Tandem Mass Tag Based Quantitative Proteomics of Developing Sea

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TMT-based Quantitative Proteomics of Developing Sea Buckthorn Berries Reveals Candidate Proteins Related to Lipid Metabolism Wei Du, Chao-Wei Xiong, Jian Ding, Hilde Nybom, Cheng-Jiang Ruan, and Hai Guo J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00764 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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Journal of Proteome Research

Corresponding Author： E-mail: [email protected]

TMT-based Developing

Quantitative Sea

Proteomics

Buckthorn

Berries

of

Reveals

Candidate Proteins Related to Lipid Metabolism Wei Du†, Chao-Wei Xiong†, Jian Ding†, Hilde Nybom , Cheng-Jiang Ruan†,* HaiGuo ‡

†

§,⊥

Institute of Plant Resources, Key Laboratory of Biotechnology and Bioresources Utilization, Ministry of Education, Dalian Nationalities University, Dalian 116600, China; ‡

Department of Plant Breeding–Balsgård, Swedish University of Agricultural Sciences, Fjälkestadsvägen 459, SE-29194 Kristianstad, Sweden. §Conseco Sea buckthorn Co. Ltd, Beijing 100038, China

⊥Inner Mongolia Hijing Environment Protection Science and Technology Co. Ltd, Inner Mongolia 017000, China ABSTRACT: Sea buckthorn (Hippophae L.) is an economically important shrub or small tree distributed in Eurasia. Most of the well-recognized medicinal and nutraceutical products are derived from its berry oil, which is rich in monounsaturated omega-7 (C16:1) fatty acid and polyunsaturated omega-6 (C18:2) and omega-3 (C18:3) fatty acids. In this study, tandem mass tags (TMT)-based quantitative analysis was used to investigate protein profiles of lipid metabolism in sea buckthorn berries harvested 30, 50 and 70 days after flowering. In total, 8626 proteins were

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identified, 6170 of which were quantified. Deep analysis results for the proteins identified and related pathways revealed initial fatty acid accumulation during whole-berry development. Abundance of most key enzymes involved in fatty acid and TAG (triacylglycerol) biosynthesis peaked at 50 days after flowering, but TAG synthesis through the PDAT (phospholipid: diacylglycerol acyltransferase) pathway mostly occurred early in berry development. In addition, the patterns of proteins involved in lipid metabolism were confirmed by combined quantitative real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and . Parallel reaction monitoring (PRM) analyses. Our data on the proteomic spectrum of sea buckthorn berries provides a scientific basic for understanding lipid metabolism and related pathways in the developing berries.

KEYWORDS: Hippophae L., proteomics, lipid metabolism, TMT-based quantitative analysis Introduction Sea buckthorn (Hippophae L.) is an important winter-hardy, woody shrub or small tree distributed in Eurasia.1 For hundreds of years, sea buckthorn berries, which contain high levels of flavonoids, vitamins, unsaturated fatty acids, organic acids, and antioxidants, have been widely used in medicine and food in China and Russia.2,3 Both the pulp and seeds of sea buckthorn berries contain oils with bioactive compounds, such as tocopherols, carotenoids, and phytosterols. Moreover, sea buckthorn berry oil is rich in omega-7 fatty acid (C16:1, up to 43%), with both cosmetic and health applications. Furthermore, high amounts of omega-6 fatty acid (C18:2; up to 42%) and -omega-3 fatty acid (C18:3; up to 39%) are present. Due to these fatty acids compositions, sea buckthorn berry oil differs from other fruit and vegetable oils in unsaturated fatty acid composition.4, 5


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Unsaturated fatty acids are synthesized and desaturated during berry development and eventually stored as triacylglycerol (TAG),6,

