Protein Secretome of Moss Plants (Physcomitrella patens) with

Article pubs.acs.org/jpr

Protein Secretome of Moss Plants (Physcomitrella patens) with Emphasis on Changes Induced by a Fungal Elicitor Mikko T. Lehtonen,† Yoshihiro Takikawa,‡ Gunilla Rönnholm,§ 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 of apoplast. Unlike vascular plants (tracheophytes), little information about ECPs is available from nonvascular plants, such as mosses (bryophytes). In this study, moss plants (Physcomitrella patens) were grown in liquid culture and treated with chitosan, a water-soluble form of chitin that occurs in cell walls of fungi and insects and elicits pathogen defense in plants. ECPs released to the culture medium were compared between chitosan-treated and nontreated control cultures using quantitative mass spectrometry (Orbitrap) and 2-DELC-MS/MS. Over 400 secreted proteins were detected, of which 70% were homologous to ECPs reported in tracheophyte secretomes. Bioinformatics analyses using SignalP and SecretomeP predicted classical signal peptides for secretion (37%) or leaderless secretion (27%) for most ECPs of P. patens, but secretion of the remaining proteins (36%) could not be predicted using bioinformatics. Cultures treated with chitosan contained 72 proteins not found in untreated controls, whereas 27 proteins found in controls were not detected in chitosan-treated cultures. Pathogen defense-related proteins dominated in the secretome of P. patens, as reported in tracheophytes. These results advance knowledge on protein secretomes of plants by providing a comprehensive account of ECPs of a bryophyte. KEYWORDS: plant secretome, proteomics, extracellular proteins, moss, bryophyte, Physcomitrella patens, defense elicitor, chitin, chitosan, quantitative mass spectrometry

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INTRODUCTION Secreted proteins play important roles in a wide range of biological processes such as cellular signaling, cell-to-cell communication and responses to biotic and abiotic stresses.1 Protein secretion can be mediated by an N-terminal signal peptide, which targets the protein to endoplasmic reticulum (ER). Following removal of the signal peptide, the mature protein is moved by secretory Golgi apparatus beyond the plasma membrane to the extracellular matrix. However, a large proportion 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, for example, “exocyst-positive organelle” mediated protein secretion or involve small Rab-type GTPases in trafficking specific secretory vesicles during cell plate formation and polarized cell expansion.3,4 It is also suggested that exosomal fusion of multivesicular bodies to plasma membrane could offer a means for protein secretion in plants.5,6 The extracellular matrix influences protein secretion. Plant cells are surrounded by a cell wall that consists of coherently aligned cellulose microfibrils and can be lignified.7,8 The outer © XXXX American Chemical Society

surface of plants is covered by an epidermis layered with cutin, suberin polymers and waxes, which form a water-impermeable cuticle.9 These characteristics of the differentiated, green, photosynthesizing parts of most tracheophyte species (vascular plants) make them less suitable for the study of protein secretion. Therefore, studies are usually done using undifferentiated plant cells or tissues grown as a suspension in liquid medium, which facilities isolation of the secreted proteins from the culture medium for analysis. Root exudates can be collected using similar approaches.1 In this respect, the characteristics of mosses, which are small bryophyte plants, offer some special opportunities for the study of protein secretome. Physcomitrella patens (Hedw.) B.S.G. is a moss species in which the haploid growth phase dominates. The haploid gametophytes initiate their growth as protonema, which are filamentous and lack a cuticule, and differentiate into stem and leaves that are not structurally complex (Figure 1A). Leaves consist of a single cell layer.10,11 P. patens is an early colonist on exposed mud and soil Received: June 10, 2013

A

dx.doi.org/10.1021/pr400827a | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Article

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.

to elevate their level of resistance.27,28 Treatment of P. patens with chitosan results in a rapid oxidative burst dependent on a secreted peroxidase (Prx34).19,29 The Prx34 knockout lines of P. patens are rendered susceptible to fungi, indicating the pivotal role of Prx34 in pathogen defense of the moss.19,29 The aim of this study was to analyze the secretome of P. patens and the possible qualitative and quantitative changes occurring in ECPs released to the culture medium upon chitosan treatment. Results were expected to advance the knowledge on how the moss extracellular proteome is affected by a fungal elicitor and to expand the knowledge on plant secretomes, which is currently limited to tracheophytes.

