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Mucilage secreted by primary roots of 3−4 day old maize seedlings was collected axenically. Shotgun nanoLC-MS/MS sequencing and annotation of the ma...
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The Mucilage Proteome of Maize (Zea mays L.) Primary Roots Wei Ma,† Nils Muthreich,‡ Chengsong Liao,† Mirita Franz-Wachtel,§ Wolfgang Schu ¨ tz,§ † ,‡ ,† Fusuo Zhang, Frank Hochholdinger,* and Chunjian Li* Department of Plant Nutrition, China Agricultural University, Beijing 100193, PR China, Center for Plant Molecular Biology, Department of General Genetics, Eberhard Karls University Tuebingen, 72076 Tuebingen, Germany, and Proteome Center Tuebingen, Interfaculty Institute for Cell Biology, University of Tuebingen, 72076 Tuebingen, Germany Received December 18, 2009

Maize (Zea mays L.) root cap cells secrete a large variety of compounds including proteins via an amorphous gel structure called mucilage into the rhizosphere. In the present study, mucilage secreted by primary roots of 3-4 day old maize seedlings was collected under axenic conditions, and the constitutively secreted proteome was analyzed. A total of 2848 distinct extracellular proteins were identified by nanoLC-MS/MS. Among those, metabolic proteins (∼25%) represented the largest class of annotated proteins. Comprehensive sets of proteins involved in cell wall metabolism, scavenging of reactive oxygen species, stress response, or nutrient acquisition provided detailed insights in functions required at the root-soil interface. For 85-94% of the mucilage proteins previously identified in the relatively small data sets of the dicot species pea, Arabidopsis, and rapeseed, a close homologue was identified in the mucilage proteome of the monocot model plant maize, suggesting a considerable degree of conservation between mono and dicot mucilage proteomes. Homologues of a core set of 12 maize proteins including three superoxide dismutases and four chitinases, which provide protection from fungal infections, were present in all three mucilage proteomes investigated thus far in the dicot species Arabidopsis, rapeseed, and pea and might therefore be of particular importance. Keywords: Maize • mucilage • proteome • shotgun • metabolism

Introduction Plant roots can sense water and nutrients by continuously producing and secreting compounds into the rhizosphere.1,2 A wide variety of carbon compounds is released from living roots to the soil, including higher molecular weight compounds such as polysaccharide mucilage and enzymes, and low molecular weight compounds such as sugars, amino acids, organic acids, fatty acids, and growth factors.3–5 The root mucilage which covers the root cap is an amorphous and uneven gel, which ranges in thickness from 50 µm to 1 mm.6 As the root extends through the soil, mucilage and associated root cap cells are left behind along the root-soil interface.7 The gel-like mucilage contributes to many interactions between the plants and soil. Mucilage facilitates soil aggregation,8,9 provides carbon for microbes,10–12 and facilitates root movement in the soil by decreasing the frictional resistance.13,14 Moreover, mucilage contributes to water holding15 and reduces the surface tension * To whom correspondence should be addressed. Chunjian Li, Department of Plant Nutrition, China Agricultural University, Yuanmingyuan West Road 2, Beijing 100193, PR China. Phone, +86 10 6273 3886, fax, +86 10 6273 1016; e-mail, [email protected]. Frank Hochholdinger, Center for Plant Molecular Biology, Department of General Genetics, Eberhard Karls University Tuebingen, 72076 Tuebingen, Germany. Phone, +49 7071 29 77024; fax, +49 761 295042; e-mail, [email protected]. † China Agricultural University. ‡ Eberhard Karls University Tuebingen. § University of Tuebingen.

2968 Journal of Proteome Research 2010, 9, 2968–2976 Published on Web 04/21/2010

of water, thus, changing the water relations of the rhizosphere.16 Furthermore, it ameliorates toxic effects of elements like Al3+,17 Cd2+,9,18 and Cu2+.18,19 Finally, mucilage contributes to a coherent sheath (rhizosheath) formation in many grasses and some dicots, which is ecologically significant and plays an important role in water and nutrient uptake.8,9 Because of the large amount of secretion, the maize mucilage has been used as a model system for decades to investigate the synthesis and secretion of the cell wall matrix.15 Chemical analyses of mucilage collected from roots of axenically grown maize revealed a protein content of 1-6% in addition to the presence of several polysaccharides and monosaccharides.20–23 These results indicate that the secretion of such carbon compounds is a natural process and not induced by environmental stress. Compounds found in the mucilage surrounding the root tip are not only secreted by the root cap, but also by border cells, which detach and differentiate from the root cap.24 A large number of border cells are present in the majority of higher plant species including maize.25 In recent years, considerable progress has been made in understanding the mechanisms and ecological significance of the secretion of small molecular weight compounds, such as organic acids induced by either phosphorus deficiency or aluminum toxicity and phytosiderophores induced by iron deficiency in grass species.26–29 In contrast, only little is known about the root mucilage proteome. Plant roots can secrete a 10.1021/pr901168v

