Protein Secretome of Moss Plants (Physcomitrella patens) withEmphasis on Changes Induced by a Fungal ElicitorMikko T. Lehtonen,† Yoshihiro Takikawa,‡ Gunilla Ronnholm,§ Motomu Akita,|| Nisse Kalkkinen,§

Elina Ahola-Iivarinen,§ Panu Somervuo,§ Markku Varjosalo,§ and Jari P. T. Valkonen*,†

†Department of Agricultural Sciences, University of Helsinki, PO Box 27, FI-00014 Helsinki, Finland‡Plant Center, Institute of Advanced Technology, Kinki University, 14-1 Minamiakasaka, Kainan, Wakayama, 642-0017, Japan§Institute of Biotechnology, University of Helsinki, PO Box 56, FI-00014 Helsinki, Finland||Department of Biotechnological Science, Kinki University, Kinokawa, Wakayama, 649-6493, Japan

*S Supporting Information

ABSTRACT: Studies on extracellular proteins (ECPs)contribute to understanding of the multifunctional nature ofapoplast. Unlike vascular plants (tracheophytes), littleinformation about ECPs is available from nonvascular plants,such as mosses (bryophytes). In this study, moss plants(Physcomitrella patens) were grown in liquid culture andtreated with chitosan, a water-soluble form of chitin thatoccurs in cell walls of fungi and insects and elicits pathogendefense in plants. ECPs released to the culture medium werecompared between chitosan-treated and nontreated control cultures using quantitative mass spectrometry (Orbitrap) and 2-DE-LC-MS/MS. Over 400 secreted proteins were detected, of which 70% were homologous to ECPs reported in tracheophytesecretomes. Bioinformatics analyses using SignalP and SecretomeP predicted classical signal peptides for secretion (37%) orleaderless secretion (27%) for most ECPs of P. patens, but secretion of the remaining proteins (36%) could not be predictedusing bioinformatics. Cultures treated with chitosan contained 72 proteins not found in untreated controls, whereas 27 proteinsfound in controls were not detected in chitosan-treated cultures. Pathogen defense-related proteins dominated in the secretomeof P. patens, as reported in tracheophytes. These results advance knowledge on protein secretomes of plants by providing acomprehensive account of ECPs of a bryophyte.

KEYWORDS: plant secretome, proteomics, extracellular proteins, moss, bryophyte, Physcomitrella patens, defense elicitor, chitin,chitosan, quantitative mass spectrometry

■ INTRODUCTION

Secreted proteins play important roles in a wide range ofbiological processes such as cellular signaling, cell-to-cellcommunication and responses to biotic and abiotic stresses.1

Protein secretion can be mediated by an N-terminal signalpeptide, which targets the protein to endoplasmic reticulum(ER). Following removal of the signal peptide, the matureprotein is moved by secretory Golgi apparatus beyond the plasmamembrane to the extracellular matrix. However, a largeproportion of the extracellular proteins (ECPs) of plants (40−70%) do not contain any classical signal peptide but use other,less-known secretory pathways.2 These pathways include, forexample, “exocyst-positive organelle”mediated protein secretionor involve small Rab-type GTPases in trafficking specificsecretory vesicles during cell plate formation and polarized cellexpansion.3,4 It is also suggested that exosomal fusion ofmultivesicular bodies to plasma membrane could offer a meansfor protein secretion in plants.5,6

The extracellular matrix influences protein secretion. Plantcells are surrounded by a cell wall that consists of coherentlyaligned cellulose microfibrils and can be lignified.7,8 The outer

surface of plants is covered by an epidermis layered with cutin,suberin polymers and waxes, which form a water-impermeablecuticle.9 These characteristics of the differentiated, green,photosynthesizing parts of most tracheophyte species (vascularplants) make them less suitable for the study of protein secretion.Therefore, studies are usually done using undifferentiated plantcells or tissues grown as a suspension in liquid medium, whichfacilities isolation of the secreted proteins from the culturemedium for analysis. Root exudates can be collected using similarapproaches.1 In this respect, the characteristics of mosses, whichare small bryophyte plants, offer some special opportunities forthe study of protein secretome.Physcomitrella patens (Hedw.) B.S.G. is a moss species in which

the haploid growth phase dominates. The haploid gametophytesinitiate their growth as protonema, which are filamentous andlack a cuticule, and differentiate into stem and leaves that are notstructurally complex (Figure 1A). Leaves consist of a single celllayer.10,11 P. patens is an early colonist on exposed mud and soil

Received: June 10, 2013

Article

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around pools of water and becomes naturally submerged in itsgrowth habitats.12 The whole plants of P. patens can be grownsubmerged in liquid culture medium in the laboratory and thesecreted proteins harvested from the culture filtrates. Theseproperties of P. patens have found industrial applications inproduction of recombinant proteins that have been tagged withthe classical secretion signal sequences and can be harvested fromthe moss culture medium.13,14 However, it appears that besides athesis whose data are not readily accessible,15 there is littleinformation about the protein secretome of wild-type P. patens orany other mosses. Secretome studies are now feasible because thewhole genome sequence of P. patens is available and facilitatesprotein identification.16 Furthermore, gel-free shotgun proteo-mics approaches based on high-resolution mass spectrometryprovide a powerful approach to characterize the proteinsecretome and obtain quantitative data by spectral counting,that is, by determining the number of copies of a given peptide inthe sample.17

P. patens is emerging as a model plant for studies on plant-pathogen interactions,18−23for which pathosystems based onfungi isolated from mosses have been established.19,20,24 Inmosses and tracheophytes the extracellular matrix of host cellsprovides a contact interface and site for recognition of fungalpathogens, followed by defense responses such as oxidative burst,rapid deposition of altered and strengthened cell walls andformation of papillae at the site of infection.20,25 Chitin is apolymer that occurs naturally in cell walls of fungi and insects andelicits host defense in P. patens19,20,24 and tracheophytes.26 Forcontrol of plant diseases, chitin is deacetylated to 90% and theresultant water-soluble compound, chitosan, is applied on plants

to elevate their level of resistance.27,28 Treatment of P. patenswith chitosan results in a rapid oxidative burst dependent on asecreted peroxidase (Prx34).19,29 The Prx34 knockout lines of P.patens are rendered susceptible to fungi, indicating the pivotalrole of Prx34 in pathogen defense of the moss.19,29

The aim of this study was to analyze the secretome of P. patensand the possible qualitative and quantitative changes occurring inECPs released to the culture medium upon chitosan treatment.Results were expected to advance the knowledge on how themoss extracellular proteome is affected by a fungal elicitor and toexpand the knowledge on plant secretomes, which is currentlylimited to tracheophytes.

■ MATERIALS AND METHODS

Plant Material

Protonemal tissue of P. patens, ecotype Gransden Wood,30 wasgrown in Petri dishes (Ø 9 cm) on a cellophane membrane(400P; Visella Oy, Valkeakoski, Finland) placed on BCDmedium [1 mM MgSO4, 1.85 mM KH2PO4 (pH 6.5, adjustedwith KOH), 10 mMKNO3, 45 μMFeSO4, 0.22 μMCuSO4, 0.19μM ZnSO4, 10 μM H3BO4, 0.10 μM Na2MoO4, 2 μM MnCl2,0.23 μMCoCl2, 0.17 μMKI]30 supplemented with 1 mMCaCl2,45 μM ethylenediaminetetraacetic acid disodium salt (Na2-EDTA), and 5 mM ammonium tartrate [(NH4)2C4H4O6], andsolidified with 0.8% agar. The cultures were grown in a growthcabinet (Model 3755, Forma Scientific, Marietta, OH, U.S.A.) at23 °C (photoperiod 12 h, light intensity 60 μmol m−2 s−1) andsubcultured weekly.

