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First published online September 11, 2003; 10.1104/pp.103.025254 Plant Physiology 133:664-682 (2003) © 2003 American Society of Plant Biologists Proteomics of Medicago truncatula Seed Development Establishes the Time Frame of Diverse Metabolic Processes Related to Reserve Accumulation1Unité de Génétique et Ecophysiologie des Légumineuses, Institut National de la Recherche Agronomique (INRA)-Dijon, Domaine d'Epoisses, 21110 Bretenières, France (K.G., C.L.S., R.D.T., J.B); and Flanders Interuniversity 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 specific stages of seed filling corresponding to the acquisition of germination capacity and protein deposition. One hundred twenty proteins differing in kinetics of appearance were subjected to matrix-assisted laser desorption ionization time of flight mass spectrometry. These analyses provided peptide mass fingerprint data that identified 84 of them. Some of these proteins had previously been shown to accumulate during seed development in legumes (e.g. legumins, vicilins, convicilins, and lipoxygenases), confirming the validity of M. truncatula as a model for analysis of legume seed filling. The study also revealed proteins presumably involved in cell division during embryogenesis (  -tubulin and annexin). Their abundance decreased 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 an accumulation of enzymes of carbon metabolism (e.g. sucrose synthase, starch synthase) and of proteins involved in embryonic photosynthesis (e.g. chlorophyll a/b binding), which may play a role in providing cofactors for protein/lipid synthesis or for CO2 refixation during seed filling. Correlated with the reserve deposition phase was the accumulation of proteins associated with cell expansion (actin 7 and reversibly glycosylated polypeptide) and of components of the precursor accumulating vesicles, which give rise to a trypsin inhibitor on maturation. Finally, we revealed a differential accumulation of enzymes involved in methionine metabolism (S-adenosyl-methionine synthetase and S-adenosylhomo-cysteine hydrolase) and propose a role for these enzymes in the transition from a highly active to a quiescent state during seed development.
The development of the angiosperm seed proceeds through histodifferentiation and seed filling and terminates with a desiccation phase after which the embryo enters into a quiescent state, thereby permitting its storage and survival in various environmental conditions (Bewley and Black, 1994  ). The storage compounds found in most mature seeds accumulate during seed filling. They are principally storage proteins, oil (often triacylglycerols) and carbohydrates (often starch; Baud et al., 2002). These reserves are of major importance for two reasons: (a) They support early seedling growth when degraded upon germination and, therefore, participate in crop establishment; and (b) they are widely used for human and animal nutrition. Seeds of legume species, such as soybean (Glycine max), pea (Pisum sativum), and fava bean (Vicia faba), are an important protein source, with 20% to as much as 40% protein content, depending on species, genotype, and environment. In contrast, seeds of graminaceous species, such as maize (Zea mays) and wheat (Triticum aestivum), are a major source of starch and contain less than 16% protein content. However, the major proteins stored in legume seeds are poor in sulfur containing amino acids and the presence of nutritionally undesirable compounds, such as protease inhibitors, remain limiting factors. Because of its nutritional and economic importance, much effort by plant breeders is directed toward the improvement of seed quality, and both plant breeding and molecular technologies can be used to produce plants carrying the desired traits (Mazur et al., 1999). Therefore, there is strong interest in identifying the processes occurring during seed filling and the proteins involved.
The use of the most agriculturally important legume crops to study legume biology is limited by the large size of their genome and the complex ploidy. Unlike the major crop legumes, Medicago truncatula is diploid, has a small genome size (approximately 500 Mb), and is currently the subject of major genomic initiatives. To date, more than 180,000 M. truncatula expressed sequence tag (EST) sequences are available in public databases, and a sequencing project for the entire genome is underway (Bell et al., 2001
To take advantage of the available genomic resources of M. truncatula, we have characterized seed development in this species at the level of its protein complement. Proteomics offers the opportunity to examine simultaneous changes in, and to classify temporal patterns of, protein accumulation occurring in complex developmental processes such as seed filling (Bove et al., 2002 Here, we provide a framework of physiological data relevant for M. truncatula seed development and report the identification by mass spectrometry (MS) of many seed proteins. This study has not only cataloged proteins but has also described their accumulation patterns at specific stages during seed development, before and during protein deposition. These findings contribute to our understanding of how metabolic networks are regulated at the protein level during reserve deposition in seeds of a legume species. This knowledge will support our attempts to engineer legume seed composition for added end user value.
