N-linked glycoproteome profiling of seedling leaf in Brachypodium distachyon L.

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N-Linked Glycoproteome Profiling of Seedling Leaf in Brachypodium distachyon L. Ming Zhang, Guan-Xing Chen, Dong-Wen Lv, Xiao-Hui Li, and Yue-Ming Yan J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr501080r • Publication Date (Web): 05 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Proteome Research

MS for J. Proteome Res.

N-Linked Glycoproteome Profiling of Seedling Leaf in Brachypodium distachyon L.

Ming Zhang1,2, Guan-Xing Chen1, Dong-Wen Lv1, Xiao-Hui Li1 and Yue-Ming Yan1,3*

1

College of Life Science, Capital Normal University, 100048 Beijing, China.

2

College of Life Science, Heze University, 274015 Shandong, China.

3

Hubei Collaborative Innovation Center for Grain Industry, 434025 Jingzhou, China

*

Corresponding author: Prof. Dr. Yueming Yan, Key Laboratory of Genetics and Biotechnology, College

of Life Science, Capital Normal University, Xisanhuan Beilu No. 105, 100048 Beijing, P. R. of China. Tel. and Fax.: +86-10-68902777 E–mail: [email protected]

1

ACS Paragon Plus Environment


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Abstract Brachypodium distachyon L., a model plant for cereal crops, has become important as an alternative and potential biofuel grass. In plants, N-glycosylation is one of the most common and important protein modifications, playing important roles in signal recognition, increase in protein activity, stability of protein structure, and formation of tissues and organs. In this study, we performed the first glycoproteome analysis in the seedling leaves of B. distachyon. Using lectin affinity chromatography enrichment and mass spectrometry-based analysis, we identified 47 glycosylation sites representing 46 N-linked glycoproteins. Motif-X analysis showed that two conserved motifs, N-X-T/S (X is any amino acid, except Pro), were significantly enriched. Further functional analysis suggested that some of these identified glycoproteins are involved in signal transduction, protein trafficking and quality control, and the modification and remodeling of cell wall components such as receptor-like kinases, protein disulfide isomerase and polygalacturonase. Moreover, transmembrane helices and signal peptide prediction showed that most of these glycoproteins could participate in typical protein secretory pathways in eukaryotes. The results provide a general overview of protein N-glycosylation modifications during the early growth of seedling leaves in B. distachyon, and supplement the glycoproteome databases of plants.

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Keywords: Brachypodium distachyon; Leaf; Lectin affinity chromatography enrichment; Glycoproteome;Glycosylation motif

Introduction N-glycosylation is an important posttranslational modification in all eukaryotes, involved in cellular localization, protein stability and solubility, signal recognition, intermolecular interactions and the development of complex multi-cellular organisms.1,2 It is commonly accepted that glycoproteins participate in protein secretory pathways via the endoplasmic reticulum (ER), Golgi apparatus, vacuole, plasma membrane, and extracellular compartments in plants. To date, various techniques, including lectin affinity chromatography,2−6 zwitterionic-hydrophilic interaction liquid chromatography (ZIC-HILIC),7−10 and filter-aided sample preparation (FASP) methods,11−13 have been developed for specific enrichment of glycopeptides and glycoproteins. Through these enrichment techniques, coupled with high-accuracy mass spectrometry (MS) and related quantitative or qualitative analyses, many large-scale glycoproteome analyses have been performed in different plant species, such as Arabidopsis thaliana,1,3 tomato,2 Vitis vinifera,7 Lotus japonicus,8 Hordeum vulgare,9 wheat,10 and cotton.13 However, our understanding of protein glycosylation in other plant species, particularly in B. distachyon belonging to the Poaceae family, remains unclear. Brachypodium distachyon L., a temperate annual grass grown in the Mediterranean and Middle East,14,15 has recently been considered as a model plant for cereal crops and a potential biofuel grass. It has a large number of specific features: a member of the Gramineae with a small diploid genome (272 Mb), short developmental cycle, self-fertilization, simple nutrient requirements, and efficient 3



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transgenosis.16 As a potential biofuel plant, scientists focus on studying the mechanisms of growth and development, specifically related to the cell wall.15,16 In the early vegetative growth phase of its seedlings, cell proliferation and differentiation results in the rapid growth of the plants, increases its photosynthetic capacity, and is required for the accumulation of biomass and yield as biofuel plants. In addition, cell expansion is involved in a series of dynamic changes, including the hydraulic pressure of cells and cell wall expansion.17 In these processes, the cell wall can be subjected to turgor pressure from the vacuoles containing water and solutes. To resist this pressure, cells must loosen the cell wall to maintain mechanical strength, and this process involves reformation and remodeling of the cell wall skeleton as well as the supplementing newly synthesized components.18,19 Recently, studies have shown that cell wall expansion and remodeling are associated with a series of proteins (enzymes) such as matrix expansins,18 glycosyl hydrolases, and glycosidases.20−23 The changes in cell wall not only enable rapid early seedling growth, but also adapt to exogenetic abiotic or biotic stress. Processes such as lignification, and factors such as cutin, waxes, and suberin of cell wall,24−26 are common adaptive strategies in plants to cope with water loss and pathogen invasion. In addition, during plant growth and development, cell walls must adapt to the rapid increases in cell numbers and size in the leaf. Although the mechanisms underlying the signaling systems of these processes are complex, recent evidence has indicated that several receptor-like kinases are involved in cell wall signaling.27,28 The rapid development of genome-wide analyses in recent years has facilitated studies on transcriptomics, proteomics, and phosphoproteomics in B.distachyon.29−31 Thousands of genes and proteins involved in plant growth, development, reproduction, and stress responses have been identified. To our knowledge, the study of protein 4


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glycosylation in Gramineae is less clear. In this study, we perform the first in vivo glycoproteome profiling in the seedling leaves of B. distachyon using lectin affinity chromatography enrichment and H218O addition1,12,32 coupled to nLC-MS/MS analysis. We focus on the effects of glycoproteins on the early seedling leaf growth in B. distachyon, which serves as a potential model species for the alternative biofuel.

Materials and Methods Plant materials Brachypodium distachyon 21 (Bd21) was used as plant material in this study. Bd21 seed surfaces were sterilized with 15% sodium hypochlorite for 3 min, and then rinsed four times in sterile distilled water. Following this, the seeds were incubated in water for 12 h at room temperature, and then transferred to a wet filter paper to germinate in darkness for 24 h (22–25°C). The seeds that germinated uniformly were selected, and grown in plastic pots containing Hoagland solution, which was changed every 2 days. In a greenhouse, the experimental conditions included daily photo cycle of 16 h light / 8 h dark (26°C/18°C) and 65–75% air humidity. Three biological replicates were performed under the same conditions. The seedling leaves at three-leaf stage were sampled for each biological replicate and frozen at −80°C for subsequent protein extraction. Protein extraction Total proteins from the leaves of Bd21 seedlings were extracted according to the procedure of Lv et al.,30 with minor modifications. Approximately 500 mg of fresh leaves from each biological replicate was ground into a fine power in liquid nitrogen. The ground power was suspended in 4 mL SDS buffer (30% sucrose, 2% SDS, 100 mM Tris-HCl, pH 8.0, 50 mM EDTA-Na2, 20 mM DTT) and 4 mL phenol (Tris-buffered, pH 8.0) in a 10 mL tube, followed by the addition of 1 mM 5



