proteomics of medicago truncatula seed development ... · during seed filling (to a final...

Proteomics of Medicago truncatula Seed DevelopmentEstablishes the Time Frame of Diverse MetabolicProcesses Related to Reserve Accumulation1

Karine Gallardo*, Christine Le Signor, Joel Vandekerckhove, Richard D. Thompson, and Judith Burstin

Unite de Genetique et Ecophysiologie des Legumineuses, Institut National de la Recherche Agronomique(INRA)-Dijon, Domaine d’Epoisses, 21110 Bretenieres, France (K.G., C.L.S., R.D.T., J.B); and FlandersInteruniversity Institute for Biotechnology and Department of Biochemistry, Gent University, Gent, Belgium(J.V.)

We utilized a proteomic approach to investigate seed development in Medicago truncatula, cv Jemalong, line J5 at specificstages of seed filling corresponding to the acquisition of germination capacity and protein deposition. One hundred twentyproteins differing in kinetics of appearance were subjected to matrix-assisted laser desorption ionization time of flight massspectrometry. These analyses provided peptide mass fingerprint data that identified 84 of them. Some of these proteins hadpreviously been shown to accumulate during seed development in legumes (e.g. legumins, vicilins, convicilins, andlipoxygenases), confirming the validity of M. truncatula as a model for analysis of legume seed filling. The study alsorevealed proteins presumably involved in cell division during embryogenesis (�-tubulin and annexin). Their abundancedecreased before the accumulation of the major storage protein families, which itself occurs in a specific temporal order:vicilins (14 d after pollination [DAP]), legumins (16 DAP), and convicilins (18 DAP). Furthermore, the study showed anaccumulation of enzymes of carbon metabolism (e.g. sucrose synthase, starch synthase) and of proteins involved inembryonic photosynthesis (e.g. chlorophyll a/b binding), which may play a role in providing cofactors for protein/lipidsynthesis or for CO2 refixation during seed filling. Correlated with the reserve deposition phase was the accumulation ofproteins associated with cell expansion (actin 7 and reversibly glycosylated polypeptide) and of components of the precursoraccumulating vesicles, which give rise to a trypsin inhibitor on maturation. Finally, we revealed a differential accumulationof enzymes involved in methionine metabolism (S-adenosyl-methionine synthetase and S-adenosylhomo-cysteine hydro-lase) and propose a role for these enzymes in the transition from a highly active to a quiescent state during seeddevelopment.

The development of the angiosperm seed proceedsthrough histodifferentiation and seed filling and ter-minates with a desiccation phase after which theembryo enters into a quiescent state, thereby permit-ting its storage and survival in various environmen-tal conditions (Bewley and Black, 1994). The storagecompounds found in most mature seeds accumulateduring seed filling. They are principally storage pro-teins, oil (often triacylglycerols) and carbohydrates(often starch; Baud et al., 2002). These reserves are ofmajor importance for two reasons: (a) They supportearly seedling growth when degraded upon germi-nation and, therefore, participate in crop establish-ment; and (b) they are widely used for human andanimal nutrition. Seeds of legume species, such assoybean (Glycine max), pea (Pisum sativum), and favabean (Vicia faba), are an important protein source,with 20% to as much as 40% protein content, depend-

ing on species, genotype, and environment. In con-trast, seeds of graminaceous species, such as maize(Zea mays) and wheat (Triticum aestivum), are a majorsource of starch and contain less than 16% proteincontent. However, the major proteins stored in le-gume seeds are poor in sulfur containing amino acidsand the presence of nutritionally undesirable com-pounds, such as protease inhibitors, remain limitingfactors. Because of its nutritional and economic im-portance, much effort by plant breeders is directedtoward the improvement of seed quality, and bothplant breeding and molecular technologies can beused to produce plants carrying the desired traits(Mazur et al., 1999). Therefore, there is strong interestin identifying the processes occurring during seedfilling and the proteins involved.

The use of the most agriculturally important le-gume crops to study legume biology is limited by thelarge size of their genome and the complex ploidy.Unlike the major crop legumes, Medicago truncatula isdiploid, has a small genome size (approximately 500Mb), and is currently the subject of major genomicinitiatives. To date, more than 180,000 M. truncatulaexpressed sequence tag (EST) sequences are availablein public databases, and a sequencing project for theentire genome is underway (Bell et al., 2001). This

1 This work was supported by the INRA-Action TransversaleStructurante program on Medicago truncatula and by INRA (post-doctoral fellowship to K.G.).

* Corresponding author; e-mail [email protected]; fax33–3– 80 – 69 –32– 63.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.103.025254.

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annual relative of alfalfa (Medicago sativa) producesseeds rich in proteins (35%–45%) and oil (approxi-mately 12%) but low in starch (�1%) at maturity (G.Duc, personal communication). Moreover, becauseM. truncatula is phylogenetically related to the majorlegume crops, its use provides the potential to trans-fer information into crop improvement.

To take advantage of the available genomic re-sources of M. truncatula, we have characterized seeddevelopment in this species at the level of its proteincomplement. Proteomics offers the opportunity toexamine simultaneous changes in, and to classifytemporal patterns of, protein accumulation occurringin complex developmental processes such as seedfilling (Bove et al., 2002). This approach was recentlyapplied to study the changes in proteins that occurduring development of starch-rich grains (Finnie etal., 2002). More recently, Watson et al. (2003) per-formed a survey of the organ-/tissue-specific pro-teomes of M. truncatula. However, to date, there hasbeen no proteomic or transcriptomic project to studyprotein or gene expression profiles during seed fill-ing in a legume species.

Here, we provide a framework of physiologicaldata relevant for M. truncatula seed development andreport the identification by mass spectrometry (MS)of many seed proteins. This study has not only cata-loged proteins but has also described their accumu-lation patterns at specific stages during seed devel-opment, before and during protein deposition. Thesefindings contribute to our understanding of howmetabolic networks are regulated at the protein levelduring reserve deposition in seeds of a legume spe-cies. This knowledge will support our attempts toengineer legume seed composition for added enduser value.

RESULTS

Physiology of M. truncatula Seed Development

To provide a framework for the proteomic study ofseed filling, a series of stages of seed development,from embryogenesis to seed dispersal, were defined.Three phases were characterized by distinct physio-logical events and the associated changes in seed dryweight and moisture status. The first phase, corre-sponding to stages preceding 12 d after pollination(DAP), was characterized by a water content of about90% of the seed fresh weight (Fig. 1). During thisphase, which corresponds to histodifferentiation orembryogenesis (Bewley and Black, 1994), the wholeseeds removed from pods were not able to germinateon water (Fig. 2A), indicating that internal seed fea-tures permitting germination are not yet developed,or that constraints imposed by the tissue surround-ing the embryo prevent germination.

The second phase was associated with a large in-crease in the seed dry matter from 12 to 36 DAP (Fig.1) and characterized by the acquisition of the ability

to germinate (Fig. 2A). The time from imbibition togermination (T1 in Fig. 2A) declined gradually fromabout 16 d at 14 DAP to about 3 d at 30 DAP,indicating that seed vigor clearly increased duringthis phase. Similarly, seedling vigor, expressed ashypocotyl elongation and early root development,increased from 14 to 22 DAP (Fig. 2B). These dataindicate that physiological and biochemical featuresof M. truncatula seeds, which allow vigorous germi-nation and subsequent growth, are established dur-ing seed filling. Germination occurred without re-moving the seed coat, indicating that in M. truncatulathe surrounding testa and endosperm are not con-straints on germination in contrast to many specieswhere these structures must be removed to allow theimmature embryo to germinate (Bewley and Black,1994).