around pools of water and becomes naturally submerged in its growth habitats.12 The whole plants of P. patens can be grown submerged in liquid culture medium in the laboratory and the secreted proteins harvested from the culture filtrates. These properties of P. patens have found industrial applications in production of recombinant proteins that have been tagged with the classical secretion signal sequences and can be harvested from the moss culture medium.13,14 However, it appears that besides a thesis whose data are not readily accessible,15 there is little information about the protein secretome of wild-type P. patens or any other mosses. Secretome studies are now feasible because the whole genome sequence of P. patens is available and facilitates protein identification.16 Furthermore, gel-free shotgun proteomics approaches based on high-resolution mass spectrometry provide a powerful approach to characterize the protein secretome and obtain quantitative data by spectral counting, that is, by determining the number of copies of a given peptide in the sample.17 P. patens is emerging as a model plant for studies on plantpathogen interactions,18−23for which pathosystems based on fungi isolated from mosses have been established.19,20,24 In mosses and tracheophytes the extracellular matrix of host cells provides a contact interface and site for recognition of fungal pathogens, followed by defense responses such as oxidative burst, rapid deposition of altered and strengthened cell walls and formation of papillae at the site of infection.20,25 Chitin is a polymer that occurs naturally in cell walls of fungi and insects and elicits host defense in P. patens19,20,24 and tracheophytes.26 For control of plant diseases, chitin is deacetylated to 90% and the resultant water-soluble compound, chitosan, is applied on plants

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MATERIALS AND METHODS

Plant Material

Protonemal tissue of P. patens, ecotype Gransden Wood,30 was grown in Petri dishes (Ø 9 cm) on a cellophane membrane (400P; Visella Oy, Valkeakoski, Finland) placed on BCD medium [1 mM MgSO4, 1.85 mM KH2PO4 (pH 6.5, adjusted with KOH), 10 mM KNO3, 45 μM FeSO4, 0.22 μM CuSO4, 0.19 μM ZnSO4, 10 μM H3BO4, 0.10 μM Na2MoO4, 2 μM MnCl2, 0.23 μM CoCl2, 0.17 μM KI]30 supplemented with 1 mM CaCl2, 45 μM ethylenediaminetetraacetic acid disodium salt (Na2EDTA), and 5 mM ammonium tartrate [(NH4)2C4H4O6], and solidified with 0.8% agar. The cultures were grown in a growth cabinet (Model 3755, Forma Scientific, Marietta, OH, U.S.A.) at 23 °C (photoperiod 12 h, light intensity 60 μmol m−2 s−1) and subcultured weekly. B



Article

Treatment with Chitosan

mass spectrometry analysis. Solvents for LC-MS separation of the digested samples were as follows: solvent A consisted of 0.1% formic acid in water (98%) and acetonitrile (2%) and solvent B consisted of 0.1% formic acid in acetonitrile (98%) and water (2%). From a thermostatic microautosampler the tryptic peptide mixture were automatically loaded onto a 15 cm fused silica analytical column with an inner diameter of 75 μm packed with C18 reversed phase material (Thermo Scientific) and the peptides were eluted from the analytical column with a 60 min gradient ranging from 5% to 35% solvent B, followed by a 10 min gradient from 35% to 80% solvent B at a constant flow rate of 300 nL/min. The analyses were performed in a data-dependent acquisition mode using a top 10 collision-induced dissociation (CID) method. Dynamic exclusion for selected ions was 30 s. No lock masses were employed. Maximal ion accumulation time allowed on the Orbitrap Elite ETD in CID mode was 100 ms for MSn in the Ion Trap and 200 ms in the FTMS. Automatic gain control was used to prevent overfilling of the ion traps and were set to 10 000 (CID) in MSn mode for the Ion Trap, and 106 ions for a full FTMS scan. Intact peptides were detected in the Orbitrap at 60 000 resolution. Peak extraction and subsequent protein identification were achieved using Proteome Discoverer software (Thermo Scientific, Waltham, MA).

−1

The stock solution of chitosan (10 mg mL ) was prepared by dissolving chitosan oligosaccharide (degree of deacetylation 90%, MW 0.5); empty cells, secretion not predictable based on bioinformatic analyses. cReported tracheophyte secretomes containing homologous proteins (FASTA ssearch36/fasta36, Evalue cutoff < 0.0001); empty cells, no homologous protein reported in tracheophyte secretomes. E