 2010 American Chemical Society

Mucilage Proteome of Maize Primary Roots 1

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battery of defense and signal proteins, which play a strategic role in the plant responses to biotic and abiotic stress.31 Because of the low protein content and the difficulty of protein purification from root mucilage, the lack of powerful tools for protein identification allowed studying only a limited number of mucilage proteins thus far. While in pea root tip exudates 124 proteins were identified by multidimensional protein identification,32 in Arabidopsis and rapeseed, 52 and 16 proteins have been identified by MudPIT and LC-MS/MS, respectively.31 In the present study, 2848 distinct proteins of the maize primary root mucilage proteome were identified and functionally characterized. This data provides a first comprehensive insight into the physiological and molecular functions involved in root interaction with the soil environment.

Experimental Procedures Plant Culture and Mucilage Collection. Seeds of the maize (Zea mays L.) hybrid Nongda 108 were surface-sterilized with 30% H2O2 for 30 min and then rinsed seven times for 1 min in sterile deionized water according to Berthelin and Leyval.33 The seeds were germinated on moist filter paper in the dark at 25 °C. After 3 days (d), seedlings with 1-2 cm long primary roots were transferred into a plastic tray with small holes on the bottom which allowed roots to grow through. This tray was used as a lid for a plastic container so that the seedling roots could contact with 1 mM axenic CaCl2 solution34 containing Micropur microorganism inhibitor (Katadyn Products, Inc., Wallisellen, Switzerland). Micropur inhibitor contains sodium dichloroisocyanurate (NaDCC) and silver chloride and was applied in order to prevent microbial degradation of the root exudation during and after collection.35 Twelve hours later, mucilage was collected from the tip of the primary roots (2-3 cm long at this stage) using a sterilized drawn glass Pasteur pipet. There was no yellowing of the root tips observed during and after mucilage collection. Potential bacterial contamination of the mucilage was tested by streaking extracted root mucilage on nutrient agar plates according to Chaboud20 and incubating the agar plates overnight. All tests for bacterial contamination of the mucilage were negative. Proteins were collected in three biological replicates with about 540 seedlings each. Biological replicate mucilage collection was repeated three times within 12 h. These mucilage samples were pooled. In this study, the maize extracellular proteins of the primary root mucilage proteome were defined as mucilage proteins released by primary root tips and border cells. The proteins released into the 1 mM axenic CaCl2 solution were not included. Seedling growth and mucilage collection were carried out in a laminar flow cabinet (1400 L × 700 W × 370 H), which provided an axenic environment. Protein Purification and SDS-PAGE. Prior to protein analyses, the mucilage samples were centrifuged at 14 000g three times for 10 min at 4 °C to remove the border cells from the mucilage according to Read and Gregory.23 The supernatant which contained the mucilage was then precipitated three times with 10% (v/v) trichloroacetic acid (TCA) containing 0.1 mM phenylmethanesulfonyl fluoride (PMSF) and subsequently purified with acetone five times as described in Damerval et al.36 Dried protein pellets were dissolved in SDS extraction buffer containing 50 mM Tris, pH 8.0, 1 mM EDTA, 2.5% (w/v) SDS, 5% (w/v) mercaptoethanol, 15% (v/v) glycerol, and 0.05% (w/v) bromophenol blue. The protein concentration was assessed with the 2-D Quant Kit (GE Healthcare, Amersham Biosciences, Piscataway, NJ). For SDS-PAGE electrophoresis,

