and postgermination development of jatropha curcas ... · proteomic analysis of oil mobilization in...

Subscriber access provided by INST OF BOTANY CAS

Journal of Proteome Research is published by the American Chemical Society.1155 Sixteenth Street N.W., Washington, DC 20036

Article

Proteomic Analysis of Oil Mobilization in Seed Germinationand Postgermination Development of Jatropha curcas

Ming-Feng Yang, Yu-Jun Liu, Yun Liu, Hui Chen, Fan Chen, and Shi-Hua ShenJ. Proteome Res., 2009, 8 (3), 1441-1451• DOI: 10.1021/pr800799s • Publication Date (Web): 16 January 2009

Downloaded from http://pubs.acs.org on April 8, 2009

More About This Article

Additional resources and features associated with this article are available within the HTML version:

• Supporting Information• Access to high resolution figures• Links to articles and content related to this article• Copyright permission to reproduce figures and/or text from this article

http://pubs.acs.org/doi/full/10.1021/pr800799s

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 andDevelopmental 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 andproteomic analysis of endosperm in germinating Jatropha curcas seeds. Results showed that the oilmobilization was initiated during germination, and then the oil was consumed for early seedlingdevelopment. The significant change in abundance of 50 protein spots during germination indicatedthat several pathways including �-oxidation, glyoxylate cycle, glycolysis, citric acid cycle, gluconeo-genesis, 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 lifecycle of seed plants, and it is a complex and multistage process.During germination, the quiescent embryonic cells shift into ametabolically active state in which complex biochemical andphysiological changes occur.1 On the basis of water uptake,germination can be divided into three phases: a rapid uptakeof water, followed by a plateau phase of water uptake, and asubsequent increase in water content coincident with radicleemergence and resumption of growth. In the past decades,many studies have been carried out on seed germination,mainly through physiological, proteomic or transcriptomicanalysis.1-11 These previous studies have provided robustinformation about several aspects during seed germination,such as the role of gibberellin acid and abscisic acid, radicleemergence, defense, endosperm weakening, and mobilizationof energy reserves. An overview of proteins present in the seedgermination has been provided by proteomic analyses in somemodel plant species, such as Arabidopsis thaliana and rice.5,8,10,12

However, proteome study in woody plant seed germination isvery limited at present. Only descriptions of European beechand Prunus campanulata were presented recently, focusing onthe process of dormancy-breaking.13,14

Germinating seeds, deprived of an efficient mineral-uptakesystem and photosynthetic apparatus, rely on reserve mobiliza-tion for germination and seedling establishment.1,2 The reservesare mainly stored in the form of lipid, protein, and starch inthe embryos or endosperm. In oilseeds, the major storagereserve is lipid, found in the form of triacylglycerol (TAG),15

which is stored in small spherical organelles called oil bodies.

TAG is initially cleaved by lipases, releasing fatty acids that aresubsequently broken down by the enzymes of �-oxidation andthe glyoxylate cycle.16,17 Efficient storage oil breakdown isessential for successful seedling establishment, which in turnis of paramount importance for plant fitness in the field.Knowledge of the underlying biochemistry and metabolism ofthe breakdown as well as the synthesis of storage oil is essentialfor the development of new and improved oilseed crops thatnot only accumulate high levels of the desired oil, but also useit efficiently to support vigorous seedling growth. Though it isan important subject in seed germination research, the previousstudies of storage reserve mobilization were mainly on thefunction of individual enzymes in the pathways throughforward and reverse genetics.18-21

Jatropha curcas is a small tree of 3-6 m in height belongingto the family of Euphorbiaceae.22 Its seed has a high contentof oil that can be reformed as biodiesel which is becomingincreasingly important as an alternative fuel for diesel en-gines.22-24 In J. curcas seed, the thin embryo is embedded ina thick endosperm that constitutes more than 90% of the totalseed 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 fewgenes, so the results were still far from comprehensivelyelucidating the mechanisms of the oil mobilization duringgermination. In this study, a canonical proteomic approach,in combination with the ultrastructural observation of en-dosperm, was applied to study oil mobilization during seedgermination and postgermination development of J. curcas. Theproteomic changes illustrated in this study clearly reflect thedynamic changes in the protein expression pattern associatedwith oil mobilization during seed germination of J. curcas.

Materials and Methods

Plant Material. Mature seeds of J. curcas were collected fromPanzhihua, Sichuan Province, China. The seeds imbibed inmoist mixture of vermiculite and peat (1:1) at 32 °C in dark.

* To whom correspondence should be addressed. E-mail, (H.C.) [email protected], (S.-H.S.) [email protected]; fax, +86-10-62596594; tel, +86-10-62836545.

† 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 Journal of Proteome Research 2009, 8, 1441–1451 1441Published on Web 01/16/2009

http://pubs.acs.org/action/showImage?doi=10.1021/pr800799s&iName=master.img-000.png&w=251&h=40

The seeds were collected at 0, 24, 48, 60, 72, 84 and 96 h afterimbibition. The endosperm tissues were stored at -80 °C untilused.

Tissue Preparation for Transmission Electron Micros-copy. For ultrastructural observation, endosperm tissues fromdry and germinated seeds were fixed in 2.5% glutaraldehydein 100 mM phosphate buffer (pH 7.0) for 4 h at roomtemperature. The samples were washed with phosphate buffer,postfixed in 1% OsO4, rinsed with phosphate buffer (3 × 15min) and dehydrated by a graded series of acetone (20%, 50%,70%, 90%, and 100% v/v). After infiltration through a gradedacetone/Epon/Spurr’s epoxy resin series, the samples wereembedded in 100% (w/v) Spurr’s epoxy resin and polymerizedat 60 °C for 24 h. The thin sections (70 nm) cut by aultramicrotome (Leica MZ6) were collected onto copper grids,poststained with supersaturated uranyl acetate and 0.4% leadcitrate, respectively, rinsed for 6 × 15 s with dH2O and viewedunder a JEOL JEM-1230 transmission electron microscope.

Oil Content and Fatty Acid Composition Analysis. Theendosperm tissues were milled using a laboratory attrition mill,and J. curcas oil was extracted with petroleum ether in a Soxhletapparatus. The solvent was removed from the oil by vacuumevaporation. Moisture and oil contents of the seeds werecarried out as described by the Association of Official AnalyticalChemists.25 The percentage yield was calculated on a dryweight basis.

