Proteomic Analysis of Lonicera japonica Thunb ... - ACS Publications

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Proteomic Analysis of Lonicera japonica Thunb. Immature Flower Buds Using Combinatorial Peptide Ligand Libraries and Polyethylene Glycol Fractionation Wei Zhu,†,‡ Xiaobao Xu,‡ Jingkui Tian,‡ Lin Zhang,*,‡ and Setsuko Komatsu*,† †

National Institute of Crop Science, National Agriculture and Food Research Organization, Tsukuba 305-8518, Japan College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou 310027, China

‡

S Supporting Information *

ABSTRACT: Lonicera japonica Thunb. flower is a well-known medicinal plant that has been widely used for the treatment of human disease. To explore the molecular mechanisms underlying the biological activities of L. japonica immature flower buds, a gel-free/label-free proteomic technique was used in combination with combinatorial peptide ligand libraries (CPLL) and polyethylene glycol (PEG) fractionation for the enrichment of low-abundance proteins and removal of high-abundance proteins, respectively. A total of 177, 614, and 529 proteins were identified in crude protein extraction, CPLL fractions, and PEG fractions, respectively. Among the identified proteins, 283 and 239 proteins were specifically identified by the CPLL and PEG methods, respectively. In particular, proteins related to the oxidative pentose phosphate pathway, signaling, hormone metabolism, and transport were highly enriched by CPLL and PEG fractionation compared to crude protein extraction. A total of 28 secondary metabolism-related proteins and 25 metabolites were identified in L. japonica immature flower buds. To determine the specificity of the identified proteins and metabolites for L. japonica immature flower buds, Cerasus flower buds were used, which resulted in the abundance of hydroxymethylbutenyl 4-diphosphate synthase in L. japonica immature flower buds being 10-fold higher than that in Cerasus flower buds. These results suggest that proteins related to secondary metabolism might be responsible for the biological activities of L. japonica immature flower buds. KEYWORDS: Lonicera japonica Thunb., immature flower buds, proteomics, combinatorial peptide ligand libraries, polyethylene glycol

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extracts of L. japonica flower buds exhibited inhibitory effects against the growth of influenza A virus.10 Among the active constituents in the whole plant of L. japonica, chlorogenic acid has been identified as a major bioactive compound with demonstrated antiviral activity.11 L. japonica flower buds also contain luteolin, which exhibits anti-inflammatory activity by suppressing the expression of 5-lipoxygenase.12 Moreover, ethanol extracts of L. japonica flower buds were found to have protective effects against acute liver injury.13 Despite the findings from these studies, the molecular mechanisms of plant growth/development and the biological activities of L. japonica flower buds at the protein level remain unclear. Protein samples of plant contained high-abundance proteins, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which comprised 30−60% of the total protein in leaves,14 and phloem filament protein/phloem lectin, which accounted for more than 80% of total protein in phloem.15 To increase the detection of low-abundance proteins in proteomic analyses, various methods have been applied including polyethylene glycol (PEG) fractionation,16 affinity column purification with RuBisCO antibody,17 organelle prefractionation,18 titanium dioxide microcolumn preconcentration for

INTRODUCTION Lonicera is a perennial twining and sprawling liana of the Caprifoliaceae family. Members of this genus are native to eastern Asia including China, Japan, and Korea1 and are widely used as traditional medicinal plants due to their various biological activities.2 For example, butanol extracts from whole plant of Lonicera japonica have anti-inflammatory activity against acute and chronic inflammation,3 and water extracts display anti-inflammatory activity against proteinase-activated receptor 2-mediated mouse paw edema.4 In addition to antiinflammatory activity, aqueous extracts of L. japonica flower buds have antiviral activity against respiratory syncytial virus,5 and ethanol extracts exhibit antifungal activity against the infectious fungal pathogens Microsporum canis and Trichophyton rubrum.6 Components within ethyl acetate extracts of Flos L. japonica flower also exhibit high antioxidant activity through the scavenging of 1,1-diphenyll-2−2-pricylhydrazyl free radicals and removal of hydroxyl radicals.7 Because of these biological activities, particularly the anti-inflammatory and antimicrobial properties of tissue extracts, Lonicera has received increasing attention as a natural source of medicinal compounds. The flower buds of L. japonica have been used in clinical practice for decades.8 Recent studies have confirmed that L. japonica flower buds have a higher content of active constituents than that of L. japonica stem and leaf.9 Methanol © XXXX American Chemical Society

Received: July 15, 2015

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DOI: 10.1021/acs.jproteome.5b00910 J. Proteome Res. XXXX, XXX, XXX−XXX

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Journal of Proteome Research

for 1 h at −20 °C with vortexing every 15 min and centrifuged at 9000 × g at 4 °C for 20 min. The supernatant was discarded, and the pellet was washed twice with 0.07% 2-mercaptoethanol in acetone. The pellet was dried using a Speed-Vac concentrator (Savant Instruments, Hickville, NY, USA). For proteomic analysis, lysis buffer consisting of 7 M urea, 2 M thiourea, 5% CHAPS, and 2 mM tributylphosphine was added and vortexed for 1 h at 25 °C. The suspension was centrifuged at 20 000 × g for 20 min at 25 °C, and the supernatant was collected as crude protein extraction. Protein concentrations were determined using the Bradford assay32 with bovine serum albumin as the standard. For sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis, SDS-sample buffer consisting of 60 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol33 was added. Protein concentrations were determined using Pierce 660 nm Protein Assay Reagent (Thermo Scientific, Rockford, IL, USA) with bovine serum albumin as the standard.

phosphoprotein enrichment,19 and fractionation of combinatorial peptide ligand libraries (CPLL).20 Of these approaches, PEG fractionation was most effective for reducing abundant proteins,21 whereas CPLL fractionation was useful for enriching low-abundance proteins.22 The CPLL method has been widely used for human and plant proteomic analyses, such as those of human platelets23 and spinach leaf,24 to enrich and increase the detection of lowabundance proteins. For example, Frohlich et al.15 reported that high-abundance proteins, such as RuBisCO, were diminished, and proteins functioning in protein transport and translation were enriched by CPLL method in Arabidopsis leaf. In addition, application of the CPLL method increased the detection of secondary metabolism-related proteins in Mahonia leaves treated with ultraviolet-B.25 By using PEG fractionation combined with immobilized metal-ion affinity chromatography, Aryal et al.26 identified low-abundance phosphoproteins in Arabidopsis leaf. PEG fractionation has also been used to detect proteins related to Pto disease resistance in tomato leaf27 and the oxidative pentose phosphate pathway in Mahonia leaf.28 Although CPLL and PEG methods are useful for detecting lowabundance proteins, no studies have directly compared these two methods. About Lonicera, metabolites were analyzed using flower buds of seven Lonicera species.29 In addition, Yuan et al.30 performed transcriptomic analysis to assess genetic differences between flowers of two Lonicera species using an RNA-sequencing method. Furthermore, Zhang et al.31 performed gel-based proteomic analysis of L. japonica flower buds under ultravioletB stress and identified 75 proteins that were potentially involved in this stress response. To date, only a limited number of studies have investigated the molecular mechanisms underlying the biological activities of L. japonica flower buds. To identify the active components that mediate the biological activities of L. japonica flower buds, various proteomic approaches have been used. In the present study, CPLL and PEG fractionation methods were combined with a gel-free/ label-free proteomic technique to increase the identification of low-abundance proteins in L. japonica immature flower buds compared to those detected in crude protein extraction. In addition, to explore the specificity of bioactive compounds in L. japonica immature flower buds, Cerasus flower buds were also analyzed using a gel-free/label-free proteomic technique.

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CPLL Fractionation

CPLL fractionation of proteins was performed according to the method of Frohlich et al.17 with minor modifications (Supplemental Figure 1). Briefly, a portion (0.5 g) of samples was homogenized in 12 mL of protein extraction buffer consisting of 50 mM Tri-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, and 1% 2-mercaptoethanol, and the resulting slurry was sonicated for 5 min. After centrifugation at 20 000 × g for 15 min at 4 °C, the supernatant was separated into three parts for buffer exchange using PD10 columns (GE Healthcare, Pittsburgh, PA, USA) following the manufacturer’s protocol. The PD10 columns were equilibrated and eluted using the following buffers: 25 mM acetate buffer (pH 4.0), 25 mM TriHCl (pH 7.0), and 25 mM Tri-HCl (pH 9.0). An amount (3.5 mL) of protein solution (pH 4, 7, and 9) was collected. Three CPLL columns containing 20 μL of ProteoMiner beads (BioRad, Hercules, CA, USA) were equilibrated with the corresponding buffer (pH 4, 7, and 9). Three parts of protein solution (pH 4, 7, and 9) were added into the CPLL columns, respectively. The protein solution was incubated with CPLL columns for 3 h at room temperature using vortex and then centrifuged at 1000 × g for 1 min. The solution was collected as flow through (FT) solution. The CPLL columns were washed with 2 mL of corresponding buffer and added solution consisting of 4% SDS and 50 mM dithiothreitol at 95 °C. These columns were incubated at 25 °C for 15 min and then centrifuged at 1000 × g for 1 min. The elution was collected as eluted solution (ES). All the FT solutions were precipitated with four volumes of cold trichloroacetic acid/acetone. All the ESs were precipitated using chloroform/methanol to remove the excess of SDS.34 The resulting pellets were mixed with the lysis buffer for proteomic analysis. For Western blotting, the resulting pellets were mixed with SDS-sample buffer.