Figure 1. (A) A plant of Physcomitrella patens grown in liquid culture medium. The plant (called gametophyte) consists of filamentous protonema (P)and a gametophore with leaves (L) that contain only a single layer of cells. (B) The main steps of the experimental workflow consisted of treatment of P.patens with chitosan for 3 h, isolation of secreted proteins from the culture medium, analysis of proteins by LC-MS and 2DE, and data analysis.

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Treatment with Chitosan

The stock solution of chitosan (10 mg mL−1) was prepared bydissolving chitosan oligosaccharide (degree of deacetylation90%, MW <5000; Kraeber GmbH & Co., Ellerbek, Germany) insterile Milli-Q water at 10 mg mL−1, prefiltered twice through 1.6μm glass fiber filter (VWR International, West Chester, PA,USA), sterile-filtered using 0.2 μm cellulose acetate filter(Whatman GmbH, Dassel, Germany) and stored at −20 °Cuntil use.Protonemal tissue was cultured in Petri dish on solid BCD

medium containing ammonium tartrate for 7 days, followed bytransfer to fresh BCD medium lacking ammonium tartrate toenhance gametophore formation, and grown on this medium for7 days. The moss tissue was harvested and transferred to a 250-mL Erlenmeyer bottle containing liquid BCD medium (40 mL)that contained 5 mM ammonium tartrate and 1% glucose. Theliquid cultures were shaken (90 rpm) in a rotary shaker at 25 °Cand illuminated from the top (photoperiod 12 h, light intensity160 μmol m−2 s−1) for 7 days. At the end of the growth period,the moss had developed gametophytes consisting of filamentousprotonema and a gametophore with leaves (Figure 1A).For each experiment, 16 replicate cultures of P. patens were

grown as described. Chitosan stock solution was added to 8 mosscultures grown in Erlenmeyer bottles to a final concentration of0.5 mg mL−1. Sterile Milli-Q water was added to the remaining 8cultures as a control treatment. Samples were collected from thechitosan-treated and control cultures 3 h after chitosanapplication. No detectable injuries or phenotypic responseswere found in moss tissues and cells, as studied with amicroscope. The experiment was carried out twice.Protein Isolation

The culture medium was filtered through 1.6 μm glass fiber filter(VWR) to exclude all plant material. Ammonium sulfate (SigmaA4915, Sigma-Aldrich Co., St. Louis, MO, U.S.A.) was slowlyadded to 70% saturation and the proteins precipitated at +4 °Covernight. The ammonium sulfate solution was centrifuged at12000× g for 30 min at +4 °C, the pellet dissolved in ice cold 100mM Tris-Cl buffer (pH 7.5) and spun at 12000 × g for 30 min at+4 °C. Supernatant was dialyzed against 100 mM Tris-Cl buffer(pH 7.5) in Spectra/Por dialysis membrane MWCO 12-14000(Spectrum Laboratories, Inc., Rancho Dominguez, CA, U.S.A.)overnight with three buffer changes at +4 °C. The dialysate wascentrifuged at 12000 × g for 30 min at +4 °C. Proteinconcentration was assayed using Bio-Rad Protein Assay (Bio-RadLaboratories, Hercules, CA, U.S.A.) and the preparations storedat −80 °C until used.Mass Spectometry

Identification of extracellular proteins by gel-free liquidchromatography tandem mass spectrometry (LC-MS/MS)(Figure 1B) was carried out with Thermo Scientific OrbitrapElite ETD mass spectrometer coupled with Proxeon EASY-nLCsystem. Three micrograms of the protein precipitate from culturemedium of 8 pooled chitosan-treated and 8 pooled controlsamples was tryptic-digested (Sequencing grade ModifiedTrypsin, V5111, Promega) to peptides in the presence of 1 Murea after reduction and alkylation with dithiothreitol (DTT)and iodoacetamide, respectively. Tryptic peptide digests werequenched with 10% trifluoroacetic acid (TFA), concentrated andpurified by reverse-phase chromatography columns [C18material, eluted with 90% acetonitrile (CH3CN), 0.1% TFA].The dried peptides were reconstituted (2% CH3CN, 0.1% TFA).Fifteen percent of the reconstituted peptides were subjected to

mass spectrometry analysis. Solvents for LC-MS separation ofthe digested samples were as follows: solvent A consisted of 0.1%formic acid in water (98%) and acetonitrile (2%) and solvent Bconsisted of 0.1% formic acid in acetonitrile (98%) and water(2%). From a thermostatic microautosampler the tryptic peptidemixture were automatically loaded onto a 15 cm fused silicaanalytical column with an inner diameter of 75 μm packed withC18 reversed phase material (Thermo Scientific) and thepeptides were eluted from the analytical column with a 60 mingradient ranging from 5% to 35% solvent B, followed by a 10 mingradient from 35% to 80% solvent B at a constant flow rate of 300nL/min. The analyses were performed in a data-dependentacquisition mode using a top 10 collision-induced dissociation(CID)method. Dynamic exclusion for selected ions was 30 s. Nolock masses were employed. Maximal ion accumulation timeallowed on the Orbitrap Elite ETD in CID mode was 100 ms forMSn in the Ion Trap and 200 ms in the FTMS. Automatic gaincontrol was used to prevent overfilling of the ion traps and wereset to 10 000 (CID) in MSn mode for the Ion Trap, and 106 ionsfor a full FTMS scan. Intact peptides were detected in theOrbitrap at 60 000 resolution. Peak extraction and subsequentprotein identification were achieved using Proteome Discoverersoftware (Thermo Scientific, Waltham, MA).

Two-Dimensional Electrophoresis (2-DE) of SecretedProteins

An IPGphor instrument (GE Healthcare, Uppsala, Sweden) wasused for isoelectric focusing (IEF) with immobilized pH gradient(IPG) strips (pH 3−10, linear gradient, 11 cm; GE Healthcare).The protein precipitate from culture medium of 8 pooledchitosan-treated and 8 pooled control samples (20−50 μgprotein) was dissolved in 8 mL ice cold 50 mMTris-Cl buffer pH7.5 and desalted and concentrated with Amicon Ultra-4 10k cutoff centrifugal filter (Millipore, Carrigtwohill, Ireland) bycentrifugation at 3220 × g at +4 °C (swing-out rotor) priorloading to IPG strips. The IPG strips were rehydrated inrehydration buffer (8 M urea, 2% 3-[(3-cholamidopropyl)-dimethylammonio]-1 propanesulfonate (CHAPS), 0.5% IPGbuffer, pH 3−10, 20mMDTT, and a trace of bromophenol blue)containing proteins from the growthmedia of chitosan-treated orcontrol plants. The IPG strips were allowed to rehydrate for 2 h,subsequently a low electric current (21 mA overnight) wasapplied to improve the entry of high-molecular-weight proteinsinto the IPG strips. The voltage settings for IEF were 500 V for 1h, gradient to 1000 V for 1 h, gradient to 6000 V for 1 h and then6000 V until 42.7 kVh. The strips were equilibrated twicesuccessively in an equilibration solution [6 M urea, 50 mM Tris-HCl, pH 6.8, 30% glycerol, 2% sodium dodecyl sulfate (SDS),and a trace of bromophenol blue], in which the first equilibrationcontained 1% DTT and the second 2.5% iodoacetamide. Thesecond dimension was done on 10−20% gradient Tris-HClpolyacrylamide gel (Criterion, Bio-Rad laboratories, Hercules,CA, U.S.A.) using a Criterion Cell apparatus (Bio-Radlaboratories). The gels were silver stained.31 Briefly, the gelswere first fixed [30% (v/v) ethanol, 0.5% (v/v) acetic acid],followed by 1× wash in 20% ethanol and 1× wash in water toreduce the background staining. The gels were sensitized in0.02% (w/v) thiosulfate, stained with silver staining solution[0.2% (w/v) silver nitrate], rinsed with water, and developed in3% (w/v) potassium carbonate, 0.05% (v/v) formaldehyde, and0.001% thiosulfate. Development was stopped using a stopsolution [5% Tris base, 2.5% (v/v) acetic acid]. The experimentwas carried out three times.