research articles 30 µg of protein extract was denatured by incubation at 100 °C for 5 min, then loaded onto a 12% SDS gel and run at 100 V using a 13 cm electrophoresis chamber (GE Healthcare; Amersham Biosciences, Piscataway, NJ). Gels were stained with Coomassie blue as described by Neuhoff et al.37 In-Gel Tryptic Digestion. The gel lane was vertically sliced into 16 pieces. Each fragment was cut into 1 mm3 cubes and washed three times with 50% 10 mM NH4HCO3/50% acetonitrile. After dehydration with 100% acetonitrile, gel pieces were incubated with 10 mM DTT in 20 mM ammonium bicarbonate for 45 min at 56 °C to reduce disulfide bonds. Alkylation of cysteines was performed by incubating the samples with 55 mM iodoacetamide in 20 mM ammonium bicarbonate for 30 min at 24 °C in the dark. Gel pieces were washed two times with 50% 10 mM ammonium bicarbonate/50% acetonitrile, then dehydrated with 100% acetonitrile, and finally dried in a vacuum concentrator. Protein digestion was performed overnight at 37 °C with 12.5 ng/µL trypsin in 20 mM ammonium bicarbonate. For protein extraction, gel pieces were incubated first with 30% acetonitrile/3% TFA, a second time with 80% acetonitrile/0.5% AcOH, and a third time with 100% acetonitrile. Supernatants were combined and desalted using RP-C18 StageTip columns.38,39 Eluted peptides were analyzed by nanoLC-MS/MS. NanoLC-MS/MS and Data Analysis. All digested peptide mixtures were separated and analyzed by online reversed-phase (RP) nanoscale capillary liquid chromatography (nanoLC) using an Eksigent nanoLC-2D system (Axel Semrau, Sprockho¨vel, Germany) coupled to a LTQ-Orbitrap-XL mass spectrometer (ThermoFisher, Bremen, Germany) equipped with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). Binding and chromatographic separation of the peptides took place in a 15 cm fused silica emitter of 75 µm inner diameter (Proxeon Biosystems, Odense, Denmark) in-house packed with reversed-phase ReproSil-Pur C18-AQ 3 µm resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). The peptide mixtures were injected onto the column with a flow of 500 nL/min for 20 min and subsequently eluted with a flow of 200 nL/min from 1.6% to 64% acetonitrile in 0.5% acetic acid in a 107 min gradient. The mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS (MS2) acquisition. Survey full scan MS spectra (from m/z 300 to 2000) were acquired in the orbitrap with a resolution of 60 000 at m/z 400 (after accumulation to a target value of 106 charges in the linear ion trap) using the lock mass option for internal calibration of each spectrum.40 The 10 most intense ions were sequentially isolated for fragmentation in the linear ion trap using collisionally induced dissociation with normalized collision energy of 35% at a target value of 5000. Target ions already selected for MS/MS were dynamically excluded for 90 s. The resulting fragment ions were recorded in the linear ion trap with unit resolution. Peak lists for database searching were generated from the raw data using MaxQuant.41 Proteins were identified by automated database searching (Mascot, Matrix Science)42 against an in-house curated version of the maize ZmProt database (www.maizegdb.org) which contains 34 958 unique maize protein sequences as of 02/05/2009. This database was complemented with frequently observed contaminants like porcine trypsin and human keratins. Carbamidomethyl-cysteine was used as a fixed modification, and variable modifications were oxidation of methionine and protein N-acetylation. Journal of Proteome Research • Vol. 9, No. 6, 2010 2969

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Ma et al. on average, 0.95 µg per root. To purify and size fraction the maize primary root mucilage proteome, ∼30 µg of mucilage proteins was separated on a 12% SDS-PAGE gel (Figure 1B).

Figure 1. (A) Maize primary root tip (left) covered with mucilage. Pipette tip (right) illustrates the viscosity of mucilage. (B) Mucilage protein extract isolated from 2-3 cm maize primary root and subsequently separated on a 12% SDS gel. For analysis, the gel was sliced into 16 fragments.

We required full tryptic specificity (cleavage at Arg-Pro and LysPro as well as Asp-Pro was included), a maximum of two miscleavages, and mass accuracies of 10 ppm for the parent ion and 0.5 Da for fragment ions. The minimum peptide size that was required was six amino acids. The maximum false discovery rates (FDR) were set to 1% for both, peptide and protein levels. Data Search and Functional Analysis. All proteins were functionally annotated via the MIPS FunCats (http://mips.gsf. de/proj/thal/db/tables/tables_gen_frame.html) catalogue. Homology analyses of pea,32 rapeseed,31 and Arabidopsis31 secretory proteins with the maize mucilage proteome were performed by downloading the protein data sets to a local stand alone blast server and performing blast analyses of all data sets against the 2848 maize proteins identified in this study (cutoff value: E < 10-10). The presence of putative signal peptides was predicted using the SignalP v3.0 (www.cbs.dtu.dk/services/ SignalP-3.0/) algorithm. Transmembrane helices were analyzed by the TMHMM server v. 2.0 (www.cbs.dtu.dk/services/TMHMM).43 Glycophosphatidylinositol (GPI) modifications were predicted with the “big-PI Plant Predictor” algorithm (http:// mendel.imp.ac.at/gpi/plant_server.html.44

