ITRAQ Based Proteomic Analysis

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Article
ITRAQ-based proteomic analysis of the metabolic
mechanisms behind lipid accumulation and degradation
during peanut seed development and post germination
Yun Wang, Xingli Ma, Xingguo Zhang, Xiaoyan He, Hemin Li, Dangqun Cui, and Dongmei Yin
J. Proteome Res., Just Accepted Manuscript DOI: 10.1021/acs.jproteome.6b00345 Publication Date (Web): 27 Sep 2016
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Page 1 of 30 Journal of Proteome Research
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4 ITRAQ-based proteomic analysis of the metabolic mechanisms behind lipid accumulation and
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6 degradation during peanut seed development and post germination
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9 Yun Wang, Xingli Ma, Xingguo Zhang, Xiaoyan He , Hemin LiDangqun CuiDongmei Yin*,
10 Henan Agricultural University, Zhengzhou 450002, China
11
12 *
13 Corresponding author:
14 Email: yindmp@yahoo.com , yindm@126.com
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17 Abstract
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19 Peanut seeds have a high oil content making it an important oil crop. During development and
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21 germination, seeds undergo complex dynamic and physiological changes. Changes in lipid metabolism
22 and underlying mechanisms during seed development have been studied extensively by DNA and RNA
23
24 sequencing; however, there are few studies on dynamic changes of proteomics during peanut seed
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26 development and germination. In this study, proteomic analyses were carried out 20, 40, 60 and 80 days
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after pollination, and 5, 10, 20 and 30 days after germination using isobaric tags for relative and
28
29 absolute quantitation (iTRAQ) technology to determine the protein profiles of lipid dynamics during
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31 peanut seed development and post germination. A total of 5,712 of 8,505 proteins were identified,
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33 quantified and divided into 23 functional groups, the largest of which was metabolism-related. Further
34 analyses of the proteins and their pathways revealed initiation of fatty acid accumulation at early stages
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36 after flowering, while lipid degradation occurred largely through the lipoxygenase-dependent pathway.
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38 Protein expression patterns related to lipid accumulation and degradation were also verified at
39
transcript levels using quantitative real-time PCR. The proteome profiles determined here will
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41 significantly enrich our understanding of the process of lipid accumulation and degradation, and the
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43 dynamic changes in metabolic networks during peanut development.
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45 Keywords: proteomics, seed development, seed germination, lipid accumulation, lipid degradation
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47
48 Introduction
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50 Peanut (Arachis hypogaea) is a major economic and edible oil crop, the seeds of which contain
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approximately 46-60% oil 1. As a result, peanut is in great demand both for human consumption and
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53 industrial use. Lipids, the major determinant of yield in oil plants, provide energy for seed germination
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55 and seedling growth. Understanding the mechanism of lipid accumulation and degradation is therefore
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57 essential for breeding new peanut species with a high yield.
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4 Triacylglycerols (TAGs) are a major form of lipid stored in seeds and fruits2,3. TAGs are hydrolyzed
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6 to generate fatty acids and glycerol, which are further broken down by -oxidation, the TCA cycle and
7 gluconeogenesis successively4. Research on the synthesis of storage oils and their biochemical and
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9 metabolic processes will help us understand the accumulation of oil and energy flow during seed
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11 development and seedling growth5-9. Seed germination and development are dynamic and complex
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processes that involve the regulation of important reserve components such as proteins, carbohydrates
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14 and oil. During seed germination, reserve components are subjected to dramatic catabolism, providing
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16 nutrients and energy for growth and development of the young embryos. Although gene expression
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18 and regulation during seed development and germination have been studied extensively, little is
19 known about the dynamic changes in corresponding proteins and enzymes during peanut seed
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21 development and germination. Guo et al. and Yin et al. identified the genes involved in lipid
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23 accumulation during seed development using transcriptome10,11. Gene transcripts of a number of lipid
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metabolic enzymes have been identified through analysis of expressed sequence tag (EST) databases,
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26 which contain a large number of non-redundant transcript sequences of coding genes1012 as well as
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28 the primary transcripts for non-coding genes such as microRNAs (miRNAs) in peanut13. In our
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30 previous study, comparative transcriptomic analysis revealed seven possible metabolic pathways
31 involved in oil accumulation during seed development. Due to the stability of translated
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33 proteins/enzymes subjected to various post-translational modifications and/or regulation, precise
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35 information on the proteins involved remains limited following transcriptome analysis. Moreover,
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transcriptome levels often fail to correlate with expressed protein levels14, thereby limiting our
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38 understanding of certain metabolic processes such as lipid metabolism.
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40 Proteomics is a powerful tool for understanding the complex regulatory mechanisms and dynamics
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42 behind protein changes. Proteomic analysis of protein dynamics during seed development and
43 germination have been addressed in Arabidopsis15,16, legumes17, rice and other species18. In peanut,
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45 using 2-DE proteomic analysis, Zhu et al. identified several candidate proteins related to pod swelling
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47 by comparing the proteomic profiles of aerial and subterranean pods19. Furthermore, Sun et al., using
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various treatments, identified 27 differentially expressed proteins in aerial gynophores, subterranean
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50 gynophores, and gynophores20. Thousands of proteins have also been detected in aerial gynophores,
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52 subterranean gynophores, and early swelling peanut pods using 1-DE with nanoLC-MS/MS
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54 approaches21. The results of these studies have provided valuable information on peanut pod swelling;
55 however, few proteins related to lipid metabolism have yet been identified. These studies also failed to
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57 identify the comprehensive molecular mechanism underlying seed development and germination in
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60 2
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4 peanuts.
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6 Isobaric tags for relative and absolute quantitation (iTRAQ) is a mass-based quantitative approach
7 that has recently become prevalent in the field of crop proteomics, allowing simultaneous
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9 identification and quantification of proteins from multiple samples with high coverage. Furthermore,
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11 iTRAQ combined with tandem mass spectrometry (MS/MS) was also developed to carry out
12
comprehensive profiling of even low-abundance proteins with more accurate quantification22. In this
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14 study, for the first time, iTRAQ-based quantitative proteomic analysis was used to understand the
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16 mobilization of protein reserves and characteristics of protein expression dynamics during seed
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18 development and germination in peanut. Quantitative reverse transcription-PCR (qRT-PCR) was also
19 carried out to correlate and validate the proteomic results related to oil accumulation and degradation.
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21 The constructed proteome profiles will significantly promote our understanding of lipid networks and
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23 lipid dynamics at different stages of seed development and germination.
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Materials and Methods
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26 Peanut seed growth conditions and treatments
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28 The peanut subline cultivar HuaU606, which originated from the parents Yuhua15 (male) and
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30 Huayu17 (female), was selected for use in the experiments. Developing seeds were manually collected
31 at different days after flowering (20, 40, 60 and 80 DAF).
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33 Mature seeds of U606 were kept at 25C with 8 h light and 16 h dark daily, in standard tissue
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35 culture flasks containing filter paper moistened with distilled water. Germination assays were
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conducted using three replicates of 40 seeds at each sampling. Cotyledons were collected at different
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38 germination stages (5, 10, 20 and 30 DAG), frozen immediately in liquid nitrogen and stored at 80C
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40 until further use.
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42 Measurements of oil content and fatty acids
43 Fresh seeds obtained at each development stages were ground into powder in liquid nitrogen. The oil
44
45 content was then calculated following the Soxhlet extraction method and the fatty acid composition
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47 analyzed by gas chromatography-mass spectrometry as described by Yang et al.23 The fresh seeds of
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different stages were ground into powder in liquid nitrogen. The oil content was calculated following
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50 soxhlet extraction method and the fatty acid composition was analyzed as follows: Taking About 0.02
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52 g powder of peanut seeds, and added 100 ul methanol with 0.2% BHT, 100 ul methanol with 0.2%
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54 BHT, 200 ul toluene along with 17:0 FA standard. The solution firstly was refluxed at 80C for 1h.
55 And then added 1.5 ml 0.9% NaCl and 2 ml hexane with 0.2% BHT and shaked for 3 min, the top
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57 phase collected for GC after centrifuged at 5000 rpm for 2 min,. The fatty acid methyl esters were
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60 3
1
2
3
4 separated by gas chromatography (DEGS-diethyl glycol succinate column). The GC conditions were:
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6 injector temperature and flame ionization detector temperature, 230C; keeping oven temperature
7 160C for 30min, then increasing at 3C/min to 230C and holding at 200C for 5 min23. The relative
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9 FA compositions were calculated by three independent biological replicates.
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11
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Protein Extraction and Digestion
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14 Protein samples obtained at 20, 40, 60 and 80 DAF, and 5, 10, 20 and 30 DAG were used for
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16 proteomic analysis. There were three sample replicates composed of a 10-seed mixture in each one.