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and studies on the synthesis, biochemistry and

metabolism of storage oil will help us understand how oils are synthesized and accumulate during berry development. The regulation of gene expression during sea buckthorn berry development has been reported.8-10 Furthermore, Fatima11 reported fatty acid compositions and transcriptomes during sea buckthorn berry development, and functional annotation of metabolic pathways indicated clear representation of those related to TAG biosynthesis and fatty acid accumulation. In addition, by comparing the lipid metabolism transcriptome in sea buckthorn berry seeds and pulp, Ding et al.12 found highly coordinated expression of GPD1 (glycerol-3-phosphate dehydrogenase), DGAT1 (diacylglycerol acyltransferase 1) and DGAT2 (diacylglycerol acyltransferase 2) genes. However, transcriptome levels are often not in accordance with protein abundance,13 which impacts our understanding of certain metabolic processes, such as fatty acid and TAG metabolism pathways. To date, there is no report on the dynamic changes of key enzymes and proteins in sea buckthorn berry development. Proteomics is a powerful method for explore protein dynamics and their complex regulatory mechanisms.14 Tandem mass tags-mass/mass spectrometry (TMT-MS/MS) is a mass-based quantitative technique for identifying and quantifying proteins that has been widely used in plant proteomics.15 Compared to the performance of two-dimensional electrophoresis, TMT-MS/MS yields a comprehensive analysis of low-abundance proteins with high accuracy.16 In this study, we applied TMT-MS/MS to identify changes in proteins involved in lipid metabolism during sea buckthorn berry development, especially proteins related to biosynthetic pathways of fatty acids and TAG. The expression patterns of candidate genes involved in these metabolic pathways were also determined.


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Materials and methods The sea buckthorn cultivar ‘XE3’ was chosen from among seedlings of Russia cultivars (Hippophae rhamnoides ssp. mongolica) planted in Heilongjiang Province, China. The ‘XE3’ garden was established in 2010 using cuttings, with one pollinating tree of cultivar ‘Wucixiong’ (ssp. mongolica) per eight ‘XE3’ plants. ‘XE3’ berries were collected at three stages of development: hard green berries (approx. 30 days after flowering, 30 DAF), green/orange berries (approx. 50 DAF) and orange/red berries (approx. 70 DAF) (Figure 1a). Determination of fatty acid components. Fatty acid components and relative percentage were determined by gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS). FAMEs (fatty acid methyl esters) were assessed according to the method of Wang et al. and SanchezSalcedo et al., with some modifications.17, 18 A sample of 20 mg of oil extracted from berries was transferred to a test tube, and 3 mL n-hexane and 4 mL 1 M methanol-potassium hydroxide solution were added; after mixing, the suspension was incubated for 30 min at 60 °C in a water bath. Afterwards, 10 mL BF3 in methanol was added, with an additional 30 min in the 60 °C water bath. Following the methyl-esterification reaction, 2 mL n-hexane and 2 mL saturated sodium chloride solution were added, and the mixture was shaken for 1 min. The mixture was centrifuged for 1 min at 8,000 x g, and the anhydrous sodium sulfate was used to dry the mixture after centrifugation. The top layer was transferred to a new vial and dried using a nitrogen stream. All sample vials were stored at -20 °C until GC-TOF/MS analysis (AxION iQT GC/MS/MS system with Clarus 680 GC, PerkinElmer, Shelton, USA). In this system, the capillary column (Agilent DB-23 60 m × 0.25 mm × 0.25 μm, USA) separates different components using carrier gas helium at a flow rate of 1.0 mL/min, with pressure of 27,600 Pa. The sample split ratio was set at 1:20. The inlet and transfer line temperatures were 230 °C and 220 °C, respectively. The heating


