were assembled with an average length
of 665 (Table S1). When the assembled transcriptome was
filtered for contigs >300 bp, the number of total contigs was
significantly reduced to 335 K with an average length of 1329
bp. The high number of contigs, especially those <300 bp, may
arise from small, noncoding RNA and/or errors in assembly.
Nevertheless, we maintained the cutoff at 200 bp for this stage
of analysis. Searching our transcriptome with tRNAscan did not
find any significant tRNA hits, and alignment against the
Arabidopsis intronic-intergenic database yielded minimal results,
indicating that our transcriptome was enriched for mRNA as
expected. When we included expression as a criterion, we
reduced the number of contigs to 532,208 contigs (>200 bp
set). Although only 31% of the expressed contigs could be
annotated using the NCBI nucleotide database, these
accounted for nearly half of the total number of reads (Table
S3). The unannotated contigs accounted for only ∼12% of total
read set, suggesting that these may be present at very low levels
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Figure 2. Taxonomic organization of the N. raf f lesiana transcriptome, providing an overview of the taxonomy and level of contamination present
based on annotation against the NCBI nt database (e-value cutoff of e-15).
by long-gradient LC−MS/MS identified 26 proteins in the
fluid. Sequence coverage was still low with only 1−4 identified
peptides matching to each protein (Table S4); these results are
similar to those of a recent study.29 As noted, the low level of
coverage could arise from a nonoptimal peptide set due to the
low frequency of lysine and arginine residues present in the
majority of the proteins identified to date. For example,
nepenthesin 2 is relatively abundant in the digestive fluid,9,13,34
but it was not detected in the tryptic digest sample due to the
absence of K/R residues in its mature active form.
Deglycosylation of the protein sample did not improve
coverage (not shown).
To circumvent the restrictions presented with trypsin, we
digested denatured N. ventrata fluid with active N. ventrata
fluid. The high activity of the fluid, and its value in generating
high sequence coverage in hydrogen/deuterium exchange MS
experiments, argued for the attempt. At our current level of
characterization, the fluid minimally contains aspartic proteases
with broad cleavage specificity.11,12,34 Upon LC−MS/MS of the
nonspecific digested fluid, we obtained substantially higher
coverage with as many as 70 matching peptides for the most
abundant proteins. We identified 19 proteins using this
approach (Table S5). Nine proteins are common with those
identified in the tryptic digestion method; therefore, in total, 36
proteins were identified in the N. ventrata fluid from the
combination of tryptic and nonspecific digestion approaches
(Table 1). The identified proteins were functionally annotated
by homology search against the NCBI nonredundant (nr)
protein sequence database or characterized biochemically. For
the relative abundance of the identified proteins based on labelfree methods, such as emPAI, to be assessed, the tryptic data is
particularly unreliable given the paucity of detectable peptides.
However, the emPAI values from the nonspecific digests are
useful given the much larger peptide set generated. The ranked
list in Table S5, therefore, approximates the order of
abundance.
Our improved digestion strategy presents a more complete
analysis of the fluid proteome when compared to a recent study
using only trypsin.29 We identified approximately 25% more
(and/or arise from error in assembly). Taxonomical annotation
of the contigs using MEGAN revealed that well over half of the
transcripts were plant-based (Streptophyta) with some minor
contamination from bacteria, fungi, and fruitflies (Figure 2 and
Table S2), indicating that our method enriched for Nepenthes
transcripts. To enrich for long transcripts and enable an
effective proteomics experiment, we filtered the translated
ORFs for polypeptides >150 aa in length, which generated
66,459 ORFs for the search.
Proteome of the Nepenthes Digestive Fluid
Visualization of the stimulated N. ventrata digestive fluid via
silver-stained SDS-PAGE showed the limited complexity of the
fluid in keeping with previous work (Figure 3). When searched
Figure 3. Silver-stained SDS-PAGE of N. ventrata digestive fluid
showing the excised bands and the major proteins identified by in-gel
tryptic digestion and mass spectrometry.