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18 The cotyledons were ground into a power in liquid nitrogen and the resulting fine powder precipitated
19 using cold 10% TCA/acetone supplemented with 50mM DTT, 2mM EDTA, protease inhibitor cocktail
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21 and PVPP powder at -20C for 2h in a 50-ml centrifuge tube. The extracts were then centrifuged at
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23 20,000g at 4C for 10 min and the supernatants discarded. The precipitate was washed three times in
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cold acetone containing 50mM DTT. After air drying, the precipitate was re-suspended in lysis buffer
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26 (8M urea, 10mM DTT, 2mM EDTA, and protease inhibitor cocktail) and processed on ice using a high
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28 intensity ultrasonic processor (Scientz) then centrifuged at 20,000g at 4C for 10 min. The
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30 supernatants were transferred to a fresh tube and the protein content determined using a 2-D Quant kit
31 (GE Healthcare) following the manufacturers instructions.
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33 The protein samples were reduced by treating the disulfide bonds with 10 mM DTT for 1 h at
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35 56C and further alkylated using 55 mM iodoacetamide for 45 min in darkness at room temperature.
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The treated protein was finally precipitated with 20% TCA for 2 h at 4C and washed with cold
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38 acetone three times. Resulting pellets were dissolved and sonicated using 0.5M TEAB and the protein
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40 suspension digested with Trypsin (Promega) at an enzyme substrate ratio of 1:50 at 37C for 12 h. To
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42 ensure complete digestion of the protein suspension, the digested protein solution was added to
43 additional trypsin at a ratio of 1:50 and incubated for a further 4h.
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45 ITRAQ Labeling and Fractionation with High-pH Reverse-Phase Chromatography
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47 After trypsin digestion, the peptides were desalinized using a Strata X C18 SPE column (Phenomenex)
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and vacuum dried. Peptides were reconstituted in 1M TEAB following the manufacturers instructions
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50 using an 8-plex iTRAQ kit (AB Sciex). Peptides were labeled as follows: at 20 DAF with iTRAQ-113,
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52 40 DAF with iTRAQ-114, 60 DAF with iTRAQ-115 and 80 DAF with iTRAQ-116; at 5 DAG with
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54 iTRAQ-117, 10 DAG with iTRAQ-118, 20 DAG with iTRAQ-119 and 30 DAG with iTRAQ-121.
55 Briefly, six units of iTRAQ reagent (per 100 g of protein) was defrosted and reconstituted in 80 L
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57 acetonitrile, and the labeled peptides from each sample incubated for 2 h at room temperature. The
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60 4
1
2
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4 peptide mixtures were then collected and vacuum dried.
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6 Labeled peptides were further separated with reverse-phase high performance liquid
7 chromatography (HPLC). The reverse-phase column (Agilent, ZORBAX Extended-C18 4.6 mm x 250
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9 mm, 5 m particle, 80 pore size) was equilibrated with 2% buffer B (10 mM ammonium formate
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11 and 90% acetonitrile, pH 10.0) and the peptide mixture in buffer A (10 mM ammonium formate in 2%
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acetonitrile, pH 10.0) loaded onto the column and eluted at a linear gradient of 5 to 8% buffer B for 5
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14 min, 8-18% B for 35 min, 18-32% B for 22 min and 32-95% B for 2 min at a constant flow rate of 1
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16 mL/min using analytical HPLC (Rigol). A total of fifty-six fractions were collected and combined
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18 equally into 14 final fractions.
19 LC-ESI-MS/MS Analysis using Q Exactive
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21 Peptides from each fraction were vacuum dried and re-suspended in buffer A (0.1% FA, 2% ACN)
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23 then centrifuged at 20,000g for 2min. The supernatant was then transferred into a sample tube and
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using EASY nLC1000 nanoUPLC (Thermo Scientific), loaded onto an Acclaim PepMap 100 C18 trap
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26 column (Dionex, 75um2cm) to elute the peptide onto a second type of Acclaim PepMap RSLC C18
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28 analytical column (Dionex, 50um15cm). A 34-min gradient was then applied at a flow rate of 300
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30 nl/min from 5 to 30% buffer B (0.1% FA, 80% ACN) followed by a linear gradient to 40% buffer B
31 for 2 min, which was then ramped to 80% buffer B in 2 min, and finally, kept at 80% buffer B for 4
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33 min.
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35 After applying the NSI source, the peptides were collected by MS/MS using Q Exactive (Thermo)
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coupled to UPLC online. Intact peptides were detected in the Orbitrap at a resolution of 70000 at 200
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38 m/z. Using 27% NCE with 12% stepped NCE, peptides were then selected for MS/MS. Ions with
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40 charge state 2-5 were allowed for fragmentation in mass spectrometers. Ion fragments were detected
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42 in the Orbitrap at a resolution of 17500 at 200 m/z. A data-dependent acquisition procedure was
43 alternated between a single MS scan followed by 20 MS/MS scans and applied to the top 20 precursor
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45 ions above a threshold ion count of 3E4 in the MS survey scan with 5.0s dynamic exclusion. The
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47 voltage of the spray was 1.8 kV. To prevent overfilling of the ion trap, automatic gain control (AGC)
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was used, with 1E5 ions accumulated to generate the MS/MS spectra. For MS scans, an m/z scan
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50 range of 350 to 1600 Da was used. Normalized collision energy was 30 eV and the underfill ratio
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52 was defined as 0.1%.
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54 Database Search
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56 The instrument data (.raw) were combined and converted into a.mgf using Proteome Discoverer (ver.
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1.3.0.339, Thermo). Peptides and protein identifications were made using the Mascot search engine
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60 5
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4 (ver. 2.3.02, Matrix Science). Protein sequence data used for MS/MS search come from
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6 Papilionoideae of UniprotKB (157,781 sequences, release time 2013.12.14). Database searches were
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limited to tryptic peptides. Carbamidomethyl (C), TMT6plex (N-term) and TMT6plex (K) were
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9 selected as fixed modifications and oxidation (M) as a variable modification, with 2 missed cleavages
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11 allowed, a precursor < 10 ppm, and fragment deviation set at 0.02 Da. The filter criteria of spectra hits
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13 given a p value <0.05 and expected cut-off of 0.05. Identified proteins with at least two unique
14 peptides at or above >95% confidence level were accepted. The FDR filter was applied at the peptide
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16 level and 1%FDR was used to filter the protein identifications. For quantifications, all quantified
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18 peptides in one protein were combined to calculate the p-values. The median of the quantified peptides
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was used as protein relative quantity. Based on protein ID, Gene Ontology (GO) annotation proteomes
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21 were derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). Differentially expressed
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23 proteins involved in lipid accumulation and degradation were subsequently clustered via hierarchical
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25 clustering and k-means clustering using EXPANDER.
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qRT-PCR Analysis
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28 Cotyledons obtained at 20, 40, 60 and 80 DAF were chosen for qRT-PCR analysis. Total RNA was
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30 extracted using the method described by Yin et al.24. RNA concentrations were measured using an
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32 ND1000 spectrophotometer and RNA quality visualized using standard agarose gel (1%, w/v).
33 RNase-free DNaseI (Fermentas, USA) was used to remove genomic DNA contaminants, and
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35 first-strand cDNA was synthesized using PrimeScript RT Master Mix (TaKaRa, Dalian, China).
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37 qRT-PCR followed the manufacturers instructions (TaKaRa, Dalian, China; primers are listed in
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Supplemental Table 1). Reactions were as follows: 95C for 30s followed by 40 cycles of 95C for 5s
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40 and 60C for 30s. All reactions were replicated three times for each sample. After amplification, the
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42 melting curve and Ct value were used to analyze the success of qRT-PCR. Relative quantification was
43 Ct
44 performed according to the 2 method.
45 Results
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47 Developmental and germinating characteristics of peanut seeds
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49 To profile the proteome dynamics during peanut seed formation and germination, we documented the
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developmental and germinating characteristics of peanut seeds in detail. Peanut needles elongate into
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52 the soil and then rapidly swell under suitable conditions. Initially, the pod developed into a chickens
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54 head shape, full of white spongy tissues. The pods then swelled rapidly into kernels, with an increase in
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56 size and decrease in spongy tissue, before finally developing into underground mature pods containing
57 mature seeds (Figure 1A). After dormancy, and after absorbing sufficient moisture (5 h imbibition), the
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60 6
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4 mature seeds started to germinate. The hypocotyl then elongated, the radicle broke through the testa, the
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6 cotyledon opened, and the germ started to grow. After germination, the radicle grew downwards
7 quickly forming taproots at 5 DAG (Figure 1A), and lateral roots started to form. Finally, the peanut
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9 germs developed into seedlings with functional stems, leaves and roots.
