Proteomic Analysis of Oil Mobilization in Seed Germination and

Proteomic Analysis of Oil Mobilization in Seed Germination and Postgermination Development of Jatropha curcas Ming-Feng Yang,†,# Yu-Jun Liu,†,# Yun Liu,† Hui Chen,*,† Fan Chen,‡ and Shi-Hua Shen*,† Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, P. R. China and Institute of Genetics and Developmental Biology, The Chinese Academy of Sciences, Beijing 100080, P. R. China Received September 19, 2008

To understand oil mobilization in germinating seeds, we performed ultrastructural observation and proteomic analysis of endosperm in germinating Jatropha curcas seeds. Results showed that the oil mobilization was initiated during germination, and then the oil was consumed for early seedling development. The significant change in abundance of 50 protein spots during germination indicated that several pathways including β-oxidation, glyoxylate cycle, glycolysis, citric acid cycle, gluconeogenesis, and pentose phosphate pathway were involved in the oil mobilization. Keywords: Jatropha curcas • seed • germination • proteome • oil • mobilization

Introduction Seed germination is a critical development stage in the life cycle of seed plants, and it is a complex and multistage process. During germination, the quiescent embryonic cells shift into a metabolically active state in which complex biochemical and physiological changes occur.1 On the basis of water uptake, germination can be divided into three phases: a rapid uptake of water, followed by a plateau phase of water uptake, and a subsequent increase in water content coincident with radicle emergence and resumption of growth. In the past decades, many studies have been carried out on seed germination, mainly through physiological, proteomic or transcriptomic analysis.1-11 These previous studies have provided robust information about several aspects during seed germination, such as the role of gibberellin acid and abscisic acid, radicle emergence, defense, endosperm weakening, and mobilization of energy reserves. An overview of proteins present in the seed germination has been provided by proteomic analyses in some model plant species, such as Arabidopsis thaliana and rice.5,8,10,12 However, proteome study in woody plant seed germination is very limited at present. Only descriptions of European beech and Prunus campanulata were presented recently, focusing on the process of dormancy-breaking.13,14 Germinating seeds, deprived of an efficient mineral-uptake system and photosynthetic apparatus, rely on reserve mobilization for germination and seedling establishment.1,2 The reserves are mainly stored in the form of lipid, protein, and starch in the embryos or endosperm. In oilseeds, the major storage reserve is lipid, found in the form of triacylglycerol (TAG),15 which is stored in small spherical organelles called oil bodies. * To whom correspondence should be addressed. E-mail, (H.C.) chenh@ ibcas.ac.cn, (S.-H.S.) [email protected]; fax, +86-10-62596594; tel, +8610-62836545. † Institute of Botany, The Chinese Academy of Sciences. # These authors contributed equally to this work. ‡ Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences. 10.1021/pr800799s CCC: $40.75

 2009 American Chemical Society

TAG is initially cleaved by lipases, releasing fatty acids that are subsequently broken down by the enzymes of β-oxidation and the glyoxylate cycle.16,17 Efficient storage oil breakdown is essential for successful seedling establishment, which in turn is of paramount importance for plant fitness in the field. Knowledge of the underlying biochemistry and metabolism of the breakdown as well as the synthesis of storage oil is essential for the development of new and improved oilseed crops that not only accumulate high levels of the desired oil, but also use it efficiently to support vigorous seedling growth. Though it is an important subject in seed germination research, the previous studies of storage reserve mobilization were mainly on the function of individual enzymes in the pathways through forward and reverse genetics.18-21 Jatropha curcas is a small tree of 3-6 m in height belonging to the family of Euphorbiaceae.22 Its seed has a high content of oil that can be reformed as biodiesel which is becoming increasingly important as an alternative fuel for diesel engines.22-24 In J. curcas seed, the thin embryo is embedded in a thick endosperm that constitutes more than 90% of the total seed weight. The oil content of endosperm is more than 60%, and can be consumed gradually for early seedling growth. Most of the foregoing researches focused on single or few genes, so the results were still far from comprehensively elucidating the mechanisms of the oil mobilization during germination. In this study, a canonical proteomic approach, in combination with the ultrastructural observation of endosperm, was applied to study oil mobilization during seed germination and postgermination development of J. curcas. The proteomic changes illustrated in this study clearly reflect the dynamic changes in the protein expression pattern associated with oil mobilization during seed germination of J. curcas.

Materials and Methods Plant Material. Mature seeds of J. curcas were collected from Panzhihua, Sichuan Province, China. The seeds imbibed in moist mixture of vermiculite and peat (1:1) at 32 °C in dark. Journal of Proteome Research 2009, 8, 1441–1451 1441 Published on Web 01/16/2009