The terminal phase of M. truncatula seed develop-ment was characterized by a decrease in seed freshweight and a drastic loss of water as the seed under-goes drying (Fig. 1). This loss of water may play arole in the switch in cellular activities from a seedformation-oriented program to an exclusivelygermination/growth-oriented program (Kermode etal., 1986). Figure 2A shows that the onset of desicca-tion tolerance is earlier than the drying phase. This isconsistent with previous studies showing a similarrapid acquisition of desiccation tolerance in develop-ing seeds of several species, such as castor bean

Figure 1. Characterization of the different phases of M. truncatula(line J5) seed development. This graph represents changes in whole-seed fresh weight (fw), dry weight (dw), and water content (wc) from8 to 44 DAP. The three following phases are indicated: I, histodif-ferentiation; II, seed filling; and III, desiccation. Data are the mean �SD of three replicates of 15 seeds. Asterisk, Pod abscission. Theaccumulation kinetics of the storage compounds during M. trunca-tula seed development were very similar to those previously de-scribed for Arabidopsis seeds (Baud et al., 2002). Early stages of seeddevelopment were characterized by a transient accumulation ofstarch; the synthesis of both proteins and triacylglycerols occurredduring seed filling (to a final concentration of about 40% and 10%,respectively), and soluble sugars belonging to the raffinose familyaccumulated to a final concentration of about 10% during the lastphase of seed development (J.-P. Boutin, personal communication,unpublished data).

Medicago truncatula Seed Development

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(Ricinus communis) or rapeseed (Brassica napus; Bew-ley and Black, 1994).

Strategy Adopted for the Proteomic Analysis ofSeed Filling

We characterized the proteome of seeds harvestedat five stages during seed filling: seeds with low dry

weight and unable to germinate (12 DAP) and seedsincreasing in dry weight and developing the abilityto germinate (14, 16, 18, and 20 DAP; Figs. 1 and 2A).The protein samples were analyzed by two-dimensional (2-D) gel electrophoresis. Seed fillingwas accompanied by an increase in the number ofprotein spots detected in Coomassie Blue gels up to18 DAP (172 � 1 spots at 12 DAP, 252 � 3 at 18 DAP,and 245 � 3 at 20 DAP). In the 2-D gels, the abun-dance of the protein spots corresponded to their vol-umes, which were determined by the ImageMaster2-D Elite software as described in “Materials andMethods.” The volume varies as a function of boththe area and the densitometry of the detected proteinspot. Therefore, spot volume is the total intensity of adefined spot and corresponds to the amount of pro-teins in that spot. We conducted some experimentsconsisting in loading five different amounts of thetotal seed protein extracts (from 2–45 �L) in 2-D gels.The results indicated that for approximately 85% ofthe studied spots, there was a strong linear relation-ship (0.90 � R2 � 0.99) between spot volume and thetotal amount of proteins loaded into the CoomassieBlue gels. For the remaining 15% of spots, the rela-tionship was slightly less linear (R2 approximately0.85). To discard experimental variations in 2-D gelsbetween the different stages, the volume of each spotwas normalized to the total volume of 20 spots,which were chosen as references because they didnot show any qualitative variation in silver- andCoomassie Blue-stained 2-D gels during all develop-mental stages analyzed.

The ANOVA of the relative abundance of the 274different spots detected over the five stages permit-ted a classification according to their accumulationpatterns (Table I). Among these polypeptides, 90 be-long to class 0 proteins, that is, spots whose level didnot significantly vary from 12 to 20 DAP. Class 1 and2 proteins were represented by spots whose abun-dance significantly increased or decreased, respec-tively, during the 12- to 20-DAP period. Class 3 and4 proteins corresponded to spots showing a transientincrease or decrease, respectively, in their abun-dance, and class 5 proteins were represented by spots

Figure 2. Viability, vigor, and dehydration tolerance of M. truncatulaseeds collected during development. A, Seed germination. T1, Startof germination (time to reach 1% of germination � SD) for seedsfreshly harvested; Gmax, final percentage of germination obtainedfor fresh and dried seeds. The arrows indicate histodifferentiation (I)and seed filling (II). B, Hypocotyl elongation (length between coty-ledons and cotyledonary node) and early root development (lengthbetween cotyledonary node and root apex) of seedlings after germi-nation of untreated seeds collected at defined developmental stagesand grown for the same time period (20 d) on water. Data are themean � SD of three replicates of 15 seeds.

Table I. Types of variation in the level of expression of the 274 individual spots reproducibly detected in 2-D gels between 12 and 20 DAP

Class Characteristics

Proteins with NV � 10,000 Proteins with NV � 10,000

No. of proteinsdetected

No. of proteinsidentified

No. of proteinsdetected

No. of proteinsidentified

0 Constant level 0 – 90 01 Increased level 32 11 61 322 Decreased level 0 – 25 133 Transiently increased level 0 – 59 234 Transiently decreased level 0 – 6 45 Varying level 0 – 1 1

Total 32 11 242 73

NV, Normalized spot volume obtained from densitometric analysis of individual spots.

Gallardo et al.

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Table II. M. truncatula polypeptides whose levels increased (class 1 proteins in Table I) between 12 and 20 DAP

These proteins were grouped according to their biological function (see Fig. 4C). Within each group, spots were classified according to theirmaximum level detected in 2-D gels between 12 and 20 DAP. No., Spot no. gi, GenBank accession no. TC, Identifier of consensus sequencesretrieved from The Institute for Genomic Research (TIGR; http://www.tigr.org/tdb/tgi/mtgi/). Cov, Coverage. At, Arabidopsis. Cj, Coptis japonica.Cm, Pumpkin. Mc, Mesembryanthemum crystallinum. Ms, Alfalfa. Mtr, M. truncatula. Os, Rice (Oryza sativa). Ps, Pea. Vf, Fava bean.

Spot 3 Homology

Group No.Spot Pattern

DAP: 12-14-16-18-20

Maximum Levela

(Stage in DAP)pI/Molecular

Mass (kD)

BestMatchingEST gi, TC

Cov Homologous ProteinSpecies/GenBank

Accession No.

pI/Mr ofHomologous

Proteins

(Table continues on following page.)




showing varying changes in their levels during thisperiod. The protein spots were further separated intwo categories: the highly abundant polypeptides

(normalized volumes ranging from 10,000–150,000),and the less abundant polypeptides (normalized vol-umes ranging from 21–10,000).

Table II. (Continued from previous page.)

Spot 3 Homology


DAP: 12-14-16-18-20

Maximum Levela


Mass (kD)



Accession No.

pI/Mr ofHomologous

Proteins


Gallardo et al.



Kinetics of Storage Protein Accumulation

All of the highly abundant polypeptides belong toclass 1 of proteins, whose abundance increased dur-ing the 12- to 20-DAP period (Table I). Only oneabundant protein spot detected in extracts from drymature seeds (stage 44 DAP in Fig. 1) accumulatedafter the 20-DAP stage (data not shown), indicatingthat storage protein deposition is an early event in M.truncatula seed formation. Eleven well-resolved spotswere analyzed by matrix-assisted laser-desorptionionization time of flight (MALDI-TOF) MS, revealingtheir identity as members of the major storage pro-tein families: the legumins, vicilins, and convicilins(group 06 in Table II; species terminology medica-gins, alfins, and conalfins not used further here forease of comparison). Each of these protein familiesappeared at different stages in seed development.Although the vicilin spots were detected in 2-D gelsat 14 DAP, the legumin spots first appeared at 16DAP, and the convicilin spots were not detected until18 DAP (Fig. 3). In pea, vicilins have also been shownto appear earlier than the other globulins, but convi-cilins started to accumulate before legumins (Wenzel

et al., 1993). Our experimental conditions did notreveal abundant proteins corresponding to the 2Salbumin family found in pea and alfalfa seeds (Hig-gins et al., 1986; Tabe et al., 1995).