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phenylmethanesulfonyl fluoride (PMSF) and 20 µL protease inhibitor cocktail (Merck KGaA, Germany) per 1 g of fresh sample to inhibit the protease activities. Samples were mixed vigorously for 15 min at room temperature, centrifuged twice at 15700 × g for 15 min each, and the supernatants were precipitated with 100 mM cold ethanolamine-methanol solution at −20°C over night. After centrifuging at 15700 × g for 15 min, the pellets were rinsed with cold acetone (−20°C) and further centrifuged three times. After freeze-drying, the pellets were added to 300 µL of solubilization buffer (7M urea, 2M Thiourea, 1% DTT (w/v) and 4% CHAPS) at room temperature for 2 h. Insoluble materials were removed by centrifugation at 15700 × g for 15 min, and protein samples were measured using a 2-D Quant Kit (Amersham Bioscience, USA). The final protein solution was stored at −80°C for later use. Protein digestion The solution containing 400 µg of protein samples and 200 µL UA buffer (8 M urea, 150 mM Tris-HCl, pH8.0) were mixed and poured into a 10-kDa filter (Millipore, Sartorius, Germany) for centrifugation at 14000 × g for 15 min. Next, 200 µL UA buffer was added again, followed by centrifugation at 14000 × g for 15 min. The filtrate was discarded. Subsequently, 100 µL IAA (50 mM IAA in UA) was added. After rotating at 2500 × g for 1 min, the solution was placed for 30 min in darkness at room temperature, and then centrifuged at 14000 × g for 10 min. One hundred microliters of UA buffer was added, followed by centrifugation at 14000 × g for 10 min. The same process was repeated two times. Following this, 100 µL of 25 mM NH4HCO3 was added, and the samples were centrifuged twice for 10 min each at 14000 × g. Then 40 µL trypsin buffer (50 µg trypsin in 100 µL 25 mM NH4HCO3) was added and rotated at 2500 × g for 1 min. The solution was incubated at 37oC for 16–18 h, followed by centrifugation for 10 min at 14 000 × g before and after the 6


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addition of 40 µL 2 ×binding buffer (40 mM Tris/HCl pH 7.6, 2 mM MnCl2, 2 mM CaCl2, 1 M NaCl). The eluates were collected in new collection tubes. Lectin

affinity

chromatography

enrichment

coupling

to

FASP

and

deglycosylation The digested peptides were enriched and processed according to the N-glyco-FASP protocol

1,11

, with minor modification. In brief, Concanavalin A (ConA, Sigma，

L7647-25MG, 6 µg/µL) was diluted by 2 × binding buffer (20 mM Tris-HCl pH 7.0, 0.5 M NaCl, 1 mM CaCl2, 1 mM MnCl2 and 1 mM MgCl2). These peptides and ConA solution (2:1, g/g) were mixed. The mixtures were transferred to a new 30-kDa filter (Millipore, Sartorius, Germany). After incubation for 1 h at room temperature, the unbound peptides were eluted by centrifugation at 14000 × g for 10 min. The captured peptides were rinsed four times with 200 µL binding buffer and each with 50 µL of 40 mM NH4HCO3 in H218O (Sigma, America). Finally, 4 µL PNGase A (Roche, 1164559001) and PNGase F (Roche，11365185001) in 40 µL of 100 mM citrate buffer solution in H218O were added to a 30-kDa filter and the samples were incubated for 3 h at 37oC. The deglycosylated peptides were eluted with 2 × 50 µL of 40 mM NH4HCO3 solution. HPLC separation and mass spectrometric analysis The deglycosylated eluted peptides were separated by capillary high performance liquid chromatography (Easy nLC), the eluted peptides were ionized by a nanoelectrospray ion source to introduce into the mass spectrometer directly. Thermo scientific Easy column (75µm× 100mm 3µm-C18, GmbH) was firstly equilibrated by

7



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0.1% (v/v) formic acid containing 2% (v/v) CH3CN. Peptides were injected into the Thermo Scientific Easy column, and then the peptides were separated with a flow rate at 250 nL/min. The related liquid phase gradient was as follows: 0-240 min gradient from 0 to 60%, 240-250 min from 60%–100%, 250-280 min from stable 100% using 0.1% (v/v) formic acid containing 84% (v/v) CH3CN. The purified peptides were analyzed on the Q-Exactive mass spectrometer (Thermo Finnigan, Germany). Setting parameters were as follows: Analysis time: 240 min; Detection mode: positive ion; Precursor scan range: 300–1800 m/z; MS1 resolution: 70000 at m/z 200; Automatic gain control (AGC) target: 3e6; MS1 maximum IT: 20 ms; Number of scan ranges: 1; Dynamic exclusion: 40.0 s. Peptide and peptide fragments m/z were sampled using higher-energy collision induced dissociation (HCD). The following conditions were used: spray voltage of 2.2 kV, heated capillary temperature of 250°C, S-lens RF level of ~55%, ion selection threshold of 1 x e6 counts for HCD, maximum ion accumulation times of 60 ms for full scans and 60 ms for HCD. The HCD fragment ion spectra were acquired in the orbitrap with resolution of 17500 at m/z 200. Ten MS2 scan fragments were collected for each full scan. Isolation window was set for 2 m/z, microscans for 1, normalized collision energy for 27 eV, and underfill ratio for 0.1 %. MS experiments were performed in triplicate for this sample. Database Query The MS raw files were analyzed using MaxQuant,33 version 1.3.0.5. Proteins were identified by searching MS and MS/MS data of peptides against B. distachyon 8


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database (NCBI_B. distachyon_25824 entries_20130320. Fasta). To increase the accuracy of identified glycoproteins, the limiting conditions were as follows. The minimal peptide length was set to 7 amino acids. The initial peptide mass tolerance in MS mode was set to 6 ppm. The fragment mass tolerance was set to 20 ppm. The false discovery rate (FDR) was set to < 1.0% for the identification of both peptides and glycosylation site. Up to two missing cleavage points were allowed. Fixed modifications were for Carbamidomethyl (C), variable modifications were Oxidation M, Deamidation 18O (N). Contaminant and reverse data were deleted from the results. All mass spectrometry data of glycopeptides were manually extracted out using a software. To reduce the false positive rate (FPR) of identified peptides, the deamidation of asparagines in a consensus sequence, Asp-Xaa-Ser/Thr motif (where Xaa is any amino acid except proline), was considered for the identification of N-glycosylation sites. The identification of glycoproteins based on Pro-Q Emerald 488 gel stain 2D Gels were stained with Pro-Q Emerald 488 glycoprotein Gel and Blot Kit Stain (Invitrogen, Oregon, USA) according to the manufacturer’s instructions. Briefly, the gels were fixed twice with 50% methanol and 5% acetic acid (1 h/each), and washed twice with 3% acetic acid (15 min/each). And then they were incubated in Pro-Q Emerald 488 staining solution in darkness for 2.5 h, and washed with 3% acetic acid for four times (30 min/each). Finally, the gels were scanned on a TyphoonTM 9400 scanner (GE Healthcare, USA) with a 488 nm excitation laser and a 532 nm long pass filter, along with a resolution of 100 microns. In addition, the same 2D gels were 9