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procedure was optimized as follows: from 50 °C to 200 °C with a gradient of 15 °C/min, increasing to 215 °C with a gradient of 3 °C/min, and up to 230 °C for ~ 10 min. For MS detection, the ion source temperature was set to 230 °C; EI ion source was used, with the same temperature as the inlet line. The MS parameters were as follows: 1.4 kV multiplier, 70 eV electron energy, 5 min solvent delay, 1 μL sample volume, and full-scan mode range from 45 to 420 Da. The different fatty acid components were identified by comparison of retention times with those of standard fatty acids, and relative percentages were calculated using the ratio of each component to the total components. Three biological replicates were performed for each sample. Oil body extraction and observation. Oil bodies were extracted using the method of Chua et al.19 Sea buckthorn berries (5 g) were ground in a mortar in cold (stored at 4 °C before use) 10 mL buffer solution A (Supplemental Table S1). The homogenate was filtered through double layers of gauze, and 15 mL solution B was added; buffer solution B was also stored at 4 °C prior to use. The homogenate was centrifuged for 30 min at 10,000 x g; a spatula was used to extract the oleaginous layer, which was dissolved in solution C. Solution D (15 mL) was transferred to the suspension, followed by centrifugation at 10,000 x g for 30 min. The oleaginous layer was dissolved in 10 mL solution A, which was mixed with 15 mL solution B. The mixture was centrifuged for 20 min at 10,000 x g. After repeating this process, the final oil layer, which was the relatively pure oil body, was dissolved in 3 ml buffer solution A and observed using an inverted microscope (Olympus IX73). Protein extraction. Sea buckthorn berries were ground in liquid nitrogen. The berry powder was dissolved in 3 mL lysis buffer (10 mM DTT, 8 mol/L urea, 30 mg Protease Inhibitor Cocktail, and 2 mM Ethylenediaminetetraacetic acid at 4°C), which was transferred to a 5-mL centrifuge tube and sonicated using a high-intensity ultrasonic processor (38 times for 3 s with a 5 s break,


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20 kHz 195 w, Scientz). After the mixture was centrifuged for 10 min at 4 °C and 20,000 x g, the supernatant was transferred to a new centrifuge tube, and 4 mL 15% trichloroacetic (TCA) was added and kept for 2 h at -20 °C to precipitate proteins. After centrifugation, the protein precipitate was washed three times with cold acetone and redissolved in buffer with 8 M urea and 100 mM tetraethylammonium tetrahydroborate (TEAB pH 8.0). The protein concentration was determined using a protein quantitative kit (Bradford method, Tiangen PA102, China). Trypsin digestion, TMT labeling, LC-MS/MS quantitative proteomic and PRM analysis. Before digestion, the protein solution was treated with 10 mM DL-dithiothreitol (DTT) for 1 h at 37 °C for disulfide reduction and 20 mM iodoacetamide (IAM) for 45 min, at 25 °C for alkylation. The protein solution was then diluted to a quarter of the original volume with 100 mM TEAB. For the first digestion reaction, 2 μg trypsin (Roche) was added to the protein solution (approximately 100 μg protein) and incubated for over 12 h; 1 μg trypsin was then added for second digestion of ~ 4 h. Before vacuum drying, peptides were desalted using a Phenomenex C18 SPE column. Individual samples (100 μg) were dissolved in 0.5 M TEAB and processed for 6-plex TMT according to the kit manual (Thermo Scientific). The peptide mixtures were mixed and incubated at 25 °C for 2 h. Finally, the peptide mixtures were loaded onto the C18 SPE column for desalting and dried using a vacuum concentrator. After labeling, peptides were redissolved in 6% ACN containing 0.1% FA (v/v) and separated by RSLC (EASY-nLC 1000) using a reverse-phase column (Acclaim PepMap RSLC, 50 μm*15 cm, 2 μm, Thermo Scientific). The gradient elution procedure was as follows: linear gradient from 6% to 22% solvent B (98% CAN, 0.1% FA v/v) for 25 min, from 22% to 36% solvent B for 10 min, ramped to 85% solvent B for 5 min, and kept for 3 min, with a constant flow rate of 400 nL/min.