Physiology of M. truncatula Seed Development
To provide a framework for the proteomic study of seed filling, a series of stages of seed development, from embryogenesis to seed dispersal, were defined. Three phases were characterized by distinct physiological events and the associated changes in seed dry weight and moisture status. The first phase, corresponding to stages preceding 12 d after pollination (DAP), was characterized by a water content of about 90% of the seed fresh weight (Fig. 1). During this phase, which corresponds to histodifferentiation or embryogenesis (Bewley and Black, 1994
The second phase was associated with a large increase 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 to germination (T1 in Fig. 2A) declined gradually from about 16 d at 14 DAP to about 3 d at 30 DAP, indicating that seed vigor clearly increased during this phase. Similarly, seedling vigor, expressed as hypocotyl elongation and early root development, increased from 14 to 22 DAP (Fig. 2B). These data indicate that physiological and biochemical features of M. truncatula seeds, which allow vigorous germination and subsequent growth, are established during seed filling. Germination occurred without removing the seed coat, indicating that in M. truncatula the surrounding testa and endosperm are not constraints on germination in contrast to many species where these structures must be removed to allow the immature embryo to germinate (Bewley and Black, 1994
The terminal phase of M. truncatula seed development was characterized by a decrease in seed fresh weight and a drastic loss of water as the seed undergoes drying (Fig. 1). This loss of water may play a role in the switch in cellular activities from a seed formation-oriented program to an exclusively germination/growth-oriented program (Kermode et al., 1986
We characterized the proteome of seeds harvested at five stages during seed filling: seeds with low dry weight and unable to germinate (12 DAP) and seeds increasing in dry weight and developing the ability to germinate (14, 16, 18, and 20 DAP; Figs. 1 and 2A). The protein samples were analyzed by two-dimensional (2-D) gel electrophoresis. Seed filling was accompanied by an increase in the number of protein spots detected in Coomassie Blue gels up to 18 DAP (172 ± 1 spots at 12 DAP, 252 ± 3 at 18 DAP, and 245 ± 3 at 20 DAP). In the 2-D gels, the abundance of the protein spots corresponded to their volumes, which were determined by the ImageMaster 2-D Elite software as described in "Materials and Methods." The volume varies as a function of both the area and the densitometry of the detected protein spot. Therefore, spot volume is the total intensity of a defined spot and corresponds to the amount of proteins in that spot. We conducted some experiments consisting in loading five different amounts of the total seed protein extracts (from 2–45 µL) in 2-D gels. The results indicated that for approximately 85% of the studied spots, there was a strong linear relationship (0.90 The ANOVA of the relative abundance of the 274 different spots detected over the five stages permitted a classification according to their accumulation patterns (Table I). Among these polypeptides, 90 belong to class 0 proteins, that is, spots whose level did not significantly vary from 12 to 20 DAP. Class 1 and 2 proteins were represented by spots whose abundance significantly increased or decreased, respectively, during the 12- to 20-DAP period. Class 3 and 4 proteins corresponded to spots showing a transient increase or decrease, respectively, in their abundance, and class 5 proteins were represented by spots showing varying changes in their levels during this period. The protein spots were further separated in two categories: the highly abundant polypeptides (normalized volumes ranging from 10,000–150,000), and the less abundant polypeptides (normalized volumes ranging from 21–10,000).