The methyl ester of the FAs present in the oil was preparedby treating 1 g of oil with 10 mL of sodium methoxide andrefluxing at a temperature of 70-90 °C for 1 h, and adding 10mL 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 waterpresent in the oil was removed by treating the oil with sodiumsulfate. FA composition of the oil was determined using gaschromatography (GC-14C Siematchu, DEGS-diethyl glycol suc-cinate 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 thenit was gradually increased at 3.0 °C/min up to 230 °C. RelativeFA compositions were calculated from three independentbiological replicates, and expressed as the percentage that eachFA represented of the total measured FAs. The difference ofoil content and fatty acid composition between time-points ofimbibition was valued by one-way ANOVA test.

Protein Extraction. Proteins were extracted using a modifiedprotocol according to Shen et al.26 For each time-point of seedimbibition, endosperm from 10 seeds was ground into finepowder in liquid nitrogen with a precooled mortar and pestle.About 500 mg of each sample was homogenized in 2 mL ofhomogenization buffer containing 20 mM Tris-HCl (pH 7.5),250 mM sucrose, 10 mM EGTA, 1 mM PMSF, 1 mM DTT, and1% Triton X-100. The homogenate was transferred into anEppendorf tube and centrifuged at 15 000g for 15 min at 4 °C.The supernatant was transferred to a new tube and proteinwas precipitated using 1/4 volume 50% cold TCA in an icy bathfor 30 min. The mixture was centrifuged at 15 000g for 15 minat 4 °C, and the supernatant was discarded. The pellet waswashed with acetone three times, centrifuged, and vacuum-dried. The dried powder was dissolved in sample buffercontaining 7 M urea, 2 M thiourea, 4% CHAPS, 2% ampholine,pH 3.5-10 (GE Healthcare Bio-Science, Little Chalf-ont, U.K.),

and 1% DTT. Three independent biological replicates werecompleted for each time-point of seed imbibition (0, 24, 48,60 and 72 h).

Gel Electrophoresis. Isoelectric focusing (IEF) was performedusing ready-to-use Immobiline Dry-Strips, linear pH gradient4-7, 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 wasrun on a Multiphor II electrophoresis unit (Amersham Bio-sciences) at 20 °C constant temperature; it was run for 1 h at300 V, 1 h at 600 V, 1 h at 1000 V, 1 h at 8000 V, finally followedby 32 000 Vh, all at 50 µA/strip. After IEF, the immobilized pHgradient (IPG) strips were incubated at room temperature for15 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 wascarried out for 15 min in the same buffer with the exceptionthat DTT was replaced by 2.5% iodoacetamide. The strips weresealed 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-mentionedequilibration 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 thebromophenol blue reached the bottom of the gel. Molecularweight markers were broad-ranged (Bio-Rad). Gels were stainedwith CBB R-250 for about 1 h followed by partial destainingwith 25% methanol and 8% acetic acid in deionized water.Reproducibility of the 2-D gels was ensured by 5 technicalreplicates for each time-point of seed imbibition.

Image and Data Analysis. The stained gels were scannedusing UMAX Power Look 2100XL scanner (UMAX, Inc., Taipei,China). The data and comparative analysis was performedusing Image Master 2D-platinum version 5.0 software (GEHealthcare BIO-Science, Little Chalf-ont, U.K.). Three imagesrepresenting three independent biological replicates for eachstate of 0, 24, 48, 60 and 72 h of seed imbibition were groupedto calculate the averaged volume of all the individual proteinspots. The abundance of spots was normalized as relativeintensity according to the normalization method provided bythe software; that is, each spot volume value was divided bythe sum of total spot volume values to obtain individual relativespot volumes. The spots that changed in abundance more than2-fold between 0 h and any time-point imbibition and passedthe Student’s t test (p < 0.05) were selected for proteinidentification.

Protein Identification. Protein spots were excised from thegels manually and cut into small pieces. Protein digestion wasperformed according to Shen et al. with slight modification.26

Each small gel piece with protein was destained with 50 mMNH4HCO3 in 50% (v/v) methanol for 1 h at 40 °C twice. Theprotein in the gel piece was reduced with 10 mM EDTA, 10mM DTT in 100 mM NH4HCO3 for 1 h at 60 °C and incubatedwith 10 mM EDTA, 40 mM iodoacetamide in 100 mM NH4HCO3

for 30 min at room temperature in the dark. The gel pieceswere minced and lyophilized, then rehydrated in 25 mMNH4HCO3 with 10 ng of sequencing grade modified trypsin(Promega, Madison, WI) at 37 °C overnight. After digestion, theprotein peptides were collected, and the gels were washed with0.1% trifluoroacetic acid (TFA) in 50% acetonitrile thrice tocollect the remaining peptides. The peptides were desalted byZipTipC 18 pipet tips (Millipore, Bedford, MA) and cocrystal-lized with 1 vol of saturated R-cyano-4-hydroxycinnamic acidin 50% (v/v) acetonitrile containing 1% TFA.

research articles Yang et al.

1442 Journal of Proteome Research • Vol. 8, No. 3, 2009

The desalted protein samples were subject to LTQ-ESI-MS/MS (ThermoFinnigan, San Jose, CA), using a surveyor high-performance liquid chromatography (HPLC) system. LC-MS/MS analysis was performed in data-dependent MS/MS scanmode controlled by BioWorks 3.1 software suite (Thermo-Finnigan). The system was fitted with a C18 RP column (0.15mm × 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 mixtureswere eluted using a gradient of 2-98% B over 60 min. Thetemperature of the heated capillary was set at 170 °C. A voltageof 3.0 kV applied to the ESI needle resulted in a distinct signal.The normalized collision energy was 35.0. The number of ionsstored in the ion trap was regulated by the automatic gaincontrol. Voltages across the capillary and the quadrupole lenseswere tuned by an automated procedure to maximize the signalfor the ion of interest. The LTQ mass spectrometer was set sothat 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 duration30 s, exclusion duration 90 s.

For protein identification, the acquired MS/MS spectra wereautomatically searched against the NCBInr database (as of May2007) using the Turbo SEQUEST program in the BioWorks 3.1software suite (Thermo-Finnigan). Search parameters were setas taxonomy, Rosids; enzyme specificity considered, trypsin;max missed cleavages, 1; fixed modifications, carbamidomethyl(C); variable modifications, oxidation (M); peptide mass toler-ance, (1.5 Da; fragment mass tolerance, (0.0 Da.

To minimize the inclusion of false positive hits, matches topeptides identified by SEQUEST were filtered according to theircharge state, cross-correlation score (Xcorr) and normalizeddifference in correlation score (deltaCn). Peptide hits wereaccepted when singly, doubly and triply charged peptides withXcorr >1.9, 2.2 and 3.75, respectively; and deltaCn > 0.1 in allcases. After the peptide sequence raw data was searched usingSEQUEST, a number of other criteria were considered in thefinal assignment of peptide and protein identifications: thenumber of matching peptides, the coverage, the Xcorr, andthe Mr and pI of the protein.

