Carnivorous Nutrition in

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Article
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
J. Proteome Res., Just Accepted Manuscript DOI: 10.1021/acs.jproteome.6b00224 Publication Date (Web): 20 Jul 2016
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Page 1 of 33 Journal of Proteome Research
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14 Carnivorous Nutrition in Pitcher Plants (Nepenthes spp.) via an Unusual
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17 Complement of Endogenous Enzymes
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20 Linda Lee, Ye Zhang, Brittany Ozar, Christoph W. Sensen, David C. Schriemer*
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28 Affiliations:
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31 Graz University of Technology, Institute of Molecular Biotechnology, Graz, Austria
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Christoph W. Sensen
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36 Department of Biochemistry and Molecular Biology and the Southern Alberta Cancer Research
37 Institute, University of Calgary, Calgary, Alberta, Canada
38 Linda Lee, Ye Zhang, Brittany Ozar, David C. Schriemer
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43 *corresponding author: D.C. Schriemer
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45 Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Canada, T2N 4N1. Ph:
46 403-210-3811 email: dschriem@ucalgary.ca
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54 Keywords:
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Nepenthes, transcriptomics, carnivory, mass spectrometry, fluid, enzymes
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ACS Paragon Plus Environment
Journal of Proteome Research Page 2 of 33
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Abstract
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6 Plants belonging to the genus Nepenthes are carnivorous, using specialized pitfall traps called
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8 pitchers that attract, capture and digest insects as a primary source of nutrients. We have used RNA
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sequencing to generate a cDNA library from the Nepenthes pitchers, and applied it to mass
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13 spectrometry-based identification of the enzymes secreted into the pitcher fluid, using a non-specific
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15 digestion strategy superior to trypsin in this application. This first complete catalog of the pitcher fluid
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18 sub-proteome includes enzymes across a variety of functional classes. The most abundant proteins
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20 present in the secreted fluid are proteases, nucleases, peroxidases, chitinases, a phosphatase and a
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22 glucanase. Nitrogen recovery involves a particularly rich complement of proteases. In addition to the
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25 two expected aspartic proteases, we discovered three novel nepenthensins, two prolyl endopeptidases
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27 that we name neprosins, and a putative serine carboxypeptidase. Additional proteins identified are
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relevant to pathogen-defense and secretion mechanisms. The full complement of acid-stable enzymes
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32 discovered in this study suggests that carnivory in the genus Nepenthes can be sustained by plant-based
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34 mechanisms alone, and does not absolutely require bacterial symbiosis.
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Introduction
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5 Carnivorous plants use specialized trapping structures to attract, capture, digest and absorb nutrients
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8 from insect prey and other sources of nitrogen, phosphorus and minerals.1-3 Trapping mechanisms
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10 include snap traps (e.g. Dionaea, also known as Venus flytrap), flypaper or adhesive traps (Drosera,
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Pinguicula), sucking bladder traps (e.g. Utricularia) and pitfall traps (e.g. Nepenthes). Plants belonging
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15 to the genus Nepenthes, also known as pitcher plants or monkey cups, are particularly intriguing. The
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genus consists of over 100 species found mostly in Southeast Asia and other tropical regions. The
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20 Nepenthes pitcher buds from the end of the leaf, and progressively forms a pitfall trap, consisting of
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22 three sections: a slippery upper rim called the peristome that is involved in attracting and trapping prey;
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24 a slippery, waxy inner wall to trap and prevent escape; and a bottom pit filled with an acidic
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27 viscoelastic fluid used to digest the trapped prey. Several structural and chemical variations of the trap
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29 seem adapted for a variety of non-invertebrate prey, including plant detritus and even tree-shrew
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feces.5, 6
The traps contribute a remarkably high percentage of the total nutrient requirement for the
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34 plant,7 and raise interesting questions regarding the processes involved, particularly those related to
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36 prey digestion.
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A common feature of all traps is the containment and presentation of the digestive fluid. Glands
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41 line the bottom section of the pitcher and perform numerous functions, including the detection of
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43 stimuli, absorption of nutrients, and the secretion of digestive enzymes and acid into the pitcher fluid.
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46 Attempts in recent years to identify and characterize the protein composition of the Nepenthes pitcher
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48 fluid yielded a surprisingly low number of enzymes. Based on this body of work, it appears that the
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50 most abundant proteins in the Nepenthes digestive fluid are Nepenthesins 1 and 2 (Nep1/2).8, 9 These
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53 are non-canonical aspartic proteases that exhibit broad cleavage specificity, high thermal stability, and
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55 optimal activities at pHs as low as 2.5.9-12 Aside from these two aspartic proteases, a handful of other
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proteins have been identified in pitcher fluid, including a -1,3-glucanase, a -D-xylosidase, chitinases,
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a peroxidase, pathogenesis-related protein PR-1, a thaumatin-like protein and a ribonuclease.13 Even
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6 this minimal composition begins to suggest a concerted mechanism for nutrient acquisition and
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8 inhibition of microbial pathogens.13-20
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Prior to modern proteomics methods, other enzymatic activities were either detected or
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13 suspected. Activities arising from cysteine proteases, phosphatases, esterases, oxidoreductases and
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15 lipases have been attributed to the Nepenthes fluid.8, 19-25 Nevertheless, the identity and source of most
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18 of these enzymes are not known. The Nepenthes fluid harbours a diverse microbial community, some
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20 of which may contribute enzymatic capacity to the fluid and form a symbiotic relationship with the
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22 plant, although their relative contribution to prey digestion is still under investigation.26-28 However,
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25 studies have questioned whether the bacterial load in the trap has a role to play in the processing of
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27 insect prey, prompting a closer inspection of fluid composition.