EXPERIMENTAL PROCEDURES

Plant Material and Growth Conditions

Lonicera japonica (Thunb.) was obtained from Pingyi cultivation base (Shandong, China). This material is the same as sample number LJ04.29 It was grown in the area without extreme drought, plant diseases, and insect pests. Cerasus yedoensis (Matsum.) was obtained from flower and tree garden company (Tsukuba, Japan). Malus spectabilis (Ait.) Borkh was obtained from flower and tree garden company (Hangzhou, China). The whole immature flower buds were collected for proteomic analysis and enzyme assay. Three independent experiments were performed as biological replicates.

PEG Fractionation

PEG fractionation of proteins was performed according to the method of Zhu et al.28 (Supplemental Figure 2). Briefly, a portion (0.2 g) of sample was homogenized in 5 mL of extraction buffer consisting of 0.5 M Tri-HCl (pH 7.8), 2% Triton X-100, 20 mM MgCl2, 2% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM EDTA. The resulting slurry was sonicated for 5 min and centrifuged at 12 000 × g for 15 min at 4 °C, and then the supernatant was collected. A 50% PEG (MW 4000) stock solution was added to the supernatant to give a final concentration of 8%. The PEG-suspended

Protein Extraction

A portion (0.1 g) of samples was ground to powder in liquid nitrogen using a mortar and pestle and was then transferred to an acetone solution containing 10% trichloroacetic acid and 0.07% 2-mercaptoethanol. The resulting mixture was vortexed and then sonicated for 10 min. The suspension was incubated B


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Journal of Proteome Research solution was placed on ice for 30 min and then centrifuged at 1500 × g for 10 min at 4 °C. The resulting pellet was collected as Fraction 1 (F1). The 50% PEG stock solution was added to the supernatant of the F1 sample to give a final concentration of 16%. The PEG suspended resulting solution was placed on ice for 30 min and then centrifuged at 12 000 × g for 15 min at 4 °C. The resulting pellet was collected as Fraction 2 (F2), and the supernatant was precipitated with four volumes of cold trichloroacetic acid/acetone at −20 °C for 1 h. After centrifugation of the solution at 12 000 × g for 15 min at 4 °C, the resulting pellet was collected as Fraction 3 (F3). The resulting pellets were mixed with the lysis buffer for proteomic analysis. For Western blotting, the resulting pellets were mixed with SDS-sample buffer.

mm) (Dionex, Germering, Germany) of an Ultimate 3000 nanoLC system (Dionex) and were then eluted with a linear acetonitrile gradient (8−30% over 150 min) in 0.1% formic acid at a flow rate of 200 nL/min. The eluted peptides were separated and sprayed on a C18 capillary tip column (75 μm ID × 120 mm) (Nikkyo Technos, Tokyo, Japan) with a spray voltage of 1.5 kV. Full-scan mass spectra were acquired in the LTQ Orbitrap mass spectrometer over 400−1500 m/z with a resolution of 30 000. A lock mass function was used for high mass accuracy.37 The ten most intense precursor ions were selected for collision-induced fragmentation in the linear ion trap at normalized collision energy of 35%. Dynamic exclusion was employed within 90 s to prevent repetitive selection of peptides.38

Western Blotting

Protein Identification from the MS Data

Proteins (10 μg) were separated on a 17% SDS-polyacrylamide gel electrophoresis gel and transferred onto a polyvinylidene difluoride membrane using a semidry transfer blotter. The blotted membrane was incubated overnight at 4 °C in blocking buffer, consisting of 20 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 5% skim milk (Difco, Sparks, MD, USA). After blocking, a membrane was incubated with a 1:3000 dilution of antiRuBisCO large subunit antibody and anti-RuBisCO small subunit antibody35 for 1 h at room temperature. Antirabbit IgG conjugated with horseradish peroxidase (Bio-Rad) was used as the secondary antibody. After 1-h incubation with the secondary antibody, signals were detected using an ECL Plus Western Blotting Detection kit (Nacalai Tesque, Kyoto, Japan) following the manufacturer’s protocol, and the signals were visualized using a LAS-3000 Luminescent Image Analyzer (Fujifilm, Tokyo, Japan). The relative intensities of bands were calculated using Quantity One software (version 4.5; Bio-Rad).

Proteins were identified using the Mascot search engine (version 2.5.1, Matrix Science, London, UK) of the UniprotPlants-Viridiplantae database (197 887 sequences) obtained from the Uniprot database (version of January 11, 2013, http:// www.uniprot.org/). The acquired raw data files were processed using Proteome Discoverer (version 1.4.0.288, Thermo Fisher Scientific). The parameters used in Mascot searches were as follows: carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine was set as a variable modification. Trypsin was specified as the proteolytic enzyme, and one missed cleavage was allowed. Peptide mass tolerance was set at 10 ppm, fragment mass tolerance was set at 0.8 Da, and peptide charge was set at +2, + 3, and +4. An automatic decoy database search was performed as part of the search. Mascot results were filtered with the Mascot Percolator to improve the accuracy and sensitivity of peptide identification.39 False discovery rates for peptide identification of all searches were less than 1.0%. Peptides with a percolator ion score of more than 13 (p < 0.05) were used for protein identification. Protein abundance was analyzed based on the emPAI value.40 Briefly, the mean of the three emPAI values was divided by the sum of the emPAI values for all identified protein and multiplied by 100. Protein content was estimated by the molar fraction percentage (mol %).

Protein Purification and Digestion for Mass Spectrometry Analysis

Proteins (100 μg) were purified with methanol and chloroform to remove any detergent from the sample solutions.36 Briefly, 400 μL of methanol was added to each sample, and the resulting solution was mixed before the further addition of 100 μL of chloroform and 300 μL of water. After mixing, the samples were centrifuged at 20 000 × g for 10 min to achieve phase separation. The upper aqueous phase was discarded, and 300 μL of methanol was added slowly to lower phase. The samples were centrifuged at 20 000 × g for 10 min, supernatants were discarded, and pellets were dried. The dried pellets were resuspended in 50 mM NH4HCO3, and proteins in the samples were reduced with 50 mM dithiothreitol for 30 min at 56 °C and alkylated with 50 mM iodoacetamide for 30 min at 37 °C in the dark. Alkylated proteins were digested with trypsin and lysyl endopeptidase (Wako, Osaka, Japan) at 1:100 enzyme/protein concentrations at 37 °C for 16 h. The resulting tryptic peptides were acidified by mixing with formic acid (pH < 3), and the resulting solution was centrifuged at 20 000 × g for 10 min. The obtained supernatant was collected, and 2 μL was applied to nanoliquid chromatography (LC)−mass spectrometry (MS).