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The peptide fractions were analyzed using a Thermo Scientific™ Q Exactive™ and hybrid quadrupole-Orbitrap mass spectrometer. The intact peptides were detected by the Orbitrap at 70,000 resolution (NCE setting = 30), and their ion fragments were selected at a resolution of 17,500. The top 20 precursor ions were chosen using a data-dependent procedure, which changed from one MS scan to 20 MS/MS scans. In the MS survey scan, dynamic exclusion was 30.0 s, and the threshold ion count was greater than 104. The following MS parameters were selected: automatic gain control (AGC) on, 2.0-kV electrospray voltage, scanning range of 350-1800 m/z, and fixed first 100 m/z mass. Using the Sea buckthorn database (Hippophae rhamnoides L. protein database.fa 46,724 sequences) 20, the MS/MS data were analyzed by combining MaxQuant with the Andromeda search engine (v.1.5.2.8). The cleavage enzyme was trypsin/P, with no more than two missing cleavages, five charges and modifications per peptide, 0.02 Da mass error, and 10ppm fragment and precursor ions. Oxidation at Met was defined as a variable modification, and carbamidomethylation at Cys was defined as a fixed modification. For TMT quantification, the ratios of the TMT reporter ion intensities in MS/MS spectra (m/z 126–131, Table 1) from raw data sets were used to calculate fold changes between samples. The minimum peptide length was seven, and an FDR (false discovery rate) threshold of 1% was used for proteins, peptides and modification sites. For each sample, the quantification was normalized using the average ratio of all the unique peptide. Protein quantitation calculated from the median ratio of protein corresponding unique peptides (at least two unique peptides per protein). Two biological replicates were performed for each sample and three technical replicates (replicate injections) were performed for each biological replicates. The results of three technical replications were averaged before the biological replications were compared. Two-sided T-tests were performed to evaluate abundance changes of corresponding protein. The one-sample two-sided t-test was a test for difference from 0 on the


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log2 scale performed with unique peptide ratio of protein. In general, a significance level of 0.05 was used for statistical testing. PRM mass spectrometric analysis was performed using a tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo). The LC parameters, electrospray voltage, scan range, Orbitrap resolution were the same as TMT methods. Automatic gain control (AGC) was set at 3E6 for full MS and 1E5 for MS/MS. The maxumum IT was set at 20 ms for full MS and auto for MS/MS. The isolation window for MS/MS was set at 2.0 m/z. After normalizing the quantitative information by the heavy isotope-labeled peptide, a relative quantitative analysis (three biological replications) was performed on the target peptides. Quantitative real-time PCR. Total RNA extracted from sea buckthorn berries (TRIzol Reagent Takara, Japan) was used to synthesize cDNA samples. The cDNA samples were mixed with Tiangen SYBR Green PCR Real Master Mix (China) and 10 μM each primer, and qRT-PCR was conducted using an Applied Biosystems 9700 RT-PCR System (USA). The program was set as follows: holding for 15 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at 58 °C and 50 s at 67 °C. qRT-PCR primers were designed using the online software Primer Quest (Table 2). The fluorescence signal was collected at the 67 °C elongation step of each cycle, and relative quantification was achieved using the 2-ΔΔCt method. Three biological and three methodological replications were performed for each of three stages of sea buckthorn berry development. Determination of ACCase and DGAT1 concentrations. The concentrations of ACCase and DGAT1 were determined using an ELISA kit according to the manufacturer’s protocol (Catalog: MBS9316012, MBS9372209. Biosource). Berry samples were pipetted into wells containing the immobilized ACCase or DGAT1 antibody. After incubating at 35 °C for 30 min, any unbound proteins were removed with wash buffer; a biotin-conjugated antibody was added, followed by