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Mass Spectrometry of Proteins Following 2-DE

Silver stained spots were cut out of the 2-DE polyacrylamide geland “in-gel” digested.32 Proteins were reduced with dithiothreitoland alkylated with iodoacetamide before digestion with trypsin.LC-MS/MS analysis was performed using an Ultimate 3000nano-LC (Dionex) and a QSTAR Elite hybrid quadrupole TOF-MS (Applied Biosystems/MDS Sciex) with nano-ES ionization.The LC-MS/MS samples were first loaded on a ProteCol C18trap column (3 μm, 10mm×150 μm, 120 Å) (SGE), followed bypeptide separation on a PepMap100 C18 analytical column (5μm, 15 cm ×75 μm, 100 Å) (LC Packings/Dionex) at 200 nL/min. The separation gradient consisted of 0−50% B in 20 min,50% B for 3 min, 50−100% B in 2 min, and 100% B for 3 min(buffer A, 0.1% formic acid; buffer B, 0.08% formic acid in 80%acetonitrile). MS data were acquired using Analyst QS 2.0software.

Protein Data Analysis

The calibrated peak files from the Orbitrap Elite were searchedagainst the genome sequence of P. patens16 (ftp://ftp.jgi-psf.org/pub/JGI_data/phytozome/v7.0/Ppatens/, version 1.6 proteinmodels) using SEQUEST. Error tolerances on the precursor andfragment ions were ±15 ppm and ±0.6 Da, respectively.Database searches were limited to fully tryptic peptides withonly one missed cleavage allowed. Carbamidomethyl cysteineand methionine oxidation were set as fixed and variablemodifications, respectively. For peptide identification the p-value < 0.05 was used. Proteins with two or more peptidematches with at least one unique peptide included wereconsidered identified.Chitosan-induced differences in the quantity of proteins

released to the culture medium by P. patens were analyzed bycomparing the amounts of the protein between chitosan-treatedand control samples. Proteins with a spectral count value higheror equal to 517 were included in the analysis with the RBioconductor package PLGEM, version 1.30.0,33 and QSPEC,version 1.2.2,34 with raw spectral counts as input values (QSPECmakes a size adjustment prior the analysis).The presence of secretory signal peptides was analyzed using

SignalP 4.0.35 Signals for nonclassical leaderless secretion werepredicted using SecretomeP 2.0.36 Gene ontologies wereretrieved using Blast2GO, version 2.6.2,37 and protein domainswere detected using Blast2GO InterProScan tool and searched atPfam Web site (http://pfam.sanger.ac.uk). The secretome datawas compared with several published tracheophyte secre-tomes.38−53 The protein accessions other than UniProt wereconverted to UniProt accessions using Protein Identifier Cross-Reference Service from EMBL-EBI, http://www.ebi.ac.uk/Tools/picr/. The protein sequence data were subsequentlyretrieved fromUniProt and compared with the proteins detectedin the secretome of P. patens using FASTA program package54

(version 36.3.5d). Tracheophyte homologues of the ECPs of P.patens were detected using Blosum62 matrix with BLASTP gap(−11/−1) penalties in the search.The 2-DE protein LC-MS/MS data were searched with the in-

house -Mascot version 2.2 against the P. patens version 1.655

protein models. Proteins with at least two peptide hits with P <0.05 were considered identified. The identified enzymes weremapped to the metabolic pathways using KEGG Mapper(http://www.genome.jp/kegg/tool/map_pathway1.html) andvisualized using iPath2.0.56

Microarray Design

The probes for a 4 × 44K Agilent oligonucleotide microarray(Agilent technologies, Santa Clara, CA, U.S.A.) were designedwith OligoArray 2.057 using P. patens virtual transcript sequencesbased on version 1.1 models16 as a template. Furthermore,version 1.1 models were used in BLAST search in the PhyscobaseEST database (http://moss.nibb.ac.jp/) for designing additionalprobes to ESTs that were not found among the version 1.1models. In OligoArray 2.0, specificity of the probe is determinedby the BLAST algorithm58 and the melting temperature (Tm)estimation. When hybridization Tm between a candidate probeand a nonspecific target exceeded the given threshold, the probewas considered nonspecific and was not selected. Probecandidates containing stable secondary structures or a stretchof more than four identical nucleotides were not accepted. Thetarget length of each probe was defined to be 60 nucleotides.

RNA extraction and microarray hybridization

P. patens was grown in liquid medium as described above. Twocultures were treated with chitosan and two cultures were grownas untreated controls. The experiment was carried out threetimes. The plant material was collected at 0, 15, 30, 90, and 180min after adding chitosan to the culture medium. Moss growthfrom the two replicate cultures of the treatment were pooled,squeeze-dried quickly between stacks of tissue paper and groundto powder in liquid nitrogen for extraction of total RNAessentially as described,59 except that the buffer did not containspermidine. mRNA from 1 μg of the total RNA was amplifiedusing Amino Allyl MessageAmpII aRNA amplification kit(Applied Biosystems, Foster City, CA, U.S.A.). The amino allylRNA (20 μg) was labeled according to manufacturer’sinstructions (Applied Biosystems) with monoreactive fluores-cent dyes Cy3 or Cy5 (GE Healthcare). Dye swaps were donebetween the replicate samples. Hybridization and washing ofmicroarrays were carried out according to the manufacturer’sinstructions (Agilent).

Microarray Analysis

Microarray slides were scanned with GenePix 4200AL(Molecular Devices, Sunnyvale, CA, U.S.A.) using 5-μmresolution. Image analysis was done using GenePix Pro 6software. Gene expression data were processed and analyzedusing Bioconductor limma package.60 Background subtractionwas carried out using normexp method with offset value of 5061

and log-ratio signals were normalized with lowess method.Differential expression of genes was assessed using moderated ttest with empirical Bayes shrinkage of standard errors asimplemented in limma package60 and correction of p-values formultiple testing by calculating the False Discovery Rate (FDR).62

Genes with FDR-values ≤ 0.05 and a minimum 2-fold change ofexpression were considered to be differentially expressed. Foldchanges were calculated from the average expression valuesdetected in treated and control samples. The probes wereremapped to the latest version of the gene models, version 1.6,55

which had become available at the time of writing. Data forselected probes were extracted from the array to visualize geneexpression of corresponding proteins whose quantity increasedin response to chitosan. When multiple probes were designed fora gene, an FDR-value ≤ 0.05 was required for including a probein further analysis, and the mean fold of signals of the probes forthe gene were used for analysis.