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stained with Coomassie brilliant blue (CBB) to visualize total proteins and used as control. Bioinformatics Protein function classifications were constructed based on the Protein family motif (Pfam) software (http://pfam.xfam.org/), EuKaryotic Orthologous Groups (KOG) database (http://eggnog.embl.de/version_3.0/) and NCBI search. Cellular localization was classified manually on the basis of WoLF PSORT (http://wolfpsort.org/), Target P (http://www.cbs.dtu.dk/services/TargetP/)34

and

UniProtKB

Gene

Ontologies

(http://www.uniprot.org/uniprot/). Significantly enriched glycopeptides motifs were extracted from glycopeptides with confidently identified glycosylation residues using the Motif-X algorithm (http://motif-x.med.harvard.edu/).35 Signal peptide of glycoproteins was searched by Signal P 4.1 Server (http://www.cbs.dtu.dk/ services /SignalP/).36 Secondary structure of glycoproteins was predicted via SABLE server version 2 (http://sable.cchmc.org/). Prediction of transmembrane helices was based on TMHMM Server v. 2.0 on-line software (http://www.cbs.dtu.dk/services/TMHMM/). The Phyre2, engine v 2.0 (http://www.sbg.Bio.ic.ac.uk/html/ page.cgi?Id=index), was used to predict the 3D structure of certain glycoproteins. The glycosylated residues were

displayed

using

Swiss-PdbViewer

(SPDBV,

version

4.1)

software

(http://spdbv.vital-it.ch/).

Results Glycoprotein identification and glycosylation site localization using nLC-MS/MS In this study, using tryptic digestion, lectin affinity chromatography enrichment, and nLC-MS/MS separation, a glycoproteome analysis was performed to explore the

10


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complex protein glycosylation modification of seedling leaves in B. distachyon (Bd21) (Fig. 1). Using screening conditions, including conserved motifs (N-X-S/T), and glycosylation site probability of no less than 0.9, 47 glycosylated sites, including 47 glycopeptides, were identified in 46 glycoproteins (Supplementary Table S1A). Notably, the proportion of 47 glycosylated sites with a glycosylation site probability of 1, accounts for approximately 92%. Moreover, of a total of 46 glycoproteins identified in this study, 36 of the total glycoproteins belonged to enzyme families such as protease/peptidase (11), glycosyl hydrolase/transferase (9), esterase/lipase (6), protein kinases (5), oxidoreductases (3) and protein trafficking and quality control related enzymes (2) (Table 1). Moreover, by manual inspection and identification of the peptide with deamidation of aspartic acid, the MS/MS maps of the identified glycopeptides in the seedling leaves of Bd21 are produced (Supplementary Fig. S1). In addition, nine glycopeptides were highly conserved among 47 glycopeptides identified in our study, which were also found in previously characterized glycoproteins

(Supplementary

Table

S1B).

Of

these

glycoproteins,

aminoacylase-1-like glycoprotein (XP_003572375.1; Supplementary Table S1C) separated by 2-DE could be stained by Pro-Q Emerald 488, further confirming that this protein was glycosylated (Supplementary Fig. S2). Functional classification and cellular localization of the identified glycoproteins in the seedling leaves of Bd21 To obtain an overview of the identified glycoproteins in the seedling leaves of B. distachyon, we carried out functional classification and cellular localization of 11



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proteins. Based on Pfam (Supplementary Table S2A), KOG (Supplementary Table S2B) and NCBI tools, 46 glycoproteins were manually classified into eight categories (Fig. 2A and Supplementary Table S2C). These included protease/peptidases (22%), glycosyl hydrolases/transferases (20%), esterases/lipases (13%), protein kinases (11%), oxidoreductases (7%), protein trafficking/folding-related enzymes (11%), and those with unknown function (16%). For cellular localization, 46 glycoproteins were classified into eight groups using WoLF PSORT and UniProtKB (GO annotation) software (Fig. 2B and Supplementary Table S2D), based on their localization to mitochondrion, chloroplast, Golgi apparatus, cytoplasm, endoplasmic reticulum, vacuole, plasma membrane, and extracellular region, which accounted for 2%, 2%, 2%, 5%, 11%, 18%, 27%, and 33%, of the total glycoproteins, respectively. It is notable that 87% of total glycoproteins belonged to the endoplasmic reticulum, Golgi apparatus, plasma membrane, vacuole, and extracellular region involved in protein secretory pathway, while only 13% belonged to other pathways related to growth and development of plants. Motif and structure analysis of glycoproteins based on bioinformatics tools To further explore the glycosylation characteristics of the identified proteins, four bioinformatics on-line tools, Motif-X, SABLE server version 2, Signal P 4.1, and TMHMM Server v. 2.0 were used in our study. Using Motif-X on-line software, two conserved motifs were enriched against the B. distachyon proteome database, namely, [NxT] and [NxS] (Fig. 3A and Supplementary Table S3), which are commonly found in animals and plants.1,12 Of 47 identified glycopeptides, [NxT] and [NxS] motifs 12


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accounted for 64% (30) and 36% (17), respectively (Fig. 3B), while no other motifs were significantly enriched in our results. Further, secondary structure analysis indicated that loop/turn, β sheet, and α helix accounted for 63% (30), 26% (12), and 11% (5) of 47 glycosylation sites, respectively (Fig. 3C and Supplementary Table S4). Moreover, through on-line SignalP software, 85% of 46 glycoproteins contained a signal peptide at the N terminus (Supplementary Fig. S3), which largely coincides with the protein secretory pathway. Glycoproteins with no signal peptide (SP) accounted for 15%. Moreover, TMHMM Server v. 2.0 analysis revealed that 50% (23) of the total glycoproteins contain transmembrane (TM) domains (Supplementary Table S5). Further, comparing with the results for SP and TM, we categorize them into four groups: (1) SP+/TM-, (2) SP-/TM+, (3) SP+/TM+, and (4) SP-/TM-. Among these four groups, 46 glycoproteins show different distribution proportions (Fig. 3D). Conservation analysis of the identified glycoproteins in the seedling leaves of Bd21 To further compare the conservation of function with other species, the sequences of the glycoproteins detected in this study were used to blast against the NCBI database, to determine the degree of conservation of glycoproteins among B. distachyon, O. sativa, and A. thaliana. The standards were set as follows: E-value < 1E-10, Score ≥ 80, and Identity ≥ 30%.30 Of 46 glycoproteins identified in the seedling leaves of Bd21, 45 (98%) showed conserved orthologs in A. thaliana, the model plant of dicots (Supplementary Table S6A). On further analysis, their identities (≥60%) accounted for 47%. On the other hand, 46 (100%) showed highly conserved orthologs in O.

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sativa, the model plant of monocots (Supplementary Table S6A). Their identity (≥60%) accounted for 91%. In addition, compared to the released literature and databases related to glycoproteins, such as tomato,2 A. thaliana,3 grape,7 and cotton,13 33 (72%) of 46 identified glycoproteins showed conserved glycosylated orthologs according to the same limiting conditions (Supplementary Table S6B). Thirteen glycoproteins showed no glycosylated orthologs in the species mentioned above， suggesting that these glycoproteins were only present in our study.