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11 Component analysis and fatty acid (FA) composition
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Next, we analyzed the major components, in particular the fatty acid composition, of the developing and
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14 germinating seeds at each stage. Protein and lipid contents were determined at 20, 40, 60 and 80 DAF
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16 and 5, 10, 20 and 30 DAG). As a result, significant changes in the oil content of the cotyledon were
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18 revealed (Figure 1B). The oil content followed an "S" curve increase during development; however,
19 during post germination stages, a sharp decrease was observed from 60.7% in the mature period to
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21 15.19% at 30 DAG. This suggests mobilization and consumption of the lipids during seed germination
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23 and post germination stages.
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The FA composition of the crude lipids at each stage were further determined in detail (Figure 1C).
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26 Among the identified FAs, two fatty acids, oleic acid (C18:1) and linoleic acid (C18:2), occupied the
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28 highest percentage followed by palmitic acid (C16:0) and stearic acid (C18:0). During seed
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30 developmental, the relative contents of oleic acid and stearic acid consistently increased. In contrast, the
31 relative content of palmitic acid was high (17.82%) at 20 DAF and then gradually decreased till
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33 maturity, while relative contents of linoleic acid, arachidic acid, and 24 carbon acids remained almost
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35 constant throughout the various development stages. Linolenic acid started to reduce quickly at an early
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stage and was almost undetectable at 40 DAF.
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38 In addition to the nine fatty acids mentioned above, a new fatty acid (C22:6) was also detected at the
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40 germination stage. At first, C22:6 increased rapidly, but then decreased quickly up till 30 DAG at which
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42 point it was undetectable. During germination, linolenic acid reappeared at a low level. The relative
43 content of C20:1 and C24:0 increased slowly at early germination stages then decreased rapidly to
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45 zero. The contents of the remaining six fatty acids (C16:0, C18:0, C18:1, C18:2, C20:0 and C22:0)
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47 changed only slightly during germination.
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Functional identification and classification of proteins identified by iTRAQ
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50 Proteins extracted from the cotyledon were initially separated by one-dimensional SDS-PAGE
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52 electrophoresis to reduce the protein complexity (Supplemental Figure 1). Protein bands showed
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54 obvious differences among samples such as those at 20 DAF, which showed protein bands obviously
55 different. That is, the proteins observed during development changed remarkably at germination.
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57 A total of 16,271 peptides were matched. The length of most (about 70%) was in the range of 8-17aa.
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60 7
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4 To determine dynamic changes in the proteomic profiles at each stage of development, relative
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6 expression levels of the proteins at 20, 40 and 60 DAF, and 5, 10, 20 and 30 DAG were investigated,
7 with those obtained at 80 DAF as a control. ITRAQ proteomic analysis identified a total of 8,505
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9 proteins across the different stages and, of these, at least 5,712 were quantified.
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11 Gene ontology (GO) categorization of the identified peptides was subsequently carried out to
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further categorize the functions of the identified proteins (Supplemental Figure 2). Cellular functions
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14 were assigned to the following cellular compartments: cell parts (40.26%), organelles (17.6%),
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16 organelle parts (15.87%) and extracellular regions (14.29%) (Supplemental Figure 2C). Biological
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18 functions were associated with the following brief pathways: metabolic processes (68.59%), biological
19 adhesion (8.96%), cellular processes (8.93%) and immune system processes (4.45%) (Supplemental
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21 Figure 2A). The most highly enriched molecular functions in the peanut seeds were catalytic proteins
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23 (68.64%), followed by binding (18.82%) and structural proteins (9.41%), respectively (Supplemental
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Figure 2B).
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26 The 5,712 quantified proteins were further classified into 23 functional categories using clusters of
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28 orthologous group (COG) classifications (Figure 2). The largest group category was found to have
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30 general functions only (11.81%) followed by proteins related to DNA replication, recombination and
31 repair (8.49%), and transcription (8.42%). Only a small fraction of the proteins were functionally
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33 related to the categories of RNA processing and modification (0.35%) and cell motility (0.05%).
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35 About 2.6% of the identified proteins (112 proteins) were related to lipid metabolism.
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Protein expression profiles of lipid pathways at different stages of peanut development
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38 Mature seeds of U606 contain approximately 60% oils, most of which are stored in oil bodies as TAGs.
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40 Approximately 241 of the identified proteins were found to be involved in lipid metabolism
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42 (Supplemental Table 2), 88 of which were differentially expressed (> 2-fold or < 0.5-fold; p<0.05)
43 (Supplemental Table 3). These differentially expressed proteins were categorized into 5 groups as
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45 follows: oil accumulation-related proteins, lipid degradation-related proteins, lipid storage-related
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47 proteins, lipid transport and some lipid binding-related proteins, respectively (Supplemental Table 3).
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A total of 36 differentially expressed proteins were related to lipid metabolism during seed
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50 development (Supplemental Table 4). A total of 72 differentially expressed proteins were observed post
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52 germination (Supplemental Table 5). During both seed development and post germination processes,
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54 the protein BON1_ARATH, which showed calcium-dependent phospholipid binding activity, was
55 up-regulated. The number of differentially expressed proteins observed during development was
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57 displayed using the Venny graphic tool (Figure 3A). Compared with the control group (80 DAF), most
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60 8
1
2
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4 differentially expressed proteins were down-regulated at 20 DAF, except ACCD_CYACA,
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6 APG_ARATH, BON1_ARATH, DGDG1_LOTJA, FACR2_ARATH, MGDG3_ARATH,
7 PATL3_ARATH, PHO13_ORYSJ, PLA1_ORYSI, STAD_SOYBN and Y3550_ARATH.
8
9 Taken together, our data suggest that enzymes involved in oil accumulation are active in lipid
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11 formation at 20 DAF. Small changes in protein expression were found at 40 and 60 DAF, except in the
12
case of four differentially expressed proteins (Figure 3A). Differentially expressed proteins observed at
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14 both 20 and 40 DAF were BON1_ARATH, HDG10_ARATH and NLTP6_AMBAR, the first two of
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16 which were up-regulated and acted as lipid binding proteins. ACCD_CYACA, BON1_ARATH and
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18 PHO13_ORYSJ were up-regulated at both 20 and 60 DAF. The number of differentially expressed
19 proteins observed post germination is also shown in Figure 3B. Five proteins, ACCA_CYAME,
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21 BON1_ARATH, OLEO3_ARATH, PAP2_BRACM and PATL3_ARATH, were all differentially
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23 expressed at these stages. A total of 72 proteins were detected as follows: oil accumulation related
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proteins (n=27), oil degradation (n=26), phospholipid transport-related proteins (n=4), lipid
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26 storage-related proteins (n=2), and other lipid binding proteins (n=13) (Supplemental Table 5). Of
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28 these, 26 proteins were involved in lipid catabolic processes as lipoxygenase, phospholipase and
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30 GDSL esterase\lipase as well as fatty acid beta-oxidation. Compared with 80 DAF, there was no
31 change at 5 DAG, except for up-regulation of 49 differentially expressed proteins involved in fatty
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33 acid beta-oxidation from 10 to 30 DAG. Up-regulation of the enzymes involved in lipase and fatty
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35 acid beta-oxidation suggests that they play an active role in post germination processes. Oleosin,
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which covers the surface of the oil body, was significantly down-regulated post germination. Four
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38 phospholipid-transport related proteins were also differentially expressed and up-regulated from 10 to
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40 30 DAG, which suggests that phospholipid-transport was active at this stage. Other lipid binding
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42 proteins showed similar changes as the phospholipid transport-related proteins. Most oil
43 accumulation-related proteins including ACCA_CYAME and STAD_SOYBN showed high abundance
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45 especially from 10 to 30 DAG.
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47 During both seed development and post germination, 66 differentially expression proteins involved
48
in lipid accumulation and degradation were observed (Table 1). K-means clustering was used to
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50 determine dynamic changes in the proteins, with 10 clusters identified (K1-K10; Figure 4). Identical
51
52 or similar expression patterns were found in each cluster at different stages. Cluster 1, which included
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54 five proteins, was down-regulated, while Clusters 5, 6, 7 and 8 contained two, three, six and six
55 proteins, respectively, and were highly abundant in terms of lipid degradation from 10 to 30 DAG.
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57 Cluster 4 included seven proteins largely concerned with lipid accumulation. These were
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60 9
1
2
3
4 down-regulated at 20 DAF, but showed no other differences at other stages. Cluster 10 contained nine
5
6 proteins and was also down-regulated at 20 DAF; however, it was up-regulated from 10 to 30 DAG.
7 Cluster 2 contained five proteins and was up-regulated at 20 DAF, while Cluster 3 contained thirteen
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9 proteins, which were up-regulated at 20 DAF and again from 10 to 30 DAG. Cluster 9 was most
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11 abundant at 20 DAG.
