TY - JOUR
AU - Thompson, R.
AB - Abstract The seed consists of several layers of specialized cell-types that divide and differentiate following a highly regulated programme in time and space. A cytological approach was undertaken in order to study the histo-differentiation at mid-embryogenesis in Medicago truncatula as a model legume, and in Pisum sativum using serial sections of embedded immature seed. Little published information is available about seed development in Medicago species. The observations from this study revealed a number of distinctive features of Medicago seed development and differentiation. Transfer cells, involved in nutrient transfer to the embryo, were clearly identified in the thin-walled parenchyma of the innermost integument. Histological Schiff–naphthol enabled carbohydrate accumulation to be followed in the different seed compartments, and revealed the storage protein bodies. Non-radioactive mRNA in situ hybridization, was carried out using mRNA probes from two highly expressed genes encoding the major vicilin and legumin A storage protein types. The timing of mRNA expression was related to that of the corresponding proteins already identified. In situ hybridization, legumin A mRNA, Medicago truncatula, mid-embryogenesis, Pisum sativum, seed, vicilin mRNA Introduction In flowering plants, seed development is a highly regulated process during which tissues from different genetic origins are differentiated (West and Harada, 1993; Wobus and Weber, 1999). Grain legumes play a key role worldwide as a source of plant proteins for feed and food. In the model legume Medicago truncatula, which is phylogenetically related to the most important European legume crops, very little information is available on seed development. However, a large programme of structural, functional, and comparative genomics is being performed (Cook, 1999). In addition, mutant populations destined for use in reverse genetics approaches such as TILLING are being generated, with a resulting need for grain phenotyping, for example, for candidate genes governing seed size, yield, and protein content (Le signor and R Thompson, personal communication). A histological approach, initiated in Medicago truncatula immature seeds at mid-embryogenesis, is reported here. The focus has been on this developmental stage in which embryo morphogenesis, after several rapid cell divisions, leads to the definition of the main organs in the seed and signals the start of the cell expansion and storage product accumulation phase. The results were compared with this study's observations in Pisum sativum seeds as a promising source of protein for feed, and to earlier data in Vicia faba and P. sativum (Borisjuk et al., 1995, 2002a, b). Moreover, P. sativum, which has both agricultural and economic importance; was also shown by macro and microsynteny genome studies to be closely related to M. truncatula (Choi et al., 2004). The expression of legumin and vicilin genes regarded as a marker of maturation was studied by mRNA in situ hybridization. The two major legume seed storage protein classes, legumin (11S) and vicilin (7S), are encoded by multi-gene families of at least 40 genes as reported for P. sativum (Casey et al., 2001). Both classes of storage proteins are normally found in legume seeds although their relative proportions may vary (Gibbs et al., 1989). The transcripts encoding storage proteins are amongst the most abundant in the embryo; their accumulation is temporally and spatially regulated during seed development (Kroj et al., 2003). The kinetics and the sites of mRNA accumulation have been investigated in relation to histo-differentiation at the early maturation phase in M. truncatula and P. sativum. Paraplast seed sections 12–14 DAP and 16–18 DAP were used to visualize and localize digoxigenin-labelled vicilin and legumin A mRNA by in situ hybridization. Materials and methods Plant material Plants of Medicago truncatula cv. Jemalong and Pisum sativum cv. Frisson were grown in growth chambers at 22/19 °C day/night temperatures and harvested at different development stages as reported by Gallardo et al. (2003). Pods were harvested between 10 and 19 d after pollination (DAP). Fixation, sectioning, and histochemical staining Immature seeds were fixed in 4% (w/v) paraformaldehyde and 50 mM of potassium phosphate buffer (pH 7.0) overnight at 4 °C, then dehydrated in a graded ethanol series, and embedded in paraplast. 