Discussion The identification and analysis of N-linked glycoproteins by nLC-MS/MS coupling to N-glyco-FASP In our study, a simple FASP method was used for N-glycopeptide enrichment, along with mannose-binding lectin (ConA). Moreover, because of specific α-1,3 core-fucosylation in plants, which is insensitive to PNGase F, PNGase A was also used for removing all N-linked glycans and coupling with the deamidation of asparagine residue under H218O condition resulting in an aspartic acid labeled with 18

O glycosylation sites in glycopeptides/glycoproteins. In nLC-MS/MS separation,

HCD was adopted for higher accuracy than that of collision induced dissociation (CID), particularly with very low glycopeptide abundance. Finally, we identified 47 conserved glycosylated sites (N-X-S/T) and 46 N-linked glycoproteins in vivo. Interestingly, of the 47 conserved glycosylated sites, most glycosylation modifications occurred in loops/turns (63%). This appears to be consistent with previous phosphorylation results.31 Compared to α helices and β fold, the preference 14


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for loops/turns in protein modification is possibly due to empty space outside the protein, which could facilitate binding to specific substrates or conformational changes. Moreover, the N-glycosylation sites of some identified glycoproteins, particularly transmembrane proteins, occur in the extracellular region, suggesting that they possibly facilitate signal recognition or the binding to substrates. In addition, of the 46 glycoproteins identified in our study, most of them possibly participate in typical and conventional secretory pathway. However, other glycoproteins having no putative SP and no TM segment were found suggesting potential atypical glycosylation mechanisms. Unexpectedly, we identified fewer glycoproteins than phosphoproteins in the seeding leaves of Bd2131. Possible reasons for this are as follows: (1) only few glycoprotein amounts were present in plants; (2) some long-glycopeptides were missing owing to the difficultly in elution; and/or (3) difficulty in enrichment of sparsely abundant glycoprotein in plants. However, this is only our initial study of glycosylation modification after protein phosphorylation. As the next step, we will focus on a study of the glycomics, which could provides important information for further studies on protein function. Glycosylated proteins (enzymes) involved in the signal transduction and signaling cascades The growth and development of plants generally involve many signaling pathways that are used to accommodate various internal and external cues. Brassinosteroids (BRs), plant hormone signal molecules, play key roles in signal transduction and 15



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signaling cascades that are required for plant growth and development.37 Receptor-like kinases (RLKs), which were identified as BR receptors in many plants (Fig. 4A), were shown to be involved in embryo development and hormone signaling, as well as in stress responses.30,38,39 Moreover, RLKs were shown to be phosphorylated in these biological processes.30,38,39 However, the knowledge regarding their glycosylation modifications in plants,2,7,12 particularly in B. distachyon, remains limited. In this study, five RLKs were found to be glycosylated and contained two conserved motifs (NXS/T).12,40 Alignment analysis showed no sequence homology except similar kinase domains. Notably, four (XP_003558467.1, XP_003558681.1, XP_003565200.1, XP_003561670.1) of five identified RLKs, possess leucine repeat regions (LRR) at the N terminus, which facilitate protein interaction,41,42 one RLKs (XP_003561504.1) contains malectin domains at the N terminus, which play an important role in the early steps of protein N-glycosylation.43 In addition, transmembrane helices were found in another 4 expect one (XP_003558681.1), suggesting that they could participate in signal recogination by forming transmembrance receptors. Besides brassinosteroid signaling, ROS signaling occurs commonly in plants during internal and external adverse cues, particularly biotic and abiotic stresses (Fig. 4A). To cope with these stresses, many peroxidases are produced in plants. Peroxidase, an important member of ROS signaling, generally possesses ferriprotoporphyrin IX and α-helices, and is involved in many biological processes such as the formation of lignin and suberin, cross-linking of cell wall components, 16


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defense against pathogen attack, and cell elongation.44,45 Moreover, through the site-directed mutagenesis method, N-glycosylation in horseradish peroxidase (HRP) improved its catalytic properties and stability to facilitate substrate binding.46 In our study, one peroxide (XP_003567636.1) was identified to show a glycosylation modification at N140. Glycoproteins (enzymes) participating in protein trafficking, processing, and quality control (QC) The rapid growth of plant depends on large numbers of newly synthesized proteins, particularly glycoproteins, which participate in the renewal of cell membranes and the formation of cell walls. N-linked glycoprotein secretion flows through a series of cellular compartments, such as the ER, ER-Golgi intermediate compartment, and Golgi apparatus, which play important roles in the synthesis, trafficking, and processing of these glycoproteins (Fig. 4B). On the ER membrane, oligosaccharides (Glc3Man9GlcNac2) are transferred to asparagine residues in new synthesized peptides by oligosaccharyltransferase (OST).47 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase, a key player of OST, is a type I integral ribophorin membrane protein (RPN1), participating in delivering high mannose oligosaccharides to asparagine residues.48 In this study, one RPN1 homolog (XP_003558910.1) was found with a glycosylation modification at N297. Through glycosyltransferase catalysis, new synthesized glycoproteins are trafficked into Golgi apparatus by coated vesicles, which involve some ER-Golgi intermediate compartment protein (ERGIC) clusters. These vesicular complexes are 17



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responsible for delivering secretory cargo from the ER to the Golgi apparatus, and contribute to the folding of the nascent proteins.49 In our work, two ERGICs (XP_003568216.1, XP_003558212.1) were found with glycosylation modifications at N289 and N209, respectively. We assume that the glycosylation in ERGICs, as a signal receptor, may contribute to its binding to the donor (cargo) and facilitate the transport of the secretory cargo (Fig. 4B). This assumption is based on the current study that ERGIC-53 is a cargo transport receptor, which is responsible for ER-to-ERGIC transport of soluble glycoprotein cargo.50 These cargoes are soon transported into the Golgi apparatus, which is responsible for the trafficking, processing, and sorting of glycoproteins within a cell.51,52 In our study, one Class I alpha-mannosidases

(LAMAN,

XP_003573734.1)

showed

a

glycosylation

modification at N662, which occurred near the C terminus. Further subcellular localization showed that it could occur in the Golgi apparatus. A previous study reported that glycosylation (N497) at the C terminus was highly conserved among LAMANs and very necessary to maintain the stability of the enzyme.53 In addition to participating in the above-mentioned protein synthesis, trafficking, and processing, these identified glycoproteins could play a key role in protein folding (Fig. 4B). Protein folding is a high-fidelity process and is subjected to adverse environmental conditions. The QC system has been developed into an environmental sensor and responder in plants, which is required for the refolding or degradation of improperly folded proteins in the secretory pathway.54 This process involves many molecular

chaperones

and

enzymes,

such

as

UDP-glucose:

18


glycoprotein

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glucosyltransferase (UGGT), calnexin, calreticulin (CRTN), Bip, Protein disulfide isomerase (PDI), and glycosidase.55,56 In our study, three glycoproteins were identified, which are related to the QC of protein folding (Table 1). UGGT, a protein sensor of the QC machinery, was responsible for the re-glycosylation of unfolded or misfolded proteins, thereby facilitating their binding to specific calreticulins on the ER

membrane

(Fig.