Functional Categorization of Identified Proteins

Protein functions were categorized using MapMan bin codes (http://mapman.gabipd.org/), as previously described.41 A small-scale prediction of the identified proteins derived from Lonicera was performed by transferring annotations from the Arabidopsis genome and consideration of orthologous genes. Pathway mapping of identified proteins was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/).42 Analysis of Metabolites

A portion (0.2 g) of samples was extracted with 3 mL of methanol and sonicated for 1.5 h. After centrifugation at 12 000 × g for 5 min, the supernatant was collected. Metabolomics analysis was performed on a Nexera UHPLC-30A (Shimadzu, Tokyo, Japan) LC system, which was coupled with a DuoSpray ion source equipped with a quadrupole time-of-flight (TOF) MS TripleTOF 5600 (AB SCIEX, Foster City, CA, USA). The separation of all samples was performed on a C18 column (2.1 mm ID × 100 mm, Waters, Dublin, Ireland) with a column temperature at 40 °C. The flow rate was 0.3 mL/min, and the mobile phase consisted of 0.1% formic acid aqueous and acetonitrile: 0−10 min (20%−100% acetonitrile) and 11−22

NanoLC−MS/MS Analysis

A nanospray LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) was operated in data-dependent acquisition mode with the installed XCalibur software (version 2.1, Thermo Fisher Scientific). Peptides in 0.1% formic acid were loaded onto a C18 PepMap trap column (300 μm ID × 5 C


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Journal of Proteome Research Statistical Analysis

min (100% acetonitrile). The sample injection volume was 2 μL. The automated calibration device system was set to perform an external calibration each sample. The source conditions were: temperature 600 °C, curtain gas 30, both GS1 and GS2 at 50, respectively, and ion-spray voltage floating at 4.5 kV. Collision energies were set to 30 eV. Date acquisition was using the information-dependent acquisition mode: TOF as survey scan (accumulation time 110 ms) and MS/MS as dependent scan with a collision energies range of 15−45 eV in eight MS/MS dependent experiments (accumulation time 110 ms). For all experiments, the TOF mass range was 100−1000 m/z. MS data acquisition was performed using Analyst TF software (version 1.5.1, AB SCIEX), and data processing was performed using PeakView software (version 1.1, AB SCIEX).

SPSS statistical software (version 22.0, IBM, Armonk, NY, USA) was used for the statistical evaluation of the results. Statistical significance was evaluated by the Student’s t-test when only two groups were compared and with the one-way analysis of variance (ANOVA) test when multiple groups were compared. All results were presented as mean ± standard deviation (SD) from three independent biological replications. A p-value less than 0.05 was considered statistically significant.

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RESULTS

Enrichment of Low-Abundance Proteins Using CPLL and Removal of High-Abundance Proteins by PEG Fractionation

Analyses of Enzyme Activities

To increase the identification of low-abundance proteins in L. japonica immature flower buds, CPLL and PEG fractionation methods were used to enrich for low-abundance proteins and remove high-abundance proteins, respectively (Figure 1). To evaluate the efficacy of the two techniques, the levels of RuBisCO proteins were evaluated by Western blot analysis (Figure 2). As indicated in the experimental workflow of the CPLL method (Supplemental Figure 1), proteins extracted from immature flower buds were incubated in CPLL columns equilibrated at different pH values (pH 4, 7, and 9). After CPLL fractionation, the abundance of RuBisCO large subunit was significantly reduced in the eluted fractions compared to the flow-through fractions (Figure 2, left). For PEG fractionation (Supplemental Figure 2), proteins from immature flower buds were treated with 8% and 16% PEG (F1, F2, and F3). After PEG fractionation, the abundances of RuBisCO large and small subunits were clearly reduced in F3 compared to F1 and F2 (Figure 2, right). Notably, peptide fragments of RuBisCO were only detected in F2. Compared to crude protein extraction, the ratio of RuBisCO large subunit to total protein was 21% (45/ 210) by the CPLL method and 18% (25/140) by PEG method.

The phenylalanine ammonia-lyase (PAL) activity was assayed according to the method of Goldson et al.43 with minor modifications. Briefly, a portion (0.2 g) of sample was homogenized in 4 mL of extraction buffer consisting of 0.1 M borate (pH 8.8) and 7 mM 2-mercaptoethanol. The resulting slurry was centrifuged at 12 000 × g for 15 min at 4 °C, and the supernatant was collected as crude enzyme extract. The reaction mixture contained 1 mL of 0.1 M borate (pH 8.8) and 0.25 mL of crude enzyme extract. The reactions were initiated by adding 0.5 mL of 20 mM L-phenylalanine. Tubes were incubated at 30 °C for 30 min, and the reaction was stopped by the addition of 0.2 mL of 6 M hydrochloric acid. PAL activity was determined from the yield of cinnamic acid by measuring the absorbance at 290 nm. The 4-coumaroyl-CoA ligase (4CL) activity was assayed using Plant 4CL enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Meilian Bioengineering Institute, Shanghai, China) according to the manufacturer’s protocol. Crude enzyme extract was achieved according to Knobloch and Hahlbrock.44 Briefly, a portion (0.5 g) of sample was homogenized in 4 mL of extraction buffer consisting of 0.2 M Tris-HCl (pH 8.0), 10% glycerol, and 15 mM 2mercaptoethanol. The levels of 4CL in samples were determined by sandwich ELISA method. The crude enzyme extract was added into the microplate with 4CL solid-phase antibody. The horseradish peroxidase-linked 4CL antibody was used as secondary antibody, and 3, 3′, 5, 5′-tetramethylbenzidine was added as substrate. Absorbance was measured by using microplate spectrophotometer at 450 nm. The 4CL activity was determined using a standard curve. The flavonol synthase (FLS) activity was assayed according to the method of Chua et al.45 with minor modification. Briefly, a portion (0.5 g) of sample was homogenized in 4 mL of extraction buffer consisting of 50 mM Tris-HCl (pH 8.9), 0.1% Triton-100, 5 mM ascorbic acid, 1 mM phenylmethanesulfonyl fluoride, and 14 mM 2-mercaptoethanol. The resulting slurry was centrifuged at 12 000 × g for 15 min at 4 °C, and then the supernatant was collected for the determination of FLS enzyme activity. The reaction mixture (total volume 500 μL) contained 200 mM sodium acetate (pH 5.0), 83 μM 2-oxoglutarate, 42 μM iron(II) sulfate, 2.5 mM sodium ascorbate, 100 μM dihydroquercetin, and 10 μg of crude enzyme extract. Subsequently, the reaction mixture was incubated at 37 °C for 10 min, and FLS activity was determined from the yield of quercetin by measuring the absorbance at 375 nm.

Proteins in L. japonica Immature Flower Buds Were Highly Enriched Using CPLL and PEG Fractionations

Proteins fractionated by the CPLL method were reduced, alkylated, digested, and analyzed using nanoLC−MS/MS. A total of 377, 397, and 407 proteins with more than two matched peptides were identified in the pH 4 (Supplemental Tables 1 and 2), pH 7 (Supplemental Tables 3 and 4), and pH 9 fractions (Supplemental Tables 5 and 6), respectively. Among the identified proteins, 98 (98/377, 26%), 75 (75/397, 19%), and 80 proteins (80/407, 20%) were specific to the pH 4, 7, and 9 fractions, respectively, and 206 proteins were commonly identified in the three fractions (Supplemental Figure 3). The main functional category of proteins in the three pH fractions was protein synthesis/posttranslational modification/folding/ degradation. The functional categories of the identified proteins were similar among the three pH fractions. The proteins that were identified in the flow-through fractions were classified as major proteins, and those present in the eluted fractions were designated as minor proteins (Supplemental Figure 1). A total of 413 and 443 proteins with more than two matched peptides were identified as major and minor proteins, respectively (Supplemental Figure 4). Of the 443 minor proteins, 201 proteins (201/443, 45%) were newly identified. The proportion of proteins classified in the functional categories of signaling (4%), transport (2%), mitochondrial electron transport (2%), and hormone metabolism (2%) was two-fold higher than that D


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Figure 1. Experimental design for the proteomic analysis of L. japonica immature flower buds. L. japonica immature flower buds were collected and ground to a powder in liquid nitrogen. One portion of the obtained powder was used for crude protein extraction by the trichloroacetic acid/acetone method. The other two portions of the obtained powder were applied to CPLL and PEG fractionation methods. In the CPLL fractionation method, FT solution and ES of pH 4, 7, and 9 were collected (Supplemental Figure 1). In the PEG fractionation method, 8% PEG pellet (F1), 16% PEG pellet (F2), and supernatant fractions (Sup, F3) were collected (Supplemental Figure 2). The protein fractions were collected and analyzed using a gel-free/ label-free proteomic technique. Three independent experiments were performed as biological replicates. Photograph courtesy of Xiaobao Xu. Copyright 2015.