Article

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

secretory signala

description

proteins with homologues reported in tracheophyte secretomes Pp1s40_134 V6.1 1-cysteine peroxiredoxin 1 Pp1s223_50 V6.1 2-oxoacid dehydrogenases acyltransferase family protein Pp1s72_290 V6.1 ABC-2 and Plant PDR ABC-type transporter family protein Pp1s89_161 V6.2 aldolase superfamily protein Pp1s159_68 V6.3 aldolase-type TIM barrel family protein Pp1s199_134 V6.1 aleurain-like protease Pp1s18_41 V6.3 AMP-dependent synthetase and ligase family protein Pp1s35_384 V6.1 ATP citrate lyase subunit B 2 Pp1s309_77 V6.1 chitinase Pp1s87_57 V6.1 clathrin light chain protein Pp1s33_110 V6.2 cobalamin-independent synthase family protein Pp1s338_47 V6.1 copper ion binding Pp1s44_277 V6.1 copper/zinc superoxide dismutase 1 Pp1s18_84 V6.1 copper/zinc superoxide dismutase 1 Pp1s40_48 V6.3 cupredoxin superfamily protein Pp1s45_111 V6.1 cytidine/deoxycytidylate deaminase family protein Pp1s15_397 V6.1 dienelactone hydrolase Pp1s35_376 V6.1 D-mannose binding lectin protein Pp1s370_29 V6.1 embryonic cell protein 63 Pp1s370_52 V6.1 embryonic cell protein 63 Pp1s223_38 V6.1 expansin A9 Pp1s22_206 V6.1 galactose mutarotase-like superfamily protein Pp1s15_292 V6.2 glutamate-1-semialdehyde-2,1-aminomutase Pp1s1_784 V6.1 GRF1-interacting factor 3 Pp1s71_207 V6.3 hemoglobin 1 Pp1s87_162 V6.2 KH domain-containing protein Pp1s1_744 V6.1 late embryogenesis abundant (LEA) protein Pp1s21_407 V6.1 lipoxygenase 1 Pp1s175_51 V6.3 LRR protein Pp1s12_44 V6.1 no functional description Pp1s181_57 V6.4 no functional description Pp1s150_51 V6.3 no functional description Pp1s9_433 V6.1 no functional description Pp1s519_3 V6.1 no functional description Pp1s16_230 V6.1 no functional description Pp1s165_12 V6.1 nucleoside diphosphate kinase family protein Pp1s26_164 V6.1 pathogenesis-related thaumatin superfamily protein Pp1s149_288 V6.1 pectinesterase Pp1s258_34 V6.1 pectinesterase Pp1s213_80 V6.1 photosystem II light harvesting complex gene 2.2 Pp1s419_7 V6.1 PLAT/LH2 domain-containing lipoxygenase family protein Pp1s218_28 V6.2 pyrophosphorylase 6 Pp1s58_148 V6.1 Rad23 UV excision repair protein family Pp1s358_60 V6.2 ribonuclease 1 Pp1s271_98 V6.1 ribosomal protein S11 family protein Pp1s406_14 V6.1 RNA helicase, ATP-dependent, SK12/DOB1 protein Pp1s131_154 V6.1 root FNR 1 Pp1s351_14 V6.2 Sec14p-like phosphatidylinositol transfer family protein Pp1s218_115 V6.1 serine protease Pp1s33_393 V6.5 SPIRAL1-like1 Pp1s12_214 V6.2 SPIRAL1-like1 Pp1s39_223 V6.2 thioredoxin Pp1s317_45 V6.2 thioredoxin Pp1s274_79 V6.1 thioredoxin Pp1s4_75 V6.1 transcriptional coactivator/pterin dehydratase Pp1s626_4 V6.1 translation initiation factor IF2/IF5 Pp1s85_94 V6.1 transmembrane proteins 14C proteins with functional domains similar to those found in secreted nonhomologous tracheophyte proteinsc Pp1s183_29 V6.1 glyoxalase/bleomycin resistance protein/dioxygenase superfamily Pp1s261_55 V6.1 glyoxalase/bleomycin resistance protein/dioxygenase superfamily F

LS LS SP LS LS SP

LS LS LS SP LS

SP LS LS LS

LS LS SP SP LS

SP LS LS LS LS LS SP LS LS LS LS LS SP LS LS

refb 40, 38, 53 38, 53 40, 38, 38, 39, 43, 38, 43, 42, 42, 43 53 48, 38, 43 43 38, 53 38, 53 38, 53 43 53 38, 43 53 53 53 53 46, 38, 38, 38, 38, 38, 53 38, 38, 43, 38, 38 43, 38, 38, 43 43 38, 38, 38, 53 53 43

47, 52, 53 40, 43, 53 40, 42, 43, 46, 47, 51, 53 42−44, 47, 51, 53 43, 53 53 42−45, 48, 49, 51−53 53 40, 42, 46−48, 51−53 53 43, 52, 53 43, 52, 53

53 39, 41, 45, 48

39, 42, 43, 49, 53 53 47, 48, 53

41−45, 47, 48, 50−53

53 40, 39, 39, 39, 52

46−48, 51, 53 42−47, 51−53 42, 47, 50, 52 42, 43, 47, 48, 50, 52

53 42, 43, 47, 50, 53 48, 51−53 53 46, 47, 52, 53 53 42−45, 47, 48, 52, 53

42−44, 46−48, 52, 53 42−44, 46−48, 52, 53 42−44, 46−48, 52, 53

53 39, 42, 44, 53



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Table 2. continued identifier

secretory signala

description

refb

c

proteins with functional domains similar to those found in secreted nonhomologous tracheophyte proteins Pp1s77_2 V6.1 Pfam:01476 LysM domain Pp1s456_19 V6.1 Pfam:06747 CHCH domain LS Pp1s111_85 V6.1 phosphatidylethanolamine-binding protein LS Pp1s172_76 V6.1 protein tyrosine phosphatase proteins with no homologues reported in tracheophyte secretomes Pp1s37_124 V6.3 Pfam:05547 immune inhibitor A peptidase M6 Pp1s108_26 V6.2 Pfam:04248 domain of unknown function (DUF427) LS Pp1s51_288 V6.1 Pfam:01048 phosphorylase superfamily SP Pp1s6_183 V6.1 Pfam:00542 ribosomal protein L7/L12 C-terminal domain LS Pp1s336_22 V6.1 no functional description LS Pp1s98_3 V6.1 no functional description Pp1s15_70 V6.2 no functional description SP Pp1s185_54 V6.1 no functional description SP Pp1s39_165 V6.2 no functional description