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Although proteomics methods have been applied to the fluid, the complement of proteins
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32 should not be considered complete. A thorough identification of all the proteins present in the
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34 Nepenthes digestive fluid is challenged by two major problems: the unusual amino acid composition of
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37 the proteins and the limited representation of carnivorous plants in the genomic/ protein databases.
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39 Many of the proteins identified in the Nepenthes digestive fluid, thus far, have a low frequency of
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41 Lys/Arg residues. These characteristics suggest that standard methods based on tryptic digestion may
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44 not be fruitful. Indeed, studies have resulted in low sequence coverage using conventional proteomic
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46 analyses.9, 13, 14, 29 Moreover, the identification of the proteins is complicated by the lack of a complete
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48 sequence of the Nepenthes genome and transcriptome. Although some transcriptomics resources are
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51 beginning to emerge, and used in a preliminary trypsin-based proteomics workflow,29 an alternative
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53 strategy is required to test for the completeness of the compositional analysis. To acquire a
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comprehensive insight into the enzymatic components secreted by the plant, we sequenced the N.
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rafflesiana transcriptome and coupled it to proteomic analysis of the fluid, using a combination of
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6 approaches. In addition to trypsin, we took advantage of the robust proteolytic capacity of the fluid
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8 itself, to increase the depth of coverage. Using these complementary approaches, we were able to
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confirm many of the known fluid proteins. We were also able to identify novel proteins that likely
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Experimental Methods
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5 RNA extraction
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8 N. rafflesiana giant plants were purchased from Urban Bog (Canada) and grown in a small terrarium in
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10 a 15:9 h light:dark photoperiod. The plants were fed with 1 or 2 Drosophila sp. per pitcher, the day
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before the pitchers were to be harvested for RNA extraction. Prior to harvesting the pitcher for RNA
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15 extraction, the digestive fluid was decanted and the pitcher was washed with deionized water to remove
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17 partially digested material and other debris. The bottom one-third of the pitcher containing the
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20 secretory glands was excised, frozen in liquid nitrogen and ground to a fine powder in the presence of
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22 liquid nitrogen. The total RNA was extracted using a modified CTAB protocol described by Meisel et
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24 al.30 In brief, the ground pitcher was lysed in warm CTAB extraction buffer (2% w/v cetrimonium
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27 bromide, 100 mM Tris-HCl pH 8, 20 mM EDTA, 1.4 M NaCl, 1% w/v polyvinylpyrrolidone MW
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29 40,000, 0.05% w/v spermidine trihydrochloride and -mercaptoethanol) and extracted several times
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with chloroform-isoamyl alcohol (24:1). The RNA was then precipitated from the aqueous phase with
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34 2.5 M LiCl, followed by chloroform extraction and precipitation in ethanol. The quality and integrity of
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36 the RNA was assessed on a 2200 TapeStation (Agilent) to have a RIN score > 8.
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Transcriptome Sequencing
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41 Illumina RNA sequencing of N. rafflesiana transcriptomes was performed by Omega Bioservices
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43 (Omega Bio-tek, Inc., Norcross, GA). RNA samples were processed using the Illumina TruSeq
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46 Stranded Total RNA Sample Prep kit (Illumina, San Diego, CA) following the manufacturers protocol.
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48 Briefly, 1 g of total RNA was treated with Ribo-Zero Plant reagents to remove the abundant
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cytoplasmic, mitochondrial and chloroplast ribosomal RNA. First strand cDNA was reverse transcribed
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53 using random primers, followed by second strand synthesis. Indexing adaptors were ligated to the
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55 dsDNA ends to generate Illumina sequencing libraries. The libraries were PCR amplified, normalized
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58 and pooled. Paired-end 100 bp sequencing was performed on an Illumina HiSeq 2500 sequencer.
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Transcriptome assembly and analysis
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6 Cleaning filters were applied to the raw sequencing data files, based on FastQC quality control
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8 statistics, and Illumina sequencing adaptors detected with Cutadapt. Reads were clipped using
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TrimmomaticPE. Reads corresponding to contaminant genomes such as all known bacteria,
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13 Tetrahymena thermophila, Fusarium graminearum and Drosophila melanogaster were removed from
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15 the transcriptome assembly. De novo transcript assembly was performed with Trinity (v2.0.6) at the
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18 default kmer size of 25 and a cutoff contig length of 200 bp. Contigs that were expressed were
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20 considered for CDS prediction in all six reading frame using Transdecoder (v2.0.1). The translated
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22 database was filtered for polypeptide >150 amino acids in length and initial annotation was based on
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25 comparison with NCBI nt, NCBI nr and SwissProt databases using Tera-Blast with an e-value cutoff of
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27 e-15. The highest scoring hit was used for the annotation.
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Plant and fluid processing
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32 N. ventrata (100 plantings in 8 pots) were grown in a dedicated greenhouse (Urban Bog, Langley, BC,
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34 Canada). On pitcher maturity, the plants were fed with 1 or 2 Drosophila per pitcher and the digestive
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37 fluid was harvested the following week as previously described.31 In brief, the fluid from
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39 approximately 1000 N. ventrata pitchers were collected, pooled, clarified through a 0.2 m filter to
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remove debris, concentrated 10-fold using an Amicon Ultra 10 kDa molecular weight cutoff centrifugal
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44 filter (Millipore), and washed with 100 mM glycine-HCl pH 2.5. The fluid was stored at 4oC for short-
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In-gel fluid proteomics analyses
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51 The N. ventrata digestive fluid was analyzed on an 8% SDS-PAGE gel and silver stained with
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53 SilverSNAP stain for mass spectrometry applications (Thermo Scientific, Rockford, USA) following
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56 the manufacturers protocol. Visible protein bands were excised, destained in 50 mM sodium
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thiosulfate and 15 mM potassium ferricyanide, reduced with 10 mM DTT followed by alkylation with
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6 50mM iodoacetamide in 100 mM ammonium bicarbonate pH 8. The gel slices were then digested with
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8 trypsin (Promega; enzyme to substrate ratio of 1:20) at 37oC overnight followed by quenching with
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0.1% trifluoroacetic acid. The digests were desalted on a C18 ZipTip (Millipore) prior to analysis by
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13 data-dependent LC-MS/MS on an Agilent 6550 iFunnel-QTOF mass spectrometer outfitted with an
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15 1260 Infinity chip cube interface, using a 25 minute reversed phase gradient run (high-capacity chip:
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18 Agilent Zorbax 300SB-C18 5 micron particles, 150 m x 75 m, using 0.1% formic acid in 3%
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20 acetonitrile for mobile phase A and 0.1% formic acid in 97% acetonitrile for mobile phase B; flow rate
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of 300 nL/min and a 2%B/min linear gradient, to 50% B). The MS/MS was configured using CID for
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25 precursor ions in the m/z range between 275 and 1700, and top-10 ion selection with a charge +2 and
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27 higher. The optimum collision energy was calculated for each precursor based on its charge and mass.