research articles The seeds were collected at 0, 24, 48, 60, 72, 84 and 96 h after imbibition. The endosperm tissues were stored at -80 °C until used. Tissue Preparation for Transmission Electron Microscopy. For ultrastructural observation, endosperm tissues from dry and germinated seeds were fixed in 2.5% glutaraldehyde in 100 mM phosphate buffer (pH 7.0) for 4 h at room temperature. The samples were washed with phosphate buffer, postfixed in 1% OsO4, rinsed with phosphate buffer (3 × 15 min) and dehydrated by a graded series of acetone (20%, 50%, 70%, 90%, and 100% v/v). After infiltration through a graded acetone/Epon/Spurr’s epoxy resin series, the samples were embedded in 100% (w/v) Spurr’s epoxy resin and polymerized at 60 °C for 24 h. The thin sections (70 nm) cut by a ultramicrotome (Leica MZ6) were collected onto copper grids, poststained with supersaturated uranyl acetate and 0.4% lead citrate, respectively, rinsed for 6 × 15 s with dH2O and viewed under a JEOL JEM-1230 transmission electron microscope. Oil Content and Fatty Acid Composition Analysis. The endosperm tissues were milled using a laboratory attrition mill, and J. curcas oil was extracted with petroleum ether in a Soxhlet apparatus. The solvent was removed from the oil by vacuum evaporation. Moisture and oil contents of the seeds were carried out as described by the Association of Official Analytical Chemists.25 The percentage yield was calculated on a dry weight basis. The methyl ester of the FAs present in the oil was prepared by treating 1 g of oil with 10 mL of sodium methoxide and refluxing at a temperature of 70-90 °C for 1 h, and adding 10 mL of water and 3 to 4 drops of concentrated sulfuric acid. The methyl esters of the oil were extracted with chloroform. The chloroform was then removed by evaporation. The water present in the oil was removed by treating the oil with sodium sulfate. FA composition of the oil was determined using gas chromatography (GC-14C Siematchu, DEGS-diethyl glycol succinate column) with a flame-ionization detector. Nitrogen, hydrogen and oxygen at flow rates of 30, 30 and 300 mL/min, respectively, were used for the analysis. The sample (0.5 µL) was injected into the system at 230 °C injector temperature. The oven temperature was kept at 160 °C for 30 min and then it was gradually increased at 3.0 °C/min up to 230 °C. Relative FA compositions were calculated from three independent biological replicates, and expressed as the percentage that each FA represented of the total measured FAs. The difference of oil content and fatty acid composition between time-points of imbibition was valued by one-way ANOVA test. Protein Extraction. Proteins were extracted using a modified protocol according to Shen et al.26 For each time-point of seed imbibition, endosperm from 10 seeds was ground into fine powder in liquid nitrogen with a precooled mortar and pestle. About 500 mg of each sample was homogenized in 2 mL of homogenization buffer containing 20 mM Tris-HCl (pH 7.5), 250 mM sucrose, 10 mM EGTA, 1 mM PMSF, 1 mM DTT, and 1% Triton X-100. The homogenate was transferred into an Eppendorf tube and centrifuged at 15 000g for 15 min at 4 °C. The supernatant was transferred to a new tube and protein was precipitated using 1/4 volume 50% cold TCA in an icy bath for 30 min. The mixture was centrifuged at 15 000g for 15 min at 4 °C, and the supernatant was discarded. The pellet was washed with acetone three times, centrifuged, and vacuumdried. The dried powder was dissolved in sample buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% ampholine, pH 3.5-10 (GE Healthcare Bio-Science, Little Chalf-ont, U.K.), 1442

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Yang et al. and 1% DTT. Three independent biological replicates were completed for each time-point of seed imbibition (0, 24, 48, 60 and 72 h). Gel Electrophoresis. Isoelectric focusing (IEF) was performed using ready-to-use Immobiline Dry-Strips, linear pH gradient 4-7, length 11 cm (Amersham Biosciences, Uppsala, Sweden), and in-gel sample rehydration method.10 Protein samples (about 600 µg) were loaded in the rehydration step. IEF was run on a Multiphor II electrophoresis unit (Amersham Biosciences) at 20 °C constant temperature; it was run for 1 h at 300 V, 1 h at 600 V, 1 h at 1000 V, 1 h at 8000 V, finally followed by 32 000 Vh, all at 50 µA/strip. After IEF, the immobilized pH gradient (IPG) strips were incubated at room temperature for 15 min in 6 M urea, 30% (w/v) glycerol, 2.5% (w/v) SDS, 1% DTT, 50 mM Tris-HCl, pH 8.6. A second equilibration step was carried out for 15 min in the same buffer with the exception that DTT was replaced by 2.5% iodoacetamide. The strips were sealed at the top of the 1.0 mm vertical second-dimension gel (Ettan Dalt six electrophoresis unit, power supply EPS 3501XL; Amersham Biosciences) with 0.8% agarose in above-mentioned equilibration buffer. SDS-PAGE was carried out on linear 15%. The running buffer contained 0.3% Tris, 1.44% glycine, 0.1% SDS, and running condition was 25 mA/gel constant until the bromophenol blue reached the bottom of the gel. Molecular weight markers were broad-ranged (Bio-Rad). Gels were stained with CBB R-250 for about 1 h followed by partial destaining with 25% methanol and 8% acetic acid in deionized water. Reproducibility of the 2-D gels was ensured by 5 technical replicates for each time-point of seed imbibition. Image and Data Analysis. The stained gels were scanned using UMAX Power Look 2100XL scanner (UMAX, Inc., Taipei, China). The data and comparative analysis was performed using Image Master 2D-platinum version 5.0 software (GE Healthcare BIO-Science, Little Chalf-ont, U.K.). Three images representing three independent biological replicates for each state of 0, 24, 48, 60 and 72 h of seed imbibition were grouped to calculate the averaged volume of all the individual protein spots. The abundance of spots was normalized as relative intensity according to the normalization method provided by the software; that is, each spot volume value was divided by the sum of total spot volume values to obtain individual relative spot volumes. The spots that changed in abundance more than 2-fold between 0 h and any time-point imbibition and passed the Student’s t test (p < 0.05) were selected for protein identification. Protein Identification. Protein spots were excised from the gels manually and cut into small pieces. Protein digestion was performed according to Shen et al. with slight modification.26 Each small gel piece with protein was destained with 50 mM NH4HCO3 in 50% (v/v) methanol for 1 h at 40 °C twice. The protein in the gel piece was reduced with 10 mM EDTA, 10 mM DTT in 100 mM NH4HCO3 for 1 h at 60 °C and incubated with 10 mM EDTA, 40 mM iodoacetamide in 100 mM NH4HCO3 for 30 min at room temperature in the dark. The gel pieces were minced and lyophilized, then rehydrated in 25 mM NH4HCO3 with 10 ng of sequencing grade modified trypsin (Promega, Madison, WI) at 37 °C overnight. After digestion, the protein peptides were collected, and the gels were washed with 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile thrice to collect the remaining peptides. The peptides were desalted by ZipTipC 18 pipet tips (Millipore, Bedford, MA) and cocrystallized with 1 vol of saturated R-cyano-4-hydroxycinnamic acid in 50% (v/v) acetonitrile containing 1% TFA.

research articles

Proteomic Analysis of Oil Mobilization

Figure 1. J. curcas seed germination process. The seeds imbibed in moisture at 32 °C in dark. Photographs were taken at 0, 24, 48, 60, 72, 84 and 96 h after imbibition. Bar equals 1.0 cm.