Figure 2. Protein abundances after CPLL and PEG fractionation of L. japonica immature flower buds. Proteins from L. japonica immature flower buds were fractionated by the CPLL and PEG methods (Figure 1). The obtained proteins (10 μg) were separated by SDSpolyacrylamide gel electrophoresis and transferred onto a polyvinylidine difluoride membrane for Western blotting with antiRuBisCO large subunit antibody and anti-RuBisCO small subunit antibody. After incubation with secondary antibody, signals were detected using ECL. The Coomassie brilliant blue (CBB) staining pattern was used as a loading control. The relative band intensities were calculated using Quantity One software. Data are shown as the mean ± SD from three independent biological replicates. Means with the same letter are not significantly different according to one-way ANOVA test (p < 0.05). Abbreviations are as follows: CE, crude protein extraction; FT, flow-through solution; ES, eluted solution; F1, 8% PEG pellet; F2, 16% PEG pellet; and F3, supernatant.

of major proteins in the respective functional categories (2, 1, 1, and 1%, respectively) (Supplemental Figure 4). Proteins fractionated by the PEG method were reduced, alkylated, digested, and analyzed using nanoLC−MS/MS. A total of 317, 73, and 230 proteins with more than two matched peptides were identified in F1 (Supplemental Table 7), F2 (Supplemental Table 8), and F3 (Supplemental Table 9), respectively. Among the identified proteins, 240 (240/317, 76%), 45 (45/75, 60%), and 163 proteins (163/230, 71%) were specific to fractions 1, 2, and 3, respectively (Supplemental Figure 5). The number of proteins related to signaling (7%, 17 proteins), redox homeostasis (6%, 14 proteins), and major carbohydrate metabolism (2%, 5 proteins) in F3 was higher than that in F1 (2%, 7 proteins; 2%, 6 proteins; and 0.4%, 1 protein). In addition, a higher number of proteins related to

secondary metabolism (3%, 7 proteins), cell wall (1%, 2 proteins), hormone metabolism (3%, 8 proteins), and the oxidative pentose phosphate pathway (2%, 4 proteins) were newly identified in F3 compared to that in F2 (Supplemental Figure 5). Proteins Related to the Oxidative Pentose Phosphate Pathway, Signaling, Hormone Metabolism, and Transport Were Highly Enriched Using CPLL and PEG Fractionations

To more deeply explore the proteins that are specific to L. japonica immature flower buds, CPLL and PEG fractionation methods were used. A total of 177, 614, and 529 proteins were E


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protein (23%), photosynthesis (9%), and cell (9%) (Figure 3, right). By using CPLL fractionation, the numbers of proteins related to signaling (3%, 21 proteins), oxidative pentose phosphate pathway (2%, 10 proteins), and hormone metabolism (2%, 12 proteins) were largely increased compared to crude protein extraction (1%, 2 proteins; 1%, 2 proteins; and 1%, 1 protein, respectively). By PEG fractionation, the numbers of proteins related to signaling (4%, 23 proteins), transport (3%, 16 proteins), oxidative pentose phosphate pathway (2%, 9 proteins), and hormone metabolism (2%, 10 proteins) were also largely increased compared to crude protein extraction (Figure 3). By using both fractionation methods, minor proteins related to signaling, oxidative pentose phosphate, and hormone metabolism were newly identified.

identified in crude protein extraction (Supplemental Table 10) and in the fractions obtained by CPLL (Supplemental Table 11) and PEG fractionation (Supplemental Table 12), respectively. Among these identified proteins, 283 (283/614, 46%) and 239 proteins (239/529, 45%) were specifically identified by the CPLL and PEG methods, respectively. Compared to crude protein extraction, 467 (467/614, 76%) and 423 proteins (423/529, 80%) were additionally identified in L. japonica immature flower buds by the CPLL and PEG methods, respectively (Figure 3, left). Among proteins in the

Twenty-eight Secondary Metabolism Related Proteins and 25 Secondary Metabolites Were Identified in L. japonica Immature Flower Buds

The proteomic analyses of L. japonica immature flower buds using the CPLL and PEG methods identified a total of 28 proteins related to secondary metabolism (Supplemental Tables 11 and 12). Among these proteins, 17 (17/28, 61%), eight (8/28, 29%), and two proteins (2/28, 7%) were involved in the biosynthesis pathways of terpenoids, phenylpropanoids, and flavonoids, respectively (Table 1). To better understand the biological activities of L. japonica immature flower buds, secondary metabolites in immature flower buds were identified by LC−TOF/MS analysis of methanol extracts. On the basis of the retention times and m/z values of the main fragment ions, a total of 25 secondary metabolites were identified. Among these metabolites, six (6/25, 24%), three (3/25, 12%), and 13 metabolites (13/25, 52%) were involved in the biosynthesis pathways of phenylpropanoids, terpenoids, and flavonoids, respectively (Table 2). Proteins and metabolites related to terpenoid and phenylpropanoid/flavonoid biosynthesis were mapped on the corresponding KEGG pathways (Figure 4). In the terpenoid biosynthesis pathway, nine proteins, consisting of six proteins related to dimethylallyl disphosphate synthesis and three proteins related to isopentenyl disphosphate synthesis, and two metabolites, including loganin and secologanate, were mapped (Figure 4A). In the penylpropanoid/flavonoid biosynthesis pathway, five proteins related to the biosynthesis of penylpropanoids and 16 metabolites were mapped. Among the identified metabolites, four metabolites, caffeic acid, ferulic acid, chlorogenic acid, and 4-coumaroyl quinic acid, were related to phenylpropanoid biosynthesis and 12 metabolites, eriodictyol, naringenin, apigenin, luteolin, apigenin 7-O-glucoside, apigenin 7-O-apioglucoside, apigenin 7-O-rutinoside, kaempferol, kaempferol 3-O-glucoside, quercetin, quercetin 3-O-glucoside, and rutin, were related to flavonoid biosynthesis (Figure 4B).

Figure 3. Functional categorization of crude protein extraction and fractionated proteins in L. japonica immature flower buds. Crude proteins extracted from L. japonica immature flower buds were reduced, alkylated, digested, and then analyzed using nanoLC−MS/ MS. Venn diagram showing the number of proteins that were commonly identified in the crude protein extraction, CPLL fractions, and PEG fractions. The identified proteins were categorized using MapMan bin codes. Pie graphs showing the percentage of proteins in each functional category. Abbreviations: protein, protein synthesis/ posttranslational modification/folding/degradation; TCA, tricarboxylic acid cycle; cell, cell organization/vesicle transport/cycle/division; redox, redox ascorbate/glutathione metabolism; CHO, carbohydrates; mitoETC, mitochondrial electron transport chains; OPP, oxidative pentose phosphate; others, containing C1-metabolism, cofactor/ vitamin metabolism, development, gluconeogenesis, metal handling binding, minor CHO metabolism, N-metabolism, RNA, and tetrapyrrole synthesis; and misc, miscellaneous.

Activities of Phenylalanine Ammonia-Lyase, 4-Coumaroyl-CoA Ligase, and Flavonol Synthase in L. japonica Immature Flower Buds Were Higher than Those in M. spectabilis

To verify differences in phenylpropanoids/flavonoids biosynthesis between medicinal and nonmedicinal flowers, the enzyme activities in L. japonica and M. spectabilis immature flower buds were measured. The activities of PAL, 4CL, and FLS involved in the biosynthesis of phenylpropanoids/ flavonoids in L. japonica immature flower buds were significantly higher than those in M. spectabilis (Figure 5). The activities of PAL and 4CL in L. japonica immature flower

crude protein extraction, the main functional categories were protein (23%), photosynthesis (8%), tricarboxylic acid cycle (8%), and glycolysis (8%). Similarly, the main functional categories for CPLL-fractionated proteins were protein (23%), photosynthesis (9%), and glycolysis (7%). However, the main functional categories for PEG-fractionated proteins were F


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Table 1. Identified Proteins Related to Secondary Metabolism in L. japonica Immature Flower Buds Using Gel-Free/Label-Free Proteomic Technique no.

protein IDa

orthologb

description

1

A1KXW2

AT5G62790

2

A7BG58

AT1G63970

3 4 5 6 7 8

A9ZMZ5 B1NYI4 B4FYM0 B5BLW2 B6UDL5 B7UCR9

AT5G48230 AT4G14210 AT5G48230 AT3G63520 AT5G60600 AT5G62790

9 10 11

K3XIH5 K4A9K9 Q8GZR6

AT2G26930 AT4G11820 AT5G60600

12 13 14 15

Q8L8H6 J9XLE5 A9ZMZ4 A9ZN09

AT4G14210 AT5G16440 AT5G48230 AT2G02500

16 17 18 19 20

B9UP05 Q84XR5 B2Z6P0 C1IC54 C1M2W0

AT4G34350 AT5G60600 AT3G21240 AT4G39330 AT4G37980

21 22 23 24 25 26 27 28

E2FYC3 E4MXV2 G8HAB1 K3YQC4 E4MW60 Q3KN68 K3XY79 C0P9J6

AT5G54160 AT2G44490 AT1G77670 AT3G53260 AT4G37980 AT4G39230 AT3G50210 AT1G74920

1-deoxy-D-xylulose 5-phosphate reductoisomerase 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase acetyl-CoA C-acetyltransferase phytoene desaturase acetyl-CoA acetyltransferase, cytosolic 2 carotenoid cleavage dioxygenase 1 hydroxymethylbutenyl 4-diphosphate synthase 1-deoxy-D-xylulose 5-phosphate reductoisomerase 4-diphosphocytidyl-2-C-methyl-erythritol kinase hydroxymethylglutaryl-CoA synthase 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase phytoene desaturase putative isopentenyl diphosphate isomerase acetyl-CoA C-acetyltransferase 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase hydroxymethylbutenyl diphosphate reductase hydroxymethylbutenyl 4-diphosphate synthase 4-coumarate:CoA ligase cinnamyl alcohol dehydrogenase somatic embryogenesis cinnamyl alcohol dehydrogenase 1 caffeic acid O-methyltransferase gentiobiase PLP-dependent aminotransferase phenylalanine ammonia-lyase cinnamyl alcohol dehydrogenase isoflavone reductase-like protein 5 uncharacterized protein aminoaldehyde dehydrogenase 1