43, 51, 53 53 53 53

a

SP, 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.

suspension cultures, root exudates and apoplastic fluids,38−53 using fasta36 and ssearch36 programs of the FASTA program package claimed to provide rigorous algorithms for identification of statistically significant sequence similarities that can be used to infer homology.54 The cutoff E-value < 0.0001 was used to identify homologous sequences. Homologues for 167 proteins (70%) of the Pp secretome were found in the reported tracheophyte secretomes (Supporting Information, Table S3). Those 71 proteins detected in Pp secretome, but which have not been reported in tracheophyte secretomes (Supporting Information, Table S3), included a CLP protease (Pp1s25_21 V6.1), a class 3 lipase (Pp1s28_217 V6.1) and also proteins related to transcriptional repression, such as a polycomb protein (Pp1s23_249 V6.1) and an SIN3B-related protein (Pp1s212_24 V6.1).65,66

Table 3. Proteins Secreted in Control Moss Cultures and Induced by Chitosan fold changec identifier Pp1s27_305 V6.1 Pp1s16_387 V6.2 Pp1s79_158 V6.3 Pp1s32_338 V6.2 Pp1s240_68 V6.1 Pp1s428_9 V6.1 Pp1s21_318 V6.2 Pp1s219_8 V6.1

Chitosan Responsive Secretory Proteins

There were 72 proteins secreted exclusively following chitosan treatment in the two experiments (Table 2). These proteins are not included in the 238 proteins of Pp secretome and are designated to group B1 in Figure 2 (and Supporting Information, Table S1). Among the 72 proteins whose secretion was chitosandependent, 12 proteins (17%) were predicted to contain a classical secretion signal peptide and 31 proteins (43%) were predicted to undergo leaderless secretion, whereas secretion of 29 proteins (40%) could not be predicted by bioinformatic analysis. For 57 proteins, homologues could be found in the published tracheophyte secretomes38−53 using fasta36 or ssearch36 (p < 0.0001) (Table 3; Supporting Information Table S3). The remaining 15 moss proteins showing chitosandependent secretion included six proteins that contained functional domains similar to those found in secreted tracheophyte proteins, but were not homologous with tracheophyte proteins at the whole protein level. Four proteins whose secretion has not been reported were an immune inhibitor A peptidase M6-domain containing protein, unknown protein DUF427, a phosphorylase superfamily protein, and a ribosomal L7/L12 C-terminal domain-containing protein. The remaining five 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

fold changea

p-valueb

disease resistance/ leucine-rich region protein Pfam: PF14368 LTP_2 calmodulin

3.8

0.008

3.3

0.0003

3.3

0.02

calmodulin

3.0

0.04

no functional description no functional description thioredoxin

2.4

0

5.3

0.003

5.1

0.02

peroxidase (Prx34)d

1.4

0.13

description

15

30 90

180

2.3

4.6

a

Chitosan-induced differences in the quantity of proteins were compared between chitosan-treated and control cultures of the moss using the R Bioconductor package PLGEM version 1.30.033 and QSPEC version 1.2.234 with raw spectral counts as input. For Pp1s428_9 V6.1 and Pp1s21_318 V6.2 the spectral count value in control cultures was lower than 5 and the fold difference cannot be considered accurate.17 bQSPEC FDR-corrected p-value. cSignificant increase (FDR ≤ 0.05) in the level of mRNA expression 15, 30, 90, or 180 min post treatment with chitosan. dInduction of Prx34 expression at 180 min post treatment with chitosan has been shown also by qPCR.19

a chitinase, two pectinesterases, a serine protease and three thioredoxins (Table 2). Both chitosan inducible pectinesterases contained 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 chitosan treated and control cultures, using ≥2-fold difference in quantity and QSPEC FDR-corrected p-value ≤ 0.05 as a threshold, G