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addition of avidin-conjugated horseradish peroxidase (HRP) and incubation at 37 °C for 1 h. After removal of any unbound avidin-enzyme by washing, a substrate solution was added. The samples were incubated for 15 min at 37 °C, and the intensity of the color was measured, indicating its proportion to the amount of bound ACCase/ DGAT1. Results Berry size and oil contents in developing berries. Berries collected at different ripening stages showed a significant increase in size as well as an obvious change in color (Figure 1a). In the first interval (30 DAF to 50 DAF), berry weight increased threefold to 21.3 g (100-fruit weight) and then decreased slightly (Figure 1b). Changes in berry oil contents were in accordance with those of the weight of berries (Figure 1c): the berry oil content increased from 9.25% (30 DAF) to 16.51% (50 DAF) and finally reached 27.46% (70 DAF); accordingly, the hundred-fruit weight increased from 9.25% (30 DAF) to 21.3% (50 DAF) and reached 30.6% (70 DAF). Our results suggest that the green berry stage might not represent the beginning of oil biosynthesis; oil biosynthesis and accumulation occurred before 30 DAF. Rapid periods for oil biosynthesis and accumulation occurred from 30 to 50 DAF. In addition, the size of oil bodies in developing berries was investigated microscopically (Supplemental Figure S1). At 30 DAF, most oil bodies were only 1 μm, though a relatively high proportion of oil bodies had a diameter larger than 10 μm at 70 DAF (Supplemental Figure S1c), indicating an obvious increase in size during berry development. Fatty acid components in sea buckthorn berry oil. The fatty acid components of extracts of the three developmental stages of sea buckthorn berries were identified using GC-TOF/MS. The primary fatty acids were palmitic (C16:0 27-35%), palmitoleic (C16:1 24-31%), linoleic (C18:2 15-21%), linolenic (C18:3 8-12%) and oleic (C18:1 8-11%) acids, which was consistent with a previous study.10 The relative contents of C16:1, C18:0 (stearic acid) and C18:1 exhibited an up-


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trend from 30 DAF to 70 DAF, whereas the relative contents of C18:2 and C18:3 saturated fatty acids decreased slightly at the late development stage. Moreover, the relative content of C16:0 first decreased from 30 DAF to 50 DAF and then increased; the relative content of C18:0 was ~ 0.52.5%. Proteomes at three berry developmental stages. In this study, 8626 proteins were detected and identified, and 6170 were quantified. Several parameters are shown in Supplemental Table S3, including the protein score, coverage percentage, number of peptides matching individual proteins, and accession number assigned to each identified protein. Pairwise comparisons of all quantified proteins were performed among three berry developmental stages, and the fold change cutoff was defined in ratios of greater than 1.5 or lower than 0.67. The MS proteomic results have been uploaded to ProteomeXchange Consortium (http://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository under dataset identifier PXD009365. The number of proteins up- or downregulated between two berry developmental stages is shown in Figure 2. Three groups were identified, with very few changes (50 vs. 70 DAF), many changes (30 vs. 70 DAF), and an intermediate number of changes (30 vs. 50 DAF) between two berry developmental stages. Changes in protein abundance displayed a trend similar to that of berry development. Cluster analysis of identified proteins. The proteins identified were classified into three types, namely, cellular component, molecular function and biological process (Figure 3). The proteins in the cellular component category are mainly related to cell (36.37%), organelle (20.75%), cell membrane (21.12%), macromolecular complex (19.80%) and membrane-enclosed lumen (1.15%). Molecular function proteins are predominantly related to binding (46.88%), catalytic activity (42.93%), nutrient reservoir activity (4.49%), transporter activity (3.17%), structural molecule activity (3.15%) and the regulation of molecular function (0.82%). Regarding biological process,


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34.10% of proteins are associated with metabolic processes, 26.49% with cellular process, 21.75% with single-organism process, 6.33% with localization, 4.12% with biological regulation, and 2.23% with cellular component organization or biogenesis. Cluster analysis of differentially quantified proteins. To deeply analyze the profiles of differentially quantified proteins between the berry developmental stages, proteins were divided into categories of biological process, molecular function and KEGG pathway (Figure 4). For biological process, the metabolic process, such as oxoacid, organic acid, and small molecule metabolic processes, was active at 30 DAF. The carbohydrate metabolic process showed a significant increase at 50 DAF. Embryo development, single-organism metabolic and cellular lipid metabolic processes were gradually enhanced after 50 DAF. In the category of molecular function, 30 DAF was the most active period of transferase activity, including transfer of acyl, one-carbon, and glycosyl and hexosyl groups; endonuclease and ribonuclease T2 activities were enhanced from 30 DAF to 50 DAF. Lastly, the important KEGG synthetic pathways occurred early in berry development, including secondary metabolites and phenylpropanoid and flavonoid biosynthesis. The cyanoamino acid metabolism pathway was most active at 50 DAF. Proteins related to fatty acid metabolism. In most plant tissues, lipids are mainly stored as TAG, with major compounds of saturated and/or unsaturated C16 and/or C18 fatty acids.21 The biosynthesis of fatty acids, in which acetyl-CoA is converted to C16 or C18 fatty acids, occurs in plastids (Figure 5); TAG synthesis occurs in the endoplasmic reticulum (Figure 6). Based on functional annotation of the sea buckthorn proteome and the KEGG database, most of the enzymes identified are involved lipid metabolism (Figure 5, Table 3). With the exception of fatty acyl-ACP thioesterase B (FATB, EC: 3.1.2.21), all known enzymes involved in fatty acid formation and accumulation were successfully quantified. Most enzymes were found to be