Results Collection and Purification of Proteins from Maize Primary Root Exudates. The goal of this study was to generate a comprehensive reference map of proteins secreted by maize primary root in order to better understand the composition and function of the maize mucilage proteome. The maize primary root mucilage proteome is defined as the complement of all proteins that are released by root cap cells and detached border cells into the mucilage which covers the root cap (Figure 1A). To analyze the composition of the maize mucilage proteome, seedlings were germinated on moist paper for 3 days and then transferred into a hydroponic system. Maize primary roots continuously secrete mucilage. Therefore, between 12-24 h after transfer into the hydroponic system, mucilage was collected from the tips of primary roots which had a length of 2-3 cm with a drawn glass Pasteur pipet three times per root. Mucilage of ∼540 maize primary roots was combined for analysis. Prior to protein isolation, mucilage samples were centrifuged and only the supernatant was subjected to protein analyses, in order to ensure that only secreted proteins were analyzed and border cells and cellular debris were excluded from further analyses. Protein extraction from mucilage yielded, 2970

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Identification and Functional Annotation of the Maize Primary Root Mucilage Proteome. To comprehensively identify proteins of the maize mucilage proteome, the SDS-PAGE gel which was used to separate proteins extracted from maize mucilage (Figure 1B) was sliced into 16 fragments. After elution of trypically digested proteins from the SDS-PAGE gel slices, 47 007 tryptic peptides were identified by nanoLC-MS/MS analyses. The sequences and additional features of these 47 007 peptides are summarized in Table S1. In total, 3462 distinct maize proteins were derived from these peptide sequences by automated database searching (Mascot, Matrix Science)42 against an in-house curated version of the maize ZmProt database (www.maizegdb.org). We chose the identification of at least two different peptide sequences per protein as minimum requirement for further analyses. This criterion was met by 2848 of the 3462 proteins which were subsequently studied in more detail. Table S2 summarizes the features of these 2848 proteins including the most likely protein accessions for each group of peptides, protein descriptions, and number of distinct peptides per protein. Moreover, the number of unique peptides (peptides that were unique to one protein ID), sequence coverage, molecular weight, and amino acid length of the leading protein in the protein group are provided. Finally, the posterior error probability (PEP) rate of all identified proteins (calculated according to Ka¨ll et al.),45 and the summed peptide intensity for the identified protein group are indicated. Protein descriptions for the 2848 proteins were provided by maizegdb.org. For 1703 of the 2848 proteins that were identified based on g2 peptide sequences, these peptide sequences can be attributed to several closely related maize proteins that share these peptide sequences. Because of the shotgun approach, it is not possible to determine if only one or all of these related proteins are present in the sample.46 While Table S2 provides only the accession of the most likely protein, Table S3 summarizes all possible protein accessions related to the 1703 groups of peptides. Subsequently, all of the 2848 proteins were assigned to functional categories via the MIPS FunCats (http://mips.gsf.de/proj/thal/db/tables/tables_ gen_frame.html) catalogue. Approximately 41% of the 2848 proteins represented by at least two peptide fragments are of unknown function (Figure 2, Table S2). The functionally annotated proteins were distributed among 11 categories (Figure 2, Table S2). Most of these proteins were related to metabolism (24.6%), but also among others to the categories: proteins with binding function (6.7%), disease/defense (4.8%), protein synthesis (4.3%), signal transduction (4.2%), and protein fate (4.1%). The remaining 614 of 3462 proteins that were represented by only one peptide identification were not further analyzed. Remarkably, ∼95% (583/614) of these proteins were of unknown function (Table S4). Mucilage Proteins Cover Many Aspects of Basal and Secondary Metabolism. The largest proportion of the functionally annotated maize root cap mucilage proteins was related to metabolism. We therefore performed a MapMan47,48 analysis to visualize the metabolic pathways represented by secreted maize root proteins (Figure 3). Each green square represents a distinct protein that was mapped to a defined metabolic pathway, while gray circles indicate subpathways for which no protein was indentified in the maize mucilage proteome. Secretory proteins identified in this study are related to basal

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Figure 2. Summary of functional classes that were attributed to the 2848 maize mucilage proteins identified in this study in percent.