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indicated that secretion of seven proteins detected in the control cultures increased significantly following chitosan treatment in both experiments (Table 3). These proteins were a leucine-rich repeat protein, a lipid transfer protein LTP_2 domain-containing protein, two calmodulin proteins, thioredoxin, and two proteins with no known function (Table 3). The amounts of these proteins increased >2.3 fold, which is a difference that could be determined at >95% confidence level.67 Expression of genes encoding the proteins, whose quantity was found to increase in the culture medium upon treatment of the moss with chitosan (Table 3), was assessed using microarray analysis. The chitosan-inducible peroxidase gene Prx34, whose expression was previously studied using quantitative real-time PCR (qPCR),19 was used as a control. Microarray analysis revealed a 4.6-fold increase in the mRNA expression of Prx34 at 180 min post-treatment with chitosan (Table 3), which is consistent with the 3.5-fold increase in the expression of Prx34 detected by qPCR at the same time post-treatment with chitosan.19 The quantity of Prx34 increased by 39−45% in the culture medium (spectral counts in control vs chitosan-treated cultures were 62 vs 86, and 66 vs 96, respectively, in the two experiments), but the difference was not statistically significant as compared with untreated control. Expression of one of the two genes encoding calmodulin proteins (i.e., the gene for Pp1s32_338 V6.2) increased by 2.3-fold in response to chitosan treatment, and the amount of the protein increased 3-fold in the culture medium. However, no significant induction of expression was observed with the other six genes encoding proteins whose quantity increased significantly in the culture medium upon chitosan treatment (Table 3). Gene ontology (GO) analysis using Blast2GO software37 and comparison of proteins classified to different GO groups between the Pp secretome and the secretome of chitosan-treated samples (group A vs group B in Figure 1) showed enrichment of proteins belonging to GO groups related to metabolism, “cellular component 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 chitosantreated cultures (Table 4). These proteins were designated to group A2 (Figure 2; Supporting Information, Table S1). One of these proteins was homologous to STIG1, which controls stigmatic exudate secretion in pistils of petunia and tobacco and is 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 a response to chitosan treatment. They included a serine protease (Pp1s48_63 V6.1 replaced by Pp1s218_115 V6.1), a leucine repeat rich protein (Pp1s35_305 V6.1 replaced by Pp1s175_51 V6.3) and a dienelactone hydrolase (Pp1s241_102 V6.1 replaced by 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 the control cultures in one experiment, their secretion could not be considered chitosan-dependent (Figure 2, group B2). Eighty proteins were secreted following chitosan treatment only in one of the two experiments. Results on these proteins were considered inconsistent (Figure 2, group A3 and Table S1). They are available in Supporting Information, Table S1 (groups A3, C1, and C3) but are not further discussed.

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

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

Three experiments were carried out on the secretome of chitosan-treated P. patens using the conventional 2-DE-LC-MS/ MS analysis to compare the results with the relatively new gelfree Orbitrap method. The three 2-DE experiments resulted in a consistent topology of protein spots, but only 30 proteins could be identified. They included 24 proteins identical to those detected with the gel-free method, and the remaining six proteins including a GDSL lipase, three glyoxal oxidases, a laccase and a leucine rich repeat protein were closely related to the proteins detected with the gel-free approach (Table 5). A larger proportion (25 out of 30) of the ECPs detected by 2-DE than found using the gel-free approach (30%) contained a classical secretion signal peptide. Five ECPs proteins detected using 2-DE were leaderless secretory proteins, as predicted with SecretomeP 2.0 (Table 5).

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DISCUSSION Results of this study show that the ECPs of P. patens include several hundred proteins that can be released when the moss is grown submerged in nutrient-containing water solution, which mimics temporary growth conditions of P. patens in its natural habitats.12 A large proportion (70%) of the ECPs of P. patens released under these conditions were homologous to the H



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Table 4. Proteins Which Were Not Found in the Culture Medium Following Treatment of Physcomitrella patens with Chitosan but Were Found in Control Cultures of P. patens identifier

description

Pp1s170_63 V6.1 Pp1s67_37 V6.1

AP2/B3-like transcriptional factor family protein basic helix−loop−helix (bHLH) DNA-binding superfamily protein dienelactone hydrolase

Pp1s241_102 V6.1 Pp1s109_205 V6.2 Pp1s6_39 V6.2

secretory signala

43

LS

53

DYNAMIN-like 1E

LS

38, 53

eukaryotic aspartyl protease family protein

SP

LS

38, 39, 42−45, 47, 48, 51−53 38, 42, 43, 51, 53 38, 41−45, 47, 48, 50−53 38, 53

SP

43, 51

peroxidase family protein

SP

Pp1s20_77 V6.1

peroxidase superfamily protein

LS

Pp1s55_121 V6.2 Pp1s66_27 V6.1


SP


SP

Pp1s65_175 V6.1 Pp1s122_57 V6.1 Pp1s309_41 V6.1 Pp1s217_58 V6.1 Pp1s201_96 V6.1 Pp1s38_59 V6.1 Pp1s441_12 V6.1 Pp1s237_75 V6.1

SP

no functional description

peroxidase superfamily protein plant protein of unknown function (DUF869) pleckstrin homology (PH) domain superfamily protein RmlC-like cupins superfamily protein serine protease stigma-specific Stig1 family protein xyloglucan endotransglucosylase/ hydrolase 5 xyloglucan endotransglucosylase/ hydrolase 9 exostosin family