Results

Seed Germination. The seeds of J. curcas imbibed inmoisture for germination. After 24 h imbibition, the rigid blacktesta ruptured, and then hypocotyl elongation and radicleprotrusion were observed after 48 h imbibition. This processis termed as germination (Figure 1). Subsequently, the hypo-cotyl and radicles continued elongation to push cotyledon outof earth, and the testa broke away from endosperm after 96 himbibition (Figure 1). To obtain an overview of oil reservemobilization, postgerminative growth until 96 h imbibition wasincluded in this work. The content of moisture increasedremarkably from 4.3% to 34.3% during the first 24 h imbibition,then experienced a stage of slow increase to 45.8% until 48 himbibition, and increased rapidly again to 81.6% until 96 himbibition (Figure 2). The S-shape curve based on water uptakeis different from that of other species such as rice, whoseplateau phase of water uptake is longer and more obvious,10

indicating the significant difference of hydration betweenoilseed and starch seed during germination.

Oil Content and FA Composition. The oil content of J. curcasendosperm underwent significant changes only at postgermi-nation stage (Figure 2). The oil content was stable at germina-

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.

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

Proteomic Analysis of Oil Mobilization research articles

Journal of Proteome Research • Vol. 8, No. 3, 2009 1443

http://pubs.acs.org/action/showImage?doi=10.1021/pr800799s&iName=master.img-001.jpg&w=399&h=222

http://pubs.acs.org/action/showImage?doi=10.1021/pr800799s&iName=master.img-002.png&w=154&h=138

tion stage. However, it decreased sharply after 60 h imbibition.Compared to 62.6% at 0 h imbibition, the oil content wasdecreased to 33.7% at 96 h imbibtion (p < 0.01) (Figure 2), and2 days later, almost all the oil in endosperm was consumed(data not shown), indicating oil was mobilized and consumedduring the germination and postgermination.

The FA composition of the crude oil from different stages ofgerminating seeds was determined (Table 1). The J. curcas seedscontained oleic acid (C18:1) and linoleic acid (C18:2) in the highestamount, followed by palmitic acid (C16:0) and stearic acid (C18:0).The content of long-chain FA (C > 20) was about 0.2%. The lowcontent of long-chain FA (C > 20), as well as the high content oflinoleic acid (C18:2), is an important characteristic of J. curcas seedoil to be used as biodiesel.27-29 Starting at 48 h imbibition, thecontent of linoleic acid (C18:2) decreased from 40.7% to 26.0% oftotal FAs at 96 h (p < 0.01), and linolenic acid was undetectableat 96 h, while no drastic decrease was observed for other FAs.These results indicate that polyunsaturated FAs, especially themain polyunsaturated FA (C18:2), were mobilized in preference toother FAs in the J. curcas seed.

Ultrastructure of Endosperm Cell. To understand themobilization of oil reserve at cytological level, endospermtissues from germinating seeds were prepared for ultrastruc-tural observation. In the endosperm cell from seeds of 0 himbibition, the most obvious morphological structures wereseveral protein storage vacuoles and many oil bodies (Figure3, 0 h). These oil bodies occupied most of the cell area, anddistributed uniformly and ranged in size from about 0.2 to 3.0µm. An obvious decrease in the amount of oil bodies wasobserved at 48 h imbibition, and this trend continued until onlya few oil bodies remained in the cell after 96 h imbibition(Figure 3, 96 h). The protein storage vacuoles enlarged after24 h imbibition, probably due to imbibition of water (Figure3, 24 h). The vacuoles kept enlarging until a large centralvacuole was formed (Figure 3, 48-96 h). The glyoxysomes andmitochondria were observed after 48 h imbibition, and theiramount increased thereafter (Figure 3, 48 and 60 h). In brief,after 48 h imbibition, the amount of oil bodies declinedcontinually, which was accompanied by the increase in thenumber of glyoxysome and mitochondria.

Proteome Analyses. Proteins extracted from the endospermof seeds imbibed for 0, 24, 48, 60 and 72 h were separated by2-D gel electrophoresis. Approximately 1000 protein spotsranged from 12 to 97 kDa were detected in each gel (Figure 4).During imbibition, a total of 138 protein spots changed morethan 2-fold in abundance and were significant statistically (p< 0.05) (Figure 4). These spots were identified through LTQ-ESI-MS/MS and NCBI database searching. Of the 138 changed

spots, 50 spots identified were categorized into 5 groups,including signal-related proteins (6 spots), oil mobilization-related proteins (17 spots), ATP synthases (6 spots), oxidativestress-related proteins (14 spots), and some other proteins (7spots) (Table 2). Enlargement of the 50 spots is displayed inFigure 5.

The changed proteins involved in signal transduction andregulation are 1-aminocyclopropane-1-carboxylate oxidases

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

samples

fatty acid 0 h 24 h 48 h 60 h 72 h 84 h 96 h

C16:0b 12.9 ( 0.1 14.3 ( 0.5 14.1 ( 0.3 14.5 ( 0.2 13.9 ( 0.1 14.4 ( 0.5 14.7 ( 0.3

C16:1 0.7 ( 0.1 0.9 ( 0.1 0.9 ( 0.2 0.9 ( 0.1 0.8 ( 0.1 0.7 ( 0.1 0.6 ( 0.1C18:0 6.4 ( 0.2 6.2 ( 0.5 5.7 ( 0.4 7.3 ( 0.6 8.3 ( 0.5 7.9 ( 0.5 9.5 ( 0.3C18:1 41.3 ( 0.6 36.9 ( 0.8 38.0 ( 0.5 38.2 ( 1.1 40.6 ( 0.5 39.7 ( 1.5 46.5 ( 0.6C18:2 38.0 ( 0.8 41.0 ( 0.9 40.4 ( 1.3 37.3 ( 0.6 34.9 ( 0.9 35.1 ( 0.6 26.3 ( 1.1C18:3 0.2 ( 0.1 0.3 ( 0.1 0.2 ( 0.1 0.3 ( 0.1 0.4 ( 0.1 0.3 ( 0.1 –C20:2 0.2 ( 0.0 0.2 ( 0.0 0.2 ( 0.1 0.4 ( 0.0 0.4 ( 0.0 0.4 ( 0.1 1.1 ( 0.3C22:1 0.2 ( 0.0 0.2 ( 0.0 0.4 ( 0.1 1.0 ( 0.2 0.5 ( 0.1 1.5 ( 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 thatdata are undetectable. Each value is the mean of three biological replicates ((SD). b The numbers denote the number of carbons and double bonds. Forexample, C18:1 stands for 18 carbons and one double bond.