Identification of Less Abundant Proteins

Many of the 242 individual less abundant proteins(normalized volumes between 21 and 10,000) showedconstant level, increased, or transiently increased be-tween 12 and 20 DAP (class 0, class 1, and class 3proteins in Table I). Some proteins showed decreasedlevels (class 2 proteins in Table I), and a few spotstransiently decreased (class 4 proteins in Table I) orshowed varying changes in their levels (class 5 pro-teins in Table I). Because the main objective of thisstudy was to reveal molecular and metabolic pro-cesses, which could play a role specifically during thephase of protein deposition, we identified by MS, inparallel to abundant seed storage proteins, those lessabundant proteins whose abundance varied between12 (stage preceding storage protein accumulation)and 20 (stage after protein deposition) DAP. One

Table II. (Continued from previous page.)

Spot 3 Homology


DAP: 12-14-16-18-20

Maximum Levela


Mass (kD)



Accession No.

pI/Mr ofHomologous

Proteins

a Higher normalized spot volume (�SD) detected between 12 and 20 DAP. b Sequence obtained by Post Source Decay analysis (PSD):TEFGPSQPFKGAR. c Best peptide matching obtained with heterologous species. d Protein identity confirmed by electrospray ionization-tandem MS (ESI-MS/MS). e Portion of the sequence covered by the matching peptides. f Three peptide masses observed for spot 389(1,266.58, 1,689.58, and 1,760.01 D), and two peptide masses observed for spot 390 (1,265.52 and 1,759.93 D) matched well with thosepredicted from PV100 of pumpkin (Cucurbita maxima): 1,759.77 D (GESLSSGAGVDHDGCVNR in positions 30–47 in the Cys-rich domain),1,266.59 D (GSPRAEYEVCR in positions 72–82 in the Cys-rich domain), and 1,689.67 D (DDEDENQRDPDWR in positions 272–284 in theArg-Glu-rich domain).




hundred nine polypeptides of low abundance, takenfrom the protein classes 1 to 5 and well resolved,were analyzed by MALDI-TOF MS, and 73 proteinswere successfully identified (Tables II to V). Amongthese proteins, 23 corresponded to minor leguminchains or vicilins, many of which accumulated at 16to 18 DAP and disappeared after this stage (TableIV). Some of these may correspond to unstable formsof storage proteins not yet fully processed or to pro-teins that were either misdirected or misfolded anddegraded. Some could also correspond to fragmentsreleased by specific proteolysis of the storage protein,as observed before Arabidopsis seed germination(Gallardo et al., 2001).

The 50 other less abundant proteins identified byMS were classified in different groups correspondingto their presumed biological function. Figure 4 showsthat many of them are presumed to be involved inenergy, disease/defense, metabolism, protein desti-nation and storage, cell growth/division, and cellstructure. Some of these proteins are nutritionallyundesirable in legume seeds, such as lipoxygenases

(spots 391, 444, and 447 and group 09 in Table II and274 in Table V) known to produce hydroperoxidesthat attack nutritionally essential components (e.g.proteins, vitamins, and polyunsaturated fatty acids)and to release off-flavors compounds (Robinson etal., 1995). Despite the low number of spots identifiedfor certain functional groups, they may also play anessential role during seed development. For example,protein spot 65 (group 01 in Table II) is involved inthe biosynthesis of thiamine that is absolutely re-quired for seed formation as shown using mutantsdefective in this biosynthetic pathway (Li and Redei,1969; Komeda et al., 1988).

Correlation of Protein Abundance withmRNA Abundance

To get a first indication of the extent of the corre-lations between mRNA and protein levels duringseed development, the frequency of occurrence of thetranscripts corresponding to the proteins identifiedwas determined in the EST data sets of the cDNA

Figure 3. Kinetics of the accumulation of some storage proteins in seeds harvested 12, 14, 16, 18, and 20 DAP. A and B,Two portions of Coomassie Blue gels show protein spots belonging to class 1 (Table II) and identified by mass spectrometryas being legumin precursors (367 in A), �-subunits of legumins (319, 321, 322, 323, 337, 359, and 369 in B), vicilins (301,302, and 385 in B), and convicilins (46 and 395 in A). Range in pI and Mr are indicated in the panels for 12-DAP seeds. C,Example of changes in the normalized volume of some spots (302, 319, and 395) between 12 and 20 DAP. D, Proportionsof the storage protein families identified by mass spectrometry.

Gallardo et al.



libraries from early and late developing M. truncatulaseeds. The Medicago EST Navigation System, previ-ously used to characterize the sets of genes expressedin roots of M. truncatula during symbiosis (Journet etal., 2002), was used to count the number of sequencesfrom the same mRNA in each EST data set. In thisprogram, the ESTs are clustered based on sequencesimilarity and assembled into contigs reflecting thetranscripts. From the proteins identified by MS, 35different clusters were identified, and 29 corre-sponded to transcripts expressed in developing

seeds. The data were then grouped according to theexpression profiles of the mRNAs during seed devel-opment (Table VI).

The comparison of the results showing significantvariations between early and late developing seedswith the proteomic data revealed some correlationsbetween transcript and protein levels. For example,the results suggest that the levels of mRNAs encod-ing proteins specifically associated with the earlystages of seed filling (class 2 and class 3 in Table I),such as Ado-Met synthetase and PDI (Table VI), were

Table III. M. truncatula polypeptides whose levels decreased (class 2 proteins in Table I) between 12 and 20 DAP

These proteins were grouped according to their biological function (see Fig. 4C). Within each group, spots were classified according to theirmaximum level detected in 2-D gels between 12 and 20 DAP. No., Spot no. DH, Dehydrogenase. gi, GenBank accession no. Cov, Coverage.At, Arabidopsis. Bn, rapeseed. Gm, soybean. Mtr, M. truncatula. Ms, Alfalfa. Os, Rice. Ps, Pea.

Spot 3 Homology

Group No.Spot PatternDAP: 12-14-

16-18-20

MaximumLevela

(Stage in DAP)

pI/MolecularMass (kD)

BestMatching

EST giCov Homologous Protein

Species/GenBankAccession No.

pI/Mr ofHomologous

Proteins

a Higher normalized volume (�SD) detected between 12 and 20 DAP. b Protein identity confirmed by ESI-MS/MS. c Sequence obtainedby PSD analysis: VIAHDPYAPADR. d Best matching obtained with heterologous species.




Table IV. M. truncatula polypeptides whose levels transiently increased (class 3 proteins in Table I) between 12 and 20 DAP

These protein spots were grouped according to their biological function (see Fig. 4C). Within each group, spots were classified according totheir maximum level detected in 2-D gels between 12 and 20 DAP. No., Spot no. gi, GenBank accession no. TC, Identifier of consensussequences retrieved in TIGR (http://www.tigr.org/tdb/tgi/mtgi/). Cov, Coverage. At, Arabidopsis. Le, Lycopersicon esculentum. Md, Malusdomestica. Ms, M. sativa. Nt, Tobacco. Ps, Pea. Vf, Fava bean.

