Carnivorous Nutrition in Pitcher Plants (Nepenthes spp.) via an

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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 Downloaded from http://pubs.acs.org on July 21, 2016

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Journal of Proteome Research

Carnivorous Nutrition in Pitcher Plants (Nepenthes spp.) via an Unusual Complement of Endogenous Enzymes Linda Lee, Ye Zhang, Brittany Ozar, Christoph W. Sensen, David C. Schriemer*

Affiliations:

Graz University of Technology, Institute of Molecular Biotechnology, Graz, Austria Christoph W. Sensen

Department of Biochemistry and Molecular Biology and the Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada Linda Lee, Ye Zhang, Brittany Ozar, David C. Schriemer

*corresponding author: D.C. Schriemer Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Canada, T2N 4N1. Ph: 403-210-3811 email: [email protected]

Keywords: Nepenthes, transcriptomics, carnivory, mass spectrometry, fluid, enzymes

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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 non-specific digestion strategy superior to trypsin in this application. This first complete catalog of the pitcher fluid sub-proteome 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.


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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.

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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 adapted for a variety of non-invertebrate prey, including plant detritus and even tree-shrew feces.5,

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The traps contribute a remarkably high percentage of the total nutrient requirement for the

plant,7 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, and the 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. Based on 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 non-canonical aspartic proteases that exhibit broad cleavage specificity, high thermal stability, and optimal activities at pH’s 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, 3 ACS Paragon Plus Environment


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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 harbours 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 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 a 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 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.



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Experimental Methods RNA extraction N. rafflesiana 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 ß-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. Transcriptome Sequencing Illumina RNA sequencing of N. rafflesiana transcriptomes was performed by Omega Bioservices (Omega Bio-tek, 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 Ribo-Zero Plant reagents to remove the abundant cytoplasmic, mitochondrial and chloroplast ribosomal RNA. 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. 6 ACS Paragon Plus Environment

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Transcriptome assembly and analysis Cleaning filters were applied to the raw sequencing data files, based on FastQC quality control statistics, and Illumina sequencing adaptors 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 frame 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). On 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 were 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 4oC for shortterm or at -80oC for long-term. In-gel fluid proteomics analyses The N. ventrata digestive fluid was analyzed on an 8% SDS-PAGE 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



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thiosulfate and 15 mM potassium ferricyanide, reduced with 10 mM DTT followed by alkylation with 50mM 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 37oC 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 an 1260 Infinity chip cube interface, using a 25 minute reversed phase gradient run (high-capacity chip: Agilent Zorbax 300SB-C18 5 micron particles, 150 µm x 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 +2 and higher. The optimum collision energy was calculated for each precursor based on its charge and mass. Ion exclusion was released after 1 spectrum and 0.2 min, to avoid re-acquiring MS/MS data for the same precursor. The data were searched against the 6-frame translation of our N. rafflesiana transcriptomes databases using Mascot. The Mascot search parameters for the tryptic digested samples were: maximum of one missed cleavage, carbamidomethyl (C) as fixed modification, methionine oxidation as a variable modification, a 20 ppm peptide mass tolerance, a 0.2 Da fragment mass tolerance. Output was filtered for peptide hits with p < 0.05 (ion score > 24). Gel-free fluid proteomic analyses To analyze the proteome of the N. ventrata pitcher fluid, the fluid was digested either with trypsin (specific digest) or N. ventrata fluid (non-specific digest) using a modified FASP protocol.32 Briefly, 15 µg of 10X 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


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exchanged to 50 mM ammonium bicarbonate pH 8 and digested with trypsin (Promega; enzyme to substrate ratio of 1:100) at 37oC overnight followed by quenching with 0.1% trifluoroacetic acid. For non-specific 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 minutes. 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 minutes 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 5.00E+05. 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 1.00E+04 .Separation were achieved using a selfpacked pico-frit column (New Objective, packed with Phenomenex 5 µm Jupiter C18 beads, 100 mm x 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. rafflesiana transcriptomes databases using Mascot. The search parameters for the tryptic digested samples were: one missed cleavage allowed, carbamidomethyl (C) as fixed modification, methionine oxidation (M) as a variable modification, a 10 ppm peptide mass tolerance, a 0.6 Da fragment mass tolerance, and the output filtered with a false discovery rate of 1% (peptide hits with p < 0.00045, an 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 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 within or overlap with another transcript, the longer assembled transcript was used for characterization.