Pp1s86_72 V6.2 Pp1s144_96 V6.1 Pp1s184_140 V6.1 Pp1s68_12 V6.1 Pp1s82_6 V6.1 Pp1s97_59 V6.2 Pp1s120_85 V6.2 Pp1s17_355 V6.1 Pp1s17_356 V6.1 Pp1s22_251 V6.1 Pp1s58_157 V6.2 Pp1s53_16 V6.1 Pp1s43_101 V6.1 Pp1s30_238 V6.1 Pp1s95_56 V6.1 Pp1s86_61 V6.2 Pp1s27_305 V6.1 Pp1s240_68 V6.1 Pp1s171_64 V6.1 Pp1s195_100 V6.1 Pp1s44_116 V6.1 Pp1s236_72 V6.1 Pp1s215_36 V6.1 Pp1s219_8 V6.1 Pp1s101_103 V6.1 Pp1s287_63 V6.1 Pp1s21_318 V6.2 Pp1s81_246 V6.1 Pp1s346_19 V6.1 Pp1s486_14 V6.1

46

SP

38, 39, 41−48, 51−53 38, 39, 41−48, 51−53 38, 39, 41−48, 51−53 38, 39, 41−48, 51−53 38, 39, 41−48, 51−53 38, 43, 53

LS

43, 53

SP

38, 40, 43−45, 49, 51−53 38, 42−45, 47, 48, 52, 53 51

LS SP SP SP LS

glyoxal oxidase-related protein (DUF1929) RNA pseudouridylate synthase no functional description

LS


SP

MW (kDa)

pI

secretorya signal

apolipoprotein

25.1

4.8

LS

chitinase

40.5

4.4

SP

chitinase class IV

31.1

5.0

SP

cupin

30.8

9.4

LS

cupin cupin

22.8 21.8

8.4 8.9

SP SP

GDSL-like lipase/ acylhydrolase GDSL-like lipase/ acylhydrolase GDSL-like lipase/ acylhydrolaseb glycerophosphoryl diester phosphodiesterase glyoxal oxidase

42.6

6.4

SP

46.1

7.6

SP

40.7

9.0

SP

65.7

4.7

LS

56.7

5.2

SP

57.8

5.1

SP

glyoxal oxidase

56.4

4.9

SP

glyoxal oxidaseb

57.0

4.9

SP

glyoxal oxidaseb

37.8

5.2

LS

63.3

4.9

SP

disease resistance/leucinerich region protein no functional description

34.8

8.6

SP

33.2

7.3

SP


23.5

7.6

SP


51.6

6.7

SP


20.3

7.3

SP

pectinesterase

40.2

6.0

SP

pectinesterase

40.8

8.3

SP

peroxidase (Prx34)

35.8

6.4

SP

purple acid phosphatase

61.7

5.3

SP

serine-threonine protein kinaseb thioredoxin

68.4

4.9

SP

13.5

6.0

LS

xyloglucan endotransglycosylase xyloglucan endotransglycosylase xyloglucan endotransglucosylase/ hydrolase 9

31.9

5.4

SP

40.4

4.9

SP

32.1

4.9

SP

identifier

38

Pp1s130_236 V6.1 Pp1s35_305 V6.1 Pp1s33_206 V6.2 Pp1s31_134 V6.1 Pp1s141_35 V6.1 Pp1s44_71 V6.1

Pp1s67_214 V6.3 Pp1s137_223 V6.1 Pp1s158_50 V6.1 Pp1s149_229 V6.1 Pp1s48_63 V6.1

refb

LS

fasciclin-like arabinogalactan family protein leucine-rich repeat (LRR) family protein NAD(P)-binding Rossmann-fold superfamily protein no functional description

Table 5. Proteins Detected in the Culture Medium of Chitosan-Treated Physcomitrella patens using 2-Dimensional Electrophoresis and LC-MS/MS

38, 39, 41−45, 48, 51, 53 38, 39, 41−45, 48, 51, 53 38c

a

SP, 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. b Reported tracheophyte secretomes containing homologous proteins (FASTA ssearch36/fasta36, E-value cutoff < 0.0001); empty cells, no homologous protein reported in tracheophyte secretomes. cPfam analysis indicated that Arabidopsis cell wall proteome38 contains a nonhomologous protein sharing a domain similar to Pp1s217_58 V6.1.

description

glyoxal oxidase

laccase

b

b

a

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

proteins detected in tracheophyte secretomes in cell suspension cultures, 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 deI