Figure 3. Visualization of metabolic pathways represented by proteins identified in the maize root secretory proteome via Mapman software. Each green square represents a protein, while gray circles indicate that no proteins matched a particular subpathway.

and secondary metabolism. Proteins involved in cell wall metabolism, energy production (electron transport, H+-ATPases, TCA circle, glycolysis), amino acid and lipid metabolism are abundant in the maize mucilage proteome. In addition, proteins involved in the secondary metabolism of terpenes, flavonoids, phenylpropanoids, and phenolics are abundantly represented in the mucilage proteome. Such secondary metabolites could play important functions in the interaction of maize roots with the environment.49 Comparison of the Monocot Maize and Dicot Arabidopsis, Rapeseed, and Pea Mucilage Proteomes. In previous studies of proteins secreted into the mucilage, only a limited number of proteins from pea (124 distinct proteins),32 Arabidopsis (52 distinct proteins),31 and rapeseed (16 distinct proteins)31 were identified. Mucilage proteins were defined in all these studies

as proteins released from root cap and border cells. To determine homologous proteins between the 2848 maize mucilage proteins identified in the present study and pea, Arabidopsis, and rapeseed mucilage proteins, we downloaded these data sets from GenBank and blasted them against the 2848 proteins identified in this study from maize. Secretory proteins were defined as homologues between maize and the three other species if blastp analyses provided an E-value < 10-10 in pairwise comparisons. While for pea all 124 secreted proteins were still available in GenBank, only 13 of 16 rapeseed proteins, and only 50 of 52 Arabidopsis proteins were still deposited in GenBank. In a first comparison, we determined the coverage of the pea, Arabidopsis, and rapeseed mucilage proteins by homologues of the considerably larger maize data set that comprised 2848 proteins. In total, between 85% and Journal of Proteome Research • Vol. 9, No. 6, 2010 2971

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Ma et al.

Table 1. Coverage of the Published Plant Mucilage Proteome Data Sets by Homologues of the Maize Mucilage Proteome Identified in This Study Pea Arabidopsis Rapeseed

86% (107/124)a 94% (47/50)b 85% (11/13)c

a

Numbers in % indicate for how many proteins of the pea,32 Arabidopsis,31 and rapeseed31 mucilage proteome homologues have been found in the maize mucilage proteome. Absolute number of proteins is indicated in parentheses. b Sequences of only 50 of 52 proteins initially identified in the Arabidopsis mucilage proteome31 are still available at NCBI. c Sequences of only 13 of 16 proteins initially identified in the rapeseed mucilage proteome31 are still available at NCBI.

Table 2. Maize Proteins for Which a Homologue Was Identified in All Three Dicot Mucilage Proteome Studied thus Far (Pea,a Arabidopsis,b and Rapeseedb)

protein ID

protein descriptions

predicted secretory proteinc

AMYB B1PEY4 B4FTS6 B4FX40 B6SIL9 B6SK54 B6T056 B6TEL0 B6TR38 B6TT00 CHIB Q6LCT7

1,4-alpha-D-glucan maltohydrolase Superoxide dismutase [Cu-Zn] Seed Chitinase A Cysteine proteinase 1 40S ribosomal protein S27a Superoxide dismutase [Cu-Zn] 4AP Superoxide dismutase [Cu-Zn] 2 Endochitinase A Basic endochitinase A Putative uncharacterized protein Endochitinase B Ubiquitin fusion protein

Yes No Yes Yes No No No Yes Yes Yes Yes No

a Wen et al.32 et al.).52

Figure 4. Overlap of unique maize proteins identified in this study that display homology with secreted proteins identified in rapeseed, pea, and Arabidopsis. In total, 117, 283, and 309 maize proteins displayed homology to rapeseed (Basu et al.),31 Arabidopsis (Basu et al.),31 and pea (Wen et al.)32 mucilage proteome, respectively. Remarkably, for 12 maize proteins, a homologue was found in all three dicot mucilage proteomes (see Table 2).