7–10 μm sections were prepared using a disposable steel knife and transferred to anti-RNase treated Dako® slides. The staining was performed using toluidine blue 1% in sodium carbonate solution (pH 7) to follow the developmental stages, fluorescent DAPI (1 μg ml−1) in McIlvaine buffer (pH 5.5) to visualize nuclear DNA, and double Schiff–naphthol staining (Fischer, 1968) to reveal polysaccharides and total protein accumulation. Soluble and insoluble proteins were stained specifically in blue with naphthol blue-black (NBB) and polysaccharides were stained in red with periodic acid–Schiff (PAS). The fast green staining followed Gallant et al. (1994). The slides were observed by light microscopy DMRX A (Leica®) using 10× and 20× power and photos were taken using a Canon® digital camera and a Sony colour video camera. DAPI-stained sections were viewed by epifluorescence. In situ hybridization The cDNA sequences were prepared by RT-PCR of total RNA sequences isolated from immature seeds of P. sativum, and M. truncatula. The primers for RT-PCR amplification shown in Table 1 were defined using consensus sequences which corresponded to the matching of the M. truncatula EST seed sequences library and known genomic pea sequences of legumin A genes (consensus of 13 EST) and vicilin (consensus of 63 EST) at http://www.tigr.org/tdb/tgi/mtgi/. The ligation of the RNA polymerase T7 and T3 promoters at the end of the cDNA fragments using T4 DNA ligase was followed by selective PCR amplification (Kit lig'n Scribe Ambion). The PCR products were used as templates for in vitro transcription to obtain the anti-sense and sense RNA probes. The 350 bp probes were labelled during in vitro transcription by the incorporation of Dig–11–UTP (Kit Maxi-Script Ambion). A riboprobe was used as a positive control. Table 1. The specific primers used in RT-PCR amplification of the legumin A and vicilin cDNA segments in Medicago truncatula and Pisum sativum Vicilin reverse  5′-GTTGAGGCTGAGCATTTG-3′  Tm 53.7 °C  Vicilin direct  5′-GCCACAGTGATAGTAGCAGT-3′  Tm 57.3 °C  Legumin reverse  5′-CATCACCTGACATCTACAACC-3′  Tm 57.9 °C  Legumin direct   5′-GGCACACCAATTAGAGTGG-3′   Tm 58.8 °C   Vicilin reverse  5′-GTTGAGGCTGAGCATTTG-3′  Tm 53.7 °C  Vicilin direct  5′-GCCACAGTGATAGTAGCAGT-3′  Tm 57.3 °C  Legumin reverse  5′-CATCACCTGACATCTACAACC-3′  Tm 57.9 °C  Legumin direct   5′-GGCACACCAATTAGAGTGG-3′   Tm 58.8 °C   View Large Slides were dewaxed, then incubated for 30 min in proteinase K (0.4% in PBS) at 37 °C. In situ hybridization was performed at 45 °C overnight in 0.5 ng μl−1 mRNA probe in 50% formamide, 1× Denhardt, 2× SSC, 10% dextran sulphate, and 1 μg μl−1E. coli tRNA. The hybridization mixture was denatured at 65 °C for 2 min before use. Post-hybridization washing at graded stringency (2× SSC, 1× SSC, 0.2× SSC) for 1 h at 50 °C was followed by immunodetection using an anti–digoxigenin antibody conjugated to alkaline phosphatase (Roche). The slides were first pretreated with a blocking solution (Roche) to prevent non-specific binding of antibodies, then incubated for 1 h at 37 °C with the anti–digoxigenin antibody (1/1000). Antigens were detected using an alkaline phosphatase assay with incubation in the NBT/BCIP substrate (Sigma) in the dark overnight following the manufacturer's instructions. Slides were kept permanently by adding 30 μl of the inSitu® mounting medium (Sigma). All glassware was sterilized and solutions were prepared in RNase-free conditions using DEPC-treated water. Results Histo-differentiation at mid-embryogenesis In 10 DAP M. truncatula seeds, the early torpedo embryo occupied a small fraction of the seed volume (Fig. 1A). In 12–14 DAP seeds the bending cotyledons embryo, fully embedded in the endosperm, had well-developed cotyledons which displayed a ramified provascular network, and an emergent embryonic axis (Fig. 1B, C, D). At this stage, a developmental gradient within the growing cotyledon was observed. The distribution of the DAPI-stained nuclei showed that the outermost abaxial cells still maintained mitotic activity. They had smaller sizes and their nuclei were in closer proximity than in the innermost adaxial region where cells have ceased dividing and started to enlarge and differentiate (Fig. 1E, F, G, H). The endosperm already cellularized by 8 DAP remained abundant between 12 and 18 DAP occupying more than half of the seed volume. A large increase in the endosperm nuclear volume indicates the occurrence of high endoploidy levels in the embryo attached endosperm (Fig. 1I). An epidermal cell-type layer, adjacent to the seed