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30 Ion exclusion was released after 1 spectrum and 0.2 min, to avoid re-acquiring MS/MS data for the
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32 same precursor. The data were searched against the 6-frame translation of our N. rafflesiana
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34 transcriptomes databases using Mascot. The Mascot search parameters for the tryptic digested samples
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37 were: maximum of one missed cleavage, carbamidomethyl (C) as fixed modification, methionine
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39 oxidation as a variable modification, a 20 ppm peptide mass tolerance, a 0.2 Da fragment mass
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tolerance. Output was filtered for peptide hits with p < 0.05 (ion score > 24).
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44 Gel-free fluid proteomic analyses
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46 To analyze the proteome of the N. ventrata pitcher fluid, the fluid was digested either with trypsin
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(specific digest) or N. ventrata fluid (non-specific digest) using a modified FASP protocol.32 Briefly,
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51 15 g of 10X concentrated N. ventrata digestive fluid was reduced with 100 mM DTT followed by
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alkylation with 50 mM iodoacetamide under denaturing condition (8 M Urea, 100 mM Tris-HCl pH 8)
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56 on a YM-10 microcon (Millipore). For digestion with trypsin, the reduced/ alkylated fluid was buffer
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exchanged to 50 mM ammonium bicarbonate pH 8 and digested with trypsin (Promega; enzyme to
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6 substrate ratio of 1:100) at 37oC overnight followed by quenching with 0.1% trifluoroacetic acid. For
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8 non-specific digestion, the reduced/ alkylated fluid was washed with 100 mM Gly-HCl pH 2.5 and
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digested with N. ventrata fluid (1:30 enzyme:substrate) at room temperature for 40 minutes. The
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13 digests were desalted on a C18 ZipTip (Millipore) prior to analysis by data-dependent LC-MS/MS on
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15 an Orbitrap Velos ETD (Thermo Scientific). The samples were analyzed twice, using 90 minutes
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18 reversed phase gradient runs, configured for top-10 ion selection using CID in a high/low
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20 configuration. One run focused on peptides with a charge state of 1+, whereas the other run focused on
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22 peptides with a charge state of 2+ and higher. MS scans were acquired over the m/z 3501800 range
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25 with a resolution of 60,000 (m/z 400). The target value was 5.00E+05. For MS/MS, ions were
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27 fragmented in the ion trap with normalized collision energy of 35%, activation q = 0.25, activation time
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of 10 ms, and one microscan. The target value was 1.00E+04 .Separation were achieved using a self-
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32 packed pico-frit column (New Objective, packed with Phenomenex 5 m Jupiter C18 beads, 100 mm x
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75 m), using 0.1% formic acid in 3% acetonitrile for mobile phase A and 0.1% formic acid in 97%
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37 acetonitrile for mobile phase B; flow rate of 300 nL/min and a 0.5%B/min linear gradient, to 45% B).
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39 The data were searched against the 6-frame translation of our N. rafflesiana transcriptomes databases
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42 using Mascot. The search parameters for the tryptic digested samples were: one missed cleavage
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44 allowed, carbamidomethyl (C) as fixed modification, methionine oxidation (M) as a variable
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46 modification, a 10 ppm peptide mass tolerance, a 0.6 Da fragment mass tolerance, and the output
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49 filtered with a false discovery rate of 1% (peptide hits with p < 0.00045, an ion score > 42). The search
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51 parameters for the fluid-digested samples were similar to those for the tryptic sample, except the search
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was configured for no enzyme specificity and the output set filtered with a false discovery rate of 1%
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(peptide hits with p < 0.009, an ion score > 49). In cases where shorter partial ORF were contained
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6 within or overlap with another transcript, the longer assembled transcript was used for characterization.
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Results
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5 Transcriptome of the stimulated Nepenthes pitcher
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8 To support the proteomics-based identification of the protein composition of Nepenthes digestive fluid,
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10 we performed Illumina RNA-sequencing to generate a comprehensive database of the Nepenthes
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transcriptome (Figure 1). We extracted the RNA from the bottom third of a N. rafflesiana giant pitcher,
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15 where the secretory glands are located,4 which had been stimulated by feeding with fruit flies to reduce
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17 fluid pH and promote the digestive state.15, 33
Because of the open nature of the Nepenthes pitcher,
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20 bacteria and fungi as well as fruit flies could be present in the pitcher. However, read pairs
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22 corresponding to these contaminating genomes accounted for less than 1% of the total read set (Figure
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24 S1, Table S1), with proteobacteria being the most dominant phylum (Figure 2). After the raw data were
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27 cleaned, including the removal of the contaminating genomes, the transcriptome was assembled from
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29 154 million paired-end reads of 100 bp length. A total of 1.36 million contigs > 200 bp were assembled
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with an average length of 665 (Table S1). When the assembled transcriptome was filtered for contigs >
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34 300 bp, the number of total contigs was significantly reduced to 335K with an average length of 1329
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36 bp. The high number of contigs, especially those < 300 bp, may arise from small, noncoding RNA
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and/or errors in assembly. Nevertheless, we maintained the cutoff at 200 bp for this stage of analysis.