All of the highly abundant polypeptides belong to class 1 of proteins, whose abundance increased during the 12- to 20-DAP period (Table I). Only one abundant protein spot detected in extracts from dry mature seeds (stage 44 DAP in Fig. 1) accumulated after the 20-DAP stage (data not shown), indicating that storage protein deposition is an early event in M. truncatula seed formation. Eleven well-resolved spots were analyzed by matrix-assisted laser-desorption ionization time of flight (MALDI-TOF) MS, revealing their identity as members of the major storage protein families: the legumins, vicilins, and convicilins (group 06 in Table II; species terminology medicagins, alfins, and conalfins not used further here for ease of comparison). Each of these protein families appeared at different stages in seed development. Although the vicilin spots were detected in 2-D gels at 14 DAP, the legumin spots first appeared at 16 DAP, and the convicilin spots were not detected until 18 DAP (Fig. 3). In pea, vicilins have also been shown to appear earlier than the other globulins, but convicilins started to accumulate before legumins (Wenzel et al., 1993
Many of the 242 individual less abundant proteins (normalized volumes between 21 and 10,000) showed constant level, increased, or transiently increased between 12 and 20 DAP (class 0, class 1, and class 3 proteins in Table I). Some proteins showed decreased levels (class 2 proteins in Table I), and a few spots transiently decreased (class 4 proteins in Table I) or showed varying changes in their levels (class 5 proteins in Table I). Because the main objective of this study was to reveal molecular and metabolic processes, which could play a role specifically during the phase of protein deposition, we identified by MS, in parallel to abundant seed storage proteins, those less abundant proteins whose abundance varied between 12 (stage preceding storage protein accumulation) and 20 (stage after protein deposition) DAP. One hundred nine polypeptides of low abundance, taken from the protein classes 1 to 5 and well resolved, were analyzed by MALDI-TOF MS, and 73 proteins were successfully identified (Tables II, III, IV, V). Among these proteins, 23 corresponded to minor legumin chains or vicilins, many of which accumulated at 16 to 18 DAP and disappeared after this stage (Table IV). Some of these may correspond to unstable forms of storage proteins not yet fully processed or to proteins that were either misdirected or misfolded and degraded. Some could also correspond to fragments released by specific proteolysis of the storage protein, as observed before Arabidopsis seed germination (Gallardo et al., 2001
The 50 other less abundant proteins identified by MS were classified in different groups corresponding to their presumed biological function. Figure 4 shows that many of them are presumed to be involved in energy, disease/defense, metabolism, protein destination and storage, cell growth/division, and cell structure. Some of these proteins are nutritionally undesirable in legume seeds, such as lipoxygenases (spots 391, 444, and 447 and group 09 in Table II and 274 in Table V) known to produce hydroperoxides that attack nutritionally essential components (e.g. proteins, vitamins, and polyunsaturated fatty acids) and to release off-flavors compounds (Robinson et al., 1995
To get a first indication of the extent of the correlations between mRNA and protein levels during seed development, the frequency of occurrence of the transcripts corresponding to the proteins identified was determined in the EST data sets of the cDNA libraries from early and late developing M. truncatula seeds. The Medicago EST Navigation System, previously used to characterize the sets of genes expressed in roots of M. truncatula during symbiosis (Journet et al., 2002
The comparison of the results showing significant variations between early and late developing seeds with the proteomic data revealed some correlations between transcript and protein levels. For example, the results suggest that the levels of mRNAs encoding proteins specifically associated with the early stages of seed filling (class 2 and class 3 in Table I), such as Ado-Met synthetase and PDI (Table VI), were higher in early developing seeds, whereas the transcripts encoding proteins that accumulated during the later stages (18–20 DAP), such as legumins and convicilins (Table VI), were preferentially expressed in the late developing seeds. The results also suggest that the levels of transcripts encoding some proteins of class 1 (Table I) decreased during the later stages of seed development. For those proteins whose abundance is maintained up to the desiccation stage, such as vicilins, the absence or decrease of the transcript at late stages suggests a high stability for these proteins throughout seed development (data not shown). Although this approach is not sufficient to establish a rigorous validation of gene expression, the results provide a basis to elucidate the mechanisms of regulation of protein accumulation and stability.
The seed occupies a central position in the life cycle of higher plants. In addition to its role in dispersal, the seed determines the success with which germination and early seedling growth occur. Moreover, seeds such as those of legumes are major food sources whose importance lies in the proteins stored during development. Our aim was to identify seed proteins characteristic of specific stages during reserve deposition in M. truncatula. These data will help elucidate the biochemical and molecular processes underlying seed filling in a legume species. Eighty-four proteins whose abundance varied during reserve deposition were identified by MS. As expected, the most abundant proteins corresponded to storage proteins. Many of the weakly abundant proteins could play a role in cell division during embryogenesis, in protein or starch deposition, in defense against herbivores, in cell expansion during reserve deposition, or in the transition from a highly active to a quiescent state during seed development. These results are discussed in the following sections.