Article

fense,39,40,42,44−46,48,52,69 metabolism,40,42,46,48 protein modification,42,45,50 cell wall modification,40,48 redox,52,69 and signaling.40,47 Proteins belonging to these functional categories were detected 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 control cultures. They included many proteins such as chitinases42,44,48,49 and thaumatin,52 which are homologous to proteins secreted by tracheophytes upon treatment with pathogens or defense elicitors. Furthermore, it was found that certain members of protein subfamilies were released to the culture medium upon chitosan treatment, in contrast to others. It is becoming evident that the responses to micro-organisms in plants are coordinated spatiotemporally based on intra- or extracellular stimuli. For example, type I pectinmethylesterases (PME) acting on pectic polysaccharides in the cell walls of Nicotiana benthamiana contain an N-terminal pectin methylesterase inhibitor (PMEI) pro region, which retains PMEs inactive in the Golgi until a yet undefined signal activates a subtilisin-type serine protease to remove the pro region.70 Hence the inactive preproteins can accumulate and be prevented from acting untimely. In our study, the N-terminal PMEI pro region was predicted by Pfam in two chitosan-responsive pectinmethylesterases of P. patens. Expression of mRNA provides evidence for likely production of the corresponding protein, but secretion of a protein is not necessarily associated with simultaneous increase in mRNA expression of the corresponding gene. Indeed, only ∼40% of the variation in protein concentration can be explained by mRNA abundances.71 For example, Prx34 is a secreted peroxidase pivotal to antifungal defense in P. patens.19 Our results show that secretion of Prx34 increases rapidly upon chitosan treatment and results in an immediate oxidative burst.29 However, qPCR analysis shows that the expression of Prx34 mRNA is enhanced only gradually over 180 min post-treatment with chitosan,19 which was reconfirmed by microarray analysis in this study. For the majority of other genes tested, no significant enhancement of transcription was observed at 180 min post treatment with chitosan, despite the fact that the quantity of the corresponding proteins increased significantly in the culture medium due to secretion. The results are characteristic of perturbed systems, in which protein quantity appears to be regulated post-translationally rather than at mRNA level.72 Peroxidases and thioredoxins were most responsive to treatment of P. patens with chitosan, suggesting that the oxidative environment is altered by the increase in number and activity of these redox enzymes. Thioredoxin was one of the seven proteins detected in the control cultures but found be present in significantly higher amounts in the cultures treated with chitosan. Furthermore, the secreted peroxidase Prx34, which is pivotal to the oxidative burst and antifungal resistance of P. patens,19,29 was found to be among the most abundant proteins in the secretome of P. patens in control cultures and its amounts increased clearly (ca. 40%) following treatment with chitosan. These results are consistent with previous studies, which revealed a rapid release of Prx34 to the culture medium upon chitosan treatment and detected a subsequent, quick oxidative burst.19,29 Taken together, it seems apparent that P. patens secretes Prx34 constitutively as a protective mechanism against fungi (and possibly insects) containing chitin as a major cell wall constituent, but the low peroxidase activity detected in control cultures in previous studies suggests that the peroxidase activity of 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 an immune inhibitor A (InA) peptidase M6 domain-containing protein, which have not been reported in the secretomes of tracheophytes challenged with pathogens or defense elicitors.39,40,42,44−46,48,52,69 Pfam and InterPro databases contain three plant protein sequences with DUF427, namely two proteins of P. patens and one protein of rice (Oryza sativa L. ssp. indica). However, a gene encoding a DUF427-containing protein exists also in barley (Hordeum vulgare L.). The gene is induced under biotic and abiotic stress conditions, but details of its functional significance remain to be elucidated.73 In mycobacteria, a DUF427-containing protein is under the control of a cyclic AMP receptor protein, which represents a regulation system needed under cell stress and in pathogenesis.74 It may be speculated that the DUF427-containing ECP of P. patens might interfere with the DUF427-containing proteins of pathogens to prevent infection, and a similar role may be hypothesized for the InA peptidase M6 domain-containing protein of the moss. InA is a metallopeptidase secreted by Bacillus thuringiensis, a bacterium pathogenic to insects, to cleave and destroy host antibacterial proteins.75 The peptidase M6 domain of InA can be found in four proteins of P. patens, but only one of them was secreted upon treatment with chitosan. Finally, there were chitosan-responsive proteins of P. patens that were not homologous to proteins detected in tracheophytes but contained similar functional domains. One of these moss proteins (Pp1s172_76 V6.1) contained a tyrosine phosphatase domain known in a mucilage protein of maize.53 This moss protein also contained the C2 domain of the tumor-suppressor protein PTEN, which is involved in lipid signaling and induced by salt and osmotic stresses in Arabidopsis.76 The studies on tracheophyte secretomes have revealed many ribosomal proteins and also DNA-binding proteins in the extracellular space, where their roles are obscure. In our study, treatment of P. patens with chitosan released a member of the ribosomal S11 protein family and a protein, which was different from the ribosomal proteins found in tracheophyte secretomes but contained the ribosomal protein L7/L12 domain. In bacteria, ribosomal protein L7/12 interacts with the bacterial elongation factor EF-Tu,77 which is an elicitor of innate immunity in plants.78 EF-Tu is recognized in plants by the serine/threonine kinase receptor EFR,79 and ethylene modulates EFR-triggered immunity by enhancing salicylic acid mediated defense.80 Hence, the extracellular moss protein containing L7/L12 domain might modulate moss-microbe interactions. Polymeric DNA excreted from the root cap in pea (Pisum sativum L.) enhances resistance to fungal infection, but whether the DNA-binding proteins of the host play a role remains to be studied.81 Extracellular ATPbinding proteins could be important because extracellular ATP plays a role in pathogen defense and programmed cell death regulation.82 The secretomes of chitosan-treated and control moss contained a few DNA-binding proteins (Supporting Information, Table S1). They also included a protein homologous to heat shock protein with DnaJ domain (Pp1s29_56 V6.1). Recent studies have demonstrated that a DnaJ domain-containing heat shock protein (HSP40.1) plays a key role in cell death and pathogen defense in soybean (Glycine max L.).83 Besides triggering the release of 72 proteins, treatment of P. patens with chitosan inhibited the release of 27 proteins. One of J