12
qRT-PCR analysis of lipid accumulation and degradation genes
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14 The value of peanut seeds depends on the lipid content. In addition to the proteomic results, 12 genes
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16 that were all successfully identified, except for DGAT, were further selected to confirm mRNA
17
18 expression. Of these, only KASII and FAD2 were not differentially expressed. KASI, KASII, KASIII,
19 ACCA, SAD and FAD2 were found to be related to fatty acid synthesis, while LOX5, GDSL, PLD and
20
21 ACOX were related to lipid degradation. Oleosin14.9 was used to reflect lipid mobility. DGAT was
22
23 chosen for further analysis of the important role of lipid synthesis. Transcriptional analyses of the 12
24
proteins showed significant changes at different stages (Figure 6). FAD2, KASII, KASIII and
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26 oleosin14.9 showed consistent patterns with the protein expression patterns throughout seed
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28 development. Although LOX5, PLD, ACOX and GDSL were not completely consistent, all were
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30 highly abundant throughout germination from 10 to 30 DAG. These results suggest that lipid
31 degradation was active during germination for seedling growth.
32
33 Discussion
34
35 The economic value of peanut is largely determined by the oil content of the seeds, which in turn is
36
related to the pathways of lipid metabolism. This study characterized the proteins involved in lipid
37
38 accumulation and degradation during seed development and post germination.
39
40 Proteins related to oil accumulation during seed development
41
42 Carbon assimilation begins with the transport of sucrose into the seeds25. During development, the
43 pentose phosphate and glycolytic pathways provide carbon source precursors for fatty acid synthesis in
44
45 plastids of peanut seeds. These precursors then participate in de novo fatty acid synthesis. In plants,
46
47 synthesis of TAG occurs in three steps: FA synthesis in the plastids, transport of FA to ER, and lastly,
48
synthesis of the TAGs. In this study, we identified various enzymes involved in each of these
49
50 processes. The first step of fatty acid synthesis depends on acetyl-CoA, which is largely provided by
51
52 plastidial pyruvate dehydrogenase (PD). PD provides acetyl-CoA and NADH for de novo fatty acid
53
54 biosynthesis26,27. Here, we identified isoforms from the subunits of PD (pyruvate dehydrogenase, E1
55 -pyruvate dehydrogenase and E3-dihydrolipoyl dehydrogenase). The PD bypass pathway has been
56
57 observed in Arabidopsis and other plants26,28. Here, in addition to PD, other enzymes involved in the
58
59
60 10
1
2
3
4 bypass pathway were also identified; for example, alcohol dehydrogenase, pyruvate carboxylase and
5
6 aldehyde dehydrogenase, suggesting existence of this pathway in developing peanut seeds.
7 A large number of proteins coding fatty acid biosynthesis enzymes were observed during peanut
8
9 seed development. Acetyl-CoA carboxylase (ACCase), which catalyzes acetyl-CoA conversion into
10
11 malonyl-CoA, is the limiting enzyme regulating fatty acid synthesis. Acetyl-CoA carboxylase is
12
comprised of four subunits: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase
13
14 subunits and 29. Eight related proteins were identified, of which three proteins corresponding to
15
16 carboxyltransferase were found to be differentially expressed during seed development.
17
18 Carboxyltransferase subunit was up-regulated during seed development, while the remaining three
19 were down-regulated at 20 DAF. This finding suggests that the four subunit proteins of acetyl-CoA
20
21 carboxylase are not equal expressed during peanut seed development. Malonyl-CoA is catalyzed by
22
23 the malonyl-CoA:acyl carrier protein S-Malonytransferase (MAT), which transfers the malonyl group
24
to a protein cofactor known as acyl carrier protein (ACP), expression of which was high at 40 DAF. In
25
26 Arabidopsis thaliana, antisense inhibition of ACP was found to reduce the content of fatty acids in the
27
28 leaves, as well as affecting the composition of fatty acids and the overall plant physiology, suggesting
29
30 an important role in fatty acid biosynthesis and the development of chloroplast membranes30. Fatty acid
31 synthase (FAS) complexes produce fatty acids, while KASIII catalyzes the initial condensation
32
33 reaction, yielding C4:0-ACP. Subsequent condensations are catalyzed by KASI and KAS for
34
35 elongation up to C16:0-ACP and C16:0-ACP to C18:0-ACP, respectively31,32. In this study, KAS
36
was highly abundant at 40 DAF. Moreover, 3-ketoacyl-ACP reductase (KAR), 3-hydroxyacyl- ACP
37
38 dehydratase and enoyl-ACP reductase (ENR) catalysis were observed at each condensation step, each
39
40 cycle increasing by two carbon chains until the synthesis of saturated fatty acids 16:0 and 18:0. Along
41
42 with synthesis, fatty acid acyl-ACP groups are hydrolyzed by acyl-ACP thioesterases, which were not
43 identified in this study, and free fatty acids are released. In this study, C16:0 decreased gradually
44
45 during peanut seed development. This findings suggests that C16:0, which is first synthesized at an
46
47 early stage of seed development, is the precursor for elongation to C18:1 and C18:2. Moreover,
48
18:0-ACP is converted to 18:1-ACP by stearoyl-ACP desaturase (SAD), the abundance of which
49
50 gradually increased with the content of C18:1, further supporting the above hypothesis. Fatty acid
51
52 extends in the form of acyl-COA, first via 3-ketoacyl-CoA synthase (KCS) catalysis33 and then
53
54 long-chain acyl-CoA synthetase (LACS), leading to the export of fatty acid acyl CoA toward the ER.
55 The assembly of TAGs takes place in the ER via enzymes such as glycerol-3-phosphateacyl
56
57 transferase (GPAT), LPAAT, phosphatidic acid phosphorylase (PAP) and diacylglycerol
58
59
60 11
1
2
3
4 acyltransferase (DGAT). These enzymes were not identified in this study, except for GPAT and
5
6 LPAAT34; however, transcripts of these enzymes were detected. GPAT catalyzes the first acylation
7 reaction, and reached a high abundance at 40 DAF. DGAT is the rate-limiting enzyme that plays an
8
9 important role in synthesis of TAGs during lipid accumulation. However, in this study, we did not
10
11 detect the protein about DGAT. Nevertheless, DGAT was detected by qRT-PCR analysis, and showed
12
a dramatic increase in expression during seed development. In this study, phospholipid:diacylglycerol
13
14 acyltransferase (PDAT), which is critical for oil accumulation in seeds, was detected35. The presence
15
16 of these enzymes suggests key roles in the glycerolipid pathway36. Synthesized TAGs are usually
17
18 stored in spherical organelles known as oil bodies. The major structural protein in these oil bodies is
19 oleosins. Oleosins attach to the oil body surface and play an important role in maintaining stability,
20
21 preventing the degradation of storage lipids until the seed germinates. In this study, oleosin 14.9
22
23 displayed down-regulation during seed development, becoming highly abundant at maturity,
24
indicating the occurrence of lipid accumulation with development.
25
26 In addition to fatty acid synthesis- and TAG synthesis-related proteins, other lipid-related proteins
27
28 and a considerable number of proteins involved in transport and -oxidation were also identified such
29
30 as ABC transport and lipoxygenase. Some members of this family are also found in other organisms
31 with oil body proteomes and have been found to take part in oil body formation37,38. Gene expression
32
33 levels in developing peanut seeds also suggested the presence of transcripts related to lipid breakdown.
34
35 In plants, -oxidation occurs widely in different tissues and at different stages, and in peanut,
36
suggesting degradation of stored oil during seed development. Moreover, the key enzymes involved in
37
38 the pentose phosphate and glycolytic pathways were also identified, suggesting an important role
39
40 during peanut seed development. Based on these data, a sketch map of mobilization of oil in peanuts
41
42 was produced (Figure 6), which were involved in the accumulation and degradation process. This
43 result will help to better understand of the metabolic processes of oil mobilization in seeds.
44
45 Lipid metabolic pathways related to oil breakdown during post germination
46
47 During seed imbibition, enzyme activity is enhanced, respiration increases, and cotyledon storage
48
materials such as fat are changed to simple soluble substances as a result of enzyme activity39. Here,
49
50 the oil content post germination showed a sharp decrease, suggesting that the oil was mobilized and
51
52 lipolyzed, resulting in a build-up of large amounts of energy and nutrition for subsequent seedling
53
54 growth. This is in line with observations showing that mobilization of storage reserves during
55 germination in oily seeds supports subsequent seedling establishment40-42. The main protein covering
56
57 oil bodies is oleosin protein. In some oily seeds, mobilization of oleosin is a prerequisite for subsequent
58
59
60 12
1
2
3
4 oil mobilization42. In this study, oleosin14.9 showed significant down-regulation with similar gene
5
6 expression during seed germination, suggesting that down-regulation of oleosin is perhaps a sign of oil
7 mobilization23.