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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 extracted the RNA from the bottom third of a N. rafflesiana 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,

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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, 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 335K 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 (and/or arise from error in assembly). Taxonomical annotation of the contigs using MEGAN revealed that well over half of the transcripts 11 ACS Paragon Plus Environment


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were plant-based (Streptophyta) with some minor contamination from bacteria, fungi and fruitfly (Figure 2, 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 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 against our assembled N. rafflesiana 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 gelfree methods. Here, gel-free tryptic digestion of the fluid followed 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 a recent study.29 As noted, the low level of coverage could arise from a non-optimal 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


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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 NCBI non-redundant (nr) protein sequence database, or characterized biochemically. To assess relative abundance of the identified proteins based on label-free methods, such as emPAI, the tryptic data is particularly unreliable given the paucity of detectable peptides. However, the emPAI values from the non-specific digests are useful, given 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 fluid proteome, when compared to a recent study using only trypsin.29 We identified approximately 25% more 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 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, two-third of the identified proteins with an intact Nterminus 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 sub-classes that aid in evaluating the mechanisms of carnivory: 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 non-canonical 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 homologs, 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. rafflesiana 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 features 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 about 100 aa (instead of the short NAP-specific insert), commonly found in vacuolar plant aspartic proteases (vAP, Figure S2). Our N. rafflesiana 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 Nep1


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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 also cleave C-terminal to proline residues, but neither Nep1 nor Nep2 possessed such prolyl-endopeptidase activity.12,

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We recently purified a fluid fraction

containing the prolyl-endopeptidase 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 homolog, which we call Npr2, but it remains to be seen if it also processes prolylendopeptidase 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 single-stranded nucleic acids.40 The acid phosphatase is closest to the purple acid phosphatases, a metallophosphoesterase found in a



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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-1,3glucanase, 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 a 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


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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 rafflesiana 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 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 3 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, 10 of which were uniquely determined using the non-specific 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: Npr1,2) 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 labs.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.



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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 anti-bacterial and/or anti-fungal properties, since they can digest pathogens and hence can be considered pathogenesis-related proteins in themselves.4 The association between carnivory and pathogenesis is further supported by the presence of several specifically pathogenesisrelated 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 peroxidase-generated 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) receptor-like 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 identify 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, since 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.


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An overlapping set of proteins were 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 a 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.



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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 and galactosidases.29 The fluid represents a steady-state harvest, therefore either these enzymes are 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 pre-filtered 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. Based on our extended proteomics analysis, it would appear that the panel of endogenous plant enzymes is sufficient to recover the nutrients needed to support growth.


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Figures

Figure 1. Transcriptomics and proteomics workflow applied to Nepenthes spp. Transcriptomics was applied to Nepenthes rafflesiana pitcher tissue and proteomics was applied to Nepenthes ventrata pitcher fluid. Photographs courtesy of Linda Lee. Copyright 2016.



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Figure 2. Taxonomic organization of the N. rafflesiana transcriptome, providing an overview of the taxonomy and level of contamination present, based on annotation against the NCBI nt database (evalue cutoff of e-15).


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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.



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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. rafflesiana transcriptome analysis (in bold) with the nepenthesins from other Nepenthes species (including an unpublished nepenthesin sequence from N. ampullaria). The percentage sequence identity relative to N. rafflesiana Nep1 is indicated in brackets. The sequence identity within the Nep2 subgroup is ~90%. B) Sequence alignment of N. rafflesiana 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.


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Figure 5. Model for the mechanism of carnivory in Nepenthes. The enzymes identified in this study are highlighted in red, while 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 release 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.



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Table 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 carboxypeptidase, SCP1 Purple acid phosphatase, PAP1 Ribonuclease, S-like, RNaseS Endonuclease 2, Endo2 β-1,3-Glucanase, Glu1 Acidic chitinase, Chit3 Chitinase, Chit1

Pathogenesis/ Host defense

Peroxidases

DOMON-like domain, Dom1 Thaumatin-like protein, TLP1 Hevein , Hev1 Cat. peroxidase 1, Prx1 Cat. peroxidase 2, Prx2