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upregulated from 30 DAF to 50 DAF, with no significant increase from 50 DAF to 70 DAF, including acetyl-CoA carboxylase (ACCase, EC: 6.4.1.2), malonyl-CoA ACP transacylase (MAT, EC: 2.3.1.39), beta-ketoacyl-ACP synthase III (KAS III, EC: 2.3.1.180), beta-ketoacyl-ACP reductase (KAR, EC: 1.1.1.100), enoyl-[acyl-carrier protein] reductase I (EAR, EC:1.3.1.9/10), Δ9-palmitoleoyl-ACP desaturase (Δ9D, EC: 1.14.1.2), stearoyl-ACP-thioesterase(FATA, EC:3.1.2.14) and long-chain acyl-CoA synthetase (LACS, EC:6.2.1.3). Four proteins, betaketoactyl-ACP synthase II (KAS II, EC: 2.3.1.179), 3-hydroxyacyl-ACP dehydratase (HAD, EC: 4.2.1.-), very-long-chain enoyl-CoA reductase (TER, EC:1.3.1.93) and palmitoyl-protein thioesterase (PPT, EC:3.1.2.22), exhibited stable abundance levels throughout berry development. Proteins involved in the TAG biosynthesis pathway. Almost all eukaryotic organisms and even some prokaryotes synthesize TAG via the G-3-P (glycerol-3-phosphate) pathway22 (Figure 6, Table 3). Almost all quantified enzymes involved in TAG biosynthesis were identified by functional annotation of the sea buckthorn transcriptome. Glycerol-3-phosphate acyltransferase (GPAT, EC: 2.3.1.15), which is the key enzyme regulating the first step of the TAG pathway, was found in small quantities at all berry developmental stages. In contrast, diacylglycerol acyltransferase 1 (DGAT1, EC: 2.3.1.75/76) and lysophosphatidylcholine acyltransferase (LPCAT, EC: 2.3.1.51) were upregulated at different berry developmental stages (Figures 6, 8). Indeed, the abundance of the desaturases omega-6 fatty acid desaturase (FAD2, EC: 1.14.19.6/22) and acyllipid omega-3 desaturase (FAD3, EC: 1.14.19.25/35/36) showed five-fold increase from 30 DAF to 50 DAF and then decreased significantly, which differed notably from the expression patterns of the main enzymes involved in lipid metabolism during berry development. Parallel Reaction Monitoring (PRM) Validation. To verify the results of TMT analysis coupled with qRT-PCR at protein level, PRM was performed among key enzymes which had