Embryogenesis starts with a morphogenesis phase during which the embryo differentiates through several distinct stages (globular, heart, and torpedo) and ends at the cotyledon stage when all embryo structures have been formed. Figure 2A shows that acquisition of ability to germinate does not occur before 14 DAP, presumably because the young embryo is not fully formed before this stage. At the end of embryogenesis, cell division arrests and the seed accumulates the storage components (Raz et al., 2001
After the cessation of cell division, the seed storage compounds are synthesized. In M. truncatula, the reserve deposition phase is characterized by a large increase in the protein content of up to 45% (G. Duc, personal communication). In our study, the most abundant protein spots, which are mainly responsible for this increase in the protein content and, thus, for the nutritional value of legume seeds, were identified as being the 7S (vicilin and convicilin) and 11S (legumin) globulins (Table II). These protein families accumulated in a specific temporal order during seed filling: vicilins (14 DAP), legumins (16 DAP), and convicilins (18 DAP; Fig. 3). The transcripts encoding the storage protein families were expressed in a similar time course when compared with protein abundance, with mRNAs encoding vicilins being preferentially expressed in the early stages, and mRNAs encoding the legumin and convicilin families being preferentially expressed in the later stages (Table VI) as observed in our proteome analysis. This suggests that the temporal accumulation of the storage protein families is likely to be transcriptionally controlled during seed development. As expected for proteins encoded by multigene families (Casey et al., 2001
The characterization of the globulin families in various dicotyledonous plants has shown that the precursor forms of these proteins are transported from the endoplasmic reticulum lumen to the protein storage vacuoles, where they are processed into specific subunits. These chains are then assembled within the protein bodies, yielding the mature forms, typically trimeric for the 7S globulins and hexameric for the 11S globulins (Gruis et al., 2002
Most of the mature products of the M. truncatula 7S and 11S globulins possessed similar masses to those found in pea and soybean seeds (Croy et al., 1980
Endoplasmic reticulum resident proteins known as molecular chaperones play important roles in the formation and assembly of the seed storage proteins (Li and Larkins, 1996
Interestingly, in addition to vicilin polypeptides of the expected 30- to 40-kD class, two protein spots of Mr approximately 120,000 (389 and 390 and group 09 in Table II) that accumulated during seed filling also possessed vicilin-related sequences. In their respective MALDI-TOF spectrums, at least eight peptide masses matched with those from vicilins. Despite their high Mrs, they were identified provisionally, therefore, as vicilins. A similar protein D of approximately 100,000 containing a vicilin-like domain was described in pumpkin (Cucurbita maxima) seeds. This protein, called PV100, is synthesized on rough endoplasmic reticulum as a precursor form consisting of a single protein chain with three domains: a vicilin-like domain, a Cys-rich domain (91 amino acids), and an Arg-/Glu-rich domain (267 amino acids; Yamada et al., 1999
In some legumes, such as soybean, starch can be present early during seed development. However, the starch level declines to about 1% and is replaced by accumulating oil reserves on maturation (Adams et al., 1980
During seed filling, the cells continue to grow by enlargement as they accumulate the storage components. Two proteins identified in this study could be involved in this cell enlargement process during protein deposition. The first corresponded to actin (ACT7 and spot 187 in Table IV), which is a fundamental component of the cytoskeleton. Its level increased specifically during protein deposition and decreased after this stage. In Arabidopsis, the gene encoding the same isoform ACT7 is preferentially expressed in vegetative tissues that contain rapidly dividing and expanding cells and appears to be the only actin gene expressed in seed tissues (McDowell et al., 1996a
The second protein (spot 206 and group 07 in Table II) showed more than 88% identity with the reversibly glycosylated polypeptide (RGP1) identified in suspension-cultured cells, roots, and leaves in Arabidopsis (Delgado et al., 1998
Interestingly, many proteins whose abundance varied during seed filling corresponded to enzymes involved in Met biosynthesis. Among the essential amino acids synthesized by plants, Met is a fundamental metabolite because it functions not only as a building block for protein but also as the precursor of Ado-Met, the primary methyl-group donor and the precursor of polyamines and the plant ripening hormone ethylene (Ravanel et al., 1998