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




(ACC oxidases, U29 and U30) and 14-3-3 proteins (U33, U36,U37 and D9). All these proteins were up-regulated except forD9.

Oil body consists of a hydrophobic matrix of TAG, sur-rounded by a half-unit phospholipid membrane containingoleosin.18,30 In the imbibition of J. curcas seed, oleosin (D17)was down-regulated significantly. After 60 h imbibition, littleoleosin was left in endosperm tissue (Figures 5 and 6). Itsuggested that the half-unit membrane of oil bodies might bedestroyed during germination and postgermination.

Oil mobilization-related proteins are mainly enzymes in-volved in the mobilization of oil. These enzymes function inglyoxylate 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. Severalenzymes 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 theimbibition (Figures 5 and 6). Among these enzymes, isocitratelyase (U32) exists only in glyoxysome and it is a key enzyme inthis pathway for catalyzing the conversion of isocitric acid intoglyoxylate and succinic acid. The citric acid cycle shares malatedehydrogenase (U26, U27 and U28) and aconitase (U47) with

glyoxylate cycle. However, isocitrate dehydrogenase (U22), aunique and key enzyme in citric acid cycle, was found up-regulated 3.9-fold (p < 0.01) after 72 h imbibition comparedto 0 h (Figure 6). The significant up-regulation of the enzymesinvolved in glyoxylate cycle and citric acid cycle during imbibi-tion, including isocitrate lyase (U32) and isocitrate dehydro-genase (U22), suggested that both pathways were getting activein the germination and postgermination process. Most of theenzymes in the gluconeogenesis pathway increased duringimbibition, as occurs for enolase (U20), phosphoglyceratemutase (U31), phosphoglycerate kinase (U23 and U49), triose-phosphate isomerase (U40 and U41), and cytosolic aldolase(U24) (Figures 5 and 6). The up-regulation of these proteinsindicated that gluconeogenesis has been activated. Two en-zymes involved in pentose phosphate pathway, 6-phospho-gluconate dehydrogenase (U21) and ribulose-5-phosphate-3-epimerase (U45), were up-regulated during imbibition. Itsuggested that pentose phosphate pathway were also active inimbibition. Two enzymes (D10 and D18) involved in oilmobilization were down-regulated. However, they are not keyenzymes in the pathways, and their isoforms were up-regulatedsignificantly.

The mitochondrial ATP synthase alpha-subunit (D1) andmitochondrial ATP synthase beta-subunit (D3, D4, D5, D6 andD7) were down-regulated. Beta-ketoacyl-ACP synthase I (D2)

Figure 4. The 2-DE maps of J. curcas seeds in the germination process. Arrows indicate the 50 proteins identified by MS/MS, whichchanged 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.




and ADP-glucose pyrophosphorylase (D8), participating in lipidand starch syntheses respectively, were down-regulated. Both70 kDa heat shock cognate protein (U19) and type IIIamembrane protein cp-wap13 (U25) have been identified asplasmodesmata-associated proteins in previous studies.31,32

Their up-regulation can facilitate the processes involved in cell-to-cell transport of macromolecules.

A total of 14 spots were oxidative stress-related proteins,including cytosolic ascorbate peroxidase (APX), superoxidedismutase (SOD), glutathione S-transferase (GST) and glu-tathione peroxidase. Most of these protein spots, 9 spots out

of 14, were up-regulated. APX (U34, U35, and U39), whichreduces hydrogen peroxide using ascorbate as an electrondonor, was up-regulated. Of 5 spots representing SOD, D12 andD13 disappeared, while U48, U42 and U43 increased duringimbibition. Their positions on the 2-DE map suggested thatthe increased ones may have resulted from the proteolysis ormodification. Two spots (U38 and U44) identified as glu-tathione S-transferase were up-regulated in the process ofimbibition. Four changed spots were identified as glutathioneperoxidase, including one up-regulated spot (U50) and threedown-regulated spots (D14, D15 and D16).

Table 2. Differential Protein Spots Identified from Seeds of J. curcas during Imbibition by LTQ-ESI-MS/MSa

spotno. Mr/pI

Mr (kDa)/pI exp P

cov(%) Xcorr protein name

accessionno.

peptide identifiedby LTQ-ESI-MS/MS

Signal-Related Proteins (6)D9 29697/4.75 30/4.89 3 15.65 4.35 14-3-3 family protein AAV50005.1 K.SAQDIALAELAPTHPIR.LU29 36034/5.57 37/5.53 5 22.96 6.18 1-aminocyclopropane-1-carboxylate oxidase AAP41850.1 K.LAEELLDLLCENLGLEK.GU30 36050/5.57 36/5.53 5 19.81 6.14 ACC oxidase 3 CAN85571.1 K.LAEELLDLLCENLGLEK.GU33 29831/4.75 33/4.75 3 9.85 4.34 14-3-3 protein AAY67798.1 K.TVDVEELTVEER.NU36 29831/4.75 31/4.60 6 34.85 5.63 14-3-3 protein AAY67798.1 R.DNLTLWTSDITDEAGDEIK.DU37 29697/4.75 30/4.68 10 35.88 4.59 14-3-3 family protein AAV50005.1 K.SAQDIALAELAPTHPIR.L

Oil Mobilization-Related Proteins (17)D10 60699/5.51 30/6.49 5 9.29 4.97 2,3-bisphosphoglycerate-independent

phosphoglycerate mutaseAAM61601.1 R.GWDAQVLGEAPHK.F

D18 15870/6.59 13/5.22 1 9.46 4.24 fructose-bisphosphate aldolase BAA76430.1 K.GILAADESTGTIGK.RU20 47912/5.56 58/5.36 5 21.35 5.73 enolase CAA82232.1 R.SGETEDTFIADLSVGLATGQIK.TU21 56373/5.55 50/6.11 7 15.26 6.08 6-phosphogluconate dehydrogenase BAA22812.1 K.GDCIIDGGNEWYENTER.RU22 47242/6.14 47/6.52 8 20 5.40 cytosolic NADP+-isocitrate dehydrogenase ABA18651.1 K.GGETSTNSIASIFAWSR.EU23 42486/5.83 45/5.79 13 35.91 5.95 cytosolic phosphoglycerate kinase 1 BAA33801.1 K.IVAEIPEGGVLLLENVR.F3