4B).56 In

our

work,

one

UGGT-like

glycoprotein

(XP_003574051.1) was identified as having a glycosylation modification at N207, and contained a KDEL motif at the C terminus, an ER resident motif. Through their re-glycosylation within a cell, these misfolded proteins were soon transferred to molecular chaperones to perform protein assembly (Fig. 4B). In our work, one molecular chaperone, protein disulfide isomerase-like 1-4-like protein (PDIL-1-4, XP_003570755.1), was found with a glycosylation modification at N220. Through sequence alignment and an NCBI search, they all found to contain a, a’, b, and b’ domains, 5 active sites (CCCCR), and one KDEL motif at the C terminus (Fig. 5A). Further, 3D structure analysis suggested that 4 domains were distributed symmetrically, and two CGHC domains and a disulfide linkage were present at the a and a’ domains, respectively (Fig. 5B). A previous study showed that ERp57, a mammalian PDI, is involved in the QC system of ER along with calnexin and calreticulin through the b’ domain interaction.57 In addition, glycosylated PDI was identified in the aleurone layers in barley to participate in the QC during ER stress.9 Possibly glycosylation at the b domain in this study facilitates the interaction with its substrates and increases the activity of enzymes to better perform the folding of the 19



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misfolded proteins. Glycoproteins (enzymes) related to the modification and remodeling of cell wall components The cell wall is highly complex and mainly consists of cellulose, hemicellulose, and pectin as well as certain proteins and phenolic components.58,59 During growth and development, the cell wall in plants is subjected to various environmental and developmental cues and has important effects on mechanical support, defense against biotic and abiotic stresses, and determination of cell expansion.28 In recent studies, certain glycosylated enzymes related to cell wall modification and remodeling were identified in tomato fruit, cotton fiber cells, and A. thaliana mature stems.2,3,9,13 In the present study, five enzymes were identified and shown to be glycosylated (Table 1). Purple acid phosphatases (PAPs) function in the formation, trafficking, and recycling of inorganic phosphorus, which is crucial for cell growth and pathogen defense.60 A previous study suggested that PAPs dephosphorylate α-xylosidase and β-glucosidase to regulate their activity, and contribute to the degradation of xyloglucan oligosaccharides and oligosaccharides in the cell walls.61 In this study, a PAP (XP_003577477.1) was identified and shown to be glycosylated at N56. Further 3D structural analysis revealed that the glycosylation site occurs at the same surface with other six active sites of PAP (Supplementary Fig. S4), which could facilitate the binding to specific substrates.62 Polygalacturonan is an important component of the pectin skeleton in plant cell walls. Its degradation, catalyzed by polygalacturonase (PG), can be crucial for cell 20


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wall elongation during the growth and development of seedling.63 In this study, two PGs (XP_003557344.1, XP_003567980.1) were identified and shown to be glycosylated at N370 and N231, respectively. The xyloglucan and cellulose complex have been considered to be responsible for controlling the extension of primary cell walls.64 Alpha-xylosidase (XYL) was shown to regulate the levels of xyloglucan oligosaccharides in the biogenesis/degradation of the cell wall.65 In Arabidopsis, XYL1 was predicted to have eight possible N-glycosylation sites.66 In our study, an XYL (XP_003568009.1), the family 31 of glycosyl hydrolases, was also identified with a glycosylation modification at N165. β-Glucosidases are a type of glycosyl hydrolase involved in several functions such as responses to biotic and abiotic stresses, lignification, and cell wall remodeling and metabolism.67 A recent study suggests that acidification of barley cell walls induced by indole-3-acetic acid causes an increase in endo-1,3:1,4-beta-glucanase and improves cell wall-bound glucanase activity.68 In our study, one glucan endo-1,3-beta-glucosidase 6-like protein (XP_003574453.1) was also identified and shown to be glycosylated at N107. Ascorbate oxidase (AO) is a cell wall-localized enzyme and contributes to the regulation of the ascorbic acid (AA) redox state. It was reported that loosening of cell walls mediated by the hydroxyl radical (OH-) could be an important process in plant development, which is required for elongation growth.69 In our study, two AOs (XP_003563398.1, XP_003563730.1) were identified and shown to be glycosylated at N333 and N377, respectively. 21



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Glycoproteins (enzymes) associated with the hydrolysis of biomacromolecule The vacuoles and cell walls of plant cells are multifunctional organelles that are involved in the maintenance of turgor pressure and the extension of cells in response to developmental and environmental factors.27,70 In our study, some glycosylated hydrolases of biomacromolecule were identified, such as serine carboxypeptidases (SCPs), β-fructofuranosidase, and proteases/ peptidase (Table 1). Serine carboxypeptidases (SCPs), members of the S10 family of serine peptidase, have been identified in many plant species, and act as secondary metabolites involved in protein degradation.71,72 Three serine carboxypeptidase-like glycoproteins (SCPLs, XP_003572138.1, XP_003572139.1, XP_003573745.1) were identified in our work, and were glycosylated at N330, N319 and N330, respectively. According to Pfam, Signal P, and sequence alignment, they showed high sequence similarity, Ser-Asp-His (S-D-H) catalytic triad67 and the same glycosylation sites (Supplementary Fig. S5A). Furthermore, 3D structure revealed that Ser-Asp-His catalytic sites and glycosylation site in each SCPL were distributed on the surface of a deep groove (Supplementary Fig. S5B), which could facilitate specific substrate binding and improve their catalytic properties. Cell wall invertases 1 (INV1) found in A. thaliana (AtcwINV1) are required for carbohydrate metabolism, growth and development of seedlings, and improved crop yield.73 Using site-directed mutagenesis, Roy et al. suggested that N-glycosylation in fructan 1-exohydrolase (1-FEH IIa) contributes to its stability and optimal conformation, facilitating substrate binding.74 In our study, β-fructofuranosidase 22


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(XP_003575078.1), named invertase 1 (INV1), and belonging to the glycosyl hydrolase 32 family, also showed a glycosylation modification. Aspartic proteases, a family of protease enzymes, have two highly conserved aspartates preferably active at acidic pH, and play an important role in protein processing and degradation.75 In our study, two aspartic proteases-like proteins (XP_003558637.1,

XP_003568979.1) were identified to show glycosylation

modifications at N276 and N246, respectively.

Conclusions In summary, this study represents the first glycoproteome analysis of the seedling leaves in Bd21. Forty six glycoproteins were identified in vivo via nLC-MS/MS. Similar to other plants, these glycoproteins were also enriched for two conserved motifs: N-X-S and N-X-T. Functional analysis indicated that these glycoproteins are required for signal transduction, protein trafficking and QC, and cell elongation and expansion. Protein glycosylation mainly occurs in three ways: (1) to facilitate signal recognition; (2) to increase the catalytic activity; and (3) to improve the stability by reducing conformational changes. Through glycosylation mechanisms, these glycoproteins possibly regulate the homeostasis of plants such that they can cope with changes caused by growth and environmental cues during the early vegetative growth stage. This study offers a new understanding of the early vegetative growth of seedling leaves from a glycoproteomics perspective.

23



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Supporting information, nformation, this material is available free of charge via the Internet at http://pubs.acs.org." Supplementary

Table

S1

Glycoproteins

identified

by

nano

liquid

chromatography-tandem mass spectrometry (nLC-MS/MS) and from other experimental data. (A) Glycoproteins identified by nLC-MS/MS. (B) Conserved glycopeptides against the glycoproteins in released literature and databases. (C) Mass spectrometry data of glycoprotein identified by Pro-Q emerald 488 glycoprotein staining. Supplementary Table S2 Functional categories and cellular localization of glycoproteins in the leaves of Bd21 seedlings based on Pfam and KOG results. (A) Protein family motif (Pfam) results. (B) EuKaryotic Orthologous Group (KOG) search results. (C) Function classification. (D) Cellular localization. Supplementary Table S3 Motif enrichment and analysis of glycopeptides in the leaves of Bd21 seedlings. Supplementary Table S4 Secondary structures of glycoproteins in the leaves of Bd21 seedlings. Supplementary Table S5 Transmembrane structure analysis of glycoproteins in the leaves of Bd21 seedlings. Supplementary Table S6 Conservation analysis of identified glycoprotein at the protein and glycoprotein level. (A) Conservation analysis of identified glycoproteins against the proteins in A. thaliana and O. sativa. (B) Conversation analysis of identified glycoproteins against the glycoproteins in released literature and databases. 24