coat and enclosing the endosperm cells, displayed a high metabolic activity with a dense cytoplasm and very small vacuoles. It should be related to a transfer cell layer (Fig. 2A, B). The seed coat was observed as a multilayered structure. Under the mucilaginous layer, the palisade epidermis layer of the outer integument had developed lignified thickenings known as macrosclereids. Then the chlorenchyma was leaning against an inner layer which accumulated anthocyanin pigment that was particularly abundant at the micropylar region. This region hosted the provascular apparatus organized in a compact group of tracheids (Fig. 2C, D, E, F). In P. sativum a similar seed coat organization was observed with starch granule accumulation in the outer integuments (Fig. 2G, H). Fig. 1. View largeDownload slide Paraplast sections in M. truncatula immature seeds at different embryo development stages: 10 DAP (A, H, I), 12–14 DAP (B, C, D, E, F, G). Toluidine blue-stained sections are in (A), (B), (C), and (D) and DAPI-stained sections are in (E), (F), (G), (H), and (I). The embryo nuclei distribution was shown in (E), (F), and (G) at 12 DAP in (E) and 14 DAP in (F) and (G). Note that nuclei distribution is dependent on the embryo region (abaxial versus adaxial). In (H) mitotic figures are shown from an enlarged embryo region. In (I) the high polyploidy of the endosperm cells became evident when nuclei were compared with the embryo cell nuclei. e, Embryo proper; en, endosperm; mi, micropyle region; sc, seed coat. Bars: (A, B, C, D) 150 μm; (E, F, G) 100 μm; (H, I) 50 μm. Fig. 1. View largeDownload slide Paraplast sections in M. truncatula immature seeds at different embryo development stages: 10 DAP (A, H, I), 12–14 DAP (B, C, D, E, F, G). Toluidine blue-stained sections are in (A), (B), (C), and (D) and DAPI-stained sections are in (E), (F), (G), (H), and (I). The embryo nuclei distribution was shown in (E), (F), and (G) at 12 DAP in (E) and 14 DAP in (F) and (G). Note that nuclei distribution is dependent on the embryo region (abaxial versus adaxial). In (H) mitotic figures are shown from an enlarged embryo region. In (I) the high polyploidy of the endosperm cells became evident when nuclei were compared with the embryo cell nuclei. e, Embryo proper; en, endosperm; mi, micropyle region; sc, seed coat. Bars: (A, B, C, D) 150 μm; (E, F, G) 100 μm; (H, I) 50 μm. Fig. 2. View largeDownload slide Mid-embryogenesis (12–14 DAP) seed sections stained with toluidine blue and showing the structure of the seed coat in M. truncatula (A, B, C, D, E, F) and P. sativum (G, H). (A) A tangential seed section through the seed coat, and endosperm. (B) An enlargement of the enclosed region where some endosperm cells surrounded by a transfer cell layer are shown. (C) A section through the micropyle illustrating the vascularization in this region. The seed epidermis with macrosclereids and the pigment layer are visualized in (D). (E) The multilayered seed coat structure is shown. (F) The seed coat and the endosperm underlined by the transfer cell layer; to be compared with (G) where the seed coat surrounds an empty endosperm vacuole in P. sativum. (H) A seed coat section in P. sativum displaying transient starch granules accumulation in the outer integument. e, Embryo proper; en, endosperm; P, palisade epidermis; mi, micropyle; ma, macrosclereid; pl, pigment layer; gp, ground parenchyma; bp, branched parenchyma; tc, transfert cells; vb, vascular bundle; cl, chlorenchyma; ev, endospermal vacuole; s, starch granule. Bars equal 100 μm. Fig. 2. View largeDownload slide Mid-embryogenesis (12–14 DAP) seed sections stained with toluidine blue and showing the structure of the seed coat in M. truncatula (A, B, C, D, E, F) and P. sativum (G, H). (A) A tangential seed section through the seed coat, and endosperm. (B) An enlargement of the enclosed region where some endosperm cells surrounded by a transfer cell layer are shown. (C) A section through the micropyle illustrating the vascularization in this region. The seed epidermis with macrosclereids and the pigment layer are visualized in (D). (E) The multilayered seed coat structure is shown. (F) The seed coat and the endosperm underlined by the transfer cell layer; to be compared with (G) where the seed coat surrounds an empty endosperm vacuole in P. sativum. (H) A seed coat section in P. sativum displaying transient starch granules accumulation in the outer integument. e, Embryo proper; en, endosperm; P, palisade epidermis; mi, micropyle; ma, macrosclereid; pl, pigment