94% of the mucilage proteins identified in the dicot species pea (Table S5a), Arabidopsis (Table S5b), and rapeseed (Table S5c) had at least one close homologue in the protein secretome of the monocot maize plant (Table 1). Therefore, we compared the overlap of the maize homologues that were identified for the three dicot species (Figure 4). For 124 distinct pea mucilage proteins, 309 maize homologues were identified. Similarly, for the 50 Arabidopsis and 13 rapeseed secreted proteins, 283 and 117 maize homologues were identified, respectively (Figure 4). In total, for 594 of the 2848 maize mucilage proteins (21%) a homologue in at least one of the much smaller dicot data sets was identified. Hence, this study provides 2254 novel maize mucilage proteins that have not been previously associated with the mucilage proteome in any other plant species. The maize proteins for which a homologue was identified in the three dicot species display a significant overlap between pea, Arabidopsis, and rapeseed (Figure 4). Remarkably, homologues for 12 maize mucilage proteins were identified in all three dicot species (Figure 4) suggesting a conserved role of these proteins in all species studies thus far. For seven of these 12 proteins, extracellular secretion is predicted by TargetP (Table 2). Structural Analysis of Maize Secretory Proteins. Extracellular secreted proteins are typically characterized by a cleavable N-terminal signal peptide which targets them to the ER, the first organelle involved in the protein translocation from the cytoplasm to the extracellular environment.50 This signal peptide is characterized by a positively charged region at the N-terminus, a central hydrophobic region followed by a polar 2972

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b

Basu et al.31 c Predicted with TargetP v. 1.1 (Emanuelsson

region containing the cleavage site. Secretory pathway signal peptides among the 2848 secreted maize proteins were predicted with TargetP v. 1.1.51 In total, 464 of the 2848 maize mucilage proteins (16%) were predicted to contain a secretory pathway signal peptide (Table S2). Another feature of extracellular proteins is the absence of transmembrane domains. Secretory proteins are translocated from the ER to the Golgi complex where they are packaged into vesicles. These vesicles then move to the plasma membrane where they are released to the extracellular matrix. Transmembrane proteins would in this last step not be released to the extracellular environment but instead be integrated into the plasma membrane. Transmembrane helices for the 2848 proteins were predicted via the TMHMM program.43 Surprisingly, TMHMM predicted for 383 of the 2848 proteins having at least one transmembrane helix, which would exclude them from being a secretory protein. However, 247 of the 383 putative transmembrane proteins were also predicted to contain a secretory N-terminal signal peptide by TargetP. Hence, in these instances, the predicted N-terminal transmembrane domain was most likely an N-terminal signal peptide. In addition, 59 of the remaining 136 putative transmembrane proteins contained only one transmembrane helix. In many instances, this transmembrane helix is located at the N-terminus of the proteins and might thus also be a signal peptide. Finally, for 43 proteins which were in part also predicted to contain transmembrane helices, the “big-PI Plant Predictor” algorithm44 suggested glycosylphosphatidylinositol (GPI) anchors. GPI anchors are glycolipids that can be posttranslationally attached to the C-terminus of a protein. GPIlinked proteins are secreted via the endoplasmic reticulum (ER) and Golgi apparatus to the extracellular space where they remain attached to the exterior leaflet of the cell membrane.

Discussion Secretion of Maize Root Extracellular Proteins. As young roots penetrate the soil environment, exudates produced by the root cap and border cells are continuously released into the rhizosphere.52 Plant roots exude a large variety of compounds into the rhizosphere including mono and polysaccharides, amino acids, secondary metabolites, and proteins.1 Despite its biological relevance, the composition and function of the root mucilage proteome remains largely unknown. In