Spot 3 Homology


16-18-20

MaximumLevela

(Stage in DAP)




Accession No.

pI/Mr ofHomologous

Proteins


Gallardo et al.



higher in early developing seeds, whereas the tran-scripts encoding proteins that accumulated duringthe later stages (18–20 DAP), such as legumins andconvicilins (Table VI), were preferentially expressedin the late developing seeds. The results also suggestthat the levels of transcripts encoding some proteinsof class 1 (Table I) decreased during the later stagesof seed development. For those proteins whose abun-dance is maintained up to the desiccation stage, suchas vicilins, the absence or decrease of the transcript at

late stages suggests a high stability for these proteinsthroughout seed development (data not shown). Al-though this approach is not sufficient to establish arigorous validation of gene expression, the resultsprovide a basis to elucidate the mechanisms of reg-ulation of protein accumulation and stability.

DISCUSSION

The seed occupies a central position in the life cycleof higher plants. In addition to its role in dispersal,

Table V. M. truncatula polypeptides whose levels transiently decreased (class 4 proteins in Table II) or showed various changes (class 5 pro-teins in Table II) between 12 and 20 DAP

These protein spots were grouped according to their biological function (see Fig. 4C). Within each group, spots were classified according totheir maximum level detected in 2-D gels between 12 and 20 DAP. No., Spot no. gi, GenBank accession no. Cov, Coverage. Bv, Beta vulgaris.Ps, Pea. Ss, Solenostemon scutellarioides.

Spot 3 Homology

Group Class No.Spot PatternDAP: 12-14-

16-18-20

MaximumLevela

(Stage in DAP)


BestMatching

EST giCov

HomologousProtein

Species/Accession

No.

pI/Mr ofHomologous

Proteins

a Higher normalized volume (�SD) detected between 12 and 20 DAP. b Protein identity confirmed by ESI-MS/MS.

Table IV. (Continued from previous page.)

Spot 3 Homology


16-18-20

MaximumLevela

(Stage in DAP)




Accession No.

pI/Mr ofHomologous

Proteins

a Higher normalized volume (�SD) detected between 12 and 20 DAP. b Protein identity confirmed by ESI-MS/MS. c Part of thesequence covered by the matching peptides. d Best matching obtained with heterologous species.




the seed determines the success with which germi-nation and early seedling growth occur. Moreover,seeds such as those of legumes are major foodsources whose importance lies in the proteins storedduring development. Our aim was to identify seedproteins characteristic of specific stages during re-serve deposition in M. truncatula. These data willhelp elucidate the biochemical and molecular pro-cesses underlying seed filling in a legume species.Eighty-four proteins whose abundance varied during

reserve deposition were identified by MS. As ex-pected, the most abundant proteins correspondedto storage proteins. Many of the weakly abundantproteins could play a role in cell division duringembryogenesis, in protein or starch deposition, indefense against herbivores, in cell expansion duringreserve deposition, or in the transition from a highlyactive to a quiescent state during seed development.These results are discussed in the followingsections.

Figure 4. Characterization of less abundant M. truncatula seed proteins whose levels vary between 12 and 20 DAP. A,Example of Coomassie Blue 2-D gel from 12-DAP seeds. Sections 1 to 3 are zoomed in C. B, Distribution of the minorproteins in the functional groups reported for Arabidopsis genes and used to classify M. truncatula proteins (Bevan et al.,1998; Watson et al., 2003). C, Variations of protein profiles in the three different sections shown in A between 12 and 20DAP. Colored labels indicate some polypeptides identified within some functional classes, and uncolored labels show thelocation of these spots when they are undetectable or present at low levels. The spots 251 and 280 decreased in their relativeabundance between 12 and 18 DAP (class 2 proteins), spot 214 transiently decreased (class 4 protein), protein spots 234,334, 363, and 428 increased (class 1), and protein spots 217, 282, 383 transiently increased (class 3 proteins). These spotswere identified by mass spectrometry as being cytosolic ascorbate peroxidase (spot 251), �-tubulin (280), GAPDHc (214 and217), oxygen-evolving enhancer protein (234), 1-aminocyclopropane-1-carboxylate (ACC) oxidase (334), chlorophylla/b-binding protein (363), glyoxalase I (428), and protein disulfide isomerase (PDI; 282). Protein spot quantitation andANOVA were carried out from four gels for each seed sample. Some spots, chosen as references to discard experimentalvariations, are shown as diamonds.

Gallardo et al.



The Abundance of Cell Division-Associated ProteinsDecreases in Transition from Embryogenesis toReserve Deposition

Embryogenesis starts with a morphogenesis phaseduring which the embryo differentiates through sev-eral distinct stages (globular, heart, and torpedo) andends at the cotyledon stage when all embryo struc-tures have been formed. Figure 2A shows that acqui-sition of ability to germinate does not occur before 14DAP, presumably because the young embryo is notfully formed before this stage. At the end of embry-ogenesis, cell division arrests and the seed accumu-lates the storage components (Raz et al., 2001). Our

results showed that the 12-DAP stage correspondedto the end of the embryogenesis phase (Fig. 1) andthat the abundance of several proteins decreased inthe transition from embryogenesis to seed filling (Ta-ble III). This was the case for �-tubulin (spot 280 inTable III) and annexin (spots 223 and 228 in Table III)that are associated with cell cycle events. Tubulinsare associated with cell division and cell enlargementaspects of the cell cycle. During cell division, theyplay an important role in separation of the organellesand daughter chromosomes (mitosis). The accumu-lation of �-tubulin has been observed during seedgermination, in relation to reactivation of cell cycleactivity (De Castro et al., 2000). In contrast, the pres-

Table VI. Relative expression level of M. truncatula transcripts based on the repetitive occurrence of sequences in the EST data sets fromimmature seeds

The cluster accession nos. of the M. truncatula ESTs identified by MS were extracted from the Medicago EST Navigation System (http://medicago.toulouse.inra.fr/) release of January 2003. The cluster accession nos. were used to search the no. of sequences from the same mRNAin two different data sets: a cDNA library of immature seeds ranging in age from 11 to 19 DAP (MtGESD, 4,525 ESTs) and a cDNA library ofimmature seeds ranging in age from 25 to 35 DAP (MtGLSD, 4,866 ESTs). The M. truncatula transcripts whose expression was found to besignificantly variable in the two different data sets (�2 test, P � 0.050) were grouped according to their expression profiles during seeddevelopment. In each class of proteins (see Table I), the corresponding M. truncatula transcripts were sorted in the descending order based ontheir EST counts in both data sets.