M.P.c

score

pI

mass (Da)

22

154

6.00

51428

terpenoids

3

38

8.78

25632

terpenoids

H. brasiliensis N. benthamiana Z. mays M. truncatula Z. mays S. miltiorrhiza

29 5 7 5 29 12

789 65 205 58 412 152

6.68 6.74 7.66 6.79 5.97 6.40

42039 65942 43450 61063 82691 51960

terpenoids terpenoids terpenoids terpenoids terpenoids terpenoids

S. italica S. italica S. lycopersicum

4 2 22

28 37 205

6.44 5.83 6.11

44308 52168 82805

terpenoids terpenoids terpenoids

T. erecta O. europaea H. brasiliensis H. brasiliensis

4 11 5 10

57 111 47 48

6.70 5.38 7.33 7.28

38216 25822 41798 34509

terpenoids terpenoids terpenoids terpenoids

Oncidium C. roseus P. trichocarpa G. hirsutum C. sativus

3 31 3 9 11

56 239 63 48 203

6.20 5.92 5.69 6.25 7.44

52389 82522 59541 39574 39462

terpenoids terpenoids phenylpropanoids phenylpropanoids phenylpropanoids

C. sinensis T. halophila P. somniferum S. italica T. halophila V. vinifera S. italica Z. mays

2 10 4 7 6 17 3 4

80 90 78 49 50 170 42 49

6.05 6.09 7.09 6.33 7.42 6.10 5.22 5.45

40149 64768 50587 78439 39457 33865 37046 55733

phenylpropanoids phenylpropanoids phenylpropanoids phenylpropanoids phenylpropanoids flavonoids flavonoids misc

species H. brasiliensis C. jambhiri

classificationd

a Protein ID, according to the Uniprot database. bOrtholog, indicates AGI code. cM.P., number of matched peptides. dClassification, according to the KEGG database; misc, miscellaneous.

acid cycle (15/177, 8%), photosynthesis (15/177, 8%), and glycolysis (14/177, 8%), whereas the main functional categories among proteins in Cerasus immature flower buds were protein (61/322, 19%), glycolysis (25/322, 8%), amino acid metabolism (23/322, 7%), cell (22/322, 7%), and transport (22/322, 7%) (Figure 6). In addition, the abundance of proteins related to photosynthesis, secondary metabolism, cell wall, oxidative pentose phosphate pathway, transport, and signaling largely differed between L. japonica and Cerasus immature flower buds (Figure 6). Notably, among the 43 commonly identified proteins, the abundance of hydroxymethylbutenyl 4-diphosphate synthase in L. japonica immature flower buds was 0.2 mol %, but this protein only comprised 0.02 mol % of proteins in Cerasus immature flower buds (protein number 21, Table 3).

buds were almost four-fold and two-fold higher than those in M. spectabilis, respectively. However, the activity of FLS in L. japonica immature flower buds, which was involved in the final step of a biosynthetic pathway for the secondary metabolite, was 1.5-fold higher than that in M. spectabilis. Abundance of Hydroxymethylbutenyl 4-diphosphate Synthase in L. japonica Immature Flower Buds Was 10-Fold Higher than That in Cerasus Immature Flower Buds

To determine differences in the biological processes between medicinal and nonmedicinal flowers, a proteomic approach was used to identify proteins in medicinal L. japonica and nonmedicinal Cerasus immature flower buds. Proteins extracted from L. japonica and Cerasus immature flower buds were reduced, alkylated, digested, and analyzed using nanoLC−MS/ MS. A total of 177 and 322 proteins with more than two matched peptides were identified in L. japonica (Supplemental Table 10) and Cerasus (Supplemental Table 13) immature flower buds, respectively. Among these proteins, 134 (134/177, 76%) and 279 (279/322, 87%) proteins were specific to L. japonica and Cerasus immature flower buds, respectively, and 43 proteins were common between the two immature flower buds (Figure 6). For L. japonica immature flower buds, the main functional categories were protein (41/177, 23%), tricarboxylic

■

DISCUSSION

CPLL and PEG Fractionation Methods Increase Detection of Low-Abundance Proteins in L. japonica Immature Flower Buds

The flowering period of L. japonica can be divided into six stages containing the juvenile bud stage, the third green stage, the second white stage, the complete white stage, the silver flowering stage, and the gold flowering stage.46 As an abundant G


Article


Table 2. Identified Secondary Metabolites in L. japonica Immature Flower Buds Using Liquid Chromatography Combined Time-of-Flight Mass Spectrometry no.

compound name

formula

RT (min)a

Mwb

parent ions (m/z)c

product ions (m/z)d

classificatione

1 2

caffeic acid umbelliferone

C9H8O4 C9H6O3

2.82 3.48

180.04226 162.03169

179.03498 161.02442

3 4

ferulic acid loganin

C10H10O4 C17H26O10

4.20 4.45

194.05791 390.15260

193.05063 389.14532

135.0448 161.0239, 133.0298, 115.0204 149.063, 69.037 339.1096, 174.0137

5 6 7 8

p-coumaroyl quinic acid quercetin eriodictyol secologanate

C16H18O8 C15H10O7 C15H12O6 C16H22O10

4.82 8.29 8.41 9.52

338.10017 302.04265 288.06339 374.12130

337.09289 301.03538 287.05611 373.15610

n.d.f 272.026, 178.999, 151.0026 n.d.f 193.1, 149.1

C16H18O9 C26H28O14

9.68 9.80

354.09508 564.14791

353.08790 563.14063

191.0559, 179.0344 269.0451

11 12 13 14 15

chlorogenic acid apigenin 7-O- arabinose (1−6) -glucoside naringenin apigenin 7-O- glucoside apigenin 7-O-rutinoside diosmetin luteolin

C15H12O5 C21H20O10 C27H30O14 C16H12O6 C15H10O6

10.84 11.07 11.33 11.79 13.91

272.06847 432.10565 578.16356 300.06339 286.04774

271.06120 431.09837 577.15628 299.05611 285.04046

flavonoids flavonoids flavonoids flavonoids flavonoids

16 17

apigenin secoxyloganin

C15H10O5 C17H24O11

14.81 16.96

270.05282 404.13186

269.04555 403.12510

18

feruloylquinic acid

C17H20O9

20.04

368.11073

367.10430

19 20 21

oleanolic acid rutin quercetin 3-O-glucoside

C30H48O3 C27H30O16 C21H20O12

20.99 23.41 25.00

456.36035 610.15339 464.09548

455.35307 609.14910 463.09010

22 23

C21H20O11 C27H30O15

25.47 25.74

448.10056 594.15847

447.09420 593.15460

24

kaempferol 3-O-glucoside kaempferol 3-O-rhamnosyl-7-Oglucoside dicaffeoylquinic acid

n.d.f 269.0451 269.0451 173.0445 285.0397, 151.0034, 133.0292 269.0451, 117.0352 371.0987, 223.0612, 179.0570 191.0557, 179.0344, 135.0446 n.d.f 300.0267 300.0278, 271.0243, 255.0299 285.0397 447.0926, 285.04

C25H24O12

30.25

516.12645

515.12200

n.d.f

25

isochlorogenic acid

C25H24O12

37.81

516.12680

515.20110

353.0872, 191.0557, 179.0344 n.d.f

9 10

phenylpropanoids phenylpropanoids phenylpropanoids terpenoids (monoterpenoids) phenylpropanoids flavonoids flavonoids terpenoids (monoterpenoids) phenylpropanoids flavonoids

flavonoids n.d.f n.d.f terpenoids (triterpenoids) flavonoids flavonoids flavonoids flavonoids

phenylpropanoids

a

RT, retention time. bMw, monoisotopic molecular weight. cParent ions, MS negative ion fragments. dProduct ions, MS/MS negative ion fragments. e Classification, according to KEGG database. fn.d., not determined.