MW

pI

Aspartic protease

47106

4.7

Signal Pep.? Yes

Aspartic protease

46625

4.1

Yes

Aspartic protease

49092

4.9

Yes

3

8

Aspartic protease

48991

4.8

Yes

23

6

Aspartic protease

48811

4.6

Yes

9

Prolyl endoprotease

43161

6.3

Yes

7

Prolyl endoprotease

>33kDa

5.2

N/A*

10

S10 acidic peptidase Metallophosphatase

>47kDa

4.3

Yes

5

5

71268

5.2

Yes

1

4

Secreted RNase/ scavenge phosphates S1-P1 nuclease/ cleave RNA & ssDNA Glycoside hydrolase 17/ pathogen defense

26224

4.1

Yes

32487

4.9

Yes

14

12

36752

4.7

Yes

6

11

14

31086

4.1

Yes

12

15

29306

4.5

Yes

13

14, 18, 52 13, 53

27995

5.5

Yes

9

23785

5.7

Yes

4

13

13

20752

6.4

Yes

10

33598

6.4

Yes

8

14

33385

4.6

Yes

21

14

Class/Function

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 Chitin binding/ antifungal activity Heme-containing/ oxidation Heme-containing/ oxidation


Dig.1 † 2

Dig.2 ‡ 1 2

3

Ref. 9 9

19, 20

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Signal transduction


Peroxidase, Prx3 Leu-rich repeat protein kinase, RPK1

Heme-containing/ oxidation Ser/ Thr Kinase/ plant dev./ pathogen resistance Possible protein kinase/ribonuclease inhibitor ADP/ATP exchange

36354

4.7

Yes

66702

5.4

Yes

Leu-rich 69848 4.8 No repeat protein, LRR1 Energy ADP/ ATP >31kDa 10. N/A* metabolism translocase, 0 ADT1 GlyceraldehydeNAD-binding/ 36664 7.1 No 3-phosphate glycolysis/ dehydrogenase, glyconeogenesis GPD1 ATP synthase/ ATP synthesis/ >18kDa 5.3 No + H -ATPase membrane-bound proton-pump subunit α, ATPA ATP synthase/ ATP synthesis/ >51kDa 4.9 No H+-ATPase membrane-bound proton-pump subunit β, ATPB Enolase, 2-phospho-D>30kDa 4.9 N/A* Eno1 glycerate conversion Protein Actin, cytoskeleton 41706 5.3 No transport Act1 Tic20-like chloroplast 31449 9.4 No protein, import protein Tic20 Protein Vesicle trafficking/ 34078 9.2 No transporter secretion Sec1 Translation/ Elongation GTP-binding 49815 9.2 No factor 1-alpha, elongation protein synthesis EF1a factor Elongation GTP-dependent 95948 6.1 No factor 2, EF2 ribosomal translocation 40S Ribosomal Protein synthesis 16367 10. No protein S14, RS14 6 Chaperone Protein folding 91593 5.0 Yes protein Hsp90 Transcription/ Histone Acetylate >86kDa 6.0 No Gene acetyltransferase, transcriptional regulation HAC1 regulators, histones Auxin response Transcription factor/ >25kDa 8.5 N/A* factor, ARF1 Auxin response factor †Trypsin diges@on. Ranking is based on the MASCOT search score. ‡Nonspeciﬁc diges@on using ac@ve Nepenthes fluid. Ranking based on MASCOT search score. *N/A, not applicable because protein fragment has a truncated N-terminus.


16 14

17

11

18

20

22

25 15 16

26

7

24 17 19 18

19


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Associated Content Supporting information Table S1 provides a summary sequence analysis of the N. rafflesiana 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 Figure 2. Tables S4S6 provide details in support of the proteomics analyses, summarized in Table 1. 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. List S1 contains the set of amino acid sequences for the proteins identified in N. rafflesiana digestive fluid.

Author Information Corresponding author Telephone: 403-210-3811 e-mail: [email protected] Author Contributions This manuscript was written with contributions from all authors. All authors have approved the final version of the manuscript. Conflict of Interest The authors declare no competing financial interest Acknowledgments The work was supported by an NSERC Discovery Grant 298351-2010 (DCS). DCS acknowledges the additional support of the Canada Research Chair program, Alberta Ingenuity - Health Solutions and the Canada Foundation for Innovation. 28 ACS Paragon Plus Environment

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Abbreviations CDS, coding sequence; CID, collisionally-induced dissociation; CTAB, cetyl trimethylammonium 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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Table of Contents Graphic

Photograph courtesy of David Schriemer. Copyright 2016.


Carnivorous Nutrition in Pitcher Plants (Nepenthes spp.) via an

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