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41 Searching our transcriptome with tRNAscan did not find any significant tRNA hits, and alignment
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43 against the Arabidopsis intronic-intergenic database yielded minimal results, indicating that our
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46 transcriptome was enriched for mRNA as expected. When we included expression as a criterion, we
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48 reduced the number of contigs to 532,208 contigs (>200 bp set). Although only 31% of the expressed
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50 contigs could be annotated using the NCBI nucleotide database, these accounted for nearly half of the
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53 total number of reads (Table S3). The unannotated contigs accounted for only ~12% of total read set,
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55 suggesting that these may be present at very low levels (and/or arise from error in assembly).
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Taxonomical annotation of the contigs using MEGAN revealed that well over half of the transcripts
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were plant-based (Streptophyta) with some minor contamination from bacteria, fungi and fruitfly
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6 (Figure 2, Table S2), indicating that our method enriched for Nepenthes transcripts. To enrich for long
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8 transcripts and enable an effective proteomics experiment, we filtered the translated ORFs for
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polypeptides > 150 aa in length, which generated 66,459 ORFs for search.
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13 Proteome of the Nepenthes digestive fluid
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15 Visualization of the stimulated N. ventrata digestive fluid via silver-stained SDS-PAGE showed the
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18 limited complexity of the fluid, in keeping with previous work (Figure 3). When searched against our
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20 assembled N. rafflesiana transcriptome, mass spectrometric data arising from in-gel tryptic digestions
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22 of the visible protein bands were able to identify major proteins present in the digestive fluid (Figure
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25 3), illustrating the utility of our cDNA library. To increase the sensitivity of our proteomic approach,
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27 we collected and concentrated large volumes of stimulated N. ventrata fluid and analyzed it using gel-
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free methods. Here, gel-free tryptic digestion of the fluid followed by long-gradient LC-MS/MS
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32 identified 26 proteins in the fluid. Sequence coverage was still low, with only 1-4 identified peptides
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34 matching to each protein (Table S4); these results are similar to a recent study.29 As noted, the low
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37 level of coverage could arise from a non-optimal peptide set, due to the low frequency of lysine and
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39 arginine residues present in the majority of the proteins identified to date. For example, nepenthesin 2
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41 is relatively abundant in the digestive fluid,9, 13, 34 but it was not detected in the tryptic digest sample,
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44 due to the absence of K/R residues in its mature active form. Deglycosylation of the protein sample did
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46 not improve coverage (not shown).
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48 To circumvent the restrictions presented with trypsin, we digested denatured N. ventrata fluid
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51 with active N. ventrata fluid. The high activity of the fluid, and its value in generating high sequence
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53 coverage in hydrogen/deuterium exchange MS experiments, argued for the attempt. At our current
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level of characterization, the fluid minimally contains aspartic proteases with broad cleavage
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specificity.11, 12, 34 Upon LC-MS/MS of the nonspecific digested fluid, we obtained substantially higher
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6 coverage, with as many as 70 matching peptides for the most abundant proteins. We identified 19
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8 proteins using this approach (Table S5). Nine proteins are common with those identified in the tryptic
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digestion method, therefore in total, 36 proteins were identified in the N. ventrata fluid from the
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13 combination of tryptic and nonspecific digestion approaches (Table 1). The identified proteins were
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15 functionally annotated by homology search against NCBI non-redundant (nr) protein sequence
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18 database, or characterized biochemically. To assess relative abundance of the identified proteins based
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20 on label-free methods, such as emPAI, the tryptic data is particularly unreliable given the paucity of
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22 detectable peptides. However, the emPAI values from the non-specific digests are useful, given much
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25 larger peptide set generated. The ranked list in Table S5, therefore, approximates the order of
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27 abundance.
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Our improved digestion strategy presents a more complete analysis of fluid proteome, when
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32 compared to a recent study using only trypsin.29 We identified approximately 25% more proteins, some
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34 of which are novel. Globally, the majority of the identified pitcher proteins have properties expected of
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37 a secreted protein functioning in an acidic fluid. Over 70% of the identified fluid proteins have a pI < 6,
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39 consistent with the acidic nature of the digestive fluid (Table 1 and Table S6). The pI is predicted from
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41 the deduced unprocessed transcript, and may not reflect that of the mature active enzymes. Acid
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44 activation of the well-characterized Nep1 and Nep2 involves autolysis at the N-terminus,35 yielding a
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46 mature enzyme with a lower pI than the proenzyme. Similar post-translational processing events may
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48 also occur for other fluid proteins, possibly resulting in even lower pI values. Consistent with the fact
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51 that the fluid proteins are secreted into the pitcher, two-third of the identified proteins with an intact N-
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53 terminus were predicted by SignalP 4.1 to have a signal peptide.