al.48 demonstrated that more proteins were detected using CPLL fractionation performed at different pH values. In the present study, extracts of L. japonica immature flower buds proteins were fractionated by CPLL at pH 4, 7, and 9. By using this approach, it was found that more new proteins were identified in the pH 4 fraction compared to the pH 7 and 9 fractions (Supplemental Figure 3). In addition, more proteins were identified in the CPLL fractions compared to that of the crude protein extraction (Supplemental Figure 4), suggesting that CPLL fractionation at different pH values might be an effective method for the enrichment of proteins in L. japonica immature flower buds. In the present study, the abundance of proteins related to photosynthesis was higher in L. japonica immature flower buds than the abundance in Creasus immature flower buds (Figure 6). In contrast, markedly fewer proteins related to transport and signaling were detected in crude protein extraction of L. japonica immature flower buds compared to Creasus immature flower buds. This latter finding may have been due to the interference of high-abundance proteins such as RuBisCO with the detection of minor proteins in L. japonica immature flower buds. Following PEG and CPLL fractionations for the removal of high-abundance proteins, the detection of proteins related to

plant protein, RuBisCO often impedes the identification of minor plants proteins14 and is also found in L. japonica flower buds.31 PEG fractionation has been widely used to reduce abundant proteins, such as RuBisCO, in plant extracts.26 In the present study, the immature flower buds with light green, which were assigned to the second white stage, were used for proteomic analysis. RuBisCO as an abundant protein was identified in crude protein extract (Figure 2); however, 16% PEG fractionation markedly reduced the level of RuBisCO (Supplemental Figure 5). This finding suggests that PEG fractionation may be an effective method for the removal of high-abundance proteins in L. japonica immature flower buds, thereby allowing for the increased detection of low-abundance proteins. The CPLL fractionation method can also be used to enrich low-abundance proteins in plant extracts.22 CPLL technology is based on the interaction of complex protein samples with a large, highly diverse library of hexapeptides bound to chromatographic supports.20 Proteins are captured by their respective specific ligand until saturation is reached. Under capacity restrained conditions, high-abundance proteins rapidly saturate their ligands, while low-abundance proteins are concentrated on their specific ligands.47 In addition, Fasoli et H


Article


Figure 4. Mapping of proteins and metabolites identified in L. japonica immature flower buds on KEGG pathways. (A) Terpenoid and (B) phenylpropanoid/flavonoid biosynthesis pathways were identified by the mapping of identified proteins and metabolites in flowers using the KEGG database. Enzymes in red boxes represent identified proteins, and the red circles indicate identified metabolites. Abbreviations: DOXP, 1-deoxy-Dxylulose 5-phosphate; MEPP, 2-C-methyl-D-erythritol 4-phosphate; 4CME, 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol; CDP-ME-2P, 2phospho-4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol; MCPP, 2-C-methyl-D-erythritol 2, 4-cyclodiphosphate; HMBDP, 1-hydroxy-2-methyl2-butenyl 4-diphosphate; HMGCoA, 3-hydroxy-3-methyl-glutaryl-CoA; GPS, geranyl diphosphate synthase; MTDP, monoterpenyl-diphosphatase; 7DOH, 7-deoxyloganin 7-hydroxylase; SLS, secologanin synthase; C4H, cinnamate 4-hydroxylase; HCT, shikimate O-hydroxycinnamoyltransferase; C3′H, coumaroylquinate 3′-monooxygenase; CCOM, caffeoyl-CoA O-methyltransferase; CHI, chalcone isomerase; F3′O, flavonoid 3′monooxygenase; FAS, flavone synthase; and FLS, flavonol synthase.

Enriched Proteins from L. japonica Immature Flower Buds Were Related to the Oxidative Pentose Phosphate Pathway, Signaling, Hormone Metabolism, and Transport

transport and signaling was markedly increased. Although the CPLL method is more suitable for the enrichment of lowabundance proteins,49 whereas PEG fractionation is suitable for the removal of high-abundance proteins,50 the proportion of RuBisCO protein removed from the CPLL and PEG fractions compared to the crude protein extraction was 79% and 83%, respectively, demonstrating that both fractionation methods are effective for removing RuBisCO. Consistent with this finding, a similar number of proteins were newly identified in L. japonica immature flower buds using the CPLL and PEG methods. These results suggest that the CPLL and PEG methods are useful for the enrichment of low-abundance proteins and removal of high-abundance proteins.

By using the CPLL and PEG methods, the numbers of proteins related to the oxidative pentose phosphate pathway, signaling, hormone metabolism, and transport in L. japonica immature flower buds were largely increased compared to that detected in the crude protein extraction (Figure 3). Additionally, proteins related to the oxidative pentose phosphate pathway were more abundant compared to Cerasus immature flower buds (Figure 6). The oxidative pentose phosphate pathway is a major source of carbon skeletons for the synthesis of phenylpropanoids and their derivatives.51 Two enzymes of the pentose phosphate I


Article


Figure 6. Functional categorization of proteins identified in L. japonica and Cerasus immature flower buds. Proteins extracted from L. japonica (black) and Cerasus (white) immature flower buds were reduced, alkylated, digested, and then analyzed using nanoLC−MS/MS. The identified proteins were categorized using MapMan bin codes. Venn diagram showing the number of proteins that were commonly identified in L. japonica and Cerasus immature flower buds. Abbreviations: protein, protein synthesis/folding/degradation/targeting; TCA, tricarboxylic acid cycle; cell, cell, cell organization/vesicle transport/cycle/division; redox, redox ascorbate/glutathione metabolism; mitoETC, mitochondrial electron transport chains; CHO, carbohydrates; OPP, oxidative pentose phosphate; others, containing C1-metabolism, cofactor/vitamin metabolism, development, fermentation, gluconeogenesis, metal handling binding, minor CHO metabolism, RNA, and tetrapyrrole synthesis; and misc, miscellaneous.

Figure 5. Enzyme activities of phenylalanine ammonia-lyase, 4coumaroyl-CoA ligase, and flavonol synthase in L. japonica and M. spectabilis immature flower buds. Immature flower buds from L. japonica and M. spectabilis were collected to analyze phenylalanine ammonia-lyase (PAL), 4-coumaroyl-CoA ligase (4CL), and flavonol synthase (FLS) activities. The column graphs show enzyme activities in L. japonica and M. spectabilis immature flower buds. Assays of PAL, 4CL, and FLS activities are described in the Experimental Procedures section. All data are presented as mean ± SD from three independent biological replicates. Asterisks (∗) indicate significant changes between L. japonica and M. spectabilis, as determined by one-way ANOVA (p < 0.05).

pathway, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, catalyze the conversion of glucose 6phosphate to ribulose 5-phosphate and generate two molecules of NADPH for amino acid and fatty acid biosynthesis.52 Transketolase catalyzes the conversion of fructose 6-phosphate to erythrose-4-phosphate,53 which is a metabolic intermediate of the shikimate pathway and is related to phenylpropanoid metabolism.54 In the present study, oxidative pentose phosphate pathway-related proteins were clearly detected in L. japonica immature flower buds, suggesting that these enzymes might be related to the biosynthesis of precursors for phenylpropanoid metabolism in L. japonica. In addition, proteins related to signaling, including 14−3−3, small GTP-binding protein, calcium-dependent protein kinase, and phospholipase C, were also identified in this study. Diaz et al.55 indicated that 14−3−3 proteins regulate the shikimate pathway and the production of aromatic compounds in Arabidopsis. Small GTP-binding proteins acting as signal transducers appear to regulate defense signal pathways and play essential roles in cytokinin biosynthesis in tobacco.56

Calcium-dependent protein kinase is an essential sensortransducer of calcium signaling pathways and was found to participate in flower morphogenesis in Pharbitis nil.57 Dowd et al.58 indicated that phospholipase C acts as a regulator of Petunia pollen tube growth. The identification of these proteins in the present study indicates that proteins related to signaling might regulate primary metabolism and growth in L. japonica immature flower buds. Several proteins related to hormone metabolism, including lipoxygenase, allene oxide synthase, 12-oxophytodienoate reductase, and snakin, were also identified in the present proteomic analysis. Lipoxygenase catalyzes the synthesis of phyto-oxylipins, which play diverse roles in plant growth, development, and senescence59 and are related to the opening of tea flower buds. 60 Allene oxide synthase and 12oxophtodienoate reductase involved in the jasmonate biosynthesis pathway catalyze the formation of 12-oxophytodienoic acid,61 a precursor of jasmonic acid, which plays a role in J