The desalted protein samples were subject to LTQ-ESI-MS/ MS (ThermoFinnigan, San Jose, CA), using a surveyor highperformance liquid chromatography (HPLC) system. LC-MS/ MS analysis was performed in data-dependent MS/MS scan mode controlled by BioWorks 3.1 software suite (ThermoFinnigan). The system was fitted with a C18 RP column (0.15 mm × 150 mm, Thermo Hypersil-Keystone). Mobile phase A (0.1% formic acid in water) and the mobile phase B (0.1% formic acid in ACN) were selected. The tryptic peptide mixtures were eluted using a gradient of 2-98% B over 60 min. The temperature of the heated capillary was set at 170 °C. A voltage of 3.0 kV applied to the ESI needle resulted in a distinct signal. The normalized collision energy was 35.0. The number of ions stored in the ion trap was regulated by the automatic gain control. Voltages across the capillary and the quadrupole lenses were tuned by an automated procedure to maximize the signal for the ion of interest. The LTQ mass spectrometer was set so that one full MS scan (m/z 400-2000) was followed by 10 MS/ MS scans on the 10 most intense ions from the MS spectrum. Dynamic Exclusion was set at repeat count 2; repeat duration 30 s, exclusion duration 90 s. For protein identification, the acquired MS/MS spectra were automatically searched against the NCBInr database (as of May 2007) using the Turbo SEQUEST program in the BioWorks 3.1 software suite (Thermo-Finnigan). Search parameters were set as taxonomy, Rosids; enzyme specificity considered, trypsin; max missed cleavages, 1; fixed modifications, carbamidomethyl (C); variable modifications, oxidation (M); peptide mass tolerance, (1.5 Da; fragment mass tolerance, (0.0 Da. To minimize the inclusion of false positive hits, matches to peptides identified by SEQUEST were filtered according to their charge state, cross-correlation score (Xcorr) and normalized difference in correlation score (deltaCn). Peptide hits were accepted when singly, doubly and triply charged peptides with Xcorr >1.9, 2.2 and 3.75, respectively; and deltaCn > 0.1 in all cases. After the peptide sequence raw data was searched using SEQUEST, a number of other criteria were considered in the final assignment of peptide and protein identifications: the number of matching peptides, the coverage, the Xcorr, and the Mr and pI of the protein.

Figure 2. Changes of moisture and oil contents (%) on dry weight basis of J. curcas endosperm in the imbibition process. Values are means of three biological replicates ((SD).

Results Seed Germination. The seeds of J. curcas imbibed in moisture for germination. After 24 h imbibition, the rigid black testa ruptured, and then hypocotyl elongation and radicle protrusion were observed after 48 h imbibition. This process is termed as germination (Figure 1). Subsequently, the hypocotyl and radicles continued elongation to push cotyledon out of earth, and the testa broke away from endosperm after 96 h imbibition (Figure 1). To obtain an overview of oil reserve mobilization, postgerminative growth until 96 h imbibition was included in this work. The content of moisture increased remarkably from 4.3% to 34.3% during the first 24 h imbibition, then experienced a stage of slow increase to 45.8% until 48 h imbibition, and increased rapidly again to 81.6% until 96 h imbibition (Figure 2). The S-shape curve based on water uptake is different from that of other species such as rice, whose plateau phase of water uptake is longer and more obvious,10 indicating the significant difference of hydration between oilseed and starch seed during germination. Oil Content and FA Composition. The oil content of J. curcas endosperm underwent significant changes only at postgermination stage (Figure 2). The oil content was stable at germinaJournal of Proteome Research • Vol. 8, No. 3, 2009 1443

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Table 1. Changes of Fatty Acid Composition (%) for J. curcas Seed Oil in the Germination and Postgermination Processa samples fatty acid

0h

24 h

48 h

60 h

72 h

84 h

96 h

C16:0b C16:1 C18:0 C18:1 C18:2 C18:3 C20:2 C22:1

12.9 ( 0.1 0.7 ( 0.1 6.4 ( 0.2 41.3 ( 0.6 38.0 ( 0.8 0.2 ( 0.1 0.2 ( 0.0 0.2 ( 0.0

14.3 ( 0.5 0.9 ( 0.1 6.2 ( 0.5 36.9 ( 0.8 41.0 ( 0.9 0.3 ( 0.1 0.2 ( 0.0 0.2 ( 0.0

14.1 ( 0.3 0.9 ( 0.2 5.7 ( 0.4 38.0 ( 0.5 40.4 ( 1.3 0.2 ( 0.1 0.2 ( 0.1 0.4 ( 0.1

14.5 ( 0.2 0.9 ( 0.1 7.3 ( 0.6 38.2 ( 1.1 37.3 ( 0.6 0.3 ( 0.1 0.4 ( 0.0 1.0 ( 0.2

13.9 ( 0.1 0.8 ( 0.1 8.3 ( 0.5 40.6 ( 0.5 34.9 ( 0.9 0.4 ( 0.1 0.4 ( 0.0 0.5 ( 0.1

14.4 ( 0.5 0.7 ( 0.1 7.9 ( 0.5 39.7 ( 1.5 35.1 ( 0.6 0.3 ( 0.1 0.4 ( 0.1 1.5 ( 0.3

14.7 ( 0.3 0.6 ( 0.1 9.5 ( 0.3 46.5 ( 0.6 26.3 ( 1.1 – 1.1 ( 0.3 1.2 ( 0.1

a Content of each fatty acid was calculated as the percentage that each fatty acid represented in the total measured fatty acids. The “-” denotes that data are undetectable. Each value is the mean of three biological replicates ((SD). b The numbers denote the number of carbons and double bonds. For example, C18:1 stands for 18 carbons and one double bond.