against our assembled N. raf f lesiana transcriptome, mass
spectrometric data arising from in-gel tryptic digestions of the
visible protein bands were able to identify major proteins
present in the digestive fluid (Figure 3), illustrating the utility of
our cDNA library. To increase the sensitivity of our proteomic
approach, we collected and concentrated large volumes of
stimulated N. ventrata fluid and analyzed it using gel-free
methods. Here, gel-free tryptic digestion of the fluid followed
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Table 1. Combined List of the Identified Proteins from the Secreted Nepenthes Fluid
process
protein metabolism
nucleic acid
metabolism
polysaccharide
metabolism
protein
Nepenthesin-1, Nep1
Nepenthesin-2, Nep2
Nepenthesin-3, Nep3
Nepenthesin-4, Nep4
Nepenthesin-5, Nep5
Neprosin-1, Npr1
Neprosin-2, Npr2
Serine carboxy-peptidase, SCP1
Purple acid phosphatase, PAP1
aspartic protease
aspartic protease
aspartic protease
aspartic protease
aspartic protease
prolyl endoprotease
prolyl endoprotease
S10 acidic peptidase
metallophosphatase
Ribonuclease, S-like, RNaseS
Endonuclease 2, Endo2
secreted RNase/scavenge phosphates
S1−P1 nuclease/cleave RNA and
ssDNA
glycoside hydrolase 17/pathogen
defense
glycoside hydrolase 18/class III
chitinase/pathogen defense
glycoside hydrolase 19/class IV
chitinase/pathogen defense
heme-binding motif/glycoside
hydrolases
antifungal activity/pathogen defense
β-1,3-Glucanase, Glu1
Acidic Chitinase, Chit3
Chitinase, Chit1
DOMON-like domain, Dom1
pathogenesis/host
defense
peroxidases
signal transduction
energy metabolism
protein transport
translation/protein
synthesis
class/function
Thaumatin-like protein, TLP1
Hevein, Hev1
Cat. peroxidase 1, Prx1
Cat. peroxidase 2, Prx2
Peroxidase, Prx3
Leu-rich repeat protein kinase,
RPK1
Leu-rich repeat protein, LRR1
chitin binding/antifungal activity
heme-containing/oxidation
heme-containing/oxidation
heme-containing/oxidation
Ser/Thr kinase/plant dev./ pathogen
resistance
possible protein kinase/ribonuclease
inhibitor
ADP/ATP translocase, ADT1
ADP/ATP exchange
Glyceraldehyde-3-phosphate
NAD-binding/glycolysis/
dehydrogenase, GPD1
glyconeogenesis
ATP synthase/H+-ATPase subunit ATP synthesis/membrane-bound
α, ATPA
proton pump
ATP synthase/H+-ATPase subunit ATP synthesis/membrane-bound
β, ATPB
proton pump
Enolase, Eno1
2-phospho-D-glycerate conversion
Actin, Act1
cytoskeleton
Tic20-like protein, Tic20
chloroplast import protein
Protein transporter Sec1
vesicle trafficking/secretion
Elongation factor 1-alpha, EF1a
GTP-binding elongation factor
Elongation factor 2, EF2
transcription/gene
regulation
GTP-dependent ribosomal
translocation
40S Ribosomal protein S14, RS14 protein synthesis
Chaperone protein Hsp90
protein folding
Histone acetyltransferase, HAC1
acetylate transcriptional regulators,
histones
Auxin response factor, ARF1
transcription factor/Auxin response
factor
MW
pI
signal
pep.?
47106
46625
49092
48991
48811
43161
>33 kDa
>47 kDa
71268
4.7
4.1
4.9
4.8
4.6
6.3
5.2
4.3
5.2
yes
yes
yes
yes
yes
yes
n/ac
yes
yes
26224
32487
4.1
4.9
36752
dig. 1a dig. 2b
2
ref
9
9
5
1
1
2
8
6
9
7
10
5
4
yes
yes
3
12
19, 20
14
4.7
yes
6
11
14
31086
4.1
yes
12
15
14, 18, 52
29306
4.5
yes
13
27995
5.5
yes
9
23785
5.7
yes
4
20752
33598
33385
36354
66702
6.4
6.4
4.6
4.7
5.4
yes
yes
yes
yes
yes
10
8
21
16
69848
4.8
no
>31 kDa
36664
10.0
7.1
n/ac
no
11
18
>18 kDa
5.3
no
20
>51 kDa
4.9
no
22
>30 kDa
41706
31449
34078
49815
4.9
5.3
9.4
9.2
9.2
n/ac
no
no
no
no
25
15
95948
6.1
no
24
16367
91593
>86 kDa
10.6
5.0
6.0
no
yes
no
17
19
>25 kDa
8.5
n/ac
3
23
13, 53
13
13
14
14
14
17
16
26
7
18
19
a
Trypsin digestion. Ranking is based on the MASCOT search score. bNonspecific digestion using active Nepenthes fluid. Ranking based on
MASCOT search score. cNot applicable (n/a) because the protein fragment has a truncated N-terminus.