Protein Class Strongest BLAST Hit EST gi Cluster Accession No. MtGESD (11–19 DAP) MtGLSD (25–35 DAP)

Transcripts preferentially expressed during early stages of seed development1 Vicilin 14986367 MtC60032.3_GC 282 551 Vicilin 14984555 MtC60159_GC 41 211 Chlorophyll a/b binding 13783768 MtC00119_GC 10 02 Vicilin 14984749 MtC60032.4_GC 280 552 Annexin 7718287 MtC20316_GC 14 12 S-adenosylmethionine synthetase 11609660 MtC00046_GC 4 03 Flavonone 3-hydroxylase 13596069 MtC93321_GC 12 03 Protein disulfide isomerase 10697964 MtC10403_GC 10 03 GAPDHc 9671653 MtC00021.1_GC 4 0

Transcripts preferentially expressed during late stages of seed development1 Legumin 14985010 MtC60042_GC 119 4511 Legumin 14986270 MtC60076_GC 67 3111 Convicilin 14985541 MtC60090_GC 71 209

Transcripts whose relative levels did not vary significantly during seed development1 Lipoxygenase 14982738 MtC60669_GC 11 151 ACC oxidase 10520454 MtC10108.2_GC 9 61 Met synthase 9669628 MtC00018_GC 4 61 Homology not assigned 13595945 MtD00176_GC 1 21 Glyoxalase 1 11897629 MtC60084_GC 3 01 Oxygen-evolving enhancer 11902222 MtC60015_GC 2 11 Starch synthase 13379977 MtC90709_GC 2 11 Reversibly glycosylated polypeptide-1 7718398 MtC10969_GC 2 01 110-kD 4SnNc-Tudor 7564626 MtC50269.1_GC 1 11 Triosephosphate isomerase 13380542 MtC00059_GC 1 01 Thiamine biosynthesis protein 7718895 MtD00077_GC 1 01 Lipoxygenase 6708795 MtC60306_GC 0 01 S-adenosyl-L-homo-Cys hydrolase 1710838 MtC30011_GC 0 02 �-Tubulin 7561503 MtC00356.1_GC 2 12 Adenosine kinase 10704292 MtC10064_GC 1 02 Pyruvate DH �-subunit 13780026 MtC30080_GC 0 02 Myo-inositol-1-P synthase 13369792 MtC60071_GC 0 03 Heat shock cognate protein 80 14882556 MtD00014_GC 5 13 Molecular chaperone BiP A 14983890 MtC00550.1_GC 3 03 Heat shock protein 90 10706885 MtC00079_GC 3 03 Putative homology not assigned 9678016 MtC30333_GC 0 03 Resistance gene analog protein 13779916 MtD02701_GC 0 04 Elongation factor EF-2 10698429 MtC00166.1_GC 2 0




ence of annexin in seeds has not been reported pre-viously. However, there is strong evidence that an-nexins are involved in cell division. For example,they accumulate during the cell cycle and peak at theend of mitosis in tobacco (Nicotiana tabacum) cells(Proust et al., 1999). Because they are localized at celljunctions and are known to bind secretory vesiclesduring exocytosis, annexins could play a role in cellwall maturation during cell division (Proust et al.,1999). Because the abundance of annexin and�-tubulin decreased significantly 14 DAP and re-mained low at the protein and mRNA levels (Fig. 4;Table VI), it is likely that these proteins participate inthe stages preceding reserve deposition, presumablyin the embryonic divisions. Their turnover reflectsthe developmental switch from embryogenesis toseed filling, i.e. a switch from cell division to reservedeposition (Figs. 3 and 4).

Synthesis and Maturation of the StorageProtein Families

After the cessation of cell division, the seed storagecompounds are synthesized. In M. truncatula, thereserve deposition phase is characterized by a largeincrease in the protein content of up to 45% (G. Duc,personal communication). In our study, the mostabundant protein spots, which are mainly responsi-ble for this increase in the protein content and, thus,for the nutritional value of legume seeds, were iden-tified as being the 7S (vicilin and convicilin) and 11S(legumin) globulins (Table II). These protein familiesaccumulated in a specific temporal order during seedfilling: vicilins (14 DAP), legumins (16 DAP), andconvicilins (18 DAP; Fig. 3). The transcripts encodingthe storage protein families were expressed in a sim-ilar time course when compared with protein abun-dance, with mRNAs encoding vicilins being prefer-entially expressed in the early stages, and mRNAsencoding the legumin and convicilin families beingpreferentially expressed in the later stages (Table VI)as observed in our proteome analysis. This suggeststhat the temporal accumulation of the storage proteinfamilies is likely to be transcriptionally controlledduring seed development. As expected for proteinsencoded by multigene families (Casey et al., 2001),several spots with similar masses but differing incharge were identified as being the same storageprotein. Because these polypeptides are broken downduring germination and used by the germinatingseedling as an initial food source (Bewley and Black,1994), it was not surprising to observe that theiraccumulation during seed filling is concomitant withthe acquisition of both seed vigor and seedling vigor(Figs. 2 and 3).

The characterization of the globulin families invarious dicotyledonous plants has shown that theprecursor forms of these proteins are transportedfrom the endoplasmic reticulum lumen to the pro-

tein storage vacuoles, where they are processed intospecific subunits. These chains are then assembledwithin the protein bodies, yielding the matureforms, typically trimeric for the 7S globulins andhexameric for the 11S globulins (Gruis et al., 2002).In 2-D gels, two spots were identified as precursorforms of legumin (Tables II and IV) and nine asacidic �-chains (Mr of approximately 40,000) or ba-sic �-chains (Mr of approximately 20,000; Table II).These polypeptides accumulated with the same timecourse during seed filling, indicating that synthesisand maturation of the 11S globulins are not devel-opmentally separated in M. truncatula. Our resultsshowed that the convicilin family was representedby polypeptides of 50 and 74 kD (Table II). Also,three protein spots with Mr of approximately 47,000were identified as vicilin and six spots as corre-sponding to the carboxyl-terminal domain (threespots with Mr of approximately 30,000) or the ami-no-terminal domain (three spots with Mr of approx-imately 17,000) of the vicilin precursor forms (TableII), suggesting that the vicilin precursor forms areprocessed to give products ranging in Mr from ap-proximately 17,000 to 47,000.

Most of the mature products of the M. truncatula 7Sand 11S globulins possessed similar masses to thosefound in pea and soybean seeds (Croy et al., 1980;Bewley and Black, 1994; Jung et al., 1998; Casey,1999), suggesting that storage protein processingmay be conserved within these species. Moreover,the BLASTP analyses of the tag consensus (TC) trans-lated sequences of these storage protein families (Ta-ble II) showed higher homology (70%–85%) withthose from agriculturally important legume crops(pea, soybean, and fava bean). Together, these datahighlight the importance of using M. truncatula as amodel to study the cellular and molecular mecha-nisms related to protein deposition in seeds of le-gume crops.

Endoplasmic reticulum resident proteins known asmolecular chaperones play important roles in theformation and assembly of the seed storage proteins(Li and Larkins, 1996; Hatano et al., 1997; Takemotoet al., 2002). Our study revealed several luminal pro-teins involved in protein folding, including a PDI anda putative chaperonin of the binding protein (BiP)class (spots 169 and 282 and group 06 in Table IV;Fig. 4C). PDI catalyzes the formation and rearrange-ment of disulfide bonds of the newly synthesizedproteins, whereas BiP, a molecular chaperone relatedto the heat shock proteins, is involved in the assem-bling of the nascent proteins by preventing theirdenaturation or aggregation and in the recognitionand disposal of misfolded polypeptides (Kainuma etal., 1995; Zhang et al., 1997). The temporal inductionof the proteins identified as BiP and PDI coincidedwith the onset of storage protein accumulation (seeFigs. 3 and 4). Therefore, these molecular chaperones

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are good candidates to study the folding and assem-bly of the storage proteins in legume seeds.