tion stage. However, it decreased sharply after 60 h imbibition. Compared to 62.6% at 0 h imbibition, the oil content was decreased to 33.7% at 96 h imbibtion (p < 0.01) (Figure 2), and 2 days later, almost all the oil in endosperm was consumed (data not shown), indicating oil was mobilized and consumed during the germination and postgermination. The FA composition of the crude oil from different stages of germinating seeds was determined (Table 1). The J. curcas seeds contained oleic acid (C18:1) and linoleic acid (C18:2) in the highest amount, followed by palmitic acid (C16:0) and stearic acid (C18:0). The content of long-chain FA (C > 20) was about 0.2%. The low content of long-chain FA (C > 20), as well as the high content of linoleic acid (C18:2), is an important characteristic of J. curcas seed oil to be used as biodiesel.27-29 Starting at 48 h imbibition, the content of linoleic acid (C18:2) decreased from 40.7% to 26.0% of total FAs at 96 h (p < 0.01), and linolenic acid was undetectable at 96 h, while no drastic decrease was observed for other FAs. These results indicate that polyunsaturated FAs, especially the main polyunsaturated FA (C18:2), were mobilized in preference to other FAs in the J. curcas seed. Ultrastructure of Endosperm Cell. To understand the mobilization of oil reserve at cytological level, endosperm tissues from germinating seeds were prepared for ultrastructural observation. In the endosperm cell from seeds of 0 h imbibition, the most obvious morphological structures were several protein storage vacuoles and many oil bodies (Figure 3, 0 h). These oil bodies occupied most of the cell area, and distributed uniformly and ranged in size from about 0.2 to 3.0 µm. An obvious decrease in the amount of oil bodies was observed at 48 h imbibition, and this trend continued until only a few oil bodies remained in the cell after 96 h imbibition (Figure 3, 96 h). The protein storage vacuoles enlarged after 24 h imbibition, probably due to imbibition of water (Figure 3, 24 h). The vacuoles kept enlarging until a large central vacuole was formed (Figure 3, 48-96 h). The glyoxysomes and mitochondria were observed after 48 h imbibition, and their amount increased thereafter (Figure 3, 48 and 60 h). In brief, after 48 h imbibition, the amount of oil bodies declined continually, which was accompanied by the increase in the number of glyoxysome and mitochondria. Proteome Analyses. Proteins extracted from the endosperm of seeds imbibed for 0, 24, 48, 60 and 72 h were separated by 2-D gel electrophoresis. Approximately 1000 protein spots ranged from 12 to 97 kDa were detected in each gel (Figure 4). During imbibition, a total of 138 protein spots changed more than 2-fold in abundance and were significant statistically (p < 0.05) (Figure 4). These spots were identified through LTQESI-MS/MS and NCBI database searching. Of the 138 changed 1444


Figure 3. Ultrastructural observation of J. curcas seeds in the imbibition process. Seeds at different stages of imbibition (0, 24, 48, 60, 72, and 96 h) were prepared for ultrastructural observation. O, oil body; PSV, protein storage vacuole; P, protein particle; V, vacuole; G, glyoxysome; M, mitochondrion.

spots, 50 spots identified were categorized into 5 groups, including signal-related proteins (6 spots), oil mobilizationrelated proteins (17 spots), ATP synthases (6 spots), oxidative stress-related proteins (14 spots), and some other proteins (7 spots) (Table 2). Enlargement of the 50 spots is displayed in Figure 5. The changed proteins involved in signal transduction and regulation are 1-aminocyclopropane-1-carboxylate oxidases


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Figure 4. The 2-DE maps of J. curcas seeds in the germination process. Arrows indicate the 50 proteins identified by MS/MS, which changed in abundance more than 2-fold between 0 h and any other time-point of imbibition. D stands for the down-regulated protein, U stands for the up-regulated protein. This is a representative figure from three biological replicates.

(ACC oxidases, U29 and U30) and 14-3-3 proteins (U33, U36, U37 and D9). All these proteins were up-regulated except for D9. Oil body consists of a hydrophobic matrix of TAG, surrounded by a half-unit phospholipid membrane containing oleosin.18,30 In the imbibition of J. curcas seed, oleosin (D17) was down-regulated significantly. After 60 h imbibition, little oleosin was left in endosperm tissue (Figures 5 and 6). It suggested that the half-unit membrane of oil bodies might be destroyed during germination and postgermination. Oil mobilization-related proteins are mainly enzymes involved in the mobilization of oil. These enzymes function in glyoxylate cycle, glycolysis, citric acid cycle, gluconeogenesis, and pentose phosphate pathway. In this study, most of them (U20, U21, U22, U23, U24, U26, U27, U28, U31, U32, U40, U41, U45, U47 and U49) were up-regulated in the seed imbibition, whereas two spots (D10 and D18) were down-regulated. Several enzymes in the glyoxylate cycle, such as malate dehydrogenase (U26, U27 and U28), isocitrate lyase (U32) and aconitase (U47) increased 3.3- to 10-fold (p < 0.05) in abundance during the imbibition (Figures 5 and 6). Among these enzymes, isocitrate lyase (U32) exists only in glyoxysome and it is a key enzyme in this pathway for catalyzing the conversion of isocitric acid into glyoxylate and succinic acid. The citric acid cycle shares malate dehydrogenase (U26, U27 and U28) and aconitase (U47) with