lower pI than the proenzyme. Similar post-translational
processing events may also occur for other fluid proteins,
possibly resulting in even lower pI values. Consistent with the
fact that the fluid proteins are secreted into the pitcher, twothirds of the identified proteins with an intact N-terminus were
predicted by SignalP 4.1 to have a signal peptide.
More specifically, the fluid proteome can be organized into a
number of functional classes and subclasses that aid in
evaluating the mechanisms of carnivory.
proteins, some of which are novel. Globally, the majority of the
identified pitcher proteins have properties expected of a
secreted protein functioning in an acidic fluid. Over 70% of
the identified fluid proteins have a pI < 6, consistent with the
acidic nature of the digestive fluid (Table 1 and Table S6). The
pI is predicted from the deduced unprocessed transcript and
may not reflect that of the mature active enzymes. Acid
activation of the well-characterized Nep1 and Nep2 involves
autolysis at the N-terminus,35 yielding a mature enzyme with a
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Figure 4. Comparative analysis of the five nepenthesins discovered based on sequence. (A) Phylogenic tree of the nepenthesins identified in our N.
raf f lesiana transcriptome analysis (in bold) with the nepenthesins from other Nepenthes species (including an unpublished nepenthesin sequence
from N. ampullaria). The percent sequence identity relative to N. raf f lesiana Nep1 is indicated in brackets. The sequence identity within the Nep2
subgroup is ∼90%. (B) Sequence alignment of N. raf f lesiana Nep1−5. The vertical lines indicate the signal peptide (SP) cleavage site predicted by
SignalP 4.1 and the proenzyme cleavage site to produce the mature active proteases. The conserved region surrounding the catalytic aspartate
residues (*) are boxed. The conserved cysteines (∧) are involved in disulfide bridges. NAP, nepenthesin-type aspartic protease-specific insertion.
The alignment and phylogeny tree were produced using ClustalW.
Proteases. The most abundant proteins in the Nepenthes
fluid are Nep1 and Nep2, which exhibit >90% homology to
Nep1 and Nep2, respectively, from other Nepenthes species.
Nep1 and Nep2 are noncanonical aspartic proteases with broad
cleavage specificity that are stable over a wide temperature
range.9−12 In addition to Nep1 and Nep2, we discovered three
additional nepenthesin homologues, which we name Nep3,
Nep4, and Nep5 (Figure 4). Nep3−5 exhibit approximately
50% homology to each other as well as to Nep1 and Nep2,
indicating they are distinct, novel aspartic proteases. Our
preliminary sequencing of the N. ampullaria transcriptome
revealed the presence of a Nep5 transcript that is 98% identical
to N. raf f lesiana Nep5 (Figure 4A and unpublished data),
suggesting that these novel nepenthesins may also be present in
other Nepenthes species. Like Nep1 and Nep2, Nep3−5 all
contain the conserved catalytic aspartate residues and the 12
cysteine residues involved in disulfide bridges (Figure 4B). The
catalytic aspartate residues in these five nepenthesins, as well as
those from other Nepenthes species, reside within two highly
conserved regions: AIMDTGSDLIWTGQ (aa110−123) and
IDSGTT (aa314−319; numbering based on Nep1 with the
catalytic aspartate underlined). These regions are more variable
and less extensive in other aspartic proteases, such as pepsin.
Another distinguishing feature of the nepenthesins is the
variable ∼20−24 aa nepenthesin-type aspartic protein (NAP)specific region, which contains four of the conserved cysteine
residues.9 Nep1−5 are different from the five aspartic proteases
AP1−5 reported by An et al.,36 which contain a plant-specific
insert of approximately 100 aa (instead of the short NAPspecific insert) commonly found in vacuolar plant aspartic
proteases (vAP, Figure S2). Our N. raf f lesiana transcriptome
does contain several transcripts homologous to these vacuolar
aspartic proteases (not shown), although we did not detect
these vAPs in the Nepenthes digestive fluids. Nep3−5 appear to
be present in the fluid in lower amounts than those of Nep1
and Nep2 (Table S5); however, the relative activity and
contribution of Nep3−5 to the digestive process remain to be
investigated.