Two 100-kD Components of the Precursor-Accumulating Vesicles (PV100) Are Synthesized duringM. truncatula Seed Filling

Interestingly, in addition to vicilin polypeptides ofthe expected 30- to 40-kD class, two protein spots ofMr approximately 120,000 (389 and 390 and group 09in Table II) that accumulated during seed filling alsopossessed vicilin-related sequences. In their respec-tive MALDI-TOF spectrums, at least eight peptidemasses matched with those from vicilins. Despitetheir high Mrs, they were identified provisionally,therefore, as vicilins. A similar protein D of approx-imately 100,000 containing a vicilin-like domain wasdescribed in pumpkin (Cucurbita maxima) seeds. Thisprotein, called PV100, is synthesized on rough endo-plasmic reticulum as a precursor form consisting of asingle protein chain with three domains: a vicilin-likedomain, a Cys-rich domain (91 amino acids), and anArg-/Glu-rich domain (267 amino acids; Yamada etal., 1999). To investigate whether the M. truncatulaprotein spots with Mr of approximately 120,000 couldbe the homologs of PV100, the unmatched peptidemasses obtained by MALDI-TOF were comparedwith those predicted from the Cys-rich and Arg-/Glu-rich domains of PV100 from pumpkin. The re-sults showed that several peptide masses for spots389 and 390 matched well with those predicted fromthese domains (Table II). Yamada et al. (1999) re-ported that the precursor form of PV100 is trans-ported by vesicles to protein storage vacuoles, whereit is processed to a mature vicilin, a trypsin inhibitor,and a basic cytotoxin-related peptide. Because theselatter two peptides might play a herbivore-deterrentrole, their elimination could improve the nutritional

quality of legume seeds for human and animal use.Such a protein has not been characterized yet inlegume seeds. Assuming a functional homology toPV100, it will be of interest, therefore, to characterizethe mature peptide products derived from the M.truncatula 120-kD proteins.

Role of Embryonic Photosynthesis during Seed Filling

In some legumes, such as soybean, starch can bepresent early during seed development. However,the starch level declines to about 1% and is replacedby accumulating oil reserves on maturation (Adamset al., 1980). As in soybean, M. truncatula seed devel-opment is characterized by a transient accumulationof starch and by the deposition of protein and lipidreserves to a final concentration of about 40% and10%, respectively (J.P. Boutin, personal communica-tion). The biochemical pathways that produce thestorage compounds are well known, but the pro-cesses that coordinate their accumulation at specificstages during seed development are not well under-stood. Our study revealed that the 16-DAP stage wascharacterized by the onset of storage protein accu-mulation (Fig. 3) and associated with an accumula-tion of starch synthase, Suc synthase, and triosephos-phate isomerase (spots 246, 272, and 297 and group01 in Table II; Fig. 5), which could be involved in thesupply of carbon substrates for the synthesis of stor-age compounds in these limiting conditions for thefixation of CO2 (Heim et al., 1993). Interestingly, anoxygen-evolving enhancer protein and two chloro-phyll a/b-binding proteins (spots 234, 249, and 363and group 02 in Table II; see also Fig. 5) accumulatedat the same stage. In accordance with this finding,embryonic photosynthesis was proposed to play arole in providing ATP and NADPH for storage com-pound synthesis during seed filling (Browse and

Figure 5. Kinetics of accumulation of proteinsinvolved in carbon and plastidial metabolismsin M. truncatula developing seeds. Some en-zymes and substrates leading to the synthesis ofstarch and/or oil are shown. Not shown areother contributions to oil metabolism, such asthe oxidative pentose phosphate pathway. FAS,Fatty acid synthesis; PEP, phosphoenolpyruvate;TAG, triacylglyceride.




Slack, 1985; Batz et al., 1995; Eastmond and Raw-sthorne, 2000; Ruuska et al., 2002). Together, thesedata suggest that photosynthesis activity in theseseeds may respond to the heavy demand by protein/oil synthesis for cofactors, such as ATP and NADPH.Although CO2 fixation has been reported to be low indeveloping seeds, we observed that the level of asubunit of Rubisco (spot 126 and group 02 in Table II)increased at 20 DAP. A similar observation was re-ported in developing Arabidopsis seeds, where a rolefor this enzyme in recycling the CO2 released duringthe biosynthesis of storage compounds during seedfilling was proposed.

Proteins Associated with Cell ExpansionAccumulate during Reserve Deposition

During seed filling, the cells continue to grow byenlargement as they accumulate the storage compo-nents. Two proteins identified in this study could beinvolved in this cell enlargement process during pro-tein deposition. The first corresponded to actin(ACT7 and spot 187 in Table IV), which is a funda-mental component of the cytoskeleton. Its level in-creased specifically during protein deposition anddecreased after this stage. In Arabidopsis, the geneencoding the same isoform ACT7 is preferentially

expressed in vegetative tissues that contain rapidlydividing and expanding cells and appears to be theonly actin gene expressed in seed tissues (McDowellet al., 1996a). Actin is involved in a number of cellu-lar processes such as cytoplasmic streaming, cellshape determination, organelle movement, and ex-tension growth (McDowell et al., 1996b). Because ofthese functions, the M. truncatula seed protein iden-tified as ACT7 could play an important role in cellexpansion during the phase of protein deposition.

The second protein (spot 206 and group 07 in TableII) showed more than 88% identity with the reversiblyglycosylated polypeptide (RGP1) identified insuspension-cultured cells, roots, and leaves in Arabi-dopsis (Delgado et al., 1998) and in pea seedlings(Dhugga et al., 1997). In these tissues, this protein isthought to have a role in cell wall polysaccharidesynthesis, possibly that of xyloglucan. A similar pro-tein was also found in wheat endosperm (Langeveldet al., 2002), but its function during seed developmenthas not been elucidated yet. The protein RGP1 iden-tified in our study accumulated significantly at thestage 18 DAP, which is characterized by a large in-crease in the abundance of the storage proteins (Fig.3). Therefore, it would be of particular interest toexamine its possible role in cell wall expansion at thisstage.

Figure 6. Differential accumulation of enzymesinvolved in Met de novo biosynthesis during M.truncatula seed filling. Reaction intermediates:ACC, AMP, S-adenosylhomo-Cys (AdoHcy), S-adenosyl-Met (Ado-Met), homo-Cys (Hcy), O-phosphohomo-Ser (OPH), and S-methyl-Met(SMM). Enzymes: 1, cystathionine �-synthase;2, cystathionine �-lyase; 3, cobalamine-independent Met synthase; 4, Ado-Met syn-thetase; 5, Ado-Met-dependent transmethy-lases; 6, AdoHcy hydrolase; 7, Ado-Met:MetS-methyltransferase (MMT); 8, SMM:Hcy S-methyltransferase (HMT); 9, ACC synthase; 10,ACC oxidase; and 11, adenosine kinase.

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Enzymes of Met Metabolism May Be Implicated in theTransition from a Highly Active State to a QuiescentState during Seed Development

Interestingly, many proteins whose abundancevaried during seed filling corresponded to enzymesinvolved in Met biosynthesis. Among the essentialamino acids synthesized by plants, Met is a funda-mental metabolite because it functions not only as abuilding block for protein but also as the precursor ofAdo-Met, the primary methyl-group donor and theprecursor of polyamines and the plant ripening hor-mone ethylene (Ravanel et al., 1998; Kim and Leus-tek, 2000; Gakiere et al., 2002).