U24 38514/6.93 44/6.24 6 16.48 5.00 cytosolic aldolase AAG21429.1 K.GILAADESTGTIGK.RU26 35526/6.32 38/6.35 4 13.55 5.27 cytosolic malate dehydrogenase AAS18241.1 K.VLVVANPANTNALILK. EU27 35675/6.33 38/6.50 6 22.89 5.57 cytosolic malate dehydrogenase BAA97412.1 K.NVIIWGNHSSTQYPDVNHATVK.TU28 35593/6.01 38/6.28 8 26.81 5.26 cytosolic malate dehydrogenase ABB36659.1 R.KLSSALSAASSACDHIR.D3

U31 60818/5.52 33/5.49 5 12.95 5.22 phosphoglycerate mutase CAA49995.1 R.SGYFNPEMEEYVEIPSDVGITFNVQPK.M3

U32 64611/6.73 33/6.27 3 4.86 3.47 isocitrate lyase CAA84632.1 R.NNGVDTLAHQK.WU40 27088/5.87 29/5.29 2 8.66 4.60 triose-phosphate isomerase CAI43251.1 K.VIACIGETLEQR.EU41 27088/5.87 29/5.51 3 13.78 4.26 triose-phosphate isomerase CAI43251.1 R.IIYGGSVNGGNCK.EU45 29898/8.3 26/5.88 2 12.46 3.06 ribulose-5-phosphate-3-epimerase AAM19354.1 K.VIEAGANALVAGSAVFGAK.DU47 98879/6.01 16/4.87 2 3.22 4.90 putative aconitase AAL13084.1 K.INPLVPVDLVIDHSVQVDVAR.SU49 23661/6.5 15/5.01 2 7.86 3.13 phosphoglycerate kinase BAA21478.1 K.KPFAAIVGGSK.V

Mitochondrial ATP Synthases (6)D1 55330/6.23 63/5.95 4 8.86 4.36 mitochondrial ATP synthase alpha-subunit Q01915 R.AAELTTLLESR.ID3 60258/5.95 61/5.51 4 9.07 5.31 mitochondrial ATP synthase beta-subunit CAA41401.1 K.CALVYGQMNEPPGAR.AD4 60258/5.95 61/5.27 7 15.84 4.88 mitochondrial ATP synthase beta-subunit CAA41401.1 R.FTQANSEVSALLGR.ID5 60258/5.95 59/5.32 10 21.00 4.88 mitochondrial ATP synthase beta-subunit CAA41401.1 K.CALVYGQMNEPPGAR.AD6 60258/5.95 59/5.38 9 23.31 5.24 mitochondrial ATP synthase beta-subunit CAA41401.1 K.CALVYGQMNEPPGAR.AD7 60258/5.95 59/5.42 4 10.32 5.38 mitochondrial ATP synthase beta-subunit CAA41401.1 R.IPSAVGYQPTLATDLGGLQER.I

Oxidative Stress-Related Proteins (14)D12 22915/6.06 25/6.28 2 7.80 4.97 IgE-binding protein MnSOD CAC13961.1 R.LVVETTANQDPLVTK.GD13 20658/5.79 19/5.30 1 5.45 2.18 superoxide dismutase AAR10812.1 R.LACGVVGLTPV.-1

D14 18531/6.13 19/6.26 1 7.14 2.26 glutathione peroxidase AAQ03092.1 R.YAPTTSPLSIEK.DD15 18581/5.72 18/5.92 1 7.19 2.50 phospholipid hydroperoxide

glutathione peroxidaseCAE46896.1 R.YAPTTSPLSIEK.D

D16 18531/6.13 18/6.11 6 27.38 3.82 glutathione peroxidase AAQ03092.1 K.GNDVDLSTYKGK.VU34 27283/5.69 31/5.37 5 26.4 4.42 cytosolic ascorbate peroxidase AAB95222.1 R.EVFGKTMGLSDQDIVALSGGHTLGR.A3

U35 27051/5.51 31/5.54 2 9.2 4.24 cytosolic ascorbate peroxidase 1 BAC92739.1 K.ALLSDPVFRPLVDK. YU38 26710/4.97 30/5.08 3 10.68 3.80 glutathione S-transferase BAC81649.1 K.LAAWIEELNK.IU39 27051/5.51 30/5.68 2 9.2 3.82 cytosolic ascorbate peroxidase 1 BAC92739.1 K.ALLSDPVFRPLVDK.YU42 25454/8.62 28/6.09 4 22.81 5.54 manganese superoxide dismutase 1 CAB56851.1 K.KLVVETTANQDPLVTK.GU43 25839/7.1 27/6.28 4 21.03 5.04 Superoxide dismutase [Mn] P35017.1 K.KLVVETTANQDPLVTK.GU44 23462/5.74 27/5.64 1 3.5 2.02 glutathione S-transferase GST 9 AAG34799.1 K.EFISIFK.Q1

U48 15195/5.47 16/6.23 3 23.03 4.29 CuZn-superoxide dismutase BAD51400.1 R.AVVVHADPDDLGK.GU50 18531/6.13 15/5.38 2 11.31 2.90 glutathione peroxidase AAQ03092.1 R.GNDVDLSTYK.G

Miscellaneous (7)D2 49773/6.86 63/6.09 7 22.65 5.55 beta-ketoacyl-ACP synthase I ABJ90468.1 K.AITTGWLHPTINQFNPEPSVEFDTVANKK.Q3

D8 56059/6.19 57/5.42 3 8.59 4.22 ADP-glucose pyrophosphorylase CAA54260.1 K.IYVLTQFNSASLNR.HD11 28099/5.81 28/5.45 6 10.20 4.45 caleosin ABB05052.1 R.CFDGSLFEYCAK.ID17 16886/9.72 16/6.29 1 6.88 2.53 16.9 kDa oleosin AAM46777.1 R.MQDMAGYVGQK.TU19 71016/5.11 65/5.02 14 24.65 6.16 70 kDa heat shock cognate protein 1 AAS57912.1 K.EQVFSTYSDNQPGVLIQVYEGER.T3

U25 39422/6.24 42/5.48 6 16.18 4.34 type IIIa membrane protein cp-wap13 AAB61672.1 K.YIYTIDDDCFVAK.NU46 26946/7.75 16/5.44 1 2.86 2.12 p-type H+-ATPase CAC28223.1 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 formolecular weight and isoelectric point; Mr (kDa)/pI exp, experimental molecular mass (in kilodaltons) and isoelectric point, which were calculated fromthe gel in Figure 4; P, number of unique matched peptides; cov (%), percentage of coverage of the identified proteins; Xcorr, SEQUEST cross-correlationscore of the peptide (for each protein, only the peptide with the highest Xcorr is presented). Superscript number (1 or 3) after peptide sequence meanssingly or triply charged state respectively, and those without superscript are doubly charged peptides. All the protein identities were from searching inNCBInr database.