We have also identified a novel class of prolyl-endoprotease
in the fluid. We previously reported that the Nepenthes fluid can
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Figure 5. Model for the mechanism of carnivory in Nepenthes. The enzymes identified in this study are highlighted in red, whereas enzymes
suggested by other studies are in blue.15,23 Receptors lining the pitcher detect the presence of prey or pathogens, resulting in the secretion of
digestive enzymes into the cavity of the pitcher. Glucanases and chitinases digest the cell wall and exoskeleton of captured prey and pathogens.
Digestion of the prey proteins and nucleic acids by the proteases, nucleases, and phosphatases releases feedstocks for the nitrogen and phosphate
requirements of the plant. Lipases, possibly from the microbiome, facilitate the digestion of lipids. The peroxidases may contribute to the host
defense against pathogen by generating ROS. The ROS can also oxidize prey proteins, which may cause them to be more susceptible to degradation
by proteases.
also cleave C-terminal to proline residues, but neither Nep1 nor
Nep2 possessed such prolyl-endopeptidase activity.12,31,37 We
recently purified a fluid fraction containing the prolylendopeptidase activity and identified a novel protease that we
called neprosin 1 (Npr1).38 In addition to Npr1, we also
detected in our present study a partial transcript corresponding
to a neprosin homologue, which we call Npr2, but it remains to
be seen if it also processes prolyl-endopeptidase activity. Both
Npr1 and Npr2 are distinct from known proline-cleaving
enzymes, each consisting of two domains of unknown function
(DUF4409 and DUF239). DUF4409 and DUF239 family
members are found in many plant species, but their biological
function has not been characterized. The presence of a serine
carboxypeptidase completes the list of proteases identified in
the Nepenthes digestive fluid. The relatively high abundance of
this enzyme (Table S5) suggests that it is significant in prey
digestion and nutrient extraction. It belongs to the S10 family
of serine proteases, which are active at low pH.39
Nucleases. We discovered two nucleases and an acid
phosphatase in the Nepenthes pitcher fluid. The predominant
nuclease appears to be a secreted S-like ribonuclease that is
>95% identical to those previously isolated from N.
bicalcarata19 and N. ventricosa.20 Like many of the acidic fluid
enzymes, the N. bicalcarata ribonuclease was found to be active
at an optimal pH of 3.5.19 We also newly identified an S1-like
endonuclease and an acid phosphatase, both of which are likely
present at moderate levels in the fluid (Table S5). The S1-like
endonuclease exhibits approximately 50% homology to other
plant endonuclease-2 enzymes, which can hydrolyze singlestranded nucleic acids.40 The acid phosphatase is closest to the
purple acid phosphatases, a metallophosphoesterase found in a
variety of plants, which is upregulated during phosphate
deficiency to scavenge phosphate by hydrolyzing various
phosphate esters.41
Glycoside Hydrolases and Pathogenesis-Related
Proteins. The three glycoside hydrolases (β-1,3-glucanase,
class III and IV chitinases), thaumatin-like protein, and two
cationic peroxidases that we identified are similar to those
characterized previously in other Nepenthes species.13,14,18
Glycoside hydrolases are involved in the digestion of
polysaccharides, such as chitin found in an insect exoskeleton,
as well as in the cell wall of pathogens and plant debris. We
discovered a protein containing a dopamine β-monooxygenase
domain (DOMON, a heme- and sugar-binding motif found in
some cytochrome and glycosyl hydrolases)42,43 as well as
hevein, a chitin-binding domain associated with antifungal
activity.44,45 Both are likely involved in aspects of glycan
recognition and play a role in plant defense against pests and
fungal infection. The two cationic peroxidases as well as a third
peroxidase that we discovered may contribute to host defense
against pathogens involving the generation of highly reactive
oxygen species (ROS).