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Supplementary Figure Legends: Supplementary Figure S1 Total tandem mass spectrometry (MS/MS) data and peptide glycosylation localization in the leaves of Bd21 seedlings. Supplementary Figure S2 The 2D gel maps shown by Coomassie brilliant blue (CBB) and Pro-Q stain methods. Supplementary Figure S3 Signal peptide maps of all identified glycoproteins in the leaves of Bd21 seedlings based on SignalP 4.1 Server. Supplementary Figure S4 Sequence alignment, three dimensional (3D) structure analysis, and MS/MS map from purple acid phosphatase (PAP). (A) Sequence alignment of homological proteins from B. distachyon against those from A. thaliana and O. sativa. (B) 3D structure analysis based on Phyre2 tool (XP_003577477.1, confidence level > 93%). (C) MS/MS map and glycosylation site of purple acid phosphatase. Supplementary Figure S5 Sequence alignment and three dimensional (3D) structure analysis in three serine carboxypeptidase-like proteins (SCPLs). (A) Sequence alignment of homological proteins from B. distachyon compared with those against A. thaliana and O. sativa. (B) 3D structure analysis of serine carboxypeptidase-like 18-like protein (XP_003572138.1, confidence level > 93%). (C) 3D structure analysis of serine carboxypeptidase-like 6 -like protein (XP_003572139.1, confidence level > 93%). (D) 3D structure analysis of serine carboxypeptidase-like 3-like protein (XP_003573745.1, confidence level > 93%).

Acknowledgements

25



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This research was financially supported by grants from the National Natural Science Foundation of China (31271703, 31471485), International Science & Technology Cooperation Program of China (2013DFG30530), Natural Science Foundation of Beijing City and the Key Developmental Project of Science and Technology, Beijing Municipal Commission of Education (KZ201410028031), and the National Key Project for Transgenic Crops in China (2011ZX08009-003-004).

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the cell using TargetP, SignalP, and related tools. Nat. Protoc. 2007, 2 (4), 953-71. (35) Meyer, L. J.; Gao, J.; Xu, D.; Thelen, J. J. Phosphoproteomic analysis of seed maturation in Arabidopsis, rapeseed, and soybean. Plant Physiol. 2012, 159 (1), 517–28. (36) Emanuelsson, O.; Brunak, S.; von Heijne, G, Nielsen, H. Locating proteins in the cell using TargetP, SignalP and related tools. Nat. Protoc. 2007, 2 (4), 953–71. (37) Wang, H.; Yang, C.; Zhang, C.; Wang, N.; Lu, D.; Wang, J.; Zhang, S.; Wang, Z. X.; Ma, H.; Wang, X. Dual role of BKI1 and 14-3-3s in brassinosteroid signaling to link receptor with transcription factors. Develop. Cell 2011, 21 (5), 825–34. (38) Schwessinger, B.; Roux, M.; Kadota, Y.; Ntoukakis, V.; Sklenar, J.; Jones, A.; Zipfel, C. Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1. PLoS Genet. 2011, 7 (4), e1002046. (39) Taylor, I.; Seitz, K.; Bennewitz, S.; Walker, J. C. A simple in vitro method to measure autophosphorylation of protein kinases. Plant Methods 2013, 9 (1), 22. (40) Parker, B. L.; Thaysen-Andersen, M.; Solis, N.; Scott, N. E.; Larsen, M. R.; Graham, M. E.; Packer, N. H.; Cordwell, S. J. Site-specific glycan-peptide analysis for determination of N-glycoproteome heterogeneity. J. Proteome Res. 2013, 12 (12), 5791–800. (41) Kobe, B. and Kajava, A. V. The leucine-rich repeat as a protein recognition motif. Curr. Opin. Struc. Biol. 2001, 11 (6), 725–32. (42) Belkhadir, Y.; Subramaniam, R.; Dang, J. L. Plant disease resistance protein signaling: NBS-LRR proteins and their partners. Curr. Opin. Plant Biol. 2004, 7 (4), 391–9. 32


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alpha-mannosidases are required for N-glycan processing and root development in Arabidopsis thaliana. Plant Cell 2009, 21 (12), 3850–67. (52) Kajiura, H.; Koiwa, H.; Nakazawa, Y.; Okazawa, A.; Kobayashi, A.; Seki, T.; Fujiyama, K. Two Arabidopsis thaliana Golgi alpha-mannosidase I enzymes are responsible for plant N-glycan maturation. Glycobiology 2010, 20 (2), 235–47. (53) Faid, V.; Evjen, G.; Tollersrud, O. K.; Michalski, J. C.; Morelle, W. Site-specific glycosylation analysis of the bovine lysosomal alpha-mannosidase. Glycobiology 2006, 16 (5), 440–61. (54) Liu, J. X. and Howell, S. H. Endoplasmic reticulum protein quality control and its relationship to environmental stress responses in plants. Plant Cell 2010, 22 (9), 2930–42. (55) Bottomley, M. J.; Batten, M. R.; Lumb, R. A.; Bulleid, N. J. Quality control in the endoplasmic reticulum: PDI mediates the ER retention of unassembled procollagen C-propeptides. Curr. Biol. 2001, 11 (14), 1114–8. (56) Ellgaard, L. and Helenius, A. Quality control in the endoplasmic reticulum. Nat Rev. Mol. Cell Biol. 2003, 4 (3), 181–91. (57) Maattanen, P.; Kozlov, G.; Gehring, K.; Thomas, D.Y. ERp57 and PDI: multifunctional protein disulfide isomerases with similar domain architectures but differing substrate-partner associations. Biochem. Cell Biol. 2006, 84 (6), 881–9. (58) Cosgrove, D. J. Growth of the plant cell wall. Nat. Rev. Mol. Cell Bio. 2005, 6 (11), 850–61. (59) Knox, J. P. Revealing the structural and functional diversity of plant cell walls. Curr. Opin. Plant Biol. 2008, 11 (3), 308–13. (60) Antonyuk, S. V.; Olczak, M.; Olczak, T.; Ciuraszkiewicz, J.; Strange, R. W. The 34


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structure of a purple acid phosphatase involved in plant growth and pathogen defence exhibits a novel immunoglobulin-like fold. IUCrJ 2014, 1 (2), 101–9. (61) Kaida, R.; Serada, S.; Norioka, N.; Norioka, S.; Neumetzler, L.; Pauly, M.; Sampedro, J.; Zarra, I.; Hayashi, T.; Kaneko, T. S. Potential role for purple acid phosphatase in the dephosphorylation of wall proteins in tobacco cells. Plant Physiol. 2010, 153 (2), 603–10. (62) Wang, U. L.; Norgård, M.; Andersson, G. N-glycosylation influences the latency and catalytic properties of mammalian purple acid phosphatase. Arch. Biochem. Biophys. 2005, 435, 147–56. (63) Xiao, C.; Somerville, C.; Anderson, C. T. POLYGALACTURONASE INVOLVED IN EXPANSION1 functions in cell elongation and flower development in Arabidopsis. Plant Cell 2014, 26 (3), 1018–35. (64) Cosgrove, D. Wall structure and wall loosening. A look backwards and forwards. Plant Physiol. 2001, 125 (1), 131–4. (65) Sánchez, M.; Gianzo, C.; Sampedro, J.; Revilla, G.; Zarra, I. Changes in α-xylosidase during intact and auxin-induced growth of pine hypocotyls. Plant Cell Physiol. 2003, 44 (2), 132–8. (66) Sampedro, J.; Sieiro, C.; Revilla, G.; González-Villa, T.; Zarra, I. Cloning and expression pattern of a gene encoding an alpha-xylosidase active against xyloglucan oligosaccharides from Arabidopsis. Plant Physiol. 2001, 126 (2), 910–20. (67) Ketudat Cairns, J. R. and Esen, A. β-Glucosidases. Cell. Mol. Life Sci. 2010, 67 (20), 3389–405. (68) Kotake, T.; Nakagawa, N.; Takeda, K.; Sakurai, N. Auxin-induced elongation growth and expressions of cell wall-bound exo- and endo-beta-glucanases in 35