Discussion

Initiation of Oil Mobilization in Germination. The seedgermination and the subsequent seedling growth need largeamounts of energy and nutrition, which can be provided onlyby seed reserves, because the germinating seed lacks a mineraluptake system and photosynthetic apparatus.2 The reserves aremainly in the form of oil, protein, and starch in embryo orendosperm, and their relative amounts vary in different species.In the endosperm of J. curcas seed, oil is the most abundantreserve (62.6%). However, the oil content decreased to 33.7%after 96 h imbibition (Figure 2), and after 6-day imbibition, littleoil was left in the wizen endosperm (data not shown). It

suggests that oil is mobilized and consumed during thegermination and postgermination stages.

In the endosperm cell, the obvious declination of the oil bodyamount was found at 48 h imbibition (Figure 3, 48 h). Itsuggested that oil mobilization had started earlier than 48 h ofimbibition. The up-regulation of some lipid catabolism-associ-ated enzymes before 48 h imbibition gave a strong support tothis notion at the molecular level. For example, the abundanceof U27 and U32 involved in glyoxylate cycle pathway increasedmore than 2.5-fold (p < 0.01) compared with 0 h imbibition(Figures 5 and 6). The major protein constituent of oil body isoleosin protein, and its mobilization is a prerequisite for

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




subsequent oil mobilization in some oilseeds.30 Thus, thedown-regulation of oleosin might be a sign of the initiation ofthe oil mobilization. In this work, oleosin (D17) displayed asignificant down-regulation from 0 to 48 h imbibition (Figures5 and 6). Thus, the mobilization of oil in endosperm of J. curcasseed is initiated during germination.

The oil content in endosperm displayed a drastic decreaseafter 60 h imbibition (Figure 2), indicating that the mobilizedoil in endosperm was transferred to embryo for building upseedlings, especially the elongation of hypocotyls and roots(Figure 1, 60 h). These results imply that large-scale lipidmobilization is initiated during germination, and the mobilizedoil is used in postgermination for establishment of seedlings.This agrees with the observations that germination is largelydriven 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 mobilizationprogress should be tightly regulated by internal factors, suchas plant hormones. Previous research has showed that ethyleneperforms a relatively vital role in dormancy release and seedgermination of many plant species.34 ACC oxidase is one ofthe key enzymes in the synthesis of ethylene. The up-regulationof ACC oxidase (U29 and U30) indicates that ethylene is anendogenous stimulator of germination of J. curcas seed. 14-3-3 proteins are ubiquitous eukaryotic regulatory proteinswhich can lead to enzyme activation or deactivation, and theyplay key regulatory roles in many cellular processes includingseed germination.35 In germinating barley seeds, the proteinlevels of all three isoforms of 14-3-3 were constant duringgermination; mRNA expression was found to be induced uponimbibition of the grains.36 In this paper, the protein level ofmost of the 14-3-3 isoforms was up-regulated significantlyduring germination. It suggests that 14-3-3s might act asregulatory proteins during the germination of J. curcas seed.

Caleosin is a Ca2+-binding oil body surface protein, and itmight participate in oil body-vacuole interactions that affectbreakdown of oil bodies during germination.18 Though no

direct evidence was observed for the participation of vacuolein the oil mobilization, vacuole might play some role in the oilmobilization, given the fact that the vacuole kept enlargingwhen the number of oil bodies decreased, and the vacuole wassurrounded tightly by oil bodies (Figure 3).

In plants, there are three possible metabolic pathwaysinvolved in the mobilization of storage lipids during seedgermination: classical glyoxysomal or peroxisomal degradationof linoleic acid, and two newfound pathways, lipoxygenase-dependent degradation of polyenoic FAs, and hypothetical FACoA-synthetase-independent pathway.37 Lipoxygenase is spe-cific for the oxidation of polyunsaturated FA in the lipoxyge-nase-dependent pathway. In some plants, it was shown thatlipase preferentially degrades the polyunsaturated FA oxidatedby lipoxygenase.38 Polyunsaturated FAs were preferentiallymobilized compared with other FAs in J. curcas endospermduring germination (Table 1). Taken together, we suggest thatlipoxygenase-dependent degradation should be an importantpathway for oil mobilization during J. curcas seed germination.

Many enzymes involved in glyoxylate cycle, glycolysis, citricacid cycle, gluconeogenesis, and pentose phosphate pathwaywere up-regulated during germination and postgermination.Especially the isocitrate lyase (U32) and isocitrate dehydroge-nase (U22), which are specific and critical enzymes in glyoxylatecycle and citric acid cycle, respectively, were both up-regulatedmore than 3.9-fold (p < 0.01) during imbibiton (Figure 6). Theseresults suggested that both glyoxylate cycle and citric acid cycleare getting active in the germination process. The glyoxylatecycle plays a fundamental role in oil mobilization and produces2-carbon compounds such as acetate or ethanol, ultimatelyproviding substrates for biosynthetic processes and respira-tion.39 Thus, this pathway is essential for postgerminativegrowth and seedling establishment in oilseed plants. The up-regulation of isocitrate lyase was also found in the germinationof Arabidopsis seed.39

The down-regulation of the alpha-subunit and beta-subunitof mitochondrial ATP synthase suggests that the up-regulation

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




of enzymes involved in citric acid cycle is not totally for energy,but for producing more substrates for subsequent gluconeo-genesis. Most of enzymes involved in gluconeogenesis were alsoup-regulated, which paved the pathway for producing glucoseas energy and nutrition source for building new seedlings. Inaddition, the activation of pentose phosphate pathway, inferredfrom the up-regulation of 6-phosphogluconate dehydrogenase(U21) and ribulose-5-phosphate-3-epimerase (U45), providesmore intermediates for biochemical reactions.

Fourteen spots were identified as oxidative stress-relatedproteins (APX, SOD, GST and glutathione peroxidase) whichfluctuated in abundance during the germinating process. Theup-regulation of APX agrees with that the level of APX mRNAin barley embryo increased in late germination.40 During theearly stage of imbibition, the rupture of J. curcas testa shouldbe accompanied by water infiltration and oxygen diffusion. Inaddition, many oxidation reactions started during the mobiliza-tion of oil should result in active oxygen species. A previousstudy showed that a great amount of hydrogen peroxide (H2O2)

generated from FA �-oxidation as a byproduct in glyoxysomesof oilseeds.41 Therefore, many oxidative stress-related proteinswere mobilized to protect enzymes and other molecules fromoxidative stress.