8
9 ACOX is a key enzyme in ethylene synthesis. During fermentation, the up-regulation of ACOX in
10
11 both protein and gene expression profiles suggests that ethylene endogenously stimulates the
12
germination of peanut seeds43. The classic pathway of storage lipid mobilization involves
13
14 triacylglycerol lipase degradation, which leads to hydrolysis of storage TAGs to free fatty acids and
15
16 glycerol44. In this study, triacylglycerol lipase was identified but not the abundance protein. A total of
17
18 84 lipid metabolic proteins were detected in this study, eight of which were lipoxygenases (LOXs).
19 LOX is a non-heme iron-containing dioxygenase that specifically catalyzes the oxidation of
20
21 polyunsaturated FA45,46. Compared with other saturated fatty acids, polyunsaturated fatty acids were
22
23 preferentially mobilized in the cotyledon of the peanut seed post germination. Combined with the
24
overall results, the LOX-dependent pathway seems to be a very important for oil mobilization during
25
26 peanut seeds post-germination.
27
28 A recent study suggested that a patatin-like phospholipase promotes lipid degradation via LOX in
29
30 cucumber cotyledons47. LOX activity is also known to be temporally involved in phospholipases and
31 lipase activity on the oil body surface48. It is possible, therefore, that phospholipases provide lipases
32
33 with access to their substrate through hydrolysis of phospholipids covering the oil body surface49. In this
34
35 study, we detected six phospholipases with similar abundances post germination. GDSL esterase/lipase
36
is thought to regulate the oil content of oil crops that change their conformation in the presence of
37
38 different substrates, resulting in broadly diverse enzymatic activities. At post germination, we identified
39
40 17 GDSL esterase/lipases, four of which were up-regulated at a similar abundance. Free fatty acids are
41
42 then transferred to glyoxosome via ABC transport50,51, most of which are highly abundant. A full set of
43 enzymes involved in fatty acid -oxidation were also detected in the present study (Figure 6). Moreover,
44
45 a number of enzymes involved in the citric acid cycle and glyoxylate cycle were up-regulated post
46
47 germination including isocitrate lyase and malate dehydrogenase, which help mobilize stocking lipids
48
during germination. These results suggest that enzymes involved in both the citric acid and glyoxylate
49
50 cycles are active in peanut seeds post germination.
51
52 Conclusions
53
54 In this study, systematic analysis of lipid metabolic processes during development and germination of
55 peanut seeds was carried out using iTRAQ labeling. From proteomic analysis, 5,712 proteins were
56
57 identified and the changes at different stages described. The findings provide important insight into the
58
59
60 13
1
2
3
4 role of lipid metabolism during peanut seed development and post germination; for example, in terms
5
6 of fatty acid synthesis, TAG synthesis and catabolism of fatty acids. In addition, other metabolic
7 pathways such as amino acid transport and metabolism, transcription and signal transduction were also
8
9 observed in these complex processes. Further studies of seed proteome processes are therefore
10
11 warranted in the future, while analysis of metabolic pathways will provide a basis for understanding
12
lipid accumulation and breakdown networks in peanut seeds.
13
14
15
16 Acknowledgements
17
18 This work was supported by grants from the National Natural Science Foundation of China (No.
19 31471525) and the Major Project of the Ministry of Science and Technology of Henan, China (No.
20
21 092101110500) .
22
23 Associated Content
24
25
26 Figure S1. SDS-PAGE scan of protein extracted from peanut seeds at different development and
27
28 post germination stages.
29
30
31 Figure S2. GO ontology of all quantified proteins at different development and post germination
32
33 stages in peanut seeds. (A) Biological processes, (B) cellular components and (C) molecular
34
35 functions.
36
37
38 Table S1. Primer sequences used in this study.
39
40
41
42 Table S2. Proteins related to lipid metabolism in peanut seeds.
43
44
45 Table S3. Categorization of differentially expressed proteins involved in lipid metabolism in
46
47 peanut seeds.
48
49
50 Table S4. Categorization of differentially expressed proteins involved in lipid metabolism during
51
52 peanut seed development.
53
54
55 Table S5. Categorization of differentially expressed proteins involved in lipid metabolism in
56
57 peanut seeds post germination.
58
59
60 14
1
2
3
4
5
6 Table S6. Annotation combine of all the identified proteins.
7
8
9 REFERENCES
10
11 (1) Wang, Y.; Zhang, X. G.; Zhao, Y. L.; Prakash, C. S.; He, G. H.;Yin, D. M. Insights into the novel
12
members of the FAD2 gene family involved in high-oleate fluxes in peanut. Genome 2015, 58 (8),
13
14 1-9.
15
16 (2) Shewry, P. R.; Napier, J. A.; Tatham, A. S. Seed storage proteins:structures and biosynthesis. Plant
17
18 Cell 1995, 7 (7), 94556.
19 (3) Voelker, T.; Kinney, A. J. Variations in the biosynthesis of seed-storage lipids. Annu Rev Plant
20
21 Physiol Plant Mol Biol 2001, 52 (1), 33561.
22
23 (4) Beevers, H. Metabolic production of sucrose from fat. Nature 1961, 191, 433-436
24
(5) Kornberg, H. L.; Beevers, H. A. Mechanism of conversion of fat to carbonhydrate in castor beans.
25
26 Nature 1957, 4575, 35-36.
27
28 (6) Poxleitner, M.; Rogers, S. W.; Lacey Samuels, A.; Browse, J.; Rogers, J. C. A role for caleosin in
29
30 degradation of oil-body storage lipid during seed germination.The Plant Journal 2006, 47 (6),
31 917933.
32
33 (7) Eastmond, P. J. SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that
34
35 initiates storage oil breakdown in germinating Arabidopsis seeds. Plant Cell 2006, 18 (3),
36
665675.
37
38 (8) Penfield, S.; Graham, S.; Graham, I. A. Storage reserve mobilization in germinating oilseeds:
39
40 Arabidopsis as a model system. Biochem. Soc. Trans 2005, 33 (2), 380383
41
42 (9) Muntz, K.; Belozersky, M. A.; Dunaevsky, Y. E.; Schlereth, A.; Tiedemann, J. Stored proteinases
43 and the initiation of storage protein mobilization in seeds during germination and seedling growth.
44
45 J. Exp. Bot 2001, 52 (362), 17411752.
46
47 (10) Guo, B. Z.; Chen, X. P.; Dang, P.; Scully, B. T.; Liang, X. Q.; Holbrook, C. C.; Yu, J. J.; Culbreath,
48
A. K. Peanut gene expression profiling in developing seeds at different reproduction stages during
49
50 Aspergillus parasiticus infection. BMC Dev Biol 2008, 8 (1), 1228.
51
52 (11) Yin, D. M.; Wang, Y.; Zhang, X. G.; Li, H. M.; Lu, X.; Zhang, J. S.; Zhang, W. K.; Chen, S. Y. De
53
54 Novo Assembly of the Peanut (Arachis hypogaeaL.)Seed Transcriptome Revealed Candidate
55 Unigenes for Oil Accumulation Pathways. PLOS ONE 2013, 8 (9), e73767.
56
57 (12) Zhang, J.; Liang, S.; Duan, J.; Wang, J.; Chen, S.; Cheng, Z. S.; Zhang, Q.; Liang, X. Q.; Li, Y. R.
58
59
60 15
1
2
3
4 Denovo assembly and characterization of the transcriptome during seed development, and
5
6 generation of genic-SSR markers in peanut (Arachis hypogaeaL.). BMC Genomics 2012, 13 (1),
7 90.
8
9 (13) Chi, X. Y.; Yang, Q. L.; Chen, X. P.; Wang, J. Y.; Pan, L. J.; Chen, M. N.; Yang, Z.; He, Y. N.;
10
11 Liang, X. Q.; Yu, S. L. Identification and characterization of microRNAs from peanut (Arachis
12
hypogaeaL.)by high-throughput sequencing. PLOS ONE 2011, 6 (11), e27530.
13
14 (14) Anderson, N. L.; Anderson, N. G. Proteome and proteomics: new technologies, new concepts, and
15
16 new words. Electrophoresis 1998, 19 (11), 18531861.
17
18 (15) Allardo, K.; Job, C.; Groot, S. P. C.; Puype, M.; Demol, H.; Vandekerckhove, J.; Job, D.
19 Proteomic analysis of Arabidopsis seed germination and priming. Plant Physiol 2001, 126 (2),
20
21 835848.
22
23 (16) Hajduch, M.; Hearne, L. B.; Miernyk, J. A.; Casteel, J. E.; Joshi, T.; Agrawal, G. K.; Song, Z.;
24
Zhou, M. Y.; Xu, D.; Thelen, J. J. Systems analysis of seed filling in Arabidopsis: using general
25
26 linear modeling to assess concordance of transcript and protein expression. Plant Physiol 2010,
27
28 152 (4), 20782087.