Article

electrophoresis (2-DE) of the extracellular proteins isolated from the liquid culture medium of Physcomitrella patens treated with chitosan, all secreted proteins of P. patens detected in this study, proteins belonging to multimember subfamilies and detected in the secretome of P. patens, and comparison of the secreted proteins of P. patens detected in this study with those reported in tracheophyte secretomes using FASTA package ssearch36 and fasta36 programs. This material is available free of charge via the Internet at http://pubs.acs.org.

them was a glyoxal oxidase containing the conserved motif DUF1929 found in eight glyoxal oxidases of P. patens. Genes encoding DUF1929-containg glyoxal oxidases are found in tracheophytes but the proteins are not known to be secreted. Their role in pathogen defense is suggested, because the DUF1929-containg glyoxal oxidase derived from a variety of grape vine (Vitis pseudoreticulata W.T. Wang), which is resistant to the powdery mildew fungus (Erysiphe necator Schwein.), increases resistance to powdery mildew when overexpressed in a susceptible variety.84 The mechanism of resistance mediated by the glyoxal oxidase is not known in Vitis85 and the putative role of the DUF1929-containing protein in, e.g., control of oxidative burst and cell death in P. patens remains to be elucidated. Chitosan treatment also prevented moss tissue from releasing a protein (Pp1s65_175v6.1) that is homologous to STIG1 and GRIM REAPER (GRI). STIG1 is secreted in rice51 and controls secretion of stigmatic exudates in pistils of petunia and tobacco.68 GRI controls generation of extracellular oxygen radicals and cell death in Arabidopsis.86 GRI is cleaved by metacaspase-9 in the extracellular space, and the resulting N-terminal peptide binds to the extracellular domain of a membrane-bound receptor-like protein kinase, which mediates the death signal into the cell.86,87 The gri mutants of Arabidopsis lacking GRI and the Arabidopsis plants overexpressing GRI are more sensitive to oxygen radicals than the wild-type plants, 86 which suggests that also Pp1s65_175v6.1 might play a role in control of redox homeostasis in P. patens. Such control is evident, because despite of the rapid oxidative burst occurring within 2.5 min from addition of chitosan to the liquid cultures of P. patens,19,29 the moss plants in our study showed no signs of cell death during the time of incubation (180 min). This study has revealed over 400 extracellular proteins of P. patens released to the liquid culture medium, including ∼100 proteins whose release was affected by treatment of the moss with chitosan, which is a plant defense elicitor. There is little information available on the protein secretomes of bryophytes. The results presented here allow the first more comprehensive comparison of bryophyte and tracheophyte secretomes and indicate many similarities. A large proportion of the extracellular proteins of P. patens could not be predicted based on bioinformatics analyses, as found in the studies on tracheophytes. Bioinformatics can be used to predict secreted proteins based on signal peptide or other conserved sequences, but prediction of leaderless secretion is based on mammalian proteins, which limits applicability because of the differences in the extracellular matrix of plants and mammalians.36 There may also be other, less-known secretory pathways in plants.2 In this study, the extracellular proteins were obtained using a noninvasive method and the moss plants did not apparently suffer from the treatment, which should largely exclude contamination of the samples with cytoplasmic proteins. Using chitosan as a defense elicitor instead of infection with fungal pathogens was motivated with the same goal, because infection with necrotrophic pathogens disrupts cells and results in release of cellular proteins.19,20,24 The data presented here lays the basis for more detailed studies on the newly identified secretory proteins and their roles in physiological processes, such as biotic and abiotic stress.

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AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Eeva Marttinen for photography. Financial support from the Academy of Finland (grant 1253126), Finland Distinguished Professor Program (FiDiPro) and The Finnish Doctoral Programme in Plant Biology is gratefully acknowledged.

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ASSOCIATED CONTENT

S Supporting Information *

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



Article

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