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Mucilage Proteome of Maize Primary Roots previous studies, protein content in mucilage of axenically grown maize has been estimated in the range of 1-6%.20–23 Maize root cap cells are hypersecretory cells.53 In particular, the lateral root cap cells of maize are rich in cisternae,54 which can exudate large amounts of mucilage.55 Moreover, detached border cells also remain metabolically active for more than a week after detachment into the soil52 and continue to secrete mucilage for days after detachment.56 In the present study, proteins were collected under axenic conditions, in order to avoid the induction of secreted proteins by microorganisms or environmental stress. Hence, the present data set comprises proteins constitutively secreted into the mucilage by living root cap and border cells.57 Most border cell specific proteins are exported into the external environment as soon as they are synthesized.58 In mucilage secreted by pea root tips, the amount of protein remained stable at approximately 1.3 µg of soluble protein per pea root tip once a full set of border cells was present.32 This is similar to the amount of 0.95 µg of protein in mucilage of maize root tips determined in this study. Identification of the Maize Primary Root Mucilage Proteome. In the present study, a comprehensive analysis of proteins secreted into the mucilage of maize primary root tips identified 2848 unique proteins with >2 different peptide fragments per protein group by using shotgun sequencing via nanoLC-MS/MS. This data set significantly exceeds previous mucilage protein data sets from rapeseed (16 proteins) based on 2-DE separation of proteins31 and from Arabidopsis (52 proteins),andpea(124proteins)basedonMudPITtechnology.31,32 For about 59% of the maize mucilage proteins, a function was provided by the protein descriptions of the maizegdb.org database. Among the annotated proteins, the largest proportion of proteins (24.6%) was related to metabolism. Remarkably, for 85-94% of the mucilage proteins identified from pea,32 Arabidopsis,31 and rapeseed,31 a maize homologue was identified (Table 1). This suggests a considerable conservation of the mucilage proteomes between evolutionary distant monocot and dicot species. On the other hand, due to the larger size of the data set identified in this study, it is likely that on average more than one maize homologue can be found for mucilage proteins of the relatively small data sets of the previously published mucilage proteomes of dicot model plants. The little overlap of mucilage proteins that was previously found between Arabidopsis, rapeseed,31 and pea32 might, therefore, not reflect the divergence of the mucilage proteomes between dicot species32 but might rather be attributed to the relatively small set of proteins identified in these studies. Carbohydrate Metabolizing and Cell Wall Proteins in the Maize Mucilage Proteomes. Plant cell walls are mainly composed of polysaccharides such as cellulose, hemicelluloses, and pectins which contain variable amounts of structural proteins.59 It has been previously demonstrated in pea that the root mucilage proteome displays certain overlap with the cell wall proteome of maize60 but that it is not strictly synonymous. In line with this observation, numerous proteins involved in cellwall metabolism have been identified (Supporting Information Table S6). Remarkably, ∼46% of the cell wall proteins identified in this study have not been previously associated with functions in the root mucilage proteome.31,32 For instance, several expansins have been identified in the maize root mucilage proteome for which no homologues were identified in dicot root mucilage proteome studies.31,32 Expansins are known to have cell-wall loosening activity during cell expansion.61 Moreover, RTH3 which encodes a monocot-specific, COBRA-like

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cell-wall protein has also been associated in this study with the mucilage proteome of maize. Furthermore, several prolinerich cell wall proteins and β-glucosidases have been identified in this study for which no homologues were identified in the dicot mucilage proteome.31,32 Many cell wall proteins are modified post-translationally. Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins that are highly glycosylated and abundant in the plant cell walls and plasma membranes.63–66 AGPs form an abundant class of plant cell surface proteoglycans with high water-holding capacity, and inherent stickiness.67 AGPs were identified in the mucilage layer by immunofluorescence localization.68 It is hypothesized that classical AGPs act as pectin plasticizers.69 Fasciclin-like arabinogalactan proteins (FLAs) are a subclass of AGPs that have, in addition to predicted AGP-like glycosylated regions, putative cell adhesion domains known as fasciclin domains.66 Five FLAs were identified in the secreted proteome of maize primary roots. Reactive Oxygen Species Scavenging Enzymes in the Maize Mucilage Proteome. Reactive oxygen species (ROS) are continuously produced under normal conditions as toxic byproduct of aerobic metabolism, but also act as signaling molecules in plants.70 Major ROS-scavenging mechanisms of plants include superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT). Additional ROS-scavageing enzymes are glutathione reductase (GR), gluthatione peroxidase (GPX), dehydroascorbate reductase, monodehydroascorbate reductase (MDAR), and other peroxidases.70 In total, 43 isoforms of these enzymes except dehydroascorbate reductase were found in the maize root mucilage proteome (Table S7). While SOD, APX, GR, MDAR, and other peroxidases were also found in dicot mucilage proteome,31,32 GPX and CAT were exclusively identified in the present study of the maize mucilage proteome. SOD acts as the first line of defense converting superoxide into hydrogen peroxide.70 Not surprisingly, SOD was therefore identified in all four mucilage proteome data sets identified so far from pea,32 rapeseed,31 Arabidopsis,31 and maize (Table 2). Maize Mucilage Proteins Related to Stress Response. The constitutively secreted proteome of maize primary roots contains a considerable number of proteins responding to abiotic (heat, salt, and drought), and biotic stress (Table S2). Proteins related to abiotic stress include a number of heat shock proteins and chaperonins, temperature-sensitive H2B, salt-inducible and water-stress proteins, and early proteins responsive to dehydration. Pathogen responsive proteins include the previously mentioned peroxidases, but also several 14-3-3 proteins, glucanases, and chitinases. Chitinases play an important role in the biocontrol of plant pathogenic fungi,71,72 and belong to the core enzymes of plant mucilage proteomes and were identified in all four plant mucilage proteomes dissected thus far (Table 2). Plant hormones play important regulatory roles in all aspects of development. Some plant stress hormones are responsible for activating cellular and environmental responses mediated by proteins regulated by these hormones including ethyleneresponsive, jasmonate-induced, brassinosteroid biosynthesislike, or abscisic stress ripening proteins identified in this study. Proteins Related to Nutrient Acquisition. Apart from the function of plant roots as organs for nutrient uptake, roots are also able to release a wide range of compounds into the root environment, including proteins.4,29 Several proteins identified in the maize primary root mucilage proteome make nutrients Journal of Proteome Research • Vol. 9, No. 6, 2010 2973