In plants, Met can be synthesized through two pathways (Fig. 6). In the de novo biosynthetic pathway, O-phosphohomo-Ser is transformed to cystathionine in a reaction catalyzed by cystathionine
Consistent with the high demand for protein synthesis between 12 and 20 DAP, two spots detected in seed extracts throughout this period corresponded to Met synthase (spots 14 and 276 and group 01 in Tables II and V), which catalyzes the last step of the de novo biosynthetic pathway of Met. In addition, one spot (156 in Table III) corresponded to Ado-Met synthetase, which catalyzes the synthesis of Ado-Met from 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 not shown). This result was supported by the observation that ESTs corresponding to Ado-Met synthetase were only found in cDNA libraries corresponding to early stages of seed development (Table VI). Ado-Met synthetase also was absent from dry mature Arabidopsis seeds (Gallardo et al., 2001
After the decrease of Ado-Met synthetase, there was an increase in the abundance of two enzymes involved in Ado-Met consumption. The first protein corresponded to AdoHcy hydrolase (spot 124 and group 01 in Table II; Fig. 6). The hypothesis that AdoHcy hydrolase is active during seed development agrees with previous results showing that Met recycling via the Ado-Met/AdoHcy and SMM cycles is not sufficient in mature seeds to maintain an appropriate pool of Met for rapid germination and seedling establishment (Gallardo et al., 2002a
In plants, Ado-Met has an important influence on cell growth and development. Beside its role in ethylene, biotin, and polyamine biosynthesis, Ado-Met is the primary methyl group donor for the methylation of amino acids, lipids, RNA, and DNA, and functions as an effector in the regulation of Thr, Lys, and Met synthesis (Ravanel et al., 1998
We utilized a proteomic approach to identify 84 M. truncatula seed proteins with characteristic developmental patterns of accumulation during protein deposition. Some of these had previously been shown to play a role during seed filling in other legume species (e.g. legumins, vicilins, and convicilins), confirming the validity of M. truncatula as a model system for analysis of legume seed filling. The present study also revealed the kinetics of storage protein accumulation in M. truncatula and new proteins to be associated with the reserve deposition process, with presumed roles in cell division (annexin), cell expansion (ACT7 and RGP1), or metabolic activities (for example, Ado-Met synthetase and AdoHcy hydrolase). Furthermore, the data revealed nutritionally undesirable components whose elimination should improve the quality of legume seeds, such as lipoxygenases and components of the precursor-accumulating vesicles (PV100), which give rise to a trypsin inhibitor on maturation. These data will facilitate further studies, which investigate the effects of genetic and environmental factors on seed quality. The role of these proteins can be further assessed by a combination of forward and reverse genetics, such as the TILLING (targeting induced local lesions in genomes) methodology (McCallum et al., 2000 ). This will provide additional information useful for understanding the complexities of seed metabolism and its control during seed development.
Plant Material A batch of 20 plants of Medicago truncatula cv Jemalong, line J5 was used for all experiments. Plants were grown in a growth chamber at 22°C/19°C day/night temperatures, under a 16-h photoperiod at 220 µE m2 s–1 light intensity with 60% to 70% relative humidity. To harvest pods at defined stages during development, individual flowers were tagged on the day of flower opening. For each stage, at least 40 flowers were labeled on the first, second, and third nodes of the main ramifications. Pods were harvested between 8 and 44 DAP. Developing seeds were removed from pods at 4°C to prevent dehydration. To determine seed fresh weight, dry weight, and water content during development, three pools of 15 randomly selected seeds were weighed (Sartorius ISO 9001 scale, Quality Control Services, Portland, OR) just after harvest and after drying at 70°C for 24 h. To assess their tolerance to dehydration, three pools of 15 fresh seeds were subjected to germination assays for seed performance analysis, and three other replicates of 15 seeds were dried for 48 h at room temperature (22 ± 3°C). These drying conditions resulted in a similar rate of water loss to that which occurred during drying at 70°C for 24 h. For protein analyses, pools of 35 seeds were frozen in liquid nitrogen and stored at –80°C.