Finally, we discovered two Leu-rich repeat (LRR) proteins,
one of which appears to be a receptor-like protein kinase. LRR
is a common motif of 20−30 amino acids rich in leucines
present in tandem repeats that mediate protein−protein
interactions. LRR receptor-like protein kinases have been
reported to be involved in plant development, germination, and
pathogen resistance.46 Two of the remaining proteins identified
correspond to the α and β subunits of ATP synthase/H+ATPase and may function as a proton pump for acidification of
the pitcher fluid.33
■
DISCUSSION
Carnivorous plants, such as Nepenthes, obtain a large fraction of
their nutrients from the digestion of captured prey and other
organic matter. We have sequenced the Nepenthes raf f lesiana
transcriptome to facilitate the identification of the enzymes
involved in Nepenthes carnivory and searched the resulting
cDNA library with proteomics data generated conventionally
(using trypsin-based methods) and by an alternative bottom-up
strategy (using the proteases contained in the pitcher plant
fluid). To our knowledge, this study is the first application of
the Nepenthes transcriptome to a detailed proteomic analysis of
the Nepenthes digestive fluid. Although a very recent study has
attempted to mine tryptic peptides using de novo MS/MS
sequencing methods, the individual peptide sequences
determined in this fashion were searched against a cDNA
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library using BLAST.29 The procedure used does not appear to
retain a statistical foundation sufficient to assess the probability
of the many proteins identified with only one or two peptides,
and misidentifications are possible. For example, nepenthesin-2
is noted as identified with three tryptic fragments, but there are
no K and R in the mature enzyme sequence in almost every
species reported.
In our study, we have identified 36 proteins in a direct search
of the cDNA library, which we prepared from RNA-seq data,
ten of which were uniquely determined using the nonspecific
digestion approach. It is clear that protein digestion represents
the most aggressively targeted functionality in steady-state
pitcher fluid. Five nepenthesins (three of which are novel: Nep
3−5) and two neprosins (both novel: Npr1, Npr2) combine to
provide a potent proteolytic digestion capacity to the fluid.
Both represent intriguing departures from their core enzymatic
classifications, as noted in recent studies from our laboratories.31,37 The newly discovered variants should prove valuable
as reagents in proteomics applications. The relatively abundant
acid-stable serine carboxypeptidase, in particular, suggests a
useful laddering enzyme to aid in peptide sequencing and will
be pursued.
We summarize the putative functions of the identified
Nepenthes fluid proteins in a model for the mechanism of
carnivory (Figure 5). The functions of the hydrolytic enzymes
in the fluid are simple to infer. Glucanases and chitinases digest
the cell wall and exoskeleton of captured prey and plant debris.
Digestion of the prey proteins and nucleic acids by the
proteases, nucleases, and phosphatases supply nitrogen and
phosphate for absorption by the plant. Many of the hydrolytic
enzymes involved in prey digestion also possess antibacterial
and/or antifungal properties because they can digest pathogens
and hence can be considered pathogenesis-related proteins
themselves.4 The association between carnivory and pathogenesis is further supported by the presence of several
specifically pathogenesis-related proteins such as the peroxidases, hevein (a chitin-binding protein), thaumatin-like
protein, and LRR proteins. The peroxidases likely contribute
to host defense against pathogens by generating ROS. It has
been proposed that oxidation of prey proteins by peroxidasegenerated ROS can enhance their susceptibility to degradation
by proteases.47 Hevein domains bind chitin and display
antifungal activity.48 Thaumatin-like protein likely serves a
similar antifungal role,49 although we note that thaumatin is a
highly potent natural sweetener, and this protein may serve as a
chemoattractant to insect prey.50 The functions of the other
proteins in the fluid are low abundance and more difficult to
ascertain with confidence. The Leu-rich repeat (LRR) receptorlike protein kinase (RPK1) could stimulate the secretion of
enzymes into the cavity of the pitcher upon sensing the
presence of prey or pathogen, and the membrane-bound H+ATPase may function as a proton pump for acidification of
pitcher fluid.33 Both proteins are membrane-associated. They
are somewhat surprising to be identified in the fluid but may
represent shed domains. The remaining proteins are involved in
energy metabolism, protein transport, protein expression, and
synthesis (Table 1). They could represent intracellular plant
proteins, because many of them lack a signal peptide, or
elements of the microbial community that share stretches of
sequence identity with entries in the Nepenthes transcriptome.