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■ RESULTS

Secretome of P. patens

A total of 238 proteins were identified in the culture medium ofnontreated P. patens in the two experiments (Figure 2). They willbe further referred to as Pp secretome (group A in Figure 2;Supporting Information, Table S1). Bioinformatics analysis usingSignalP and SecretomeP programs identified a putative signal

peptide (SP) for classical secretion in 88 identified proteins(37%) and predicted leaderless secretion (LS) of 64 proteins(27%), whereas the secretion of the remaining 86 proteins (36%)was not predicted by bioinfomatics. There were 103 proteinsdetected in control cultures in only one of the two experiments.They will not be further considered (group B2, Figure 2; andgroups C1 and C2 in Supporting Information, Table S1).The most abundant 5% of the proteins corresponded to 30%

of the total tandem mass spectra (Table 1). Many abundantproteins belonged to multimember subfamilies (SupportingInformation, Table S2), for example, enzymes, such ashydrolases, oxidoreductases, and transferases. The mostabundant enzymes homologous to those associated withpathogen defense in vascular plants were GDSL lipases,50

chitinases,63 peroxidases,19 and glyoxal oxidases (Table 1;Supporting Information, Table S2). The proteins related tosignal transduction included calcium binding proteins, leucin richreceptor-like kinases and GTP-binding proteins. Atypicalapoplastic enzymes involved in amino acid biosynthesis includedmethionine synthase and adenosylhomocysteinase. Manyglycolytic enzymes47,64 such as aldose 1-epimerase, fructose-bisphosphate aldolase, phosphoglucose isomerase, phosphogly-cerate kinase, and triosephosphate isomerase were also detected.The enzymes detected in the secretome were mapped tometabolic pathways using KEGG Mapper, which revealed thatthey were mainly related to nucleotide, carbohydrate and energymetabolism and involved in purine metabolism, glycolysis,tricarboxylic acid cycle (citric acid) cycle and carbon fixation(Supporting Information, Figure S1).Pp secretome was compared with the available tracheophyte

secretome data, including secreted proteins detected in cell

Figure 2.Grouping of the extracellular proteins of Physcomitrella patens,which were secreted to the culture medium, according to treatment ofmoss plants. The number of proteins identified in the liquid culturemedium of (A) control plants, and (B) chitosan treated plants isindicated in the figure. Group A proteins were found in all controls,whereas group A1 and group B proteins were found in all chitosantreated cultures. Proteins of group A2 were found in controls only,whereas proteins of group B1 were detected only in chitosan-treatedsamples. Group A3 proteins were found in both controls and in onechitosan-treated sample. Group B2 proteins were present in bothchitosan-treated samples and in one control (see also SupportingInformation Table S1).

Table 1. Most Abundant Proteins in the Secretome of Physcomitrella patens

rankinga

identifier control chitosan description secretory signalb refc

Pp1s17_355 V6.1 1 2 GDSL-like lipase/acylhydrolase SP 38, 39, 43−45, 50, 52, 53Pp1s486_14 V6.1 2 4 xyloglucan endotransglycosylase SP 38, 39, 41−45, 48, 51, 53Pp1s44_116 V6.1 3 6 no functional description SPPp1s219_8 V6.1 4 3 peroxidase (Prx34) SP 38, 39, 42−48, 51−53Pp1s184_140 V6.1 5 5 chitinase class I SP 39, 42−45, 48, 49, 51−53Pp1s240_68 V6.1 6 1 no functional description SP 51Pp1s97_59 V6.2 7 (20) cupin SP 38, 40, 43−45, 47, 49, 51−53Pp1s23_249 V6.1 8 7 VEFS-box of polycomb protein LSPp1s81_246 V6.1 9 15 xyloglucan endotransglycosylase SP 38, 39, 41−45, 48, 51, 53Pp1s39_149 V6.1 10 14 AAA+ type ATPase with peptidase M41 domain 38, 43, 47, 53Pp1s44_345 V6.1 11 (39) Pfam: PF06830 Root_cap SP 51, 53Pp1s276_72 V6.1 (12) 12 Pfam: PF00847 AP2 LS 43Pp1s136_71 V6.1 (15) 8 protease inhibitor/seed storage/LTP family SP 43, 47Pp1s22_119 V6.1 (16) 13 no functional descriptionPp1s236_72 V6.1 (17) 16 pectinesterase SP 38, 39, 42, 43, 47, 52Pp1s44_147 V6.1 (18) 11 no functional description 38, 43, 53Pp1s179_68 V6.1 (27) 17 GDSL-like lipase/acylhydrolase SP 38, 39, 43−45, 50, 52, 53Pp1s106_4 V6.1 (29) 9 uncharacterized conserved protein LS 53Pp1s464_17 V6.1 (34) 18 phosphate-induced protein 1 conserved region LS 38, 43, 51, 53Pp1s16_387 V6.2 (81) 10 Pfam: PF14368 LTP_2 SP 43, 47, 51, 53

aThe most abundant 5% of the secreted moss proteins corresponding to 30% of the total tandem mass spectra detected in the control cultures(control) or chitosan-treated cultures (chitosan) are ranked according to the highest scoring tandem mass spectra. The rankings of proteins notbelonging to the most abundant 5% of the secreted proteins in the respective treatment (control or chitosan) are shown in parentheses. bSP,secretory signal peptide as predicted with SignalP 4.0; LS, leaderless secretion as predicted with SecretomeP 2.0 (value >0.5); empty cells, secretionnot predictable based on bioinformatic analyses. cReported tracheophyte secretomes containing homologous proteins (FASTA ssearch36/fasta36, E-value cutoff < 0.0001); empty cells, no homologous protein reported in tracheophyte secretomes.

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Table 2. Proteins of Physcomitrella patens Secreted Exclusively in Response to Treatment with Chitosan