Article

Journal of Proteome Research Table 3. Common Proteins Identified between L. japonica and Cerasus Immature Flower Buds

mol (%)d M.P.b

score

pI

mass (Da)

L. japonica

Cerasus

87 95 102 14

1529 965 2113 258

4.86 4.76 6.29 6.58

51166 50728 59987 50770

cell cell mitoETC photosynthesis

1.32 1.13 0.94 0.87

1.24 0.86 0.88 0.28

C. melo

74

1109

6.61

36471

glycolysis

0.64

0.63

E. grandis S. italica H. vulgare V. baccifera

74 37 2 15

642 859 58 198

4.94 6.09 12.12 6.64

50615 35803 7602 48739

cell TCA protein not assigned

0.62 0.46 0.41 0.38

0.59 0.28 0.33 0.23

T. halophila S. chacoense P. trifoliata S. latifolia C. annuum S. italica O. ramosa

47 14 68 10 13 9 53

1110 278 1368 230 164 384 823

5.21 5.99 5.78 6.58 7.39 6.16 6.4

73890 27251 47986 51352 66376 64225 84862

0.36 0.34 0.34 0.30 0.27 0.26 0.25

0.41 0.14 0.60 0.13 0.10 0.15 0.22

S. lycopersicum S. italica S. italica M. sativa S. lycopersicum

6 15 14 9 6

112 195 200 271 106

7.94 7.46 7.06 8.29 6.11

38007 66448 42409 38397 82805

0.24 0.23 0.23 0.23 0.20

0.12 0.15 0.18 0.13 0.02

S. italica A. thaliana C. melo S. commersonii A. thaliana

14 6 2 16 29

238 167 120 445 472

6.62 4.84 10.64 7.27 4.53

80203 30656 15761 63333 48385

0.19 0.19 0.18 0.18 0.18

0.10 0.15 0.14 0.16 0.17

D6PAY2 K3Z913 B3TLQ5 K3ZX77 O78327 K3Y9Y1 D2KZ81

glyoxisomal malate dehydrogenase uncharacterized protein uncharacterized protein malate dehydrogenase hydroxymethylbutenyl 4-diphosphate synthase uncharacterized protein 60S acidic ribosomal protein P0−2 40S ribosomal protein S24 beta chaperonin 60 putative calcium-binding protein, calreticulin peptidyl-prolyl cis−trans isomerase uncharacterized protein 60S ribosomal protein L11 uncharacterized protein transketolase 1 uncharacterized protein S-adenosylmethionine synthase

stress glycolysis glycolysis photosynthesis TCA protein amino acid metabolism gluconeogenesis TCA glycolysis gluconeogenesis secondary metabolism photosynthesis protein protein protein signaling

Vanda S. italica E. guineensis S. italica C. annuum S. italica M. domestica

20 4 5 2 12 2 14

40 136 162 77 167 68 424

8.46 9.47 9.92 11.34 6.62 10.07 4.94

18582 26793 20950 21045 80398 24396 24831

0.15 0.14 0.13 0.13 0.13 0.11 0.11

0.12 0.08 0.28 0.10 0.11 0.09 0.22

34 36

E4MXR5 Q7XAE2

mRNA, clone: RTFL01−40-M04 putative fructokinase 2

T. halophila P. integrifolia

4 13

218 240

5.9 5.35

73489 35181

0.11 0.10

0.06 0.12

35 37 38 39 40 41

K4A6 V8 B6TNX7 O04946 F6HGZ1 A9P9A2 B9RI89

S. italica Z. mays N. tabacum V. vinifera P. trichocarpa R. communis

12 4 5 4 4 4

210 123 32 102 128 67

7.03 5.4 8.78 7.49 9.36 8.18

70549 30582 41925 63329 46272 40375

0.10 0.09 0.09 0.08 0.08 0.07

0.13 0.07 0.05 0.06 0.04 0.05

42 43

B9HJ23 F2DIS4

uncharacterized protein proteasome subunit alpha type enoyl-ACP reductase pectinesterase putative uncharacterized protein serine-threonine protein kinase, planttype, putative predicted protein malic enzyme

cell cell protein protein OPP protein amino acid metabolism stress major CHO metabolism glycolysis protein lipid metabolism cell wall RNA not assigned

P. trichocarpa H. vulgare

8 6

40 109

6.54 7.4

101700 68411

misc TCA

0.06 0.04

0.02 0.03

no.

protein IDa

description

1 2 3 4

B5M4B1 K3Z679 C6GFP3 D6C638

5

E5GBV9

6 7 8 9

A7KQH0 K4ACE3 F2EG92 Q8MCY2

10 11 12 13 14 15 16

E4MXI2 Q6T379 D7NHW9 G5DVX2 Q9AXR6 K3YR06 B2VQE0

beta-tubulin uncharacterized protein ATP synthase subunit beta ribulose-1,5-bisphosphate carboxylase/ oxygenase large subunit glyceraldehyde-3-phosphate dehydrogenase beta-tubulin uncharacterized protein predicted protein ribulose bisphosphate carboxylase large chain mRNA, clone: RTFL01−39-D20 triosephosphate isomerase 2-phospho-D-glycerate hydrolase phosphoglycerate kinase ATP:citrate lyase uncharacterized protein methionine synthase

17 18 19 20 21

Q645M9 K3XFR6 K3XXC5 O48903 Q8GZR6

22 23 24 25 26

K3XVH0 A8MQR4 E5GBS0 Q0W9E2 Q8LC80

27 28 29 30 31 32 33

species S. tuberosum S. italica G. hirsutum Psychotria

functionc

a

Protein ID, according to the Uniprot database. bM.P., number of matched peptides. cFunction, function categorized using MapMan bin codes. Mol (%), protein abundance; cell, cell organization/vesicle transport/cycle/division; mitoETC, mitochondrial electron transport chains; TCA, tricarboxylic acid cycle; protein, protein synthesis/folding/degradation/targeting; OPP, oxidative pentose phosphate; RNA, RNA processing/ regulation of transcription; and misc, miscellaneous.

d

metabolism-related proteins in L. japonica immature flower buds might regulate flower growth and opening. Furthermore, several proteins related to transport, such as vacuolar type ATPase, voltage-dependent anion channel, and

controlling the timing of anther dehiscence in Arabidopsis.62 Snaking-1, which is a member of the gibberellic acid stimulated in Arabidopsis protein family, functions in regulating growth and development in potato.63 The identified hormone K


Article

Journal of Proteome Research aquaporin, were also identified in L. japonica immature flower buds. Vacuolar-type ATPase is the dominant proton pump in the endomembranes of most plant cells64 and plays a central role in plant growth and development by energizing the transport of metabolites and facilitating vesicle fusion.65 The voltage-dependent anion channel, which is a major outer mitochondrial membrane protein, is important for various physiological functions such as leaf development and growth in Arabidopsis.66 Aquaporins, which are membrane channels for water transport, are correlated with petal growth by cell expansion in rose flowers.67 The findings from the present and previous studies suggest that proteins related to transport play crucial roles in the growth of L. japonica immature flower buds.

related to phenylpropanoid secondary metabolism catalyze the synthesis of precursors for flavonoid biosynthesis and may be involved in the antiviral, anti-inflammatory, and antioxidant activities in L. japonica immature flower buds. Biosynthesis of Secondary Metabolites in L. japonica Immature Flower Buds