Metabolisms of Oil Mobilization. On the basis of the resultsin this study and extant data,20 a sketch map of the metabolicprocesses of oil mobilization is shown in Figure 7. After J. curcasseed imbibition in moisture, ethylene is induced to break thedormancy of the seeds. Some regulatory proteins such as 14-3-3 might be involved in the activation or deactivation ofenzymes. Proteinase and lipase are induced to break the oleosinand the lipids of the half-unit membrane of oil body, leadingto breakdown of the membrane. Then, triacylglycerol is hy-drolyzed by lipases through classical glyoxysomal (Figure 7a)and/or lipoxygenase-dependent pathways (Figure 7b) intoglycerol and FAs. After oxidation to dyhydroxyacetone, theglycerol can be fed into the gluconeogenesis pathway (Figure7c). Because of the preference of lipoxygenase to polyunsatu-rated FAs (Figure 7b-1) and the preference of lipase to hydro-

Figure 7. Mobilization of storage oil during germination and postgermination. The triacylglycerols in the oil bodies are hydrolyzedunder catalyzation of lipases into fatty acids and glycerol (a), or are oxidated under catalyzation of lipoxygenase into hydroperoxidesfirst (b-1) and then hydrolyzed under catalyzation of lipases into fatty acids and glycerol (b-2). The glycerol can be fed into thegluconeogenesis pathway after oxidation to dyhydroxyacetone (c). Fatty acids are activated in the glyoxysomes as CoA-thioesters anddegraded by �-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 cytosolis a precursor for the synthesis of hexoses via the gluconeogenesis pathway (g). The intermediates formed in pentose phosphatepathway (h) can be used for biosyntheses of other compounds. The identified protein spots related to oil mobilization, pathwaysinvolved in oil mobilization, and key steps of the oil mobilization are highlighted by red, green, and blue, respectively.




peroxides (Figure 7b-2), which is a product from oxidation ofpolyunsaturated FAs under lipoxygenase catalyzation, thepolyunsaturated FAs are preferably released from TAG duringgermination (Figure 7b-2). The released free FAs are degradedto acetyl CoA by �-oxidation in glyoxysomes (Figure 7d). Twomolecules of acetyl CoA generate one molecule of succinatein glyoxysome (Figure 7e). The succinate is transferred to themitochondria and converted there into malate by a partialreaction of the citric cycle (Figure 7f). Then, the malate isreleased from mitochondria to citosol where it is converted intophosphoenolpyruvate. Phosphoenolpyruvate is a precursor forthe synthesis of hexoses by the gluconeogenesis pathway(Figure 7g). The intermediates formed in pentose phosphatepathway (Figure 7h) can be used for biosyntheses of othercompounds such as RNA, protein, and so forth. The macro-molecules from gluconeogenesis and other processes can betransported to the growing embryo under the help of plas-modesmata-associated proteins.

Concluding Remarks

Several pathways, such as �-oxidation, glyoxylate cycle,glycolysis, citric acid cycle, gluconeogenesis, pentose phosphatepathway, were involved in the mobilization. The process wasprobably fulfilled by the cooperation of many parts of theendosperm cell, such as oil body, glyoxysome, mitochondrionand vacuole. In addition, signal transduction, transport andactive-oxygen-cleaning systems were also involved in thecomplex processes. These results would benefit to furtherunderstand 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 theKnowledge Innovation Program of the Chinese Academy ofSciences (KSCX2-YW-G-027-2, KSCX2-YW-G-035), the NationalNatural Science Foundation of China (U0733005).

Supporting Information Available: SupplementaryTable S1, a comprehensive list of the differential protein spotsidentified from the seeds of J. curcas during imbibition by LTQ-ESI-MS/MS. This material is available free of charge via theInternet 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 tomatoseeds. 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. Spatio-temporal changes in germination and radical elongation of barleyseeds tracked by proteome analysis of dissected embryo, aleuronelayer, 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.; Vande-kerckhove, J.; Job, D. Proteomic analysis of Arabidopsis seedgermination and priming. Plant Physiol. 2001, 126, 835–848.

(6) Muller, K.; Tintelnot, S.; Leubner-Metzger, G. Endosperm-limitedBrassicaceae seed germination: abscisic acid inhibits embryo-induced endosperm weakening of Lepidium sativum (cress) andendosperm rupture of cress and Arabidopsis thaliana. Plant CellPhysiol. 2006, 47, 864–877.

(7) Nakabayashi, K.; Okamoto, M.; Koshiba, T.; Kamiya, Y.; Nambara,E. Genome-wide profiling of stored mRNA in Arabidopsis thalianaseed germination: epigenetic and genetic regulation of transcrip-tion in seed. Plant J. 2005, 41, 697–709.

(8) Rajjou, L.; Belghazi, M.; Huguet, R.; Robin, C.; Moreau, A.; Job, C.;Job, D. Proteomic investigation of the effect of salicylic acid onArabidopsis seed germination and establishment of early defensemechanisms. Plant Physiol. 2006, 141, 910–923.

(9) Sreenivasulu, N.; Usadel, B.; Winter, A.; Radchuk, V.; Scholz, U.;Stein, N.; Weschke, W.; Strickert, M.; Close, T. J.; Stitt, M.; Graner,A.; Wobus, U. Barley grain maturation and germination: metabolicpathway and regulatory network commonalities and differenceshighlighted by new MapMan/PageMan Profiling Tools. PlantPhysiol. 2008, 146, 1738–1758.

(10) Yang, P.; Li, X.; Wang, X.; Chen, H.; Chen, F.; Shen, S. Proteomicanalysis of rice (Oryza sativa) seeds during germination. Proteomics2007, 7, 3358–3368.

(11) Zhang, H.; Sreenivasulu, N.; Weschke, W.; Stein, N.; Rudd, S.;Radchuk, V.; Potokina, E.; Scholz, U.; Schweizer, P.; Zierold, U.;Langridge, P.; Varshney, R. K.; Wobus, U.; Graner, A. Large-scaleanalysis of the barley transcriptome based on expressed sequencetags. Plant J. 2004, 40, 276–290.

(12) Gallardo, K.; Job, C.; Groot, S. P.; Puype, M.; Demol, H.; Vande-kerckhove, J.; Job, D. Proteomics of Arabidopsis seed germination.A comparative study of wild-type and gibberellin-deficient seeds.Plant Physiol. 2002, 129, 823–837.