identifier description secretory signala refb

proteins with homologues reported in tracheophyte secretomesPp1s40_134 V6.1 1-cysteine peroxiredoxin 1 40, 47, 52, 53Pp1s223_50 V6.1 2-oxoacid dehydrogenases acyltransferase family protein 38, 40, 43, 53Pp1s72_290 V6.1 ABC-2 and Plant PDR ABC-type transporter family protein LS 53Pp1s89_161 V6.2 aldolase superfamily protein 38, 40, 42, 43, 46, 47, 51, 53Pp1s159_68 V6.3 aldolase-type TIM barrel family protein LS 53Pp1s199_134 V6.1 aleurain-like protease SP 40, 42−44, 47, 51, 53Pp1s18_41 V6.3 AMP-dependent synthetase and ligase family protein LS 38, 43, 53Pp1s35_384 V6.1 ATP citrate lyase subunit B 2 LS 38, 53Pp1s309_77 V6.1 chitinase SP 39, 42−45, 48, 49, 51−53Pp1s87_57 V6.1 clathrin light chain protein 43, 53Pp1s33_110 V6.2 cobalamin-independent synthase family protein 38, 40, 42, 46−48, 51−53Pp1s338_47 V6.1 copper ion binding LS 43, 53Pp1s44_277 V6.1 copper/zinc superoxide dismutase 1 LS 42, 43, 52, 53Pp1s18_84 V6.1 copper/zinc superoxide dismutase 1 LS 42, 43, 52, 53Pp1s40_48 V6.3 cupredoxin superfamily protein SP 43Pp1s45_111 V6.1 cytidine/deoxycytidylate deaminase family protein LS 53Pp1s15_397 V6.1 dienelactone hydrolase 48, 53Pp1s35_376 V6.1 D-mannose binding lectin protein 38, 39, 41, 45, 48Pp1s370_29 V6.1 embryonic cell protein 63 43Pp1s370_52 V6.1 embryonic cell protein 63 43Pp1s223_38 V6.1 expansin A9 SP 38, 39, 42, 43, 49, 53Pp1s22_206 V6.1 galactose mutarotase-like superfamily protein LS 53Pp1s15_292 V6.2 glutamate-1-semialdehyde-2,1-aminomutase LS 38, 53Pp1s1_784 V6.1 GRF1-interacting factor 3 LS 53Pp1s71_207 V6.3 hemoglobin 1 38, 47, 48, 53Pp1s87_162 V6.2 KH domain-containing protein 53Pp1s1_744 V6.1 late embryogenesis abundant (LEA) protein LS 43Pp1s21_407 V6.1 lipoxygenase 1 LS 53Pp1s175_51 V6.3 LRR protein SP 38, 41−45, 47, 48, 50−53Pp1s12_44 V6.1 no functional description 43Pp1s181_57 V6.4 no functional description SP 53Pp1s150_51 V6.3 no functional description 53Pp1s9_433 V6.1 no functional description LS 53Pp1s519_3 V6.1 no functional description 53Pp1s16_230 V6.1 no functional description 46, 53Pp1s165_12 V6.1 nucleoside diphosphate kinase family protein 38, 40, 46−48, 51, 53Pp1s26_164 V6.1 pathogenesis-related thaumatin superfamily protein SP 38, 39, 42−47, 51−53Pp1s149_288 V6.1 pectinesterase LS 38, 39, 42, 47, 50, 52Pp1s258_34 V6.1 pectinesterase LS 38, 39, 42, 43, 47, 48, 50, 52Pp1s213_80 V6.1 photosystem II light harvesting complex gene 2.2 LS 38, 52Pp1s419_7 V6.1 PLAT/LH2 domain-containing lipoxygenase family protein 53Pp1s218_28 V6.2 pyrophosphorylase 6 LS 38, 53Pp1s58_148 V6.1 Rad23 UV excision repair protein family LS 38, 42, 43, 47, 50, 53Pp1s358_60 V6.2 ribonuclease 1 SP 43, 48, 51−53Pp1s271_98 V6.1 ribosomal protein S11 family protein LS 38, 53Pp1s406_14 V6.1 RNA helicase, ATP-dependent, SK12/DOB1 protein 38Pp1s131_154 V6.1 root FNR 1 LS 43, 46, 47, 52, 53Pp1s351_14 V6.2 Sec14p-like phosphatidylinositol transfer family protein 38, 53Pp1s218_115 V6.1 serine protease LS 38, 42−45, 47, 48, 52, 53Pp1s33_393 V6.5 SPIRAL1-like1 LS 43Pp1s12_214 V6.2 SPIRAL1-like1 LS 43Pp1s39_223 V6.2 thioredoxin SP 38, 42−44, 46−48, 52, 53Pp1s317_45 V6.2 thioredoxin LS 38, 42−44, 46−48, 52, 53Pp1s274_79 V6.1 thioredoxin 38, 42−44, 46−48, 52, 53Pp1s4_75 V6.1 transcriptional coactivator/pterin dehydratase LS 53Pp1s626_4 V6.1 translation initiation factor IF2/IF5 53Pp1s85_94 V6.1 transmembrane proteins 14C 43proteins with functional domains similar to those found in secreted nonhomologous tracheophyte proteinsc

Pp1s183_29 V6.1 glyoxalase/bleomycin resistance protein/dioxygenase superfamily 53Pp1s261_55 V6.1 glyoxalase/bleomycin resistance protein/dioxygenase superfamily 39, 42, 44, 53

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suspension cultures, root exudates and apoplastic fluids,38−53

using fasta36 and ssearch36 programs of the FASTA programpackage claimed to provide rigorous algorithms for identificationof statistically significant sequence similarities that can be used toinfer homology.54 The cutoff E-value < 0.0001 was used toidentify homologous sequences. Homologues for 167 proteins(70%) of the Pp secretome were found in the reportedtracheophyte secretomes (Supporting Information, Table S3).Those 71 proteins detected in Pp secretome, but which have notbeen reported in tracheophyte secretomes (SupportingInformation, Table S3), included a CLP protease (Pp1s25_21V6.1), a class 3 lipase (Pp1s28_217 V6.1) and also proteinsrelated to transcriptional repression, such as a polycomb protein(Pp1s23_249 V6.1) and an SIN3B-related protein (Pp1s212_24V6.1).65,66

Chitosan Responsive Secretory Proteins

There were 72 proteins secreted exclusively following chitosantreatment in the two experiments (Table 2). These proteins arenot included in the 238 proteins of Pp secretome and aredesignated to group B1 in Figure 2 (and Supporting Information,Table S1). Among the 72 proteins whose secretion was chitosan-dependent, 12 proteins (17%) were predicted to contain aclassical secretion signal peptide and 31 proteins (43%) werepredicted to undergo leaderless secretion, whereas secretion of29 proteins (40%) could not be predicted by bioinformaticanalysis. For 57 proteins, homologues could be found in thepublished tracheophyte secretomes38−53 using fasta36 orssearch36 (p < 0.0001) (Table 3; Supporting InformationTable S3). The remaining 15 moss proteins showing chitosan-dependent secretion included six proteins that containedfunctional domains similar to those found in secretedtracheophyte proteins, but were not homologous withtracheophyte proteins at the whole protein level. Four proteinswhose secretion has not been reported were an immune inhibitorA peptidase M6-domain containing protein, unknown proteinDUF427, a phosphorylase superfamily protein, and a ribosomalL7/L12 C-terminal domain-containing protein. The remainingfive proteins could not be placed to any functional category(Table 2).Secretion of specific members of certain protein subfamilies

was found to be dependent on chitosan treatment. They included

a chitinase, two pectinesterases, a serine protease and threethioredoxins (Table 2). Both chitosan inducible pectinesterasescontained an N-terminal pectin methylesterase inhibitor (PMEI)domain pro region, as identified by Pfam search.Chitosan secretome overlapped with the control secretome by

182 proteins in both experiments (Figure 2, group A1).Comparison of spectral counts of peptides between chitosantreated and control cultures, using ≥2-fold difference in quantityand QSPEC FDR-corrected p-value ≤ 0.05 as a threshold,

Table 2. continued

identifier description secretory signala refb

proteins with functional domains similar to those found in secreted nonhomologous tracheophyte proteinsc

Pp1s77_2 V6.1 Pfam:01476 LysM domain 43, 51, 53Pp1s456_19 V6.1 Pfam:06747 CHCH domain LS 53Pp1s111_85 V6.1 phosphatidylethanolamine-binding protein LS 53Pp1s172_76 V6.1 protein tyrosine phosphatase 53proteins with no homologues reported in tracheophyte secretomesPp1s37_124 V6.3 Pfam:05547 immune inhibitor A peptidase M6Pp1s108_26 V6.2 Pfam:04248 domain of unknown function (DUF427) LSPp1s51_288 V6.1 Pfam:01048 phosphorylase superfamily SPPp1s6_183 V6.1 Pfam:00542 ribosomal protein L7/L12 C-terminal domain LSPp1s336_22 V6.1 no functional description LSPp1s98_3 V6.1 no functional descriptionPp1s15_70 V6.2 no functional description SPPp1s185_54 V6.1 no functional description SPPp1s39_165 V6.2 no functional description

aSP, secretory signal peptide as predicted with SignalP 4.0; LS, leaderless secretion as predicted with SecretomeP 2.0 (value >0.5); empty cells,secretion not predictable based on bioinformatic analyses. bReported tracheophyte secretomes containing homologous proteins (FASTA ssearch36/fasta36, E-value cutoff < 0.0001). cResults based on Pfam analysis.