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significant differences between sea buckthorn berry development stages. Eight proteins of interest, namely ACCase, KAS III, LPCAT, FAD2, FAD3, GPAT, PDAT and DGAT1 were selected for analysis. The results of the PRM showed that the eight candidate proteins exhibited similar trends between different development stages in the TMT results, which supports the plausibility and reliability of the TMT data (Table 4). Discussion Only a few plants contain high levels of palmitoleic acid, for instance, the oils of macadamia nut and sea buckthorn berry (Figure 1d).23 The high nutritive value of sea buckthorn berry oil is mainly due to the optimal ratio of monounsaturated (C16:1 and C18:1) to polyunsaturated fatty acids. Our study is the first to characterize the enzymes and oleosin related to TAG biosynthesis and fatty acid accumulation in sea buckthorn berry. These new data will improve our understanding of lipid metabolism during sea buckthorn berry development. Enzymes related to fatty acid synthesis during berry development. De novo fatty acid synthesis starts from acetyl-CoA, with continuous extension to C16 and/or C18 chains, as initially catalyzed by ACCase,24 one of the main enzymes regulating carbon flux toward fatty acid formation and accumulation.25 It has been reported that ACCase overexpression increases the lipid content.26, 27 In this study, ACCase enzymes containing α-subunits were successfully identified and quantified and found to be upregulated from 30 DAF to 50 DAF. Our data indicate that a rapid accumulation of ACCase was very important for fatty acid formation and accumulation early in berry development. The enzyme KAS III catalyzes the original condensation reaction, in which the acetyl group of acetyl-CoA is transferred to malonyl-ACP, ultimately producing β-ketoacylACP with a four-carbon chain.28 The kas3 mutation leads to a partial loss of the fatty acid synthesis pathway in plastids.29 Similar to ACCase, KAS III was also upregulated at 50 DAF. However, the


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expression pattern of KAS III was different from the abundance profile of the protein, indicating that KAS III expression might enhance the protein content, KAS III expression occurred before protein synthesis, or the secondary structure of the protein was changed by post translational modification.30 The abundance of KAR was similar to that of EAR. In particular, these results show that regulation of the condensation reaction was in accordance with that of the reduction reaction. The enzymes FATB and FATA play essential roles in fatty acid synthesis and accumulation and in carbon flux trends of woody oilcrops.31, 32 FATB catalyzes the thioesterification of C16:0-ACP to C16:0 acid, and remaining acyl-ACPs (C16:1, C18:0 and C18:1-ACP) are converted to acylacids via FATA catalysis.33 Although FATB was not detected in this study, FATB mRNA expression occurred later than that of FATA (Figure 7), in accordance with results reported by Fatima11. Low expression of FATB indirectly prompted the conversion of C16:0-ACP to C18:0ACP, the source substance for C18:1, C18:2 and C18:3 syntheses. In addition, SAD is the key enzyme for determining the relative percentages of unsaturation in total fatty acids, catalyzing the primary desaturation reaction in fatty acid metabolism.34 Increasing abundance of SAD and low expression of FATB contributed to a strong increase in unsaturated fatty acid contents during berry development (Figure 1D). Previous reports have shown that in developing rapeseed embryos, SAD knockdown resulted in significantly increased levels of stearic acid.35 Furthermore, almost all fatty acid synthase subunits, which catalyze carbon chain extension, were upregulated at 50 DAF, showing that fatty acids were rapidly synthesized around 50 DAF. Enzymes related to TAG synthesis during berry development. In the endoplasmic reticulum, TAG is produced under a series of reactions. First, acylation of G3P by GPAT produces LPA, which is further transformed to PA by LPAAT.36, 37 In our study, both mRNA and protein levels