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More specifically, the fluid proteome can be organized into a number of functional classes and
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6 sub-classes that aid in evaluating the mechanisms of carnivory:
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8 Proteases - The most abundant proteins in the Nepenthes fluid are Nep1 and Nep2, which exhibit
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>90% homology to Nep1 and Nep2, respectively, from other Nepenthes species. Nep1 and Nep2 are
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13 non-canonical aspartic proteases with broad cleavage specificity that are stable over a wide temperature
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15 range.9-12 In addition to Nep1 and Nep2, we discovered three additional nepenthesin homologs, which
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18 we name Nep3, Nep4 and Nep5 (Figure 4). Nep3-5 exhibit approximately 50% homology to each other
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20 as well as to Nep1 and Nep2, indicating they are distinct, novel aspartic proteases. Our preliminary
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22 sequencing of the N. ampullaria transcriptome revealed the presence of a Nep5 transcript that is 98%
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25 identical to N. rafflesiana Nep5 (Figure 4A and unpublished data), suggesting that these novel
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27 nepenthesins may also be present in other Nepenthes species. Like Nep1 and Nep2, Nep3-5 all contain
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the conserved catalytic aspartate residues, and the 12 cysteine residues involved in disulfide bridges
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32 (Figure 4B). The catalytic aspartate residues in these five nepenthesins, as well as those from other
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34 Nepenthes species, reside within two highly conserved regions: AIMDTGSDLIWTGQ (aa110-123)
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37 and IDSGTT (aa314-319; numbering based on Nep1, with the catalytic aspartate underlined). These
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39 regions are more variable and less extensive in other aspartic proteases, such as pepsin. Another
40
41 distinguishing features of the nepenthesins is the variable ~20-24 aa nepenthesin-type aspartic protein
42
43
44 (NAP)-specific region, which contains four of the conserved cysteine residues.9 Nep1-5 are different
45
46 from the five aspartic proteases AP1-5 reported by An et al.,36 which contain a plant-specific insert of
47
48 about 100 aa (instead of the short NAP-specific insert), commonly found in vacuolar plant aspartic
49
50
51 proteases (vAP, Figure S2). Our N. rafflesiana transcriptome does contain several transcripts
52
53 homologous to these vacuolar aspartic proteases (not shown), although we did not detect these vAPs in
54
55
56
the Nepenthes digestive fluids. Nep3-5 appear to be present in the fluid in lower amounts than Nep1
57
58
59
60 14
1
2
3
and Nep2 (Table S5), however the relative activity and contribution of Nep3-5 to the digestive process
4
5
6 remain to be investigated.
7
8 We have also identified a novel class of prolyl-endoprotease in the fluid. We previously
9
10
11
reported that the Nepenthes fluid can also cleave C-terminal to proline residues, but neither Nep1 nor
12
13 Nep2 possessed such prolyl-endopeptidase activity.12, 31, 37
We recently purified a fluid fraction
14
15 containing the prolyl-endopeptidase activity and identified a novel protease that we called neprosin 1
16
17
18 (Npr1).38 In addition to Npr1, we also detected in our present study a partial transcript corresponding to
19
20 a neprosin homolog, which we call Npr2, but it remains to be seen if it also processes prolyl-
21
22 endopeptidase activity. Both Npr1 and Npr2 are distinct from known proline-cleaving enzymes, each
23
24
25 consisting of two domains of unknown function (DUF4409 and DUF239). DUF4409 and DUF239
26
27 family members are found in many plant species, but their biological function has not been
28
29
characterized. The presence of a serine carboxypeptidase completes the list of proteases identified in
30
31
32 the Nepenthes digestive fluid. The relatively high abundance of this enzyme (Table S5) suggests that it
33
34 is significant in prey digestion and nutrient extraction. It belongs to the S10 family of serine proteases,
35
36
37 which are active at low pH.39
38
39 Nucleases We discovered two nucleases and an acid phosphatase in the Nepenthes pitcher fluid. The
40
41 predominant nuclease appears to be a secreted S-like ribonuclease that is >95% identical to those
42
43
44 previously isolated from N. bicalcarata19 and N. ventricosa.20 Like many of the acidic fluid enzymes,
45
46 the N. bicalcarata ribonuclease was found to be active at an optimal pH of 3.5.19 We also newly
47
48 identified an S1-like endonuclease and an acid phosphatase, both of which are likely present at
49
50
51 moderate levels in the fluid (Table S5). The S1-like endonuclease exhibits approximately 50%
52
53 homology to other plant endonuclease-2 enzymes, which can hydrolyze single-stranded nucleic acids.40
54
55
56
The acid phosphatase is closest to the purple acid phosphatases, a metallophosphoesterase found in a
57
58
59
60 15
1
2
3
variety of plants, which is upregulated during phosphate deficiency to scavenge phosphate by
4
5
6 hydrolyzing various phosphate esters.41
7
8 Glycoside hydrolases and pathogenesis-related proteins The three glycoside hydrolases (-1,3-
9
10
11 glucanase, class III and IV chitinases), thaumatin-like protein and two cationic peroxidases that we
12
13 identified are similar to those characterized previously in other Nepenthes species.13, 14, 18 Glycoside
14
15
hydrolases are involved in the digestion of polysaccharides, such as chitin found in an insect
16
17
18 exoskeleton as well as in the cell wall of pathogens and plant debris. We discovered a protein
19
20 containing a dopamine -monooxygenase domain (DOMON, a heme- and sugar-binding motif found in
21
22
23
some cytochrome and glycosyl hydrolases),42, 43 as well as hevein, a chitin-binding domain associated
24
25 with antifungal activity.44, 45 Both are likely involved in aspects of glycan recognition and play a role in
26
27 plant defense against pests and fungal infection. The two cationic peroxidases as well as a third
28
29
30 peroxidase that we discovered may contribute to a host defense against pathogens involving the
31
32 generation of highly-reactive oxygen species (ROS).