Table 3. Proteins Secreted in Control Moss Cultures andInduced by Chitosan

fold changec

identifier descriptionfold

changea p-valueb 15 30 90 180

Pp1s27_305V6.1

diseaseresistance/leucine-richregion protein

3.8 0.008

Pp1s16_387V6.2

Pfam: PF14368LTP_2

3.3 0.0003

Pp1s79_158V6.3

calmodulin 3.3 0.02

Pp1s32_338V6.2

calmodulin 3.0 0.04 2.3

Pp1s240_68V6.1

no functionaldescription

2.4 0

Pp1s428_9V6.1

no functionaldescription

5.3 0.003

Pp1s21_318V6.2

thioredoxin 5.1 0.02

Pp1s219_8V6.1

peroxidase(Prx34)d

1.4 0.13 4.6

aChitosan-induced differences in the quantity of proteins werecompared between chitosan-treated and control cultures of the mossusing the R Bioconductor package PLGEM version 1.30.033 andQSPEC version 1.2.234 with raw spectral counts as input. ForPp1s428_9 V6.1 and Pp1s21_318 V6.2 the spectral count value incontrol cultures was lower than 5 and the fold difference cannot beconsidered accurate.17 bQSPEC FDR-corrected p-value. cSignificantincrease (FDR ≤ 0.05) in the level of mRNA expression 15, 30, 90, or180 min post treatment with chitosan. dInduction of Prx34 expressionat 180 min post treatment with chitosan has been shown also byqPCR.19

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indicated that secretion of seven proteins detected in the controlcultures increased significantly following chitosan treatment inboth experiments (Table 3). These proteins were a leucine-richrepeat protein, a lipid transfer protein LTP_2 domain-containingprotein, two calmodulin proteins, thioredoxin, and two proteinswith no known function (Table 3). The amounts of theseproteins increased >2.3 fold, which is a difference that could bedetermined at >95% confidence level.67

Expression of genes encoding the proteins, whose quantity wasfound to increase in the culture medium upon treatment of themoss with chitosan (Table 3), was assessed using microarrayanalysis. The chitosan-inducible peroxidase gene Prx34, whoseexpression was previously studied using quantitative real-timePCR (qPCR),19 was used as a control. Microarray analysisrevealed a 4.6-fold increase in the mRNA expression of Prx34 at180 min post-treatment with chitosan (Table 3), which isconsistent with the 3.5-fold increase in the expression of Prx34detected by qPCR at the same time post-treatment withchitosan.19 The quantity of Prx34 increased by 39−45% in theculture medium (spectral counts in control vs chitosan-treatedcultures were 62 vs 86, and 66 vs 96, respectively, in the twoexperiments), but the difference was not statistically significant ascompared with untreated control. Expression of one of the twogenes encoding calmodulin proteins (i.e., the gene forPp1s32_338 V6.2) increased by 2.3-fold in response to chitosantreatment, and the amount of the protein increased 3-fold in theculture medium. However, no significant induction of expressionwas observed with the other six genes encoding proteins whosequantity increased significantly in the culture medium uponchitosan treatment (Table 3).Gene ontology (GO) analysis using Blast2GO software37 and

comparison of proteins classified to different GO groups betweenthe Pp secretome and the secretome of chitosan-treated samples(group A vs group B in Figure 1) showed enrichment of proteinsbelonging to GO groups related to metabolism, “cellularcomponent organization” (e.g., cell wall modification) and“response to stress” in chitosan-treated samples (Figure 3).There were 27 proteins, which were detected in Pp secretome

(control cultures), but were not found to be secreted in chitosan-treated cultures (Table 4). These proteins were designated togroup A2 (Figure 2; Supporting Information, Table S1). One ofthese proteins was homologous to STIG1, which controlsstigmatic exudate secretion in pistils of petunia and tobacco andis secreted in rice (Oryza sativa L.)51,68 (Table 4).A few proteins found in Pp secretome were replaced with

another member of the same protein subfamily secreted as aresponse to chitosan treatment. They included a serine protease(Pp1s48_63 V6.1 replaced by Pp1s218_115 V6.1), a leucinerepeat rich protein (Pp1s35_305 V6.1 replaced by Pp1s175_51V6.3) and a dienelactone hydrolase (Pp1s241_102 V6.1 replacedby Pp1s15_397 V6.1) (Tables 2 and 4).There were 62 proteins secreted following chitosan treatment

in both experiments, but because they were found also in thecontrol cultures in one experiment, their secretion could not beconsidered chitosan-dependent (Figure 2, group B2). Eightyproteins were secreted following chitosan treatment only in oneof the two experiments. Results on these proteins wereconsidered inconsistent (Figure 2, group A3 and Table S1).They are available in Supporting Information, Table S1 (groupsA3, C1, and C3) but are not further discussed.

Proteins Detected Using 2-Dimensional Electrophoresis(2-DE)

Three experiments were carried out on the secretome ofchitosan-treated P. patens using the conventional 2-DE-LC-MS/MS analysis to compare the results with the relatively new gel-free Orbitrap method. The three 2-DE experiments resulted in aconsistent topology of protein spots, but only 30 proteins couldbe identified. They included 24 proteins identical to thosedetected with the gel-free method, and the remaining six proteinsincluding a GDSL lipase, three glyoxal oxidases, a laccase and aleucine rich repeat protein were closely related to the proteinsdetected with the gel-free approach (Table 5). A largerproportion (25 out of 30) of the ECPs detected by 2-DE thanfound using the gel-free approach (30%) contained a classicalsecretion signal peptide. Five ECPs proteins detected using 2-DEwere leaderless secretory proteins, as predicted with SecretomeP2.0 (Table 5).

■ DISCUSSIONResults of this study show that the ECPs of P. patens includeseveral hundred proteins that can be released when the moss isgrown submerged in nutrient-containing water solution, whichmimics temporary growth conditions of P. patens in its naturalhabitats.12 A large proportion (70%) of the ECPs of P. patensreleased under these conditions were homologous to the

Figure 3.Number of secreted proteins in different gene ontology (GO)groups in controls and chitosan-treated samples of Physcomitrella patensaccording to GO-Slim plant subset at GO level 3.

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proteins detected in tracheophyte secretomes in cell suspensioncultures, root exudates and apoplastic fluids.38−53 The proteins,

which are typically abundant in the secretomes of tracheophytes,belong to functional groups related to pathogen de-

Table 4. Proteins Which Were Not Found in the CultureMedium Following Treatment of Physcomitrella patens withChitosan but Were Found in Control Cultures of P. patens

identifier descriptionsecretorysignala refb

Pp1s170_63V6.1

AP2/B3-like transcriptional factorfamily protein

38

Pp1s67_37 V6.1 basic helix−loop−helix (bHLH)DNA-binding superfamilyprotein

LS 43

Pp1s241_102V6.1

dienelactone hydrolase LS 53

Pp1s109_205V6.2

DYNAMIN-like 1E LS 38, 53

Pp1s6_39 V6.2 eukaryotic aspartyl protease familyprotein

SP 38, 39, 42−45,47, 48,51−53

Pp1s130_236V6.1

fasciclin-like arabinogalactanfamily protein

38, 42, 43, 51,53

Pp1s35_305V6.1

leucine-rich repeat (LRR) familyprotein

SP 38, 41−45, 47,48, 50−53

Pp1s33_206V6.2

NAD(P)-binding Rossmann-foldsuperfamily protein

LS 38, 53

Pp1s31_134V6.1

no functional description SP 43, 51

Pp1s141_35V6.1

no functional description 46

Pp1s44_71 V6.1 peroxidase family protein SP 38, 39, 41−48,51−53

Pp1s20_77 V6.1 peroxidase superfamily protein LS 38, 39, 41−48,51−53

Pp1s55_121V6.2

peroxidase superfamily protein SP 38, 39, 41−48,51−53

Pp1s66_27 V6.1 peroxidase superfamily protein SP 38, 39, 41−48,51−53

Pp1s67_214V6.3

peroxidase superfamily protein SP 38, 39, 41−48,51−53

Pp1s137_223V6.1

plant protein of unknown function(DUF869)