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indicated that GPAT was synthesized in abundance only at the beginning of berry development. Compared to other traditional high-oil-content crops, the decreased abundance of GPAT may lead to a relatively low amount of TAGs in sea buckthorn berries. LPIN catalyzes the release of phosphate from PA, generating DAG, which then serves as a substrate for biosynthesis of membrane lipids or for TAG formation. DGAT catalyzes final acylation of DAG to produce TAG. Alternatively, the sn-2 acyl chain of PC can be transferred to DAG by PDAT, forming lyso-PC and TAG. Although these two pathways have similar effects with regard to TAG synthesis in higher plants, LPCAT was upregulated early in berry development. Thus, LPACT, together with several fold upregulation of FAD2 and FAD3 (30-50 DAF), presents a mechanism for enriching the acyl-CoA pool, whereby polyunsaturated fatty acids can be synthesized using PC.38 In addition, the correspondingly high abundance of all detected desaturases involved in lipid biosynthesis (SAD, FAD2 and FAD3) in the early and middle periods indicates that monounsaturated and polyunsaturated fatty acids were mainly formed during the middle stage of berry development (Figure 1D), possibly in response to different regulators such as plant endogenous hormones and transcription factors39. In woody oil trees, DGATs are key regulatory enzymes determining the last step of TAG assembly in lipidaccumulation.40 Although DGAT1 and DGAT2 have been characterized in many plants, these two enzymes are not very similar at the DNA level or in protein sequence. DGAT2 is related to formation of different fatty acids (saturated and unsaturated) in TAG production in different organs.41 In our study, due to low DGAT2 expression or a lack of a peptide match in the database, only DGAT1 was detected in abundance during sea buckthorn berry development. Nonetheless, expression of DGAT1 and DGAT2 was quantified by qRT-PCR (Figure 7). The protein abundance of DGAT1 was evidenced by ELISA (Figure 8), which showed significant


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increases during berry development, reaching a maximum at 70 DAF. In contrast, oil contents did not increase significantly late in berry development, which may be due to other crucial or downregulated enzymes. DGAT1 appears to be upregulated at the late berry development stage, which may contribute to promoting the conversion of intermediates into TAG and thus complete final oil accumulation.42 Oleosins and oil bodies. TAG is generally stored in spherical organelles called oil bodies.43 Oleosins are the main proteins of oil bodies, forming a steric barrier surface for maintaining lipid droplet structure in the cytoplasm.44 Oleosins in the cruciferous oilseed plants mustard and rapeseed have been reported to stabilize lipid droplets and offer a large surface area for TAG per unit, accelerating oil mobilization by lipases after germination.45 In this study, six types of accumulated oleosins were detected during berry development in sea buckthorn. These oleosins were successively upregulated from 30 DAF to 70 DAF and exhibited a correlation with lipid accumulation, whereby the berry oil content gradually increased during berry development. The amount of large oil bodies (>10 μm) also increased gradually during berry development (Supplemental Figure S1). It should be noted that during oil body extraction, normal-sized oil bodies did not fuse to generate larger oil bodies. With an irregular shape and no detectable oleosins, the larger oil bodies do not act as a long-term storage organ but as a bait for attract animal vectors, which is favorable for seed spread.46

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge at ACS website http://pubs.acs.org: Figure S1 Oil bodies in sea buckthorn berries at three stages of berry development.


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Figure S2 Analysis of reproducibility for two biological repeated experiments by Pearson correlation coefficient. Figure S3. Cluster analysis of three technical replication. Mass spectrogram of eight qRT-PCR verified proteins. Table S1 Buffer solution for oil body extraction. Supplementary file: Table S2 Protein MS identified information.xlsx

AUTHOR INFORMATION Corresponding Author *Tel: 86-411-87652536. Fax: 86-411-87618179. E-mail: [email protected] Funding Sources This research was supported by the National Natural Science Foundation of China (C.J. Ruan, No.31570681 and J. Ding, No. 31800574). ACKNOWLEDGMENT Jingjie PTM BioLab Co. Ltd. (Hangzhou, China) helps for analysis of the mass spectrometry. REFERENCES (1) Zheng, J.; Kallio, H.; Yang, B., Sea buckthorn (Hippophae rhamnoides ssp. rhamnoides) berries in Nordic environment: compositional response to latitude and weather conditions. J. Agric. Food Chem. 2016, 64 (24), 5031-5044. (2) Ruan, C. J.; Rumpunen, K.; Nybom, H., Advances in improvement of quality and resistance in a multipurpose crop: sea buckthorn. Crit. Rev. Biotechnol. 2013, 33 (2), 126-144.


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Figure legends Figure 1. Changes in morphology and oil content during sea buckthorn berry development. (A) Berries at 30, 50 and 70 DAF. (B) Hundred-fruit weight at 30, 50 and70 DAF (p

Tandem Mass Tag Based Quantitative Proteomics of Developing Sea

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