research articles available for plant uptake such as legumin-like and germinlike proteins, and acid phosphatase. Phosphorus (P) is one of the major limiting factors for plant growth in many soils. Beside exudation of carboxylates to mobilize sparingly soluble P sources in the soil, secretion of acid phosphatase may contribute to P acquisition by hydrolysis of organic P esters in the rhizosphere, which can compose up to 30-80% of the total soil P.29 Iron (Fe) is another major limiting factor for plant growth in terrestrial ecosystems. About one-third of the world’s soils are Fe-deficient for plant growth because the Fe is poorly soluble.28 A group of proteins contributes to iron binding, including ferredoxin-dependent glutamate synthase, aconitate hydratase, frataxin, hemoglobin-like protein, aldehyde oxidase, cytochrome, ferritin, heme-binding protein, and ZmNAS1 protein. Some proteins, such as zinc ion binding protein, NO3high affinity nitrate transporter, potassium ion binding, and molybdenum cofactor biosynthesis protein, can be found in maize extracellular proteome to contribute to plant nutrition. Cytosolic Marker Proteins in the Mucilage Proteome. Among the mucilage proteins of maize, a large array of cytosolic markers including, for example, 78 distinct ribosomal proteins, 36 translation initiation and elongation factors, 16 histones, and 35 enzymes of glycolysis covering all 10 enzymatic reactions of glycolysis (Table S8) have been identified. In the previously studied dicot mucilage proteome data sets, only four of the 10 enzymatic steps of glycolysis were represented.31,32 If these cytosolic proteins have a function in the maize mucilage or represent leakage that occurs during cell separation from the root cap remains to be elucidated in the future.

Conclusion Root exudation plays an important role in plant growth, by helping plants to create a favorable rhizospheric environment. Root exudates, by controlling rhizosphere community structure, significantly influence plant health, development, and productivity. In the present study, 2848 distinct mucilage proteins of primary root tips were identified. This comprehensive data set provides novel insights into the composition and function of plant mucilage proteomes and helps to better understand the biological processes proceeding in the root-soil interface.

Acknowledgment. We thank the National Natural Science Foundation of China (No: 30671237), the State Key Basic Research and Development Plan of China (No. 2007CB109302) and the Innovative Group Grant of National Natural Science Foundation of China (No: 30821003) for financial support to C.L. This project was supported in part by grant HO2249/8 of the Deutsche Forschungsgemeinschaft (DFG) to F.H. The Proteome Centrum Tu ¨bingen is supported by the Ministerium fu ¨r Wissenschaft und Kunst, Landesregierung Baden-Wu ¨rttemberg. We would like to thank Marc Lohse (Max Planck Institute for Molecular Plant Physiology, Golm, Germany) for providing maize mapping files for the Mapman analysis. Supporting Information Available: Table S1, sequences and description of all peptides identified in this study. Table S2, protein groups represented by at least two distinct peptides. Table S3, multiple protein IDs identified by a unique set of peptide fragments. Table S4, protein groups that were represented by only one peptide identification and were not further analyzed. Table S5a, pea mucilage proteins blasted against the 2848 maize secretory proteins. Table S5b, Arabi2974

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Ma et al. dopsis mucilage proteins blasted against the 2848 maize secretory proteins. Table S5c, unique rapeseed mucilage proteins blasted against the 2848 maize secretory proteins. Table S6, proteins involved in cell wall metabolism identified in the maize root mucilage proteome. Table S7, enzymes involved in the scavenging of reactive oxygen species identified in the maize root mucilage proteome. Table S8, enzymes of all 10 steps of glycolysis were identified in the maize root mucilage proteome. This material is available free of charge via the Internet at http://pubs.acs.org.

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