38, 43, 53

Pp1s158_50V6.1

pleckstrin homology (PH)domain superfamily protein

LS 43, 53

Pp1s149_229V6.1

RmlC-like cupins superfamilyprotein

SP 38, 40, 43−45,49, 51−53

Pp1s48_63 V6.1 serine protease LS 38, 42−45, 47,48, 52, 53

Pp1s65_175V6.1

stigma-specific Stig1 familyprotein

SP 51

Pp1s122_57V6.1

xyloglucan endotransglucosylase/hydrolase 5

SP 38, 39, 41−45,48, 51, 53

Pp1s309_41V6.1

xyloglucan endotransglucosylase/hydrolase 9

SP 38, 39, 41−45,48, 51, 53

Pp1s217_58V6.1

exostosin family LS 38c

Pp1s201_96V6.1

glyoxal oxidase-related protein(DUF1929)

LS

Pp1s38_59 V6.1 RNA pseudouridylate synthase

Pp1s441_12V6.1

no functional description

Pp1s237_75V6.1

no functional description SP

aSP, secretory signal peptide as predicted with SignalP 4.0; LS,leaderless secretion as predicted with SecretomeP 2.0 (value > 0.5);empty cells, secretion not predictable based on bioinformatic analyses.bReported tracheophyte secretomes containing homologous proteins(FASTA ssearch36/fasta36, E-value cutoff < 0.0001); empty cells, nohomologous protein reported in tracheophyte secretomes. cPfamanalysis indicated that Arabidopsis cell wall proteome38 contains anonhomologous protein sharing a domain similar to Pp1s217_58V6.1.

Table 5. Proteins Detected in the Culture Medium ofChitosan-Treated Physcomitrella patens using 2-DimensionalElectrophoresis and LC-MS/MS

identifier descriptionMW(kDa) pI

secretorya

signal

Pp1s86_72V6.2

apolipoprotein 25.1 4.8 LS

Pp1s144_96V6.1

chitinase 40.5 4.4 SP

Pp1s184_140V6.1

chitinase class IV 31.1 5.0 SP

Pp1s68_12V6.1

cupin 30.8 9.4 LS

Pp1s82_6 V6.1 cupin 22.8 8.4 SPPp1s97_59V6.2

cupin 21.8 8.9 SP

Pp1s120_85V6.2

GDSL-like lipase/acylhydrolase

42.6 6.4 SP

Pp1s17_355V6.1

GDSL-like lipase/acylhydrolase

46.1 7.6 SP

Pp1s17_356V6.1

GDSL-like lipase/acylhydrolaseb

40.7 9.0 SP

Pp1s22_251V6.1

glycerophosphoryl diesterphosphodiesterase

65.7 4.7 LS

Pp1s58_157V6.2

glyoxal oxidase 56.7 5.2 SP

Pp1s53_16V6.1

glyoxal oxidaseb 57.8 5.1 SP

Pp1s43_101V6.1

glyoxal oxidase 56.4 4.9 SP

Pp1s30_238V6.1

glyoxal oxidaseb 57.0 4.9 SP

Pp1s95_56V6.1

glyoxal oxidaseb 37.8 5.2 LS

Pp1s86_61V6.2

laccaseb 63.3 4.9 SP

Pp1s27_305V6.1

disease resistance/leucine-rich region protein

34.8 8.6 SP

Pp1s240_68V6.1

no functional description 33.2 7.3 SP

Pp1s171_64V6.1

no functional description 23.5 7.6 SP

Pp1s195_100V6.1

no functional description 51.6 6.7 SP

Pp1s44_116V6.1

no functional description 20.3 7.3 SP

Pp1s236_72V6.1

pectinesterase 40.2 6.0 SP

Pp1s215_36V6.1

pectinesterase 40.8 8.3 SP

Pp1s219_8V6.1

peroxidase (Prx34) 35.8 6.4 SP

Pp1s101_103V6.1

purple acid phosphatase 61.7 5.3 SP

Pp1s287_63V6.1

serine-threonine proteinkinaseb

68.4 4.9 SP

Pp1s21_318V6.2

thioredoxin 13.5 6.0 LS

Pp1s81_246V6.1

xyloglucanendotransglycosylase

31.9 5.4 SP

Pp1s346_19V6.1

xyloglucanendotransglycosylase

40.4 4.9 SP

Pp1s486_14V6.1

xyloglucanendotransglucosylase/hydrolase 9

32.1 4.9 SP

aSP, secretory signal peptide as predicted with SignalP 4.0; LS,leaderless secretion as predicted with SecretomeP 2.0 (value > 0.5).bProtein not detected in Orbitrap analysis.

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fense,39,40,42,44−46,48,52,69 metabolism,40,42,46,48 protein modifica-tion,42,45,50 cell wall modification,40,48 redox,52,69 and signal-ing.40,47 Proteins belonging to these functional categories weredetected also in the secretome of P. patens.Treatment of P. patens with chitosan released 72 new proteins

to the culture medium, which were not found in the controlcultures. They included many proteins such as chiti-nases42,44,48,49 and thaumatin,52 which are homologous toproteins secreted by tracheophytes upon treatment withpathogens or defense elicitors. Furthermore, it was found thatcertain members of protein subfamilies were released to theculture medium upon chitosan treatment, in contrast to others. Itis becoming evident that the responses to micro-organisms inplants are coordinated spatiotemporally based on intra- orextracellular stimuli. For example, type I pectinmethylesterases(PME) acting on pectic polysaccharides in the cell walls ofNicotiana benthamiana contain an N-terminal pectin methyl-esterase inhibitor (PMEI) pro region, which retains PMEsinactive in the Golgi until a yet undefined signal activates asubtilisin-type serine protease to remove the pro region.70 Hencethe inactive preproteins can accumulate and be prevented fromacting untimely. In our study, the N-terminal PMEI pro regionwas predicted by Pfam in two chitosan-responsive pectinmethy-lesterases of P. patens.Expression of mRNA provides evidence for likely production

of the corresponding protein, but secretion of a protein is notnecessarily associated with simultaneous increase in mRNAexpression of the corresponding gene. Indeed, only ∼40% of thevariation in protein concentration can be explained by mRNAabundances.71 For example, Prx34 is a secreted peroxidasepivotal to antifungal defense in P. patens.19 Our results show thatsecretion of Prx34 increases rapidly upon chitosan treatment andresults in an immediate oxidative burst.29 However, qPCRanalysis shows that the expression of Prx34 mRNA is enhancedonly gradually over 180 min post-treatment with chitosan,19

which was reconfirmed by microarray analysis in this study. Forthe majority of other genes tested, no significant enhancement oftranscription was observed at 180 min post treatment withchitosan, despite the fact that the quantity of the correspondingproteins increased significantly in the culture medium due tosecretion. The results are characteristic of perturbed systems, inwhich protein quantity appears to be regulated post-translation-ally rather than at mRNA level.72