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barley coleoptiles. Plant Cell Physiol. 2000, 41 (11), 1272–8. (69) Müller, K.; Linkies, A.; Vreeburg, R. A.; Fry, S. C.; Krieger-Liszkay, A.; Leubner-Metzger, G. In vivo cell wall loosening by hydroxyl radicals during cress seed germination and elongation growth. Plant Physiol. 2009, 150 (4), 1855–65. (70) Marty, F. Plant vacuoles. Plant Cell 1999, 11 (4), 587–99. (71) Fraser, C. M.; Rider, L. W.; Chapple, C. An expression and bioinformatics analysis of the Arabidopsis serine carboxypeptidase-like gene family. Plant Physiol. 2005, 138 (2), 1136–48. (72) Rawlings, N. D.; Barrett, A. J.; Bateman, A. MEROPS: the peptidase database. Nucleic Acids Res. 2010, 38 (D), D227–33. (73) Lammens, W.; Le Roy, K.; Van Laere, A.; Rabijns, A.; Van den Ende, W. Crystal structures of Arabidopsis thaliana cell-wall invertase mutants in complex with sucrose. J. Mol. Biol. 2008, 377 (2), 378–85. (74) Roy, K. L.; Verhaest, M.; Rabijns, A.; Clerens, S.; Van Laere, A.; Van den Ende, W.

N-glycosylation

affects

substrate

specificity

of

chicory

fructan

1-exohydrolase: evidence for the presence of an inulin binding cleft. New Phytol. 2007, 176 (2), 317–24. (75) Mazorra-Manzano, M. A.; Yada, R. Y. Expression and characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana. Phytochemistry 2008, 69 (13), 2439–48.

Abbreviations ABA, abscisic acid; AGC, Automatic gain control; AO, ascorbate oxidase; APL, aspartic proteinase-like protein; BR, Brassinosteroid; ConA, concanavalin; ER, 36


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endoplasmic reticulum; ERLEC1, endoplasmic reticulum lectin 1-like protein; ERGIC, ER-Golgi intermediate compartment protein; FASP, filter-aided sample preparation; FDR, false discovery rate; HCD, higher-energy collisional dissociation; HPLC, high performance liquid chromatography; INV1, invertase 1; KOG, euKaryotic

orthologous

groups;

LAMAN,

alpha-mannosidase;

MS,

mass

spectrometry; OST, oligosaccharyltransferase; PAP, purple acid phosphatase; PDI, protein disulphide isomerase; Pfam, Protein family motif; PG, Polygalacturonase; PMSF, phenylmethanesulfonyl fluoride; QC, quality control; RLK, receptor-like kinases; ROS, reactive oxygen species; SCPL, serine carboxypeptidase-like proteins; SPDBV, Swiss-PdbViewer; UGGT, UDP-glucose: glycoprotein glucosyltransferase; XYL, alpha-xylosidase; ZIC-HILIC, zwitterionic-hydrophilic interaction liquid chromatography

37



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Table 1 The identified glycoproteins (enzymes) with conserved motifs from seedling leaves of Bd21 Accession ID