29
30 (17) Chen, S. X.; Harmon, A. C. Advances in plant proteomics. Proteomics 2006, 6, 5504 -5516.
31 (18) Kim, S. T.; Wang, Y. M.; Kang, Y.; Kim, S. G.; Rakwal, R.; Kim, Y. C.; Kang, K. Y. Developing
32
33 rice embryo proteomics reveals essential role for embryonic proteins in regulation of seed
34
35 germination. J Proteome Res 2009, 8 (7), 35983605.
36
(19) Zhu, W.; Zhang, E.; Li, H.; Chen, X.; Zhu, F.; Hong, Y. B.; Liao, B. S.; Liu, S. Y.; Liang, X. Q.
37
38 Comparative proteomics analysis of developing peanut aerial and subterranean pods identifies pod
39
40 swelling related proteins. Journal of Proteomic 2013, 91,172187.
41
42 (20) Sun, Y.; Wang, Q.; Li, Z.; Hou, L.; Dai, S.; Liu, W. Comparative Proteomics of Peanut Gynophore
43 Development under Dark and Mechanical Stimulation. J Proteome Res 2013, 12 (12), 55025511.
44
45 (21) Zhao, C. Z.; Zhao, S. Z.; Hou, L.; Xia, H.; Wang, J. S.; Li, C. S.; Li, A. Q.; Li, T. T.; Zhang, X. Y.;
46
47 Wang, x. j. Proteomics analysis reveals differentially activated pathways that operate in peanut
48
gynophores at different developmental stages. BMC Plant Biology 2015, 15 (1), 188.
49
50 (22) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.;
51
52 Pillai, S.; Dey, S.; Daniels, S., Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.;
53
54 Jacobson, A.; Pappin, D. J. Multiplexed protein quantitation in Saccharomyces cerevisiae using
55 amine-reactive isobaric tagging reagents.Mol. Cell Proteomics 2004, 3 (12), 11541169.
56
57 (23) Yang, M. F.; Liu, Y. J.; Liu, Y.; Chen, H.; Chen, F.; Shen, S. H. Proteomic analysis of oil
58
59
60 16
1
2
3
4 mobilization in seed germination and postgermination development of Jatropha curcas. J
5
6 Proteome Res 2009, 8(3), 1441-1451.
7 (24) Yin, D. M.; Liu, H. Y.; Zhang, X. G.; Cui, D. Q.; A protocol for extraction of high-quality RNA and
8
9 DNA from peanut plant tissues. Mol. Biotechnol 2011, 49 (2), 186191.
10
11 (25) Dennis, D. T.; Miernyk, J. A. Compartmentation of Nonphotosynthetic Carbohydrate Metabolism.
12
Ann. Rev. Plant Physiol 1982, 33 (1), 2750.
13
14 (26) Lin, M.; Oliver, D. J. The role of acetyl-Coenzyme A synthetase in Arabidopsis. Plant Physiol
15
16 2008, 147 (4), 18221829.
17
18 (27) Tovar-Mendez, A.; Miernyk, J. A.; Randall, D. D. Regulation of pyruvate dehydrogenase
19 complex activity in plant cells. Eur. J. Biochem 2003, 270 (6), 10431049
20
21 (28) Oliver, D. J.; Nikolau, B. J.; Wurtele, E. S. Acetyl-CoA Life at the metabolic nexus. Plant Sci
22
23 2009, 176 (5), 597601.
24
(29) Shorrosh, B. S.; Savage, L. J.; Soll, J.; Ohlrogge, J. B. The pea chloroplast membrane- associated
25
26 protein, IEP96, is a subunit of acetyl-CoA carboxylase. Plant J 1996, 10 (2), 261-268.
27
28 (30) Branen, J. K.; David, K.; Shintani.; Nicki, J. Engeseth.Expression of Antisense Acyl Carrier
29
30 Protein-4 Reduces Lipid Content in Arabidopsis Leaf Tissue. Plant physiology 2003, 132 (2),
31 748-756.
32
33 (31) Wu, G. Z.; Xue, H. W. Arabidopsisbeta-ketoacyl-[acyl carrier protein] synthase I is crucial for
34
35 fatty acid synthesis and plays a role in chloroplast division and embryo development. Plant Cell
36
2010, 22 (2), 37263744.
37
38 (32) Clough, R. C.; Matthis, A. L.; Barnum, S. R.; Jaworski, J. G. Purification and characterization of
39
40 3-ketoacyl-acyl carrier protein synthase III from spinach. A condensing enzyme utilizing
41
42 acetyl-coenzyme A to initiate fatty acid synthesis. J BiolChem 1992, 267 (29), 2099220998.
43 (33) Wu, G.; Wu, Y.; Xiao, L.; Li, X. D.; Lu, C. M. Zero erucic acid trait of rapeseed (Brassica napus
44
45 L.) results from a deletion of four base pairs in the fatty acid elongase 1 gene. Theoretical and
46
47 Applied Genetics 2008, 116 (4), 491-499.
48
(34) Furumoto, T.; Yamaguchi, T.; Ohshima-Ichie, Y.; Nakamura, M.; Tsuchida-Iwata, Y.;
49
50 Shimamura, M.; Ohnishi, J.; Hata, S.; Gowik, U.; Westhoff, P.; Brautigam, A.; Weber, A. P. M.;
51
52 Lzui, K. A plastidial sodium-dependent pyruvate transporter. Nature 2011, 476 (7361), 472475.
53
54 (35) Zhang, M.; Fan, J.; Taylor, D. C.; Ohlrogge, J. B. DGAT1 and PDAT1 acyltransferases have
55 overlapping functions in arabidopsis triacylglycerol biosynthesis and are essential for normal
56
57 pollen and seed development. Plant Cell 2009, 21 (12), 3885- 3901.
58
59
60 17
1
2
3
4 (36) Kennedy, E. P. The biological synthesis of phospholipids. Can. J. Biochem. Physiol 1956, 34 (2),
5
6 334-348.
7 (37) Athenstaedt, K.; Jolivet, P.; Boulard, C.; Zivy, M.; Negroni, L.; Nicaud, J. M.; Chardot, T. Lipid
8
9 particle composition of the yeast Yarrowia lipolytica depends on the carbon source. Proteomics
10
11 2006, 6 (5), 1450- 1459.
12
(38) Guo, Y.; Walther, T. C.; Rao, M.; Stuurman, N.; Goshima, G.; Terayama, K.; Wong, J. S.; Vale, R.
13
14 D.; Walter, P.; Farese, R. V. Functional genomic screen reveals genes involved in lipiddroplet
15
16 formation and utilization. Nature 2008, 453 (7195), 657-661.
17
18 (39) Bewley, J. D. Seed germination and dormancy. Plant Cell 1997, 9 (7), 10551066.
19 (40) Cernac, A.; Andre, C.; Hoffmann-Benning, S.; Benning, C. WRI1 is required for seed
20
21 germination and seedling establishment. Plant Physiol 2006, 141 (2), 745-757.
22
23 (41) Penfield, S.; Graham, S.; Graham, I. A. Storage reserve mobilization in germinating oilseeds:
24
Arabidopsis as a model system. Biochem.Soc. Trans 2005, 33 (2), 380-383.
25
26 (42) Graham, I. A. Seed storage oil mobilization. Annual Review of Plant Biology 2008, 59, 115-142.
27
28 (43) Sadeghipour, H. R.; Bhatla, S. C. Differential sensitivity of oleosins to proteolysis during oil body
29
30 mobilization in sunflower seedlings. Plant Cell Physiol 2002, 43 (10), 1117-1126.
31 (44) Quettier, A. L.; Eastmond, P. J. Storage oil hydrolysis during early seedling growth. Plant
32
33 Physiology and Biochemistry 2009, 47 (6), 485-490.
34
35 (45) Feussner, I.; Kuhn, H.; Wasternack, C. Lipoxygenase-dependent degradation of storage lipids.
36
Trends Plant Sci 2001, 6 (6), 268-273.
37
38 (46) Adlercreutz, P.; Gitlesen, T.; Ncube, I.; READ, J. S. Vernonia lipase:a plant lipase with strong
39
40 fatty acid selectivity. Methods Enzymol 1997, 284, 220-232.
41
42 (47) Rudolph, M.; Schlereth, A.; Krner, M.; Feussner, K.; Berndt, E.; Melzer, M.; Hornung, E.;
43 Feussner, I. The lipoxygenasedependent oxygenation of lipid body membranes is promoted by a
44
45 patatin-type phospholipase in cucumber cotyledons. Journal of Experimental Botany 2011, 62 (2),
46
47 749-760.