glyoxylate cycle. However, isocitrate dehydrogenase (U22), a unique and key enzyme in citric acid cycle, was found upregulated 3.9-fold (p < 0.01) after 72 h imbibition compared to 0 h (Figure 6). The significant up-regulation of the enzymes involved in glyoxylate cycle and citric acid cycle during imbibition, including isocitrate lyase (U32) and isocitrate dehydrogenase (U22), suggested that both pathways were getting active in the germination and postgermination process. Most of the enzymes in the gluconeogenesis pathway increased during imbibition, as occurs for enolase (U20), phosphoglycerate mutase (U31), phosphoglycerate kinase (U23 and U49), triosephosphate isomerase (U40 and U41), and cytosolic aldolase (U24) (Figures 5 and 6). The up-regulation of these proteins indicated that gluconeogenesis has been activated. Two enzymes involved in pentose phosphate pathway, 6-phosphogluconate dehydrogenase (U21) and ribulose-5-phosphate-3epimerase (U45), were up-regulated during imbibition. It suggested that pentose phosphate pathway were also active in imbibition. Two enzymes (D10 and D18) involved in oil mobilization were down-regulated. However, they are not key enzymes in the pathways, and their isoforms were up-regulated significantly. The mitochondrial ATP synthase alpha-subunit (D1) and mitochondrial ATP synthase beta-subunit (D3, D4, D5, D6 and D7) were down-regulated. Beta-ketoacyl-ACP synthase I (D2) Journal of Proteome Research • Vol. 8, No. 3, 2009 1445

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Table 2. Differential Protein Spots Identified from Seeds of J. curcas during Imbibition by LTQ-ESI-MS/MS spot no.

Mr/pI

D9 U29 U30 U33 U36 U37

29697/4.75 36034/5.57 36050/5.57 29831/4.75 29831/4.75 29697/4.75

Mr (kDa)/ pI exp P

30/4.89 37/5.53 36/5.53 33/4.75 31/4.60 30/4.68

3 5 5 3 6 10

cov (%) Xcorr

15.65 22.96 19.81 9.85 34.85 35.88

4.35 6.18 6.14 4.34 5.63 4.59

protein name

accession no.

Signal-Related Proteins (6) 14-3-3 family protein 1-aminocyclopropane-1-carboxylate oxidase ACC oxidase 3 14-3-3 protein 14-3-3 protein 14-3-3 family protein

AAV50005.1 AAP41850.1 CAN85571.1 AAY67798.1 AAY67798.1 AAV50005.1

peptide identified by LTQ-ESI-MS/MS

K.SAQDIALAELAPTHPIR.L K.LAEELLDLLCENLGLEK.G K.LAEELLDLLCENLGLEK.G K.TVDVEELTVEER.N R.DNLTLWTSDITDEAGDEIK.D K.SAQDIALAELAPTHPIR.L

D18 U20 U21 U22 U23 U24 U26 U27 U28 U31 U32 U40 U41 U45 U47 U49

15870/6.59 47912/5.56 56373/5.55 47242/6.14 42486/5.83 38514/6.93 35526/6.32 35675/6.33 35593/6.01 60818/5.52 64611/6.73 27088/5.87 27088/5.87 29898/8.3 98879/6.01 23661/6.5

13/5.22 58/5.36 50/6.11 47/6.52 45/5.79 44/6.24 38/6.35 38/6.50 38/6.28 33/5.49 33/6.27 29/5.29 29/5.51 26/5.88 16/4.87 15/5.01

1 5 7 8 13 6 4 6 8 5 3 2 3 2 2 2

9.46 21.35 15.26 20 35.91 16.48 13.55 22.89 26.81 12.95 4.86 8.66 13.78 12.46 3.22 7.86

Oil Mobilization-Related Proteins (17) 4.97 2,3-bisphosphoglycerate-independent AAM61601.1 phosphoglycerate mutase 4.24 fructose-bisphosphate aldolase BAA76430.1 5.73 enolase CAA82232.1 6.08 6-phosphogluconate dehydrogenase BAA22812.1 5.40 cytosolic NADP+-isocitrate dehydrogenase ABA18651.1 5.95 cytosolic phosphoglycerate kinase 1 BAA33801.1 5.00 cytosolic aldolase AAG21429.1 5.27 cytosolic malate dehydrogenase AAS18241.1 5.57 cytosolic malate dehydrogenase BAA97412.1 5.26 cytosolic malate dehydrogenase ABB36659.1 5.22 phosphoglycerate mutase CAA49995.1 3.47 isocitrate lyase CAA84632.1 4.60 triose-phosphate isomerase CAI43251.1 4.26 triose-phosphate isomerase CAI43251.1 3.06 ribulose-5-phosphate-3-epimerase AAM19354.1 4.90 putative aconitase AAL13084.1 3.13 phosphoglycerate kinase BAA21478.1

D1 D3 D4 D5 D6 D7

55330/6.23 60258/5.95 60258/5.95 60258/5.95 60258/5.95 60258/5.95

63/5.95 61/5.51 61/5.27 59/5.32 59/5.38 59/5.42

4 4 7 10 9 4

8.86 9.07 15.84 21.00 23.31 10.32

4.36 5.31 4.88 4.88 5.24 5.38

D12 D13 D14 D15

22915/6.06 20658/5.79 18531/6.13 18581/5.72

25/6.28 19/5.30 19/6.26 18/5.92

2 1 1 1

7.80 5.45 7.14 7.19

4.97 2.18 2.26 2.50

D16 U34 U35 U38 U39 U42 U43 U44 U48 U50

18531/6.13 27283/5.69 27051/5.51 26710/4.97 27051/5.51 25454/8.62 25839/7.1 23462/5.74 15195/5.47 18531/6.13