An overlapping set of proteins was observed by Schulze and
colleagues in the acidic digestive fluid of the Venus flytrap,51
suggesting that these two different carnivorous plants use
similar mechanisms for prey digestion. However, the major
proteases in the Venus flytrap secretions are cysteine proteases,
whereas the main proteases in Nepenthes are aspartic proteases.
Cysteine protease activity has been suggested to be present in
the Nepenthes fluid, as activity was inhibited by cysteine
protease inhibitor E-64.20 Although numerous transcripts
corresponding to putative cysteine proteases are present in
our cDNA library, we did not detect any in the Nepenthes fluid.
It remains possible that such determinations are species
dependent; however, we note that a high degree of consistency
has emerged in the identification of several enzymes across
many varieties (aspartic proteases, glucanases, and chitinases,
for example). The enzymatic properties do not appear to vary
significantly.
■
CONCLUSIONS
Our improved proteomics workflow did not identify other
proteins that have been reported to be present in the Nepenthes
fluid and native to the plant. Pathogenesis-related protein 1
(PR-1) and an oxidoreductase14,16 were detected, but their
scores were below our significance threshold. We could detect
no lipases, xylosidases, or galactosidases.29 The fluid represents
a steady-state harvest; therefore, these enzymes are either
transient members produced during fluid maturation or, more
likely, were contributed by the plant microbiome. Although the
acidity and antimicrobial properties of the Nepenthes fluid
suppress the growth of pathogens, the fluid still hosts a diverse
microbial community, which may form a mutualistic relationship with the host plant.26,28 A recent metagenomics study of
the Nepenthes microbiome revealed as many as 18 bacteria
phyla with proteobacteria representing the predominant class.26
This bacterial class was also found in our prefiltered
transcriptome (Figure 2). Bacteria isolated from Nepenthes
fluid have been reported to possess protease, chitinase, and
lipase activities;23,26 however, it remains an open question
whether these contribute meaningfully to mechanisms of
carnivory. It is possible that bacterial colonization is mostly
parasitic in nature; the topic requires further study. If the
unusually broad and robust activity profile of the nepenthesins
is any indication, the other acid-stable enzymes may present an
equally broad substrate profile sufficient to supply amino acids,
sugars, and phosphate from a range of sources. On the basis of
our extended proteomics analysis, it would appear that the
panel of endogenous plant enzymes is sufficient to recover the
nutrients needed to support growth.
■
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jproteome.6b00224.
Figure S1 provides an overview of the transcriptome
analysis procedures in support of Figure 1; Figure S2
provides a phylogenetic analysis of nepenthesins relative
to vacuolar aspartic proteases; and List S1 contains the
set of amino acid sequences for the proteins identified in
N. raf f lesiana digestive fluid (PDF)
Table S1 provides a summary sequence analysis of the N.
raf flesiana transcriptome; Table S2 provides a taxonomic
analysis and assessment of the level of contaminations in
the transcriptome; and Table S3 provides a summary of
the Nepenthes transcriptome annotation all in support of
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■
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Figure 2; Tables S4−S6 provide details in support of the
proteomics analyses summarized in Table 1 (XLSX)
AUTHOR INFORMATION
Corresponding Author
*Faculty of Medicine, University of Calgary, 3330 Hospital
Drive NW, Calgary, Canada, T2N 4N1. Ph: 403-210-3811. Email: [email protected].
Author Contributions
This manuscript was written with contributions from all
authors. All authors have approved the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The work was supported by an NSERC Discovery Grant
298351-2010 to D.C.S., who acknowledges the additional
support of the Canada Research Chair program, Alberta
Ingenuity−Health Solutions and the Canada Foundation for
Innovation.
■
ABBREVIATIONS
CDS, coding sequence; CID, collisionally induced dissociation;
CTAB, cetyltrimethylammonium bromide; EDTA, ethylene
diamine tetraacetate; emPAI, exponentially modified protein
abundance index; ETD, electron transfer dissociation; FASP,
filter-aided sample preparation; LC−MS/MS, liquid chromatography tandem mass spectrometry; MS, mass spectrometry;
Nep, nepenthesin; Npr, neprosin; ORF, open reading frame
■
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