33
34 Finally, we discovered two Leu-rich repeat (LRR) proteins, one of which appears to be a
35
36
37 receptor-like protein kinase. LRR is a common motif of 20-30 amino acids rich in leucines, present in
38
39 tandem repeats that mediate protein-protein interactions. LRR receptor-like protein kinases have been
40
41
reported to be involved in plant development, germination and pathogen resistance.46 Two of the
42
43
44 remaining proteins identified correspond to the and subunits of ATP synthase/ H+-ATPase and may
45
46 function as a proton-pump for acidification of the pitcher fluid.33
47
48
49
50
51
52
53
54
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56
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59
60 16
1
2
3
Discussion
4
5
6 Carnivorous plants, such as Nepenthes, obtain a large fraction of their nutrients from the digestion of
7
8 captured prey and other organic matter. We have sequenced the Nepenthes rafflesiana transcriptome to
9
10
11
facilitate the identification of the enzymes involved in Nepenthes carnivory, and searched the resulting
12
13 cDNA library with proteomics data generated conventionally (using trypsin-based methods) and by an
14
15 alternative bottom-up strategy (using the proteases contained in the pitcher plant fluid). To our
16
17
18 knowledge, this study is the first application of the Nepenthes transcriptome to a detailed proteomic
19
20 analysis of the Nepenthes digestive fluid. Although a very recent study has attempted to mine tryptic
21
22 peptides using de novo MS/MS sequencing methods, the individual peptide sequences determined in
23
24
25 this fashion were searched against a cDNA library using BLAST.29 The procedure used does not appear
26
27 to retain a statistical foundation sufficient to assess the probability of the many proteins identified with
28
29
only one or two peptides, and misidentifications are possible. For example, nepenthesin-2 is noted as
30
31
32 identified with 3 tryptic fragments, but there are no K and R in the mature enzyme sequence in almost
33
34 every species reported.
35
36
37 In our study, we have identified 36 proteins in a direct search of the cDNA library, which we
38
39 prepared from RNA-seq data, 10 of which were uniquely determined using the non-specific digestion
40
41 approach. It is clear that protein digestion represents the most aggressively targeted functionality in
42
43
44 steady-state pitcher fluid. Five nepenthesins (three of which are novel: Nep 3-5) and two neprosins
45
46 (both novel: Npr1,2) combine to provide a potent proteolytic digestion capacity to the fluid. Both
47
48 represent intriguing departures from their core enzymatic classifications, as noted in recent studies from
49
50
51 our labs.31, 37
The newly-discovered variants should prove valuable as reagents in proteomics
52
53 applications. The relatively abundant acid-stable serine carboxypeptidase, in particular, suggests a
54
55
56
useful laddering enzyme to aid in peptide sequencing, and will be pursued.
57
58
59
60 17
1
2
3
We summarize the putative functions of the identified Nepenthes fluid proteins in a model for
4
5
6 the mechanism of carnivory (Figure 5). The functions of the hydrolytic enzymes in the fluid are simple
7
8 to infer. Glucanases and chitinases digest the cell wall and exoskeleton of captured prey and plant
9
10
11
debris. Digestion of the prey proteins and nucleic acids by the proteases, nucleases and phosphatases
12
13 supply nitrogen and phosphate for absorption by the plant. Many of the hydrolytic enzymes involved in
14
15 prey digestion also possess anti-bacterial and/or anti-fungal properties, since they can digest pathogens
16
17
18 and hence can be considered pathogenesis-related proteins in themselves.4 The association between
19
20 carnivory and pathogenesis is further supported by the presence of several specifically pathogenesis-
21
22 related proteins such as the peroxidases, hevein (a chitin-binding protein), thaumatin-like protein, and
23
24
25 LRR proteins. The peroxidases likely contribute to host defense against pathogens by generating ROS.
26
27 It has been proposed that oxidation of prey proteins by peroxidase-generated ROS can enhance their
28
29
susceptibility to degradation by proteases.47 Hevein domains bind chitin and display antifungal
30
31
32 activity.48 Thaumatin-like protein likely serves a similar antifungal role,49 although we note that
33
34 thaumatin is a highly potent natural sweetener and this protein may serve as a chemoattractant to insect
35
36
37 prey.50 The functions of the other proteins in the fluid are low abundance and more difficult to ascertain
38
39 with confidence. The Leu-rich repeat (LRR) receptor-like protein kinase (RPK1) could stimulate the
40
41 secretion of enzymes into the cavity of the pitcher, upon sensing the presence of prey or pathogen, and
42
43
44 the membrane-bound H+-ATPase may function as a proton-pump for acidification of pitcher fluid.33
45
46 Both proteins are membrane-associated. They are somewhat surprising to identify in the fluid, but may
47
48 represent shed domains. The remaining proteins are involved in energy metabolism, protein transport,
49
50
51 protein expression and synthesis (Table 1). They could represent intracellular plant proteins, since
52
53 many of them lack a signal peptide, or elements of the microbial community that share stretches of
54
55
56
sequence identity with entries in the Nepenthes transcriptome.
57
58
59
60 18
1
2
3
An overlapping set of proteins were observed by Schulze and colleagues in the acidic digestive
4
5
6 fluid of the Venus flytrap,51 suggesting that these two different carnivorous plants use similar
7
8 mechanisms for prey digestion. However, the major proteases in the Venus flytrap secretions are
9
10
11
cysteine proteases, whereas the main proteases in Nepenthes are aspartic proteases. Cysteine protease
12
13 activity has been suggested to be present in the Nepenthes fluid, as activity was inhibited by a cysteine
14
15 protease inhibitor E-64.20 Although numerous transcripts corresponding to putative cysteine proteases
16
17
18 are present in our cDNA library, we did not detect any in the Nepenthes fluid. It remains possible that
19
20 such determinations are species dependent, however we note that a high degree of consistency has
21
22 emerged in the identification of several enzymes across many varieties (aspartic proteases, glucanases,
23
24
25 and chitinases, for example). The enzymatic properties do not appear to vary significantly.