18/6.11 31/5.37 31/5.54 30/5.08 30/5.68 28/6.09 27/6.28 27/5.64 16/6.23 15/5.38

6 5 2 3 2 4 4 1 3 2

27.38 26.4 9.2 10.68 9.2 22.81 21.03 3.5 23.03 11.31

3.82 4.42 4.24 3.80 3.82 5.54 5.04 2.02 4.29 2.90

Oxidative Stress-Related Proteins (14) IgE-binding protein MnSOD CAC13961.1 R.LVVETTANQDPLVTK.G superoxide dismutase AAR10812.1 R.LACGVVGLTPV.-1 glutathione peroxidase AAQ03092.1 R.YAPTTSPLSIEK.D phospholipid hydroperoxide CAE46896.1 R.YAPTTSPLSIEK.D glutathione peroxidase glutathione peroxidase AAQ03092.1 K.GNDVDLSTYKGK.V cytosolic ascorbate peroxidase AAB95222.1 R.EVFGKTMGLSDQDIVALSGGHTLGR.A3 cytosolic ascorbate peroxidase 1 BAC92739.1 K.ALLSDPVFRPLVDK. Y glutathione S-transferase BAC81649.1 K.LAAWIEELNK.I cytosolic ascorbate peroxidase 1 BAC92739.1 K.ALLSDPVFRPLVDK.Y manganese superoxide dismutase 1 CAB56851.1 K.KLVVETTANQDPLVTK.G Superoxide dismutase [Mn] P35017.1 K.KLVVETTANQDPLVTK.G glutathione S-transferase GST 9 AAG34799.1 K.EFISIFK.Q1 CuZn-superoxide dismutase BAD51400.1 R.AVVVHADPDDLGK.G glutathione peroxidase AAQ03092.1 R.GNDVDLSTYK.G

D2 D8 D11 D17 U19 U25 U46

49773/6.86 56059/6.19 28099/5.81 16886/9.72 71016/5.11 39422/6.24 26946/7.75

63/6.09 57/5.42 28/5.45 16/6.29 65/5.02 42/5.48 16/5.44

7 3 6 1 14 6 1

22.65 8.59 10.20 6.88 24.65 16.18 2.86

5.55 4.22 4.45 2.53 6.16 4.34 2.12

Miscellaneous (7) beta-ketoacyl-ACP synthase I ADP-glucose pyrophosphorylase caleosin 16.9 kDa oleosin 70 kDa heat shock cognate protein 1 type IIIa membrane protein cp-wap13 p-type H+-ATPase

D10 60699/5.51 30/6.49

5 9.29

mitochondrial mitochondrial mitochondrial mitochondrial mitochondrial mitochondrial

Mitochondrial ATP Synthases ATP synthase alpha-subunit ATP synthase beta-subunit ATP synthase beta-subunit ATP synthase beta-subunit ATP synthase beta-subunit ATP synthase beta-subunit

(6) Q01915 CAA41401.1 CAA41401.1 CAA41401.1 CAA41401.1 CAA41401.1

ABJ90468.1 CAA54260.1 ABB05052.1 AAM46777.1 AAS57912.1 AAB61672.1 CAC28223.1

R.GWDAQVLGEAPHK.F K.GILAADESTGTIGK.R R.SGETEDTFIADLSVGLATGQIK.T K.GDCIIDGGNEWYENTER.R K.GGETSTNSIASIFAWSR.E K.IVAEIPEGGVLLLENVR.F3 K.GILAADESTGTIGK.R K.VLVVANPANTNALILK. E K.NVIIWGNHSSTQYPDVNHATVK.T R.KLSSALSAASSACDHIR.D3 R.SGYFNPEMEEYVEIPSDVGITFNVQPK.M3 R.NNGVDTLAHQK.W K.VIACIGETLEQR.E R.IIYGGSVNGGNCK.E K.VIEAGANALVAGSAVFGAK.D K.INPLVPVDLVIDHSVQVDVAR.S K.KPFAAIVGGSK.V R.AAELTTLLESR.I K.CALVYGQMNEPPGAR.A R.FTQANSEVSALLGR.I K.CALVYGQMNEPPGAR.A K.CALVYGQMNEPPGAR.A R.IPSAVGYQPTLATDLGGLQER.I

K.AITTGWLHPTINQFNPEPSVEFDTVANKK.Q3 K.IYVLTQFNSASLNR.H R.CFDGSLFEYCAK.I R.MQDMAGYVGQK.T K.EQVFSTYSDNQPGVLIQVYEGER.T3 K.YIYTIDDDCFVAK.N K.IHAIIDK.F1

a The spot numbers correspond those given in Figures 4, 5, 6 and 7. U, up-regulated protein; D, down-regulated protein; Mr/pI, theoretical values for molecular weight and isoelectric point; Mr (kDa)/pI exp, experimental molecular mass (in kilodaltons) and isoelectric point, which were calculated from the gel in Figure 4; P, number of unique matched peptides; cov (%), percentage of coverage of the identified proteins; Xcorr, SEQUEST cross-correlation score of the peptide (for each protein, only the peptide with the highest Xcorr is presented). Superscript number (1 or 3) after peptide sequence means singly or triply charged state respectively, and those without superscript are doubly charged peptides. All the protein identities were from searching in NCBInr database.

and ADP-glucose pyrophosphorylase (D8), participating in lipid and starch syntheses respectively, were down-regulated. Both 70 kDa heat shock cognate protein (U19) and type IIIa membrane protein cp-wap13 (U25) have been identified as plasmodesmata-associated proteins in previous studies.31,32 Their up-regulation can facilitate the processes involved in cellto-cell transport of macromolecules. A total of 14 spots were oxidative stress-related proteins, including cytosolic ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione S-transferase (GST) and glutathione peroxidase. Most of these protein spots, 9 spots out 1446


of 14, were up-regulated. APX (U34, U35, and U39), which reduces hydrogen peroxide using ascorbate as an electron donor, was up-regulated. Of 5 spots representing SOD, D12 and D13 disappeared, while U48, U42 and U43 increased during imbibition. Their positions on the 2-DE map suggested that the increased ones may have resulted from the proteolysis or modification. Two spots (U38 and U44) identified as glutathione S-transferase were up-regulated in the process of imbibition. Four changed spots were identified as glutathione peroxidase, including one up-regulated spot (U50) and three down-regulated spots (D14, D15 and D16).

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Figure 5. Enlargement parts of Figure 4 to compare the abundance of the 50 protein spots.