Protein description

Modification peptides

Site probability

Glycosite

Second structure

Glycosyl hydrolase/transferase XP_003557344.1

Probable polygalacturonase-like

_NALPIISN(de)ITIK_

1

N370

β strand

XP_003562496.1

Xylose isomerase-like

_IGFN(de)GTLLIEPKPQEPTK_

1

N269

Coil/loop

XP_003567980.1

Probable polygalacturonase At1g80170-like

_VTAPVDSPGTVGALLVN(de)SSDVR_

1

N231

Coil/loop

XP_003568510.1

Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A-like

_DHDVVN(de)GSATTTQMYR_

1

N555

Coil/loop

XP_003573734.1

Lysosomal alpha-mannosidase-like

_SVLLN(de)VTR_

1

N662

β strand

XP_003575078.1

Beta-fructofuranosidase, insoluble isoenzyme 1-like

_VLFGWAN(de)ESDSVPHDK_

1

N333

Coil/loop

XP_003574453.1

Predicted: glucan endo-1,3-beta-glucosidase 6-like

_N(de)VSNYLNDGCNIR_

1

N107

α helix

XP_003568009.1

Alpha-xylosidase-like

_STGQTLFN(de)TSHGGPLVFK_

1

N165

β strand

XP_003574051.1

UDP-glucose:glycoprotein glucosyltransferase-like

_SN(de)VTAPVAILYGAVGTK_

1

N207

Coil/loop

Protease/peptidase XP_003558522.1

Uncharacterized protein LOC100841880

_SVLPDSN(de)ITVTSQK_

1

N144

Coil/loop

XP_003558637.1

Aspartic proteinase nepenthesin-1-like

_SSLYYVN(de)VTGLSVGR_

1

N276

β strand

XP_003568979.1

Aspartic proteinase-like

_GN(de)HTYVPVSR_

1

N246

Coil/loop

XP_003569899.1

Lysosomal Pro-X carboxypeptidase-like

_WITTEFGGHN(de)ISAVLEK_

1

N413

Coil/loop

XP_003572138.1

Serine carboxypeptidase-like 18-like

_N(de)STYFLSEVWTNNEAVR_

1

N330

Coil/loop

XP_003572139.1


_N(de)STYFLSEVWANDEAVR_

1

N319

Coil/loop

XP_003570941.1

Kynurenine formamidase-like

_HTN(de)ITAEAMESLNIPK_

1

N157

Coil/loop

XP_003572375.1

Aminoacylase-1-like

_N(de)LTFEFK_

1

N324

α helix

XP_003564110.1

Aminoacylase-1-like

_N(de)LTYQLMK_

1

N327

Coil/loop

XP_003570941.1

Kynurenine formamidase-like

_ESMEN(de)GSEYNLSELR_

0.997

N99

Coil/loop

0.994

N206

Coil/loop

1

N330

Coil/loop

XP_003577703.1

Uncharacterized hydrolase HI_0588-like

_TVDNQN(de)LSFVDAADSAGYK_

XP_003573745.1


_N(de)ATYFLSELWTNDK_ Esterase/lipase

XP_003568079.1

Lecithin-cholesterol acyltransferase-like 1-like

_N(de)TTAPEPDAPCFADQLR_

1

N81

Coil/loop

XP_003575252.1

Probable glycerophosphoryl diester phosphodiesterase 2-like

_VMIQSTN(de)SSVLMK_

1

N533

Coil/loop

XP_003563753.1

Uncharacterized protein LOC100842729

_AVVPVGVVN(de)VTR_

1

N325

β strand

38


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XP_003577477.1

Purple acid phosphatase 4-like

_AGQYN(de)QTLVAEQMGVVGEK_

XP_003579004.1

Nucleoside-triphosphatase-like

_SVVPWWQM(ox)N(de)R_

XP_003577495.1

Protein AUXIN RESPONSE 4-like

_SLN(de)QSFDLR_

N56

Coil/loop

1

N123

Coil/loop

1

N306

α helix

1

Protein trafficking/quality control related enzymes XP_003570755.1

Protein disulfide isomerase-like 1-4-like

_LGPGVHN(de)VTTVDEAEK_

1

N220

Coil/loop

XP_003558910.1

Dolichyl-diphosphooligosaccharide- glycosyltransferase subunit 1-like

_DEIGN(de)ISTSHLWSDSK_

1

N297

Coil/loop

Protein kinasis XP_003558467.1

Probable inactive receptor kinase At1g48480-like

_LDLPTLEQFN(de)VSYNK_

1

N193

β strand

XP_003558681.1

Leucine-rich repeat receptor-like -protein kinase BAM1-like

_ILNYLN(de)LSR_

1

N558

β strand

XP_003561504.1

Receptor-like protein kinase HERK 1-like

_EYSLN(de)ITR_

1

N183

β strand

XP_003561670.1

Probable LRR receptor-like protein kinase At5g49770-like

_IGFDLSN(de)QTFKPPR_

1

N524

Coil/loop

XP_003565200.1

Probable LRR receptor-like -protein kinase At1g06840-like

_GDPCVGN(de)WSR_

1

N63

Coil/loop

Oxidordeuctase XP_003563398.1

L-ascorbate oxidase homolog

_WN(de)LTASGPR_

XP_003563730.1

L-ascorbate oxidase-like

_TLPATALLN(de)YTNSR_

XP_003567636.1

Peroxidase 1-like

_DSVN(de)LTGTNSFYQVPSGR_

Note: (de) in the bracket shows the deamidatio

39


1

N333

Coil/loop

0.981

N377

β strand

1

N140

α helix


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 45

Figure 1. Experimental workflow of glycoproteome analysis in seedling leaves of B. distachyon (Bd21) during the early vegetative growth stage. (1) Protein extraction from seedlings for three biological replicates; (2) Tryptic digestion; (3) Glycopeptide enrichment through lectin affinity chromatography; (4) Gaining of glycopeptides (18O) by PNGase A and addition of H218O; (5) Purification and determination of peptides (18O) by nLC-MS/MS analysis; (6) Glycosylation residue localization of peptides (18O, the addition of 2.98Da) by MaxQuant software; (7) Bioinformatics analysis. LAC, lectin affinity chromatography; LC, liquid chromatography; MS, mass spectrum. :α-1,3-Fucose;

:β-1,2-Xylose; :HexNAc.

40


:Mannose; :GlcNAc;

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Figure 2. Functional categories and cellular localization of all glycoproteins identified in the seedling leaves of Bd21. (A) Functional categories. (B) Cellular localization.

41



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Figure

3.

The

numbers

and

proportion

of

motifs,

secondary

Page 42 of 45

structures,

and

signal

peptides/transmembrane (TM) of glycoproteins in the seedling leaves of Bd21. (A) Two conserved motifs (N-X-S/T) were enriched by Motif-X. (B) The proportion of N-X-S was lower than N-X-T. (C) The proportion of loop/turn was higher than β sheet and α helix. (D) The proportion of various combinations of SP/TM.

42


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Figure 4. Signal transduction, protein trafficking, and protein processing and folding of glycoproteins in seedling leaves of Bd21. (A) ROS (H2O2) and RLK-mediated signaling pathways on the plasma membrane. (B) Protein transporting, quality control, and protein processing occurring in the ER, cytoplasm, and Golgi complex. Reactive oxides species (ROS); Hydrogen peroxide (H2O2); Peroxidase 1

(PER1);

Brassinosteroids

Dolichyl-diphosphooligosaccharide-protein

(BRs);

Receptor-like

glycosyltransferase

subunit

kinases (RPN1);

(RLKs); Endoplasmic

reticulum lectin 1 (ERLEC1); UDP-glucose:glycoprotein glucosyltransferase (UGGT); Calreticulin (CRTN); Protein disulfide isomerase-like protein (PDIL); Endoplasmic reticulum-Golgi intermediate compartment protein (ERGIC); Lysosomal alpha-mannosidase (LAMAN).

43



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Page 44 of 45

Figure 5. Sequence alignment, structural composition, three dimensional (3D) structure, and tandem mass spectrometry (MS/MS) map of PDIL-4 in seedling leaves. (A) Protein disulfide isomerase like protein1-4 (PDIL1-4) sequence alignment against O. sativa, T. aestivum, and Z. mays. The purple balls show five active sites (C136, C139, C478, C481, R543), the five-pointed stars show KDEL motif. (B) 3D structure (confidence level>91%); the glycosylation site was displayed at N220. (C) Glycopeptide MS/MS data.

Table of content synopsis 44


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45


First Comprehensive Proteome Analyses of Lysine Acetylation and Succinylation in Seedling Leaves of Brachypodium distachyon L.

Large-scale phosphoproteome analysis in seedling leaves of Brachypodium distachyon L.

Molecular characterization and expression profiling of the protein disulfide isomerase gene family in Brachypodium distachyon L.

Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

Molecular characterisation and evolution of HMW glutenin subunit genes in Brachypodium distachyon L.

Comparative analysis and modeling of superoxide dismutases (SODs) in Brachypodium distachyon L.

Genetic Architecture of Flowering-Time Variation in Brachypodium distachyon.

The arrangement of Brachypodium distachyon chromosomes in interphase nuclei.

Long noncoding RNAs in the model species Brachypodium distachyon.

Ultrastructure of microsporogenesis and microgametogenesis in Brachypodium distachyon.

Quantitative Trait Loci Associated with Drought Tolerance in Brachypodium distachyon.

Transcriptomic Profiling of the Maize (Zea mays L.) Leaf Response to Abiotic Stresses at the Seedling Stage.

The α-gliadin genes from Brachypodium distachyon L. provide evidence for a significant gap in the current genome assembly.

Use of Agrobacterium rhizogenes Strain 18r12v and Paromomycin Selection for Transformation of Brachypodium distachyon and Brachypodium sylvaticum.

Genomic architecture and functional relationships of intronless, constitutively- and alternatively-spliced genes in Brachypodium distachyon.

EREBP superfamily in Brachypodium distachyon.

Enhanced oxidative stress resistance through activation of a zinc deficiency transcription factor in Brachypodium distachyon.

Genome-wide Analysis and Expression Profile of the MYB genes in Brachypodium distachyon.

Spatial distribution of epigenetic modifications in Brachypodium distachyon embryos during seed maturation and germination.

Range of cell-wall alterations enhance saccharification in Brachypodium distachyon mutants.

Brachypodium distachyon - A Useful Model in the Qualification of Mutagen-Induced Micronuclei Using Multicolor FISH.

Genome-wide analysis of basic helix-loop-helix (bHLH) transcription factors in Brachypodium distachyon.

Regulation of FLOWERING LOCUS T by a microRNA in Brachypodium distachyon.

Daily changes in temperature, not the circadian clock, regulate growth rate in Brachypodium distachyon.