48
(48) Zienkiewicz, A.; Zienkiewicz, K.; Rejon, J. D.; Dios Alche, J.; Castro, A.; Rodriguez-Garcia,
49
50 M. I. Olive seed protein bodies store degrading enzymes involved in mobilization of oil bodies.
51
52 Journal of Experimental Botany 2014, 65 (1), 103-115.
53
54 (49) Bhatla, S. C.; Vandana, S.; Kaushik, V. Recent development in the oil body- associated signaling
55 molecules during lipolysis in oil seeds. Plant Signaling and Behavior 2009, 4 (3), 176-182.
56
57 (50) Zolman, B. K.; Silva, I. D.; Bartel, B. The Arabidopsis pxa1 mutant is defective in an ATP-
58
59
60 18
1
2
3
4 binding cassette transporter-like protein required for peroxisomal fatty acid -oxidation. Plant
5
6 Physiology 2001, 127 (3), 1266-1278.
7 (51) Hayashi, M.; Nito, K.; Takei-Hoshi, R.; Yagi, M.; Kondo, M. Ped3p is a peroxisomal ATP-
8
9 binding cassette transporter that might supply substrates for fatty acid -oxidation. Plant and Cell
10
11 Physiology 2002, 43 (1), 1-11.
12
13
14
15
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18
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20
21
22
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24
25
26
27
28
29
30
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34
35
36
37
38
39
40
41
42
43
44
45
46
47
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60 19
1
2
3
4 Tables
5
6
7 Table 1 Differentially expressed proteins involved in lipid accumulation and degradation in peanut
8 seeds.
9
10 20DAF 40DAF 60DAF 5DAG 10DAG 20DAG/ 30DAG/
Protein ID Protein Name
11 /80DAF /80DAF /80DAF /80DAF /80DAF 80DAF 80DAF
12 ACC2_ARATH Acetyl-CoA carboxylase 2 1.9 1.2 1.3 1.0 2.8 2.3 2.3
13 Oil
Acetyl-coenzyme A carboxylase carboxyl
14 accumulation *ACCA_CYAME 0.4 1.2 1.4 2.7 7.1 4.7 4.8
transferase subunit alpha
15 related proteins
16 ACCC2_POPTR Biotin carboxylase 2, chloroplastic 1.1 1.5 1.2 1.0 2.9 2.4 3.2
17 Acetyl-coenzyme A carboxylase carboxyl
18 ACCD_ANTFO 1.2 1.3 1.2 1.1 1.2 0.5 0.9
transferase subunit beta
19
20 ACCD_CALFG 1.6 1.0 1.1 1.9 6.5 5.2 7.4
21
22 ACCD_CUSGR
1.1 1.4 1.2 1.1 1.6 1.8
2.4
23 transferase subunit beta
24 Acetyl-coenzyme A carboxylase carboxyl
25 ACCD_CYACA 4.0 2.2 2.3 0.8 1.8 1.8 1.8
26
27 ACCD_LOTJA 0.2 1.7 1.3 1.2 3.1 3.0 3.7
28 transferase subunit beta
29 ACP_CYAPA Acyl carrier protein 0.2 1.3 1.0 1.2 2.6 2.0 3.2
30 ACP1_CASGL Acyl carrier protein 1 0.8 1.9 1.0 0.5 0.8 0.6 0.7
31 BCH_GENLU Beta-carotene 3-hydroxylase 0.9 1.0 0.8 1.6 1.2 1.3
0.3
32
C86B1_ARATH Cytochrome P450 86B1 0.3 0.7 0.9 1.1 2.2 1.9 2.4
33
34 DGDG1_LOTJA Digalactosyldiacylglycerol synthase 1 2.0 1.6 1.6 1.2 3.5 2.3 2.8
35 DGDG2_SOYBN Digalactosyldiacylglycerol synthase 2 0.0 1.4 1.2 0.8 1.3 1.2 1.2
36 *FABH_SPIOL 3-oxoacyl-[acyl-carrier-protein] synthase III 0.3 1.0 1.0 1.0 1.7 1.0 1.3
37
FACR2_ARATH Fatty acyl-CoA reductase 2 2.3 1.5 1.1 0.8 1.9 1.8 2.0
38
39 FACR3_ARATH Fatty acyl-CoA reductase 3 7.7 1.2 1.2 0.9 2.1 1.9 2.2
40 FACR8_ARATH Fatty acyl-CoA reductase 8 1.7 1.5 1.3 1.2 3.3 2.9 2.8
41 Probable glycerol-3-phosphate
42 GPAT3_ARATH 0.6 1.1 1.2 0.8 2.1 1.9 2.1
acyltransferase
43
44 *KASC1_HORVU 3-oxoacyl-[acyl-carrier-protein] synthase I 0.6 1.1 1.0 1.4 4.5 3.4 4.6
45 KCS8_ARATH 3-ketoacyl-CoA synthase 8 0.4 1.6 1.3 1.2 3.4 2.7 3.7
46 SNF1-related protein kinase regulatory
47 KING1_ARATH 5.9 1.4 1.6 1.8 6.7 4.2 4.0
subunit gamma-1
48
LACS1_ARATH Long chain acyl-CoA synthetase 1 0.1 0.7 0.7 0.6 1.3 1.0 1.4
49
50 LACS4_ARATH Long chain acyl-CoA synthetase 4 0.3 1.0 0.9 0.7 1.5 0.8 1.1
51 1-acyl-sn-glycerol-3-phosphate
LPAT3_ARATH 0.9 0.9 0.7 1.3 3.2 2.2 2.6
52 acyltransferase
53 Probable monogalactosyldiacylglycerol
54 MGDG_SOYBN 0.3 1.1 1.6 1.0 2.2 1.7 1.9
synthase
55
56 MGDG_SPIOL Monogalactosyldiacylglycerol synthase 0.4 0.7 0.9 0.8 1.1 1.1 1.5
57 MGDG3_ARATH Monogalactosyldiacylglycerol synthase 3 2.9 1.4 1.0 0.7 4.8 3.3 2.9
58
59
60 20
1
2
3 PHO13_ORYSJ Phosphate transporter PHO1-3 2.2 2.0 2.4 3.7 17.5 8.8 9.1
4
PLSB_CUCSA Glycerol-3-phosphate acyltransferase 1.0 1.4 1.4 1.0 2.2 1.6 1.8
5
6 PLSB_SPIOL Glycerol-3-phosphate acyltransferase 0.3 1.3 1.1 0.9 2.0 1.4 1.2
7 STAD_HELAN Acyl-[acyl-carrier-protein] desaturase 1.1 1.1 1.1 0.7 3.8 4.6 2.6
8 STAD_SOYBN Acyl-[acyl-carrier-protein] desaturase 2.4 1.2 1.1 1.1 2.6 2.1 2.6
9
*STAD6_ARATH Acyl-[acyl-carrier-protein] desaturase 6 0.2 1.0 1.2 1.0 1.4 0.8 1.0
10
11 Y3684_ARATH Acyltransferase-like protein At3g26840 1.7 0.9 1.0 0.9 2.3 2.5 2.2
12 Lipid Putative peroxisomal acyl-coenzyme A
ACO12_ARATH 1.5 0.9 1.1 1.1 2.4 1.4 1.6
13 degradation oxidase
14
-related *ACOX2_CUCMA Acyl-coenzyme A oxidase 0.7 1.3 1.2 0.9 2.4 3.1 2.3
15
proteins APG_ARATH Anter-specific proline-rich protein APG 3.2 1.1 1.3 1.0 2.0 1.9 1.9
16
17 CISY3_ARATH Citrate synthase 3 0.9 0.9 1.0 0.9 3.7 2.0 2.8
18 ECH2_ARATH Enoyl-CoA hydratase 2 1.5 2.0 1.3 1.0 2.4 1.6 2.1
19 GDL10_ARATH GDSL esterase\lipase At1g28610 0.2 1.0 1.1 0.7 0.6 0.4 0.6
20
GDL35_ARATH GDSL esterase\lipase At2g19010 0.5 1.8 2.1 1.3 3.1 2.7 3.1
21
22 *GDL76_ARATH GDSL esterase\lipase At5g14450 0.9 1.0 0.9 1.4 2.7 3.2 3.0
23 GDL90_ARATH GDSL esterase\lipase At5g42170 0.8 1.4 1.1 1.1 2.9 2.0 2.4
24 GLIP2_ARATH GDSL esterase\lipase 2 0.3 0.8 0.7 0.8 1.7 0.7 0.8
25
LCAT4_ARATH Lecithine-cholesterol acyltransferase-like 4 1.1 1.6 1.2 1.0 2.4 1.9 2.5
26
27 LOX1_LENCU Linoleate 9S-lipoxygenase 1.2 1.4 1.1 1.3 3.0 2.5 3.6
28 LOX2_PEA Seed linoleate 9S-lipoxygenase-2 1.4 1.5 1.1 1.1 2.2 2.1 2.6
29 LOX3_SOYBN Seed linoleate 9S-lipoxygenase-3 1.2 1.4 1.5 1.0 2.6 2.3 2.5
30
LOX4_ARATH Lipoxygenase 4 2.4 1.1 1.2 0.9 2.4 2.0 2.1
31
32 LOX4_ORYSJ Probable linoleate 9S-lipoxygenase 4 1.1 1.1 1.0 0.5 0.6 0.5 0.4
33 LOX4_SOYBN Linoleate 9S-lipoxygenase-4 2.0 1.2 1.3 0.9 2.6 2.3 2.4
34 *LOX5_ARATH Linoleate 9S-lipoxygenase 5 0.8 1.1 1.2 1.8 4.1 3.1 4.0
35
LOXX_SOYBN Seed linoleate 9S-lipoxygenase 0.9 1.1 1.1 1.0 2.2 1.9 2.1
36
37 Glyoxysomal fatty acid beta-oxidation
MFPA_BRANA 2.4 1.2 0.9 1.2 3.6 4.0 3.6
38 multifunctional protein MFP-a
39 Glyoxysomal fatty acid beta-oxidation
40 MFPA_CUCSA 0.8 1.3 1.2 1.2 3.7 2.4 2.8
multifunctional protein MFP-a
41
PEX1_ARATH Peroxisome biogenesis protein 1 2.4 1.4 1.2 1.1 2.6 2.3 2.9
42
43 PEX10_ARATH Peroxisome biogenesis factor 10 0.4 0.7 0.7 0.7 1.9 1.3 1.9
44 PLA1_ORYSI Phospholipase A1-II 1 2.9 1.3 1.7 1.3 4.1 2.9 3.3
45 PLCD1_ARATH Phosphoinositide phospholipase C 1 2.6 2.0 1.3 0.7 1.8 1.7 2.3
46
*PLCD3_ARATH Phosphoinositide phospholipase C 3 1.8 1.1 1.1 1.1 2.7 2.3 2.9
47
48 PLCD4_ARATH Phosphoinositide phospholipase C 4 0.7 2.6 1.0 1.1 2.8 2.1 2.9