26
27
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60 19
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Conclusions
4
5
6 Our improved proteomics workflow did not identify other proteins that have been reported to be
7
8 present in the Nepenthes fluid, and native to the plant. Pathogenesis-related protein 1 (PR-1) and an
9
10
11
oxidoreductase14, 16 were detected, but their scores were below our significance threshold. We could
12
13 detect no lipases, xylosidases and galactosidases.29 The fluid represents a steady-state harvest, therefore
14
15 either these enzymes are transient members produced during fluid maturation or, more likely, were
16
17
18 contributed by the plant microbiome. Although the acidity and antimicrobial properties of the
19
20 Nepenthes fluid suppress the growth of pathogens, the fluid still hosts a diverse microbial community,
21
22 which may form a mutualistic relationship with the host plant.26, 28 A recent metagenomics study of the
23
24
25 Nepenthes microbiome revealed as many as 18 bacteria phyla, with proteobacteria representing the
26
27 predominant class.26 This bacterial class was also found in our pre-filtered transcriptome (Figure 2).
28
29
Bacteria isolated from Nepenthes fluid have been reported to possess protease, chitinase and lipase
30
31
32 activities,23, 26
however it remains an open question whether these contribute meaningfully to
33
34 mechanisms of carnivory. It is possible that bacterial colonization is mostly parasitic in nature; the
35
36
37 topic requires further study. If the unusually broad and robust activity profile of the nepenthesins is any
38
39 indication, the other acid-stable enzymes may present an equally broad substrate profile, sufficient to
40
41 supply amino acids, sugars and phosphate from a range of sources. Based on our extended proteomics
42
43
44 analysis, it would appear that the panel of endogenous plant enzymes is sufficient to recover the
45
46 nutrients needed to support growth.
47
48
49
50
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60 20
1
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Figures
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48 Figure 1. Transcriptomics and proteomics workflow applied to Nepenthes spp. Transcriptomics was
49
50 applied to Nepenthes rafflesiana pitcher tissue and proteomics was applied to Nepenthes ventrata
51
52
53 pitcher fluid. Photographs courtesy of Linda Lee. Copyright 2016.
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60 21
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30 Figure 2. Taxonomic organization of the N. rafflesiana transcriptome, providing an overview of the
31 taxonomy and level of contamination present, based on annotation against the NCBI nt database (e-
32 value cutoff of e-15).
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34
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21 Figure 3. Silver-stained SDS-PAGE of N. ventrata digestive fluid, showing the excised bands and the
22 major proteins identified by in-gel tryptic digestion and mass spectrometry.
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41 Figure 4. Comparative analysis of the five nepenthesins discovered, based on sequence. A) Phylogenic
42 tree of the nepenthesins identified in our N. rafflesiana transcriptome analysis (in bold) with the
43 nepenthesins from other Nepenthes species (including an unpublished nepenthesin sequence from N.
44 ampullaria). The percentage sequence identity relative to N. rafflesiana Nep1 is indicated in brackets.
45
46 The sequence identity within the Nep2 subgroup is ~90%. B) Sequence alignment of N. rafflesiana
47 Nep1-5. The vertical lines indicate the signal peptide (SP) cleavage site predicted by SignalP 4.1, and
48 the proenzyme cleavage site to produce the mature active proteases. The conserved region surrounding
49 the catalytic aspartate residues (*) are boxed. The conserved cysteines (^) are involved in disulfide
50 bridges. NAP, nepenthesin-type aspartic protease-specific insertion. The alignment and phylogeny tree
51
52
were produced using ClustalW.
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37 Figure 5. Model for the mechanism of carnivory in Nepenthes. The enzymes identified in this study are
38 highlighted in red, while enzymes suggested by other studies are in blue.15, 23 Receptors lining the
39 pitcher detect the presence of prey or pathogens, resulting in the secretion of digestive enzymes into the
40 cavity of the pitcher. Glucanases and chitinases digest the cell wall and exoskeleton of captured prey
41
42
and pathogens. Digestion of the prey proteins and nucleic acids by the proteases, nucleases and
43 phosphatases release feedstocks for the nitrogen and phosphate requirements of the plant. Lipases,
44 possibly from the microbiome, facilitate the digestion of lipids. The peroxidases may contribute to the
45 host defense against pathogen by generating ROS. The ROS can also oxidize prey proteins, which may
46 cause them to be more susceptible to degradation by proteases.
47
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60 25
1
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Table
6
7
8 Table 1. Combined list of the identified proteins from the secreted Nepenthes fluid.
9
10 Signal Dig.1 Dig.2
11 Process Protein Class/Function MW pI Ref.
Pep.?