Discussion Initiation of Oil Mobilization in Germination. The seed germination and the subsequent seedling growth need large amounts of energy and nutrition, which can be provided only by seed reserves, because the germinating seed lacks a mineral uptake system and photosynthetic apparatus.2 The reserves are mainly in the form of oil, protein, and starch in embryo or endosperm, and their relative amounts vary in different species. In the endosperm of J. curcas seed, oil is the most abundant reserve (62.6%). However, the oil content decreased to 33.7% after 96 h imbibition (Figure 2), and after 6-day imbibition, little oil was left in the wizen endosperm (data not shown). It

suggests that oil is mobilized and consumed during the germination and postgermination stages. In the endosperm cell, the obvious declination of the oil body amount was found at 48 h imbibition (Figure 3, 48 h). It suggested that oil mobilization had started earlier than 48 h of imbibition. The up-regulation of some lipid catabolism-associated enzymes before 48 h imbibition gave a strong support to this notion at the molecular level. For example, the abundance of U27 and U32 involved in glyoxylate cycle pathway increased more than 2.5-fold (p < 0.01) compared with 0 h imbibition (Figures 5 and 6). The major protein constituent of oil body is oleosin protein, and its mobilization is a prerequisite for Journal of Proteome Research • Vol. 8, No. 3, 2009 1447

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Yang et al.

Figure 6. Relative abundance of the 50 protein spots in Figure 5. For each spot, the maximum abundance during imbibition process was set as 1, and other time-points were expressed as a proportion of the maximum abundance. Each value is from an average of three independent biological replicates ((SD).

subsequent oil mobilization in some oilseeds.30 Thus, the down-regulation of oleosin might be a sign of the initiation of the oil mobilization. In this work, oleosin (D17) displayed a significant down-regulation from 0 to 48 h imbibition (Figures 5 and 6). Thus, the mobilization of oil in endosperm of J. curcas seed is initiated during germination. The oil content in endosperm displayed a drastic decrease after 60 h imbibition (Figure 2), indicating that the mobilized oil in endosperm was transferred to embryo for building up seedlings, especially the elongation of hypocotyls and roots (Figure 1, 60 h). These results imply that large-scale lipid mobilization is initiated during germination, and the mobilized oil is used in postgermination for establishment of seedlings. This agrees with the observations that germination is largely driven by the metabolism of storage reserves other than lipids, while seed oil is used for subsequent seedling establishment.20,33 Enzymes Involved in Oil Mobilization. The oil mobilization progress should be tightly regulated by internal factors, such as plant hormones. Previous research has showed that ethylene performs a relatively vital role in dormancy release and seed germination of many plant species.34 ACC oxidase is one of the key enzymes in the synthesis of ethylene. The up-regulation of ACC oxidase (U29 and U30) indicates that ethylene is an endogenous stimulator of germination of J. curcas seed. 143-3 proteins are ubiquitous eukaryotic regulatory proteins which can lead to enzyme activation or deactivation, and they play key regulatory roles in many cellular processes including seed germination.35 In germinating barley seeds, the protein levels of all three isoforms of 14-3-3 were constant during germination; mRNA expression was found to be induced upon imbibition of the grains.36 In this paper, the protein level of most of the 14-3-3 isoforms was up-regulated significantly during germination. It suggests that 14-3-3s might act as regulatory proteins during the germination of J. curcas seed. Caleosin is a Ca2+-binding oil body surface protein, and it might participate in oil body-vacuole interactions that affect breakdown of oil bodies during germination.18 Though no 1448


direct evidence was observed for the participation of vacuole in the oil mobilization, vacuole might play some role in the oil mobilization, given the fact that the vacuole kept enlarging when the number of oil bodies decreased, and the vacuole was surrounded tightly by oil bodies (Figure 3). In plants, there are three possible metabolic pathways involved in the mobilization of storage lipids during seed germination: classical glyoxysomal or peroxisomal degradation of linoleic acid, and two newfound pathways, lipoxygenasedependent degradation of polyenoic FAs, and hypothetical FA CoA-synthetase-independent pathway.37 Lipoxygenase is specific for the oxidation of polyunsaturated FA in the lipoxygenase-dependent pathway. In some plants, it was shown that lipase preferentially degrades the polyunsaturated FA oxidated by lipoxygenase.38 Polyunsaturated FAs were preferentially mobilized compared with other FAs in J. curcas endosperm during germination (Table 1). Taken together, we suggest that lipoxygenase-dependent degradation should be an important pathway for oil mobilization during J. curcas seed germination. Many enzymes involved in glyoxylate cycle, glycolysis, citric acid cycle, gluconeogenesis, and pentose phosphate pathway were up-regulated during germination and postgermination. Especially the isocitrate lyase (U32) and isocitrate dehydrogenase (U22), which are specific and critical enzymes in glyoxylate cycle and citric acid cycle, respectively, were both up-regulated more than 3.9-fold (p < 0.01) during imbibiton (Figure 6). These results suggested that both glyoxylate cycle and citric acid cycle are getting active in the germination process. The glyoxylate cycle plays a fundamental role in oil mobilization and produces 2-carbon compounds such as acetate or ethanol, ultimately providing substrates for biosynthetic processes and respiration.39 Thus, this pathway is essential for postgerminative growth and seedling establishment in oilseed plants. The upregulation of isocitrate lyase was also found in the germination of Arabidopsis seed.39 The down-regulation of the alpha-subunit and beta-subunit of mitochondrial ATP synthase suggests that the up-regulation


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Figure 7. Mobilization of storage oil during germination and postgermination. The triacylglycerols in the oil bodies are hydrolyzed under catalyzation of lipases into fatty acids and glycerol (a), or are oxidated under catalyzation of lipoxygenase into hydroperoxides first (b-1) and then hydrolyzed under catalyzation of lipases into fatty acids and glycerol (b-2). The glycerol can be fed into the gluconeogenesis pathway after oxidation to dyhydroxyacetone (c). Fatty acids are activated in the glyoxysomes as CoA-thioesters and degraded by β-oxidation into acetyl CoA (d). From two molecules of acetyl CoA, the glyoxylate cycle forms one molecule of succinate (e), which is converted by the citrate cycle in the mitochondria to malate (f). Phosphoenolpyruvate formed from malate in the cytosol is a precursor for the synthesis of hexoses via the gluconeogenesis pathway (g). The intermediates formed in pentose phosphate pathway (h) can be used for biosyntheses of other compounds. The identified protein spots related to oil mobilization, pathways involved in oil mobilization, and key steps of the oil mobilization are highlighted by red, green, and blue, respectively.