Peroxidases and thioredoxins were most responsive totreatment of P. patens with chitosan, suggesting that the oxidativeenvironment is altered by the increase in number and activity ofthese redox enzymes. Thioredoxin was one of the seven proteinsdetected in the control cultures but found be present insignificantly higher amounts in the cultures treated with chitosan.Furthermore, the secreted peroxidase Prx34, which is pivotal tothe oxidative burst and antifungal resistance of P. patens,19,29 wasfound to be among the most abundant proteins in the secretomeof P. patens in control cultures and its amounts increased clearly(ca. 40%) following treatment with chitosan. These results areconsistent with previous studies, which revealed a rapid release ofPrx34 to the culture medium upon chitosan treatment anddetected a subsequent, quick oxidative burst.19,29 Takentogether, it seems apparent that P. patens secretes Prx34constitutively as a protective mechanism against fungi (andpossibly insects) containing chitin as a major cell wallconstituent, but the low peroxidase activity detected in controlcultures in previous studies suggests that the peroxidase activityof Prx34 is not long-lasting.19,29 It may be for that reason that

quick release of fresh, active Prx34 is needed at fungal attack,which was mimicked with chitosan treatment in our experiments.Chitosan-responsive ECPs of P. patens included a protein

containing a domain of unidentified function (DUF427) and animmune inhibitor A (InA) peptidase M6 domain-containingprotein, which have not been reported in the secretomes oftracheophytes challenged with pathogens or defense elic-itors.39,40,42,44−46,48,52,69 Pfam and InterPro databases containthree plant protein sequences with DUF427, namely twoproteins of P. patens and one protein of rice (Oryza sativa L.ssp. indica). However, a gene encoding a DUF427-containingprotein exists also in barley (Hordeum vulgare L.). The gene isinduced under biotic and abiotic stress conditions, but details ofits functional significance remain to be elucidated.73 Inmycobacteria, a DUF427-containing protein is under the controlof a cyclic AMP receptor protein, which represents a regulationsystem needed under cell stress and in pathogenesis.74 It may bespeculated that the DUF427-containing ECP of P. patens mightinterfere with the DUF427-containing proteins of pathogens toprevent infection, and a similar role may be hypothesized for theInA peptidase M6 domain-containing protein of the moss. InA isa metallopeptidase secreted by Bacillus thuringiensis, a bacteriumpathogenic to insects, to cleave and destroy host antibacterialproteins.75 The peptidaseM6 domain of InA can be found in fourproteins of P. patens, but only one of them was secreted upontreatment with chitosan. Finally, there were chitosan-responsiveproteins of P. patens that were not homologous to proteinsdetected in tracheophytes but contained similar functionaldomains. One of these moss proteins (Pp1s172_76 V6.1)contained a tyrosine phosphatase domain known in a mucilageprotein of maize.53 This moss protein also contained the C2domain of the tumor-suppressor protein PTEN, which isinvolved in lipid signaling and induced by salt and osmoticstresses in Arabidopsis.76

The studies on tracheophyte secretomes have revealed manyribosomal proteins and also DNA-binding proteins in theextracellular space, where their roles are obscure. In our study,treatment of P. patens with chitosan released a member of theribosomal S11 protein family and a protein, which was differentfrom the ribosomal proteins found in tracheophyte secretomesbut contained the ribosomal protein L7/L12 domain. In bacteria,ribosomal protein L7/12 interacts with the bacterial elongationfactor EF-Tu,77 which is an elicitor of innate immunity inplants.78 EF-Tu is recognized in plants by the serine/threoninekinase receptor EFR,79 and ethylene modulates EFR-triggeredimmunity by enhancing salicylic acid mediated defense.80 Hence,the extracellular moss protein containing L7/L12 domain mightmodulate moss-microbe interactions. Polymeric DNA excretedfrom the root cap in pea (Pisum sativum L.) enhances resistanceto fungal infection, but whether the DNA-binding proteins of thehost play a role remains to be studied.81 Extracellular ATP-binding proteins could be important because extracellular ATPplays a role in pathogen defense and programmed cell deathregulation.82 The secretomes of chitosan-treated and controlmoss contained a few DNA-binding proteins (SupportingInformation, Table S1). They also included a proteinhomologous to heat shock protein with DnaJ domain(Pp1s29_56 V6.1). Recent studies have demonstrated that aDnaJ domain-containing heat shock protein (HSP40.1) plays akey role in cell death and pathogen defense in soybean (Glycinemax L.).83

Besides triggering the release of 72 proteins, treatment of P.patens with chitosan inhibited the release of 27 proteins. One of

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them was a glyoxal oxidase containing the conserved motifDUF1929 found in eight glyoxal oxidases of P. patens. Genesencoding DUF1929-containg glyoxal oxidases are found intracheophytes but the proteins are not known to be secreted.Their role in pathogen defense is suggested, because theDUF1929-containg glyoxal oxidase derived from a variety ofgrape vine (Vitis pseudoreticulataW.T. Wang), which is resistantto the powdery mildew fungus (Erysiphe necator Schwein.),increases resistance to powdery mildew when overexpressed in asusceptible variety.84 The mechanism of resistance mediated bythe glyoxal oxidase is not known inVitis85 and the putative role ofthe DUF1929-containing protein in, e.g., control of oxidativeburst and cell death in P. patens remains to be elucidated.Chitosan treatment also prevented moss tissue from releasing aprotein (Pp1s65_175v6.1) that is homologous to STIG1 andGRIM REAPER (GRI). STIG1 is secreted in rice51 and controlssecretion of stigmatic exudates in pistils of petunia and tobacco.68

GRI controls generation of extracellular oxygen radicals and celldeath in Arabidopsis.86 GRI is cleaved by metacaspase-9 in theextracellular space, and the resulting N-terminal peptide binds tothe extracellular domain of a membrane-bound receptor-likeprotein kinase, which mediates the death signal into the cell.86,87

The gri mutants of Arabidopsis lacking GRI and the Arabidopsisplants overexpressing GRI are more sensitive to oxygen radicalsthan the wild-type plants,86 which suggests that alsoPp1s65_175v6.1 might play a role in control of redoxhomeostasis in P. patens. Such control is evident, because despiteof the rapid oxidative burst occurring within 2.5 min fromaddition of chitosan to the liquid cultures of P. patens,19,29 themoss plants in our study showed no signs of cell death during thetime of incubation (180 min).This study has revealed over 400 extracellular proteins of P.

patens released to the liquid culture medium, including ∼100proteins whose release was affected by treatment of the mosswith chitosan, which is a plant defense elicitor. There is littleinformation available on the protein secretomes of bryophytes.The results presented here allow the first more comprehensivecomparison of bryophyte and tracheophyte secretomes andindicate many similarities. A large proportion of the extracellularproteins of P. patens could not be predicted based onbioinformatics analyses, as found in the studies on tracheophytes.Bioinformatics can be used to predict secreted proteins based onsignal peptide or other conserved sequences, but prediction ofleaderless secretion is based on mammalian proteins, whichlimits applicability because of the differences in the extracellularmatrix of plants and mammalians.36 There may also be other,less-known secretory pathways in plants.2 In this study, theextracellular proteins were obtained using a noninvasive methodand the moss plants did not apparently suffer from the treatment,which should largely exclude contamination of the samples withcytoplasmic proteins. Using chitosan as a defense elicitor insteadof infection with fungal pathogens was motivated with the samegoal, because infection with necrotrophic pathogens disruptscells and results in release of cellular proteins.19,20,24 The datapresented here lays the basis for more detailed studies on thenewly identified secretory proteins and their roles inphysiological processes, such as biotic and abiotic stress.

■ ASSOCIATED CONTENT

*S Supporting Information

Enzymes detected in the secretome of Physcomitrella patens andplaced to the KEGG global metabolic map, two-dimensional

electrophoresis (2-DE) of the extracellular proteins isolated fromthe liquid culture medium of Physcomitrella patens treated withchitosan, all secreted proteins of P. patens detected in this study,proteins belonging to multimember subfamilies and detected inthe secretome of P. patens, and comparison of the secretedproteins of P. patens detected in this study with those reported intracheophyte secretomes using FASTA package ssearch36 andfasta36 programs. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author

*Tel. +358-9-19158387. Fax. +358-9-19158727. E-mail: jari.valkonen@helsinki.fi.Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Eeva Marttinen for photography. Financial supportfrom the Academy of Finland (grant 1253126), FinlandDistinguished Professor Program (FiDiPro) and The FinnishDoctoral Programme in Plant Biology is gratefully acknowl-edged.

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