49 PLD_PHYIN Phospholipase D 0.4 0.6 0.8 0.9 2.0 1.2 1.1
50 PLDA1_PIMBR Phospholipase D alpha 1 0.6 1.7 1.3 0.9 2.6 2.3 2.4
51
PLDP2_ARATH Phospholipase D p2 0.9 1.3 1.0 1.1 2.9 2.1 2.5
52
53 *OLEO3_ARATH Oleosin 14.9 kDa 0.1 0.9 0.8 0.2 0.1 0.1 0.1
54 ITRAQ ratios of lipid accumulation and degradation are shown. The ratios represent relative expression levels of the
55 proteins at 20, 40and 60 DAF, and 5, 10, 20 and 30 DAG compared with 80 DAF. Numbers in bold represent differentially
56 expressed proteins. * Represents the protein chosen for qRT-PCR.
57
58
59
60 21
1
2
3
4
5
6 Figure Legends
7 Figure 1. Morphological and dynamic changes in fatty acids during peanut seed development and post
8
9 germination. (A) Peanut seed development and post germination processes. The seeds germinated in
10
11 moisture at 272C. Photographs were taken at 20, 40, 60 and 80 DAF, and 5, 10 , 20 and 30 DAG
12
after imbibition. Bar: 1.0 cm. (B) Changes in oil content (%) in peanut seeds during development and
13
14 germination stages. Values represent the means of three biological replicates (SD). (C) Compositions
15
16 and changes in fatty acids. Values represent means of three biological replicates (SD). C16:0,
17
18 palmitic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid; C20:0,
19 arachidic acid; C20:1, arachidonic acid; C22:0, behenic acid; C22:6, docosahexaenoic acid; C24:0,
20
21 tetracosanoic acid.
22
23
24
Figure 2. Histogram showing clusters of orthologous groups (COG) classification. A total of 5712
25
26 proteins were classified within the 23 categories.
27
28
29
30 Figure 3. Differentially expressed proteins involved in lipid metabolism in peanut seeds. Venn
31 diagrams showing the number of proteins differentially expressed at (A) each development stage and
32
33 (B) at each post germination stage.
34
35
36
Figure 4. K-means clustering (A) and protein expression profiles (B) of all proteins involved in lipid
37
38 accumulation and degradation. The nine clusters are shown in B (K1 - K10).
39
40
41
42 Figure 5. Expression profiles of genes involved in lipid accumulation and degradation at each stage of
43 peanut seed development. qRT-PCR analysis of cDNA isolated from developing seeds at 20, 40, 60
44
45 and 80 DAF. Relative expression ratios of each sample were compared with samples obtained at 80
46
47 DAF. Data represent the means SD of three replicates. The actin gene from peanut was used as an
48
internal standard. Abbreviations of gene names are as described in Table 1. ACCA, FAD2, SAD, KAS
49
50 I, KAS II, KAS III, oleosin, LOX, PLD, ACOX, GDSL represent ACCA_CYAME, FAD12_CREAL,
51
52 STAD6_ARATH, KASC1_HORVU, KASC2_ARATH, FABH_SPIOL, OLEO3_ARATH,
53
54 LOX5_ARATH, PLCD3_ARATH, ACOX2_CUCMA and GDL76_ARATH, respectively.
55
56
57 Figure 6. The metabolic processes of oil mobilization in development and postgermination stages in
58
59
60 22
1
2
3
4 peanut seeds. The identified proteins found in these pathways. The icons beside each protein name
5
6 show the expression profiles of protein during different stages. FA, fatty acid; G3P,Glyceraldehydes-3-
7 phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid ; DAG, diacylglycerol; TAG,
8
9 triacylglycerol.
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26 Figure 1. Morphological and dynamic changes in fatty acids during peanut seed development and post
27 germination. (A) Peanut seed development and post germination processes. The seeds germinated in
28 moisture at 272C. Photographs were taken at 20, 40, 60 and 80 DAF, and 5, 10 , 20 and 30 DAG after
29 imbibition. Bar: 1.0 cm. (B) Changes in oil content (%) in peanut seeds during development and
30 germination stages. Values represent the means of three biological replicates (SD). (C) Compositions and
changes in fatty acids. Values represent means of three biological replicates (SD). C16:0, palmitic acid;
31
C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid; C20:0, arachidic acid;
32 C20:1, arachidonic acid; C22:0, behenic acid; C22:6, docosahexaenoic acid; C24:0, tetracosanoic acid.
33 Figure 1
34 338x190mm (300 x 300 DPI)
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26 Figure 2. Histogram showing clusters of orthologous groups (COG) classification. A total of 5712 proteins
27 were classified within the 23 categories.
28 Figure 2
29 190x107mm (300 x 300 DPI)
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26 Figure 3. Differentially expressed proteins involved in lipid metabolism in peanut seeds. Venn diagrams
27 showing the number of proteins differentially expressed at (A) each development stage and (B) at each
28 post germination stage.
29 Figure 3
30 190x107mm (300 x 300 DPI)
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26 Figure 4. K-means clustering (A) and protein expression profiles (B) of all proteins involved in lipid
27 accumulation and degradation. The nine clusters are shown in B (K1 - K10).
28
29 Figure 4
30 190x107mm (300 x 300 DPI)
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26 Figure 5. Expression profiles of genes involved in lipid accumulation and degradation at each stage of peanut
27 seed development. qRT-PCR analysis of cDNA isolated from developing seeds at 20, 40, 60 and 80 DAF.
28 Relative expression ratios of each sample were compared with samples obtained at 80 DAF. Data represent
29 the means SD of three replicates. The actin gene from peanut was used as an internal standard.
30 Abbreviations of gene names are as described in Table 1. ACCA, FAD2, SAD, KAS I, KAS II, KAS III, oleosin,
LOX, PLD, ACOX, GDSL represent
31
ACCA_CYAME, FAD12_CREAL, STAD6_ARATH, KASC1_HORVU, KASC2_ARATH, FABH_SPIOL,
32 OLEO3_ARATH, LOX5_ARATH, PLCD3_ARATH, ACOX2_CUCMA and GDL76_ARATH, respectively.
33 Figure 5
34 190x107mm (300 x 300 DPI)
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26 Figure 6. The metabolic processes of oil mobilization in development and postgermination stages in peanut
27 seeds. The identified proteins found in these pathways. The icons beside each protein name show the
28 expression profiles of protein during different stages. FA, fatty acid; G3P,Glyceraldehydes-3-
29 phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid ; DAG, diacylglycerol; TAG, triacylglycerol.
30 Figure 6
338x190mm (300 x 300 DPI)
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26 TOC
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28 338x190mm (300 x 300 DPI)
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