In plants, Met can be synthesized through two path-ways (Fig. 6). In the de novo biosynthetic pathway,O-phosphohomo-Ser is transformed to cystathioninein a reaction catalyzed by cystathionine �-synthase,then to Hcy in a reaction catalyzed by cystathionine�-lyase, and finally to Met in the presence of thecobalamin-independent Met synthase. In the Met-recycling pathway (Hanson and Roje, 2001), SMM, acompound unique to plants, is synthesized by amethyl transfer from Ado-Met to Met, in a reactioncatalyzed by Ado-Met:Met S-methyltransferase. SMMcan then be reconverted to Met by transferring amethyl group to Hcy in a reaction catalyzed by SMM:Hcy S-methyltransferase. These reactions, togetherwith the reactions catalyzed by Ado-Met synthetaseand AdoHcy hydrolase, constitute the SMM cycle,which may be the main mechanism in plants for short-term control of Ado-Met level. This cycle consumeshalf of the Ado-Met produced (Ranocha et al., 2001).

Consistent with the high demand for protein syn-thesis between 12 and 20 DAP, two spots detected inseed extracts throughout this period corresponded toMet synthase (spots 14 and 276 and group 01 inTables II and V), which catalyzes the last step of thede novo biosynthetic pathway of Met. In addition,one spot (156 in Table III) corresponded to Ado-Metsynthetase, which catalyzes the synthesis of Ado-Metfrom Met and ATP. Interestingly, the level of Ado-Met synthetase fell sharply at the 16-DAP stage (Fig.6) and remained low up to desiccation (data notshown). This result was supported by the observa-tion that ESTs corresponding to Ado-Met synthetasewere only found in cDNA libraries corresponding toearly stages of seed development (Table VI). Ado-Met synthetase also was absent from dry matureArabidopsis seeds (Gallardo et al., 2001) but accumu-lated in the transition from a quiescent to a highlyactive state during germination (Gallardo et al.,2002a). In contrast to germination, during seed de-velopment, there is a switch from a period of highlyactive metabolism associated with cell expansion,differentiation, and accumulation of storage productsto a period during which the overall biosyntheticactivity decreases as the embryo prepares for quies-cence. Assuming a functional homology to Ado-Metsynthetase found in germinating Arabidopsis seeds,

the accumulation pattern of the M. truncatula Ado-Met synthetase may also reflect the metabolic shift indeveloping seeds.

After the decrease of Ado-Met synthetase, therewas an increase in the abundance of two enzymesinvolved in Ado-Met consumption. The first proteincorresponded to AdoHcy hydrolase (spot 124 andgroup 01 in Table II; Fig. 6). The hypothesis thatAdoHcy hydrolase is active during seed develop-ment agrees with previous results showing that Metrecycling via the Ado-Met/AdoHcy and SMM cyclesis not sufficient in mature seeds to maintain an ap-propriate pool of Met for rapid germination andseedling establishment (Gallardo et al., 2002a).Knowing that AdoHcy hydrolase is being subjectedto feedback inhibition by adenosine, it was interest-ing to note that developing seeds also containedadenosine kinase (spot 209, Table III; Fig. 6) cata-lyzing the phosphorylation of adenosine to adeninemonophosphate (Moffatt et al., 2002). The secondprotein corresponded to ACC oxidase (spot 334 andgroup 03 in Table II; Fig. 6). This enzyme is involvedin the synthesis of the plant ripening hormone eth-ylene, which has been shown to control cotyledonexpansion during embryo development in rapeseed(Hays et al., 2000), and is implicated generally inripening processes.

In plants, Ado-Met has an important influence oncell growth and development. Beside its role in eth-ylene, biotin, and polyamine biosynthesis, Ado-Metis the primary methyl group donor for the methyl-ation of amino acids, lipids, RNA, and DNA, andfunctions as an effector in the regulation of Thr, Lys,and Met synthesis (Ravanel et al., 1998). The disap-pearance of Ado-Met synthetase and the accumula-tion of Ado-Met-consuming enzymes are likely todecrease Ado-Met levels. Given the important regu-lating influence of Ado-Met, this may promote therepression of the metabolic activities leading to aquiescent state. This suggests that the same mecha-nism may be implicated in the repression of meta-bolic activities during seed development and theirresumption during germination (Gallardo et al.,2002a).

CONCLUSION

We utilized a proteomic approach to identify 84 M.truncatula seed proteins with characteristic develop-mental patterns of accumulation during protein dep-osition. Some of these had previously been shown toplay a role during seed filling in other legume species(e.g. legumins, vicilins, and convicilins), confirmingthe validity of M. truncatula as a model system foranalysis of legume seed filling. The present studyalso revealed the kinetics of storage protein accumu-lation in M. truncatula and new proteins to be asso-ciated with the reserve deposition process, with pre-sumed roles in cell division (annexin), cell expansion




(ACT7 and RGP1), or metabolic activities (for exam-ple, Ado-Met synthetase and AdoHcy hydrolase).Furthermore, the data revealed nutritionally undesir-able components whose elimination should improvethe quality of legume seeds, such as lipoxygenasesand components of the precursor-accumulating ves-icles (PV100), which give rise to a trypsin inhibitor onmaturation. These data will facilitate further studies,which investigate the effects of genetic and environ-mental factors on seed quality. The role of theseproteins can be further assessed by a combination offorward and reverse genetics, such as the TILLING(targeting induced local lesions in genomes) method-ology (McCallum et al., 2000). This will provide ad-ditional information useful for understanding thecomplexities of seed metabolism and its control dur-ing seed development.

MATERIALS AND METHODS

Plant Material

A batch of 20 plants of Medicago truncatula cv Jemalong, line J5 was usedfor all experiments. Plants were grown in a growth chamber at 22°C/19°Cday/night temperatures, under a 16-h photoperiod at 220 �E m2 s�1 lightintensity with 60% to 70% relative humidity. To harvest pods at definedstages during development, individual flowers were tagged on the day offlower opening. For each stage, at least 40 flowers were labeled on the first,second, and third nodes of the main ramifications. Pods were harvestedbetween 8 and 44 DAP. Developing seeds were removed from pods at 4°Cto prevent dehydration. To determine seed fresh weight, dry weight, andwater content during development, three pools of 15 randomly selectedseeds were weighed (Sartorius ISO 9001 scale, Quality Control Services,Portland, OR) just after harvest and after drying at 70°C for 24 h. To assesstheir tolerance to dehydration, three pools of 15 fresh seeds were subjectedto germination assays for seed performance analysis, and three other rep-licates of 15 seeds were dried for 48 h at room temperature (22 � 3°C). Thesedrying conditions resulted in a similar rate of water loss to that whichoccurred during drying at 70°C for 24 h. For protein analyses, pools of 35seeds were frozen in liquid nitrogen and stored at �80°C.

Germination Assays

Germination assays were carried out in a growth chamber under condi-tions described above. Two seed samples were subjected to germinationassays: fresh developing seeds and developing seeds dried at room temper-ature. For each assay, three replicates of 15 developing seeds were incubatedon three sheets of absorbent paper and a black membrane filter with a whitegrid (45 mm diameter, Schleicher & Schull, Dassel, Germany) wetted with1.5 mL of distilled water, in covered plastic boxes (50 mm diameter).Germination was scored when the primary root protruded through thesurrounding structures.

Preparation of Total Protein Extracts

Total protein extracts were prepared from immature seeds at differentstages of seed development. For each stage, a batch of 35 seeds was groundin liquid nitrogen using mortar and pestle. Total proteins were extracted at4°C in 20 �L mg�1 seed dry matter (see Fig. 1) of a thiourea/urea lysis bufferused previously for Arabidopsis seeds (Gallardo et al., 2002b). After 10 minat 4°C, 14 mm dithiothreitol (Amersham Biosciences, Orsay, France) wasadded, and the protein extracts were stirred for 20 min at 4°C and thencentrifuged (35,000g for 10 min) at 4°C. The supernatant was submitted to asecond clarifying centrifugation step as above. The final supernatant corre-sponded to the total protein extract. Protein concentration was measuredaccording to Bradford (1976).