To determine the specificity of the mechanisms underlying the biological activities of L. japonica as a medicinal plant, the activities of enzymes related to phenylpropanoids/flavonoids biosynthesis were determined in L. japonica and M. spectabilis immature flower buds. Additionally, a proteomic approach was used to identify and compare the proteins in L. japonica and Cerasus immature flower buds. The activities of PAL, 4CL, and FLS in L. japonica immature flower buds were higher than those in M. spectabilis immature flower buds (Figure 5). PAL is the rate-limiting enzyme, which catalyzes the first step in the biosynthesis of phenylpropanoids such as lignins, flavonoids, and coumarins.73 4CL is a provider of activated thioester substrates and catalyzes the formation of hydroxycinnamoylCoA for phenylpropanoid biosynthesis.81 FLS is a key enzyme in the flavonoid pathway, which plays an important role in the final step of kaempferitrin biosynthesis.82 In the present study, the high activities of PAL, 4CL, and FLS in L. japonica immature flower buds might be more beneficial to the biosynthesis of flavonoids such as kaempferol and quercetin in L. japonica compared to M. spectabilis. Among the 43 commonly identified proteins, the abundance of hydroxymethylbutenyl 4-diphosphate synthase was 10-fold higher in L. japonica immature flower buds compared to that in Cerasus immature flower buds (Table 3). Hydroxymethylbutenyl 4-diphosphate synthase, which is required for function of the methylerythritol phosphate pathway,83 catalyzes the conversion of methylerythritol 2,4-cyclodiphosphate into hydroxymethylbutenyl 4-diphosphate, which is the precursor of isopentenyl diphosphate and dimethylallyl diphosphate synthesis.84 In addition, geranyl diphosphate, which is a common precursor of monoterpenoids, is synthesized from isophentenyl diphosphate and dimethylallyl diphosphate.70 The high abundance of hydroxymethylbutenyl 4-diphosphate synthase in L. japonica immature flower buds might promote the synthesis of precursors for the biosynthesis monoterpenoids, which are the main active constituents in L. japonica. The results of the present proteomic analysis revealed that L. japonica immature flower buds contain more proteins related to secondary metabolism and fewer proteins related to the cell wall compared to Cerasus immature flower buds (Figure 6). Notably, PAL, which catalyzes the synthesis of cinnamic acid, was identified in L. japonica immature flower buds. Cinnamic acid is an important precursor for flavonoid and lignin biosynthesis.85 Salvador et al.86 reported that exogenously applied cinnamic acid increased the production of lignin in soybean. Lignin is crucial for the structural integrity of cell walls and plays a role in protecting plants.87 Expansins, which are involved in cell wall modifications, catalyze the long-term expansion of cell walls.88 Ma et al.89 demonstrated that overexpression of the expansin gene in rice plants promoted cell growth and also decreased the content of lignin. In immature flower buds of the medicinal plant L. japonica, cinnamic acid might be used to synthesize phenylpropanoids and flavonoids, which are the active constituents. In contrast, in immature flower buds of the nonmedicinal plant Cerasus,

Proteins Related to Secondary Metabolism May Mediate the Biological Activities in L. japonica Immature Flower Buds

The identified proteins related to secondary metabolism in L. japonica immature flower buds were mainly categorized as enzymes of terpenoid, phenylpropanoid, and flavonoid biosynthetic pathways (Figure 4). The biosynthesis of terpenoids proceeds via two main biosynthetic pathways, the mevalonic acid and methylerythritol phosphate pathways.68 Hydroxymethylglutaryl-CoA synthase and acetyl-CoA Cacetyltransferase of the mevalonic acid pathway catalyze the condensation of acetyl-CoA and acetoacetyl-CoA to form 3hydroxy-3-methylglutaryl-CoA,69 which is the precursor of isopentenyl diphosphate. The enzymes involved in the methylerythritol phosphate pathway were identified in the present study, which are responsible for the biosynthesis of dimethylallyl diphosphate.70 Additionally, isopentenyl diphosphate and dimethylallyl diphosphate are used by geranyl diphosphate synthase to catalyze the synthesis of geranyl diphosphate,71 which is the precursor of monoterpenoids loganin and secologanate. Oku et al.72 reported that loganin and secologanate, which were isolated from L. japonica flower buds, had allergy-preventive activity. In the present study, numerous proteins related to terpenoid biosynthesis and metabolites related to monoterpenoid production, such as loganin and secologanated, were identified in L. japonica immature flower buds. This finding suggests that proteins related to terpenoid secondary metabolism are responsible for the biosynthesis of monoterpenoids, which are the main active compounds in L. japonica immature flower buds. As a key enzyme in phenylpropanoid metabolism, PAL catalyzes the conversion of L-phenylalanine to cinnamic acid,73 which is an important precursor of numerous phenylpropanoid compounds. Cinnamic acid is converted to ferulic acid by the consecutive action of cinnamate 4-hydroxylase and caffeic acid 3-O-methyltransferase.74 Ferulic acid has both anti-inflammatory and antioxidative activities.75 Cinnamate 4-hydroxylase, together with 4CL, also catalyzes the conversion of cinnamic acid to 4-coumaroyl-CoA, which is the precursor for chlorogenic acid76 and flavonoids.77 Chlorogenic acid is a major bioactive compound in the whole plant of L. japonica and has antiviral activity.12 Flavonoids such as naringenin, apigenin, and eriodictyol, which were previously isolated from L. japonica, exhibit antioxidative, anticancer, and anti-inflammatory activities.78−80 In the present study, five proteins and four metabolites involved in the phenylpropanoid pathway were identified. In addition, 12 flavonoids that function as active compounds in L. japonica immature flower buds were also identified. Taken together, these results suggest that proteins L


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japonica immature flower buds using CPLL fractionation; proteins identified in L. japonica immature flower buds using PEG fractionation; proteins identified in Cerasus flower buds; schematic workflow of CPLL fractionation of proteins in L. japonica immature flower buds; schematic workflow for PEG fractionation of proteins in L. japonica immature flower buds; proteins identified in L. japonica immature flower buds using the CPLL fractionation method with different elution pHs; major and minor proteins identified in L. japonica immature flower buds using the CPLL fractionation method; proteins identified in L. japonica immature flower buds using PEG fractionation (PDF)

cinnamic acid might be used to synthesize lignin, which is related to the cell wall protein expansin.

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CONCLUDING REMARKS Lonicera is a medicinal plant with antiviral, anti-inflammatory, and antioxidant activities.2 The L. japonica immature flower buds are widely used in clinical practice because of the higher content of active constituents compared to the stem and leaf.8,9 To investigate the molecular mechanisms underlying the biological activities of L. japonica immature flower buds, a gel-free/label-free proteomic approach was used in combination with CPLL and PEG methods, which were used to enrich low-abundance proteins and reduce high-abundance proteins, respectively.21,22 The proteomic analysis revealed that proteins related to the oxidative pentose phosphate pathway, signaling, hormone metabolism, and transport were largely enriched, and 28 proteins related to secondary metabolism were identified in L. japonica immature flower buds using the CPLL and PEG methods. It is indicated that the CPLL and PEG methods were found to be effective tools for detecting low-abundance proteins in plant tissue. Furthermore, a total of 25 metabolites containing terpenoids, phenylpropanoids, and flavonoids were identified in L. japonica immature flower buds. The activities of PAL, 4CL, and FLS involved in the biosynthesis of flavonoids in L. japonica immature flower buds were higher than those in M. spectabilis. In addition, hydroxymethylbutenyl 4-diphosphate synthase involved in the biosynthesis of terpenoids was 10-fold more abundant in L. japonica immature flower buds compared to Cerasus. Taken together, the present findings suggest that the abundances of proteins related to secondary metabolism in L. japonica immature flower buds are higher than those in Cerasus and M. spectabilis, which are responsible for its medicinal property.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: skomatsu@affrc.go.jp. Phone: +81-29-838-8693. Fax: +81-29-838-8694. *E-mail: [email protected]. Phone: +86-571-87951301. Fax: +86-571-87951676. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank X. Wang, X. Yin, and G. Mustafa at the National Institute of Crop Science for experimental support and guidance during this research. This work was also supported by the National Science Foundation of China (Grant No. 81473182).

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ABBREVIATIONS LC, liquid chromatography; MS, mass spectrometry; TOF, time-of-flight; CPLL, combinatorial peptide ligand libraries; PEG, polyethylene glycol; RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; PAL, phenylalanine ammonia-lyase; 4CL, 4-coumarate-CoA ligase; FLS, flavonol synthase

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b00910. The MS proteomics data have been deposited with the ProteomeXchange Consortium (http:// proteomecentral.proteomexchange.org) via the PRIDE partner repository90 with the data set identifier PXD002230. Proteins identified in L. japonica immature flower buds at fraction of elution of pH 4 fraction using CPLL fractionation; proteins identified in L. japonica immature flower buds in the flow-through pH 4 fraction using CPLL fractionation; proteins of L. japonica immature flower buds identified in the pH 7 elution fraction from CPLL fractionation; proteins of L. japonica immature flower buds identified in the flow-through fraction at pH 7 using CPLL fractionation; proteins of L. japonica immature flower buds identified in the pH 9 elution fraction using CPLL fractionation; proteins of L. japonica immature flower buds identified in in the flow-through pH 9 fraction using CPLL fractionation; proteins of L. japonica immature flower buds identified in fraction 1 using PEG fractionation; proteins of L. japonica immature flower buds identified in fraction 2 using PEG fractionation; proteins of L. japonica immature flower buds identified in fraction 3 using PEG fractionation; crude extract proteins identified in L. japonica immature flower buds; proteins identified in L.

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