Germination assays were carried out in a growth chamber under conditions described above. Two seed samples were subjected to germination assays: fresh developing seeds and developing seeds dried at room temperature. For each assay, three replicates of 15 developing seeds were incubated on three sheets of absorbent paper and a black membrane filter with a white grid (45 mm diameter, Schleicher & Schull, Dassel, Germany) wetted with 1.5 mL of distilled water, in covered plastic boxes (50 mm diameter). Germination was scored when the primary root protruded through the surrounding structures.
Total protein extracts were prepared from immature seeds at different stages of seed development. For each stage, a batch of 35 seeds was ground in liquid nitrogen using mortar and pestle. Total proteins were extracted at 4°C in 20 µL mg–1 seed dry matter (see Fig. 1) of a thiourea/urea lysis buffer used previously for Arabidopsis seeds (Gallardo et al., 2002b
Proteins were first separated by isoelectrofocusing (IEF). For the preparation of 2-D gels for Coomassie Blue staining, IEF was carried out with 30 µL of the various protein extracts; for silver-stained 2-D gels, IEF was performed with 15 µL of protein extracts. Proteins were separated using gel strips forming an immobilized nonlinear 3 to 10 pH gradient (Immobiline DryStrip, 24 cm; Amersham Biosciences). Strips were rehydrated in the IPGphor 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, and the protein extracts. IEF was performed at 20°C in the IPGphor system for 7 h at 50 V, 1 h at 300 V, 2 h at 3,500 V, and 7 h at 8,000 V. Before the second dimension, each gel strip was incubated at room temperature for 2 x 15 min in 2 x 15 mL equilibration solution as described by Gallardo et al. (2002a
Gels were stained with either Coomassie Brilliant Blue G-250 (Bio-Rad, Hercules, CA) according to Mathesius et al. (2001
Spots of interest were excised from Coomassie Blue 2-D gels and digested by sequence grade trypsin (Promega, Madison, WI). After digestion, the supernatant-containing peptides were concentrated by batch adsorption on beads (POROS 50 R2; Roche Molecular Biochemicals, Basel) and analyzed on a MALDI-TOF mass spectrometer (Reflex II; Bruker, Bremen, Germany) after on-target desorption with matrix solution (Gevaert et al., 1998
The relative expression level of the mRNAs encoding the identified proteins was studied by using the Medicago EST Navigation System (http://medicago.toulouse.inra.fr/) release of January 2003. In this program, the ESTs are clustered based on sequence similarity and assembled into contigs reflecting the transcripts. The cluster accession numbers corresponding to the ESTs identified by mass spectrometry were extracted from the sequence retrieval system (SRS search) and used to determine the EST frequencies by electronic northern in two different data sets: a cDNA library of immature seeds collected from pods ranging in age from 11 to 19 DAP (MtGESD, 4,525 ESTs) and a cDNA library of immature seeds collected from pods ranging in age from 25 to 35 DAP (MtGLSD, 4,866 ESTs). The data were then compared with the proteomic results.
We thank Gerard Duc (Unité de Gènetique et Ecophysiologie des Legumineuses, INRA, Dijon, France) for initiating this project, for his constant support, and for critical reading of the manuscript. We are grateful to Françoise Moussy (Unité et Ecophysiologie des Legumineuses, INRA, DIJON, FRANCE) for her valuable help in the collection of the plant material. We thank H. Demol and M. Puype (Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Gent University, Belgium) for excellent work regarding MALDI-TOF mass spectrometry analysis. We sincerely thank Luc Negroni (INRA, Gif-sur-Yvette, France) for ESI-MS/MS analyses. We also thank Dominique Job (Bayer CropScience Joint Laboratory Unité Mixte de Recherche Centre National de la Recherche Scientifique, Lyon, France) for helpful discussions, in particular regarding Met metabolism. Received April 30, 2003; returned for revision May 27, 2003; accepted July 1, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.025254.
1 This work was supported by the INRA-Action Transversale Structurante program on Medicago truncatula and by INRA (postdoctoral fellowship to K.G.). * Corresponding author; e-mail gallardo{at}epoisses.inra.fr; fax 33–3–80–69–32–63.
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ABSTRACT
RESULTS
R2 
-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, Proportions of the storage protein families identified by mass spectrometry.
2 test, P < 0.050) were grouped according to their expression profiles during seed development. In each class of proteins (see 
-synthase, then to Hcy in a reaction catalyzed by cystathionine 