2-D Electrophoresis

Proteins were first separated by isoelectrofocusing (IEF). For the prepa-ration of 2-D gels for Coomassie Blue staining, IEF was carried out with 30�L of the various protein extracts; for silver-stained 2-D gels, IEF wasperformed with 15 �L of protein extracts. Proteins were separated using gelstrips forming an immobilized nonlinear 3 to 10 pH gradient (ImmobilineDryStrip, 24 cm; Amersham Biosciences). Strips were rehydrated in theIPGphor system (Amersham Biosciences) for 7 h at 20°C with the thiourea/urea lysis buffer containing 2% (v/v) Triton X-100, 20 mm dithiothreitol, andthe protein extracts. IEF was performed at 20°C in the IPGphor system for7 h at 50 V, 1 h at 300 V, 2 h at 3,500 V, and 7 h at 8,000 V. Before the seconddimension, each gel strip was incubated at room temperature for 2 � 15 minin 2 � 15 mL equilibration solution as described by Gallardo et al. (2002a).Proteins were then separated in vertical polyacrylamide gels according toGallardo et al. (2002a). For each stage analyzed, at least four replicated 2-Dgels were done.

Protein Staining and Analysis of 2-D Gels

Gels were stained with either Coomassie Brilliant Blue G-250 (Bio-Rad,Hercules, CA) according to Mathesius et al. (2001) or with silver nitrateaccording to Blum et al. (1987) using the Hoefer Automated Gel Stainerapparatus from Amersham Biosciences. Image acquisition was done using aSharp JX-330 scanner (Amersham Biosciences) with a resolution of 300microns pixel�1 and an optical density range from 0.05 to 3.05. Imageanalysis was carried out on Coomassie Blue gels with the ImageMaster 2-DElite version 3.1 software (Amersham Biosciences), which allows spot de-tection and quantification, background subtraction (non-spot mode), andspot matching across the different gels. Protein spots were selected forquantitative analysis if they were consistently visible in the four replicatesfor at least one stage. Because seed development is associated with manymorphological (e.g. embryo size), physiological (e.g. increase in seed dryweight), and molecular (e.g. a high proportion of proteins accumulated)changes, the choice of a reference or normalization procedure to correctexperimental variations in 2-D gels was complex. For this purpose, thevolume of each spot (i.e. spot abundance) was normalized in the differentgels by using several methods. A first procedure, provided by the Image-Master 2-D Elite software, divided each spot volume value by the sum of allspot volume values to obtain relative spot abundances. Because the mostabundant proteins were not present in 2-D gels at 12 DAP and account formore than 30% of the total spot volume at 20 DAP, this method did notreduce any experimental variations in 2-D gels between the different stages.Therefore, we tested two other normalization procedures based either on asingle spot (procedure provided by the ImageMaster 2-D Elite software) oron 20 spots showing constant levels during the different stages subjected toproteomics. These spots were selected by comparing qualitatively silver-and Coomassie Blue-stained 2-D gels across developmental stages. Thequantitative data showed a greater reduction of experimental variationsbetween replicated 2-D gels and between the different developmental stagesby using the scaling procedure based on the 20 reference spots. Therefore,the volume of each spot was normalized to the total volume of these 20reference spots whose abundance was qualitatively constant from 12 to 20DAP (Fig. 4B, diamonds). To compare differences in protein abundanceamong the different samples, a one-way ANOVA and a Student-Newman-Keuls test were performed for each spot, using the SAS software package(SAS Institute, 1999).

Protein Identification by MS

Spots of interest were excised from Coomassie Blue 2-D gels and digestedby sequence grade trypsin (Promega, Madison, WI). After digestion, thesupernatant-containing peptides were concentrated by batch adsorption onbeads (POROS 50 R2; Roche Molecular Biochemicals, Basel) and analyzedon a MALDI-TOF mass spectrometer (Reflex II; Bruker, Bremen, Germany)after on-target desorption with matrix solution (Gevaert et al., 1998). Beforeeach analysis, the instrument was externally calibrated using two syntheticpeptides spotted as near as possible to the biological sample. All searcheswere done using MASCOT (http://www.matrixscience.com) against the M.truncatula EST (approximately 181,000 entries in September 2002) from theNational Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/Entrez/) and TIGR (http://www.tigr.org/tdb/tgi/mtgi/). To qualify

Gallardo et al.



as a positive identification, the following criteria were used: protein scoresshould have P � 0.05, coverage of the protein by the matching peptidesmust reach a minimum of 10%, and at least four independent peptidesshould match within a stringent 10-ppm maximum deviation of mass accu-racy. In some cases, protein identities were further confirmed from PSDspectra generated from selected peptides. A number of protein spots withuncertain identities were selected and analyzed by electrospray ionization-tandem MS (ESI-MS/MS) at the Unite Mixte de Recherches de GénétiqueVégétale, INRA/Universite de Paris-Sud/Institut National AgronomiquePares-Grignon (Gif-sur-Yvette, France). Search for protein sequence homol-ogy was carried out by submitting the EST (or TC) translation product to aBLASTP search (http://www.ncbi.nlm.nih.gov/blast/). Theoretical massesand isoelectric points of the homologous proteins were predicted by enter-ing the sequence at http//:www.expasy.ch/tools/peptide-mass.html/.

EST Counting in Data Sets of Two cDNALibraries from Developing Seeds

The relative expression level of the mRNAs encoding the identifiedproteins was studied by using the Medicago EST Navigation System (http://medicago.toulouse.inra.fr/) release of January 2003. In this program, theESTs are clustered based on sequence similarity and assembled into contigsreflecting the transcripts. The cluster accession numbers corresponding tothe ESTs identified by mass spectrometry were extracted from the sequenceretrieval system (SRS search) and used to determine the EST frequencies byelectronic northern in two different data sets: a cDNA library of immatureseeds collected from pods ranging in age from 11 to 19 DAP (MtGESD, 4,525ESTs) and a cDNA library of immature seeds collected from pods ranging inage from 25 to 35 DAP (MtGLSD, 4,866 ESTs). The data were then comparedwith the proteomic results.

ACKNOWLEDGMENTS

We thank Gerard Duc (Unite de Genetique et Ecophysiologie des Legu-mineuses, INRA, Dijon, France) for initiating this project, for his constantsupport, and for critical reading of the manuscript. We are grateful toFrancoise Moussy (Unite et Ecophysiologie des Legumineuses, INRA,DIJON, FRANCE) for her valuable help in the collection of the plantmaterial. We thank H. Demol and M. Puype (Flanders InteruniversityInstitute for Biotechnology, Department of Biochemistry, Gent University,Belgium) for excellent work regarding MALDI-TOF mass spectrometryanalysis. We sincerely thank Luc Negroni (INRA, Gif-sur-Yvette, France) forESI-MS/MS analyses. We also thank Dominique Job (Bayer CropScienceJoint Laboratory Unite Mixte de Recherche Centre National de la RechercheScientifique, Lyon, France) for helpful discussions, in particular regardingMet metabolism.

Received April 30, 2003; returned for revision May 27, 2003; accepted July 1,2003.

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