of enzymes involved in citric acid cycle is not totally for energy, but for producing more substrates for subsequent gluconeogenesis. Most of enzymes involved in gluconeogenesis were also up-regulated, which paved the pathway for producing glucose as energy and nutrition source for building new seedlings. In addition, the activation of pentose phosphate pathway, inferred from the up-regulation of 6-phosphogluconate dehydrogenase (U21) and ribulose-5-phosphate-3-epimerase (U45), provides more intermediates for biochemical reactions. Fourteen spots were identified as oxidative stress-related proteins (APX, SOD, GST and glutathione peroxidase) which fluctuated in abundance during the germinating process. The up-regulation of APX agrees with that the level of APX mRNA in barley embryo increased in late germination.40 During the early stage of imbibition, the rupture of J. curcas testa should be accompanied by water infiltration and oxygen diffusion. In addition, many oxidation reactions started during the mobilization of oil should result in active oxygen species. A previous study showed that a great amount of hydrogen peroxide (H2O2)

generated from FA β-oxidation as a byproduct in glyoxysomes of oilseeds.41 Therefore, many oxidative stress-related proteins were mobilized to protect enzymes and other molecules from oxidative stress. Metabolisms of Oil Mobilization. On the basis of the results in this study and extant data,20 a sketch map of the metabolic processes of oil mobilization is shown in Figure 7. After J. curcas seed imbibition in moisture, ethylene is induced to break the dormancy of the seeds. Some regulatory proteins such as 143-3 might be involved in the activation or deactivation of enzymes. Proteinase and lipase are induced to break the oleosin and the lipids of the half-unit membrane of oil body, leading to breakdown of the membrane. Then, triacylglycerol is hydrolyzed by lipases through classical glyoxysomal (Figure 7a) and/or lipoxygenase-dependent pathways (Figure 7b) into glycerol and FAs. After oxidation to dyhydroxyacetone, the glycerol can be fed into the gluconeogenesis pathway (Figure 7c). Because of the preference of lipoxygenase to polyunsaturated FAs (Figure 7b-1) and the preference of lipase to hydroJournal of Proteome Research • Vol. 8, No. 3, 2009 1449

research articles peroxides (Figure 7b-2), which is a product from oxidation of polyunsaturated FAs under lipoxygenase catalyzation, the polyunsaturated FAs are preferably released from TAG during germination (Figure 7b-2). The released free FAs are degraded to acetyl CoA by β-oxidation in glyoxysomes (Figure 7d). Two molecules of acetyl CoA generate one molecule of succinate in glyoxysome (Figure 7e). The succinate is transferred to the mitochondria and converted there into malate by a partial reaction of the citric cycle (Figure 7f). Then, the malate is released from mitochondria to citosol where it is converted into phosphoenolpyruvate. Phosphoenolpyruvate is a precursor for the synthesis of hexoses by the gluconeogenesis pathway (Figure 7g). The intermediates formed in pentose phosphate pathway (Figure 7h) can be used for biosyntheses of other compounds such as RNA, protein, and so forth. The macromolecules from gluconeogenesis and other processes can be transported to the growing embryo under the help of plasmodesmata-associated proteins.

Concluding Remarks Several pathways, such as β-oxidation, glyoxylate cycle, glycolysis, citric acid cycle, gluconeogenesis, pentose phosphate pathway, were involved in the mobilization. The process was probably fulfilled by the cooperation of many parts of the endosperm cell, such as oil body, glyoxysome, mitochondrion and vacuole. In addition, signal transduction, transport and active-oxygen-cleaning systems were also involved in the complex processes. These results would benefit to further understand the reserve mobilization of oilseeds. Abbreviations: TAG, triacylglycerol; FA, fatty acid; SOD, superoxide dismutase; GST, glutathione S-transferase; APX, ascorbate peroxidase.

Acknowledgment. This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-G-027-2, KSCX2-YW-G-035), the National Natural Science Foundation of China (U0733005). Supporting Information Available: Supplementary Table S1, a comprehensive list of the differential protein spots identified from the seeds of J. curcas during imbibition by LTQESI-MS/MS. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Sheoran, I. S.; Olson, D. J.; Ross, A. R.; Sawhney, V. K. Proteome analysis of embryo and endosperm from germinating tomato seeds. Proteomics 2005, 5, 3752–3764. (2) Bewley, J. D. Seed germination and dormancy. Plant Cell 1997, 9, 1055–1066. (3) Bonsager, B. C.; Finnie, C.; Roepstorff, P.; Svensson, B. Spatiotemporal changes in germination and radical elongation of barley seeds tracked by proteome analysis of dissected embryo, aleurone layer, and endosperm tissues. Proteomics 2007, 7, 4528–4540. (4) Bradford, K. J. A water relations analysis of seed germination rates. Plant Physiol. 1990, 94, 840–849. (5) Gallardo, K.; Job, C.; Groot, S. P.; Puype, M.; Demol, H.; Vandekerckhove, J.; Job, D. Proteomic analysis of Arabidopsis seed germination and priming. Plant Physiol. 2001, 126, 835–848. (6) Muller, K.; Tintelnot, S.; Leubner-Metzger, G. Endosperm-limited Brassicaceae seed germination: abscisic acid inhibits embryoinduced endosperm weakening of Lepidium sativum (cress) and endosperm rupture of cress and Arabidopsis thaliana. Plant Cell Physiol. 2006, 47, 864–877. (7) Nakabayashi, K.; Okamoto, M.; Koshiba, T.; Kamiya, Y.; Nambara, E. Genome-wide profiling of stored mRNA in Arabidopsis thaliana seed germination: epigenetic and genetic regulation of transcription in seed. Plant J. 2005, 41, 697–709.

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