12 Protein Nepenthesin-1, Aspartic protease 47106 4.7 Yes 2 1 9
13 metabolism Nep1
14
Nepenthesin-2, Aspartic protease 46625 4.1 Yes 2 9
15
Nep2
16
Nepenthesin-3, Aspartic protease 49092 4.9 Yes 3 8
17
18 Nep3
19 Nepenthesin-4, Aspartic protease 48991 4.8 Yes 23 6
20 Nep4
21 Nepenthesin-5, Aspartic protease 48811 4.6 Yes 9
22 Nep5
23 Neprosin-1, Prolyl endoprotease 43161 6.3 Yes 7
24 Npr1
25 Neprosin-2, Prolyl endoprotease >33kDa 5.2 N/A* 10
26 Npr2
27 Serine carboxy- S10 acidic >47kDa 4.3 Yes 5 5
28 peptidase, SCP1 peptidase
29
Nucleic Purple acid Metallophosphatase 71268 5.2 Yes 1 4
30
acid phosphatase,
31
metabolism PAP1
32
33 Ribonuclease, Secreted RNase/ 26224 4.1 Yes 3 19,
34 S-like, RNaseS scavenge 20
35 phosphates
36 Endonuclease 2, S1-P1 nuclease/ 32487 4.9 Yes 14 12
37 Endo2 cleave RNA & ssDNA
38 Polysaccharide -1,3-Glucanase, Glycoside hydrolase 36752 4.7 Yes 6 11 14
39 metabolism Glu1 17/ pathogen defense
40
41 Acidic chitinase, Glycoside hydrolase 31086 4.1 Yes 12 15 14,
42 Chit3 18/ class III chitinase/ 18,
43 pathogen defense 52
44 Chitinase, Glycoside hydrolase 29306 4.5 Yes 13 13,
45
Chit1 19/ class IV chitinase/ 53
46
pathogen defense
47
DOMON-like Heme-binding motif/ 27995 5.5 Yes 9
48
49 domain, Dom1 glycoside hydrolases
50 Pathogenesis/ Thaumatin-like Antifungal activity/ 23785 5.7 Yes 4 13 13
51 Host defense protein, TLP1 pathogen defense
52 Hevein , Chitin binding/ 20752 6.4 Yes 10
53 Hev1 antifungal activity
54 Peroxidases Cat. peroxidase 1, Heme-containing/ 33598 6.4 Yes 8 14
55 Prx1 oxidation
56 Cat. peroxidase 2, Heme-containing/ 33385 4.6 Yes 21 14
57
Prx2 oxidation
58
59
60 26
1
2
3 Peroxidase, Heme-containing/ 36354 4.7 Yes 16
4 Prx3 oxidation
5
Signal Leu-rich repeat Ser/ Thr Kinase/ 66702 5.4 Yes 14
6
transduction protein kinase, plant dev./
7
RPK1 pathogen
8
9 resistance
10 Leu-rich Possible protein 69848 4.8 No 17
11 repeat protein, kinase/ribonuclease
12 LRR1 inhibitor
13 Energy ADP/ ATP ADP/ATP >31kDa 10. N/A* 11
14 metabolism translocase, exchange 0
15 ADT1
16 Glyceraldehyde- NAD-binding/ 36664 7.1 No 18
17 3-phosphate glycolysis/
18 dehydrogenase, glyconeogenesis
19 GPD1
20 ATP synthase/ ATP synthesis/ >18kDa 5.3 No 20
21 +
H -ATPase membrane-bound
22 proton-pump
subunit , ATPA
23
ATP synthase/ ATP synthesis/ >51kDa 4.9 No 22
24
H+-ATPase membrane-bound
25
26 subunit , ATPB proton-pump
27 Enolase, 2-phospho-D- >30kDa 4.9 N/A* 25
28 Eno1 glycerate conversion
29 Protein Actin, cytoskeleton 41706 5.3 No 15
30 transport Act1
31 Tic20-like chloroplast 31449 9.4 No 16
32 protein, import protein
33 Tic20
34 Protein Vesicle trafficking/ 34078 9.2 No 26
35 transporter secretion
36 Sec1
37 Translation/ Elongation GTP-binding 49815 9.2 No 7
38
protein factor 1-alpha, elongation
39
synthesis EF1a factor
40
41 Elongation GTP-dependent 95948 6.1 No 24
42 factor 2, EF2 ribosomal translocation
43 40S Ribosomal Protein synthesis 16367 10. No 17
44 protein S14, RS14 6
45 Chaperone Protein folding 91593 5.0 Yes 19
46 protein Hsp90
47 Transcription/ Histone Acetylate >86kDa 6.0 No 18
48 Gene acetyltransferase, transcriptional
49 regulation HAC1 regulators, histones
50 Auxin response Transcription factor/ >25kDa 8.5 N/A* 19
51
factor, ARF1 Auxin response factor
52
53 Trypsin diges@on. Ranking is based on the MASCOT search score.
54 Nonspecic diges@on using ac@ve Nepenthes fluid. Ranking based on MASCOT search score.
55 *N/A, not applicable because protein fragment has a truncated N-terminus.
56
57
58
59
60 27
1
2
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4
5
6
Associated Content
7
8 Supporting information
9
10 Table S1 provides a summary sequence analysis of the N. rafflesiana transcriptome, Table S2 provides
11
12
13 a taxonomic analysis and assessment of the level of contaminations in the transcriptome, and Table S3
14
15 provides a summary of the Nepenthes transcriptome annotation, all in support of Figure 2. Tables S4-
16
17 S6 provide details in support of the proteomics analyses, summarized in Table 1. Figure S1 provides an
18
19
20 overview of the transcriptome analysis procedures, in support of Figure 1. Figure S2 provides a
21
22 phylogenetic analysis of nepenthesins relative to vacuolar aspartic proteases. List S1 contains the set of
23
24
amino acid sequences for the proteins identified in N. rafflesiana digestive fluid.
25
26
27
28
29 Author Information
30
31
32
Corresponding author
33
34 Telephone: 403-210-3811
35
36 e-mail: dschriem@ucalgary.ca
37
38 Author Contributions
39
40 This manuscript was written with contributions from all authors. All authors have approved the final
41
42 version of the manuscript.
43
44
45 Conflict of Interest
46
47 The authors declare no competing financial interest
48
49
50
Acknowledgments
51
52 The work was supported by an NSERC Discovery Grant 298351-2010 (DCS). DCS acknowledges the
53
54 additional support of the Canada Research Chair program, Alberta Ingenuity - Health Solutions and the
55
56
57 Canada Foundation for Innovation.
58
59
60 28
1
2
3
Abbreviations
4
5
6 CDS, coding sequence; CID, collisionally-induced dissociation; CTAB, cetyl trimethylammonium
7
8 bromide; EDTA, ethylene diamine tetraacetate; emPAI, exponentially modified protein abundance
9
10
11
index; ETD, electron transfer dissociation; FASP, filter aided sample preparation; LCMS/MS, liquid
12
13 chromatography tandem mass spectrometry; MS,mass spectrometry; Nep, nepenthesin; Npr, neprosin;
14
15 ORF, open reading frame.
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