(13) Pawlowski, T. A. Proteomics of European beech (Fagus sylvaticaL.) seed dormancy breaking: influence of abscisic and gibberellicacids. Proteomics 2007, 7, 2246–2257.

(14) Lee, C. S.; Chien, C. T.; Lin, C. H.; Chiu, Y. Y.; Yang, Y. S. Proteinchanges between dormant and dormancy-broken seeds of Prunuscampanulata Maxim. Proteomics 2006, 6, 4147–4154.

(15) Bewley, J. D.; Black, M. Seeds: Physiology of Development andGermination, 2nd ed.; Plenum: New York, 1994.

(16) Beevers, H. Metabolic production of sucrose from fat. Nature 1961,191, 433–436.

(17) Kornberg, H. L.; Beevers, H. A mechanism of conversion of fat tocarbohydrate in castor beans. Nature 1957, 4575, 35–36.

(18) Poxleitner, M.; Rogers, S. W.; Lacey Samuels, A.; Browse, J.; Rogers,J. C. A role for caleosin in degradation of oil-body storage lipidduring seed germination. Plant J. 2006, 47, 917–933.

(19) Eastmond, P. J. SUGAR-DEPENDENT1 encodes a patatin domaintriacylglycerol lipase that initiates storage oil breakdown ingerminating Arabidopsis seeds. Plant Cell 2006, 18, 665–675.

(20) Penfield, S.; Graham, S.; Graham, I. A. Storage reserve mobilizationin germinating oilseeds: Arabidopsis as a model system. Biochem.Soc. Trans. 2005, 33, 380–383.

(21) Muntz, K.; Belozersky, M. A.; Dunaevsky, Y. E.; Schlereth, A.;Tiedemann, J. Stored proteinases and the initiation of storageprotein mobilization in seeds during germination and seedlinggrowth. J. Exp. Bot. 2001, 52, 1741–1752.

(22) Openshaw, K. A review of Jatropha curcas : an oil plant ofunfulfilled promise. Biomass Bioenergy 2000, 19, 1–15.

(23) Kandpal, J. B.; Madan, M. Jatropha curcus: a renewable source ofenergy for meeting future energy needs. Renewable Energy 1995,6, 159–160.

(24) Berchmans, H. J.; Hirata, S. Biodiesel production from crudeJatropha curcas L. seed oil with a high content of free fatty acids.Bioresour. Technol. 2008, 99, 1716–1721.

(25) AOAC Official Methods of Analysis, 16th ed.; Association of OfficialAnalytical Chemists: Arilington, VA, 1995; Vol. 4, pp 1-45.

(26) Shen, S.; Jing, Y.; Kuang, T. Proteomics approach to identifywound-response related proteins from rice leaf sheath. Proteomics2003, 3, 527–535.

(27) Adamska, E.; Cegielska-Taras, T.; Kaczmarek, Z.; Szala, L. Multi-variate approach to evaluating the fatty acid composition of seedoil in a doubled haploid population of winter oilseed rape (Brassicanapus L.). J. Appl. Genet. 2004, 45, 419–425.

(28) Goodrum, J. W.; Geller, D. P. Influence of fatty acid methyl estersfrom hydroxylated vegetable oils on diesel fuel lubricity. Bioresour.Technol. 2005, 96, 851–855.

(29) Comlekcioglu, N.; Karaman, S.; Ilcim, A. Oil composition and somemorphological characters of Crambe orientalis var. orientalis andCrambe tataria var. tataria from Turkey. Nat. Prod. Res. 2008, 22,525–532.

(30) Sadeghipour, H. R.; Bhatla, S. C. Differential sensitivity of oleosinsto proteolysis during oil body mobilization in sunflower seedlings.Plant Cell Physiol. 2002, 43, 1117–1126.

(31) Epel, B. L.; Lent, J. W.M. v.; Cohen, L.; Kotlizky, G.; Katz, A.;Yahalom, A. A 41 kDa protein isolated from maize mesocotyl cellwalls immunolocalizes to plasmodesmata. Protoplasma 1996, 191,70–78.

(32) Aoki, K.; Kragler, F.; Xoconostle-Cazares, B.; Lucas, W. J. A subclassof plant heat shock cognate 70 chaperones carries a motif that



facilitates trafficking through plasmodesmata. Proc. Natl. Acad. Sci.U.S.A. 2002, 99, 16342–16347.

(33) Cernac, A.; Andre, C.; Hoffmann-Benning, S.; Benning, C. WRI1 isrequired for seed germination and seedling establishment. PlantPhysiol. 2006, 141, 745–757.

(34) Koornneef, M.; Bentsink, L.; Hilhorst, H. Seed dormancy andgermination. Curr. Opin. Plant Biol. 2002, 5, 33–36.

(35) Fulgosi, H.; Soll, J.; de Faria Maraschin, S.; Korthout, H. A. A. J.;Wang, M.; Testerink, C. 14-3-3 proteins and plant development.Plant Mol. Biol. 2002, 50, 1019–1029.

(36) Testerink, C.; van der Meulen, R. M.; Oppedijk, B. J.; de Boer, A. H.;Heimovaara-Dijkstra, S.; Kijne, J. W.; Wang, M. Differences inspatial expression between 14-3-3 isoforms in germinating barleyembryos. Plant Physiol. 1999, 121, 81–87.

(37) Feussner, I.; Kuhn, H.; Wasternack, C. Lipoxygenase-dependentdegradation of storage lipids. Trends Plant Sci. 2001, 6, 268–273.

(38) Adlercreutz, P.; Gitlesen, T.; Ncube, I.; READ, J. S. Vernonia lipase:a plant lipase with strong fatty acid selectivity. Methods Enzymol.1997, 284, 220–232.

(39) Eastmond, P. J.; Germain, V.; Lange, P. R.; Bryce, J. H.; Smith, S. M.;Graham, I. A. Postgerminative growth and lipid catabolism inoilseeds lacking the glyoxylate cycle. Proc. Natl. Acad. Sci. U.S.A.2000, 97, 5669–5674.

(40) Potokina, E.; Sreenivasulu, N.; Altschmied, L.; Michalek, W.; Graner,A. Differential gene expression during seed germination in barley(Hordeum vulgare L.). Funct. Integr. Genomics 2002, 2, 28–39.

(41) Graham, I. A.; Eastmond, P. J. Pathways of straight and branchedchain fatty acid catabolism in higher plants. Prog. Lipid Res. 2002,41, 156–181.

PR800799S



and postgermination development of jatropha curcas ... · proteomic analysis of oil mobilization in...

Documents