layer; gp, ground parenchyma; bp, branched parenchyma; tc, transfert cells; vb, vascular bundle; cl, chlorenchyma; ev, endospermal vacuole; s, starch granule. Bars equal 100 μm. In M. truncatula a transient starch accumulation was observed in the internal integument of the seed coat as early as 8 DAP (Fig. 3A). Later, in 12–14 DAP seed sections, the periodic acid–Schiff staining (PAS) revealed, only in the seed coat, a high level of carbohydrate accumulation mainly as starch granules (Fig. 3B). During seed filling at 16 DAP, the cotyledon cells displayed several storage vacuoles which replaced the large central vacuole. Protein accumulation was revealed as dark-blue cytoplasmic strands by naphthol histochemical staining, which identified a multitude of dense storage protein bodies in the cotyledon cells. However, starch granules were never detected in the cotyledon cells (Fig. 3C). These dense bodies (1–5 μm diameter) were fast green positive (Fig. 3D) and also immunolabelled by vicilin antibodies (data not shown). Fig. 3. View largeDownload slide Immature seed sections showing storage accumulation in M. truncatula (A, B, C, D) and in P. sativum (E, F, G, H). (A) Starch accumulation in the seed coat 8 DAP after toluidine blue staining. (B, C) Schiff–naphthol staining. (B) There is a high level of carbohydrate accumulation in the seed coat at 14 DAP and (C) shows the storage protein bodies in embryo cells at 16 DAP. At the same stage, protein bodies were revealed as green granules by fast green in (D). Note that starch granules are absent during protein storage accumulation in the M. truncatula embryo. (E, F) Schiff–naphthol staining. (E) Starch accumulation in 12 DAP pea embryo cells. (F) Protein bodies and starch in 19 DAP embryo cells. (G, H) Protein bodies stained by fast green and starch by iodide in embryo cells at 14 DAP (G) and 19 DAP (H). Bars equal 50 μm. e, Embryo proper; en, endosperm; n, cell nucleus; pb, protein bodies; sc, seed coat; s, starch. Arrows in (D), (G), and (H) point to protein bodies. Fig. 3. View largeDownload slide Immature seed sections showing storage accumulation in M. truncatula (A, B, C, D) and in P. sativum (E, F, G, H). (A) Starch accumulation in the seed coat 8 DAP after toluidine blue staining. (B, C) Schiff–naphthol staining. (B) There is a high level of carbohydrate accumulation in the seed coat at 14 DAP and (C) shows the storage protein bodies in embryo cells at 16 DAP. At the same stage, protein bodies were revealed as green granules by fast green in (D). Note that starch granules are absent during protein storage accumulation in the M. truncatula embryo. (E, F) Schiff–naphthol staining. (E) Starch accumulation in 12 DAP pea embryo cells. (F) Protein bodies and starch in 19 DAP embryo cells. (G, H) Protein bodies stained by fast green and starch by iodide in embryo cells at 14 DAP (G) and 19 DAP (H). Bars equal 50 μm. e, Embryo proper; en, endosperm; n, cell nucleus; pb, protein bodies; sc, seed coat; s, starch. Arrows in (D), (G), and (H) point to protein bodies. In developing pea cotyledons at 12 DAP, the early maturation stage, Schiff–naphthol revealed starch accumulation but no storage protein bodies were observed (Fig. 3E). Protein bodies began to differentiate in 14 DAP cotyledon cells, and became abundant in 19 DAP seeds. As shown in Fig. 3F, G, and H, the naphthol blue-black-stained protein bodies were also fast green positive. They were observed with an accumulation of numerous starch granules. Vicilin and legumin A mRNA accumulation by in situ hybridization In M. truncatula, vicilin and legumin A mRNA accumulation detected by in situ hybridization was very abundant in seed sections at 16 DAP. Probe binding was evident as a strong blue-black colour observed in the cytoplasm surrounding the nuclei and the large vacuole. Hybridization signals were revealed in all the cotyledon cells, showing a gradient toward enlarged adaxial cotyledon cells (Fig. 4A, B, C). The legumin A anti-sense probe was also faintly detected in the embryonic axis cells (Fig. 4D). In seed sections at 12–14 DAP, vicilin and legumin A mRNA accumulation was evident when compared with the sense probe; however, cell labelling was uniformly weaker throughout the cotyledon at this stage and particularly so for the legumin A mRNA probe (Fig. 4E, F, G). The embryo epidermis and the provascular tissue did not express either of the transcripts, neither did the seed coat nor the endosperm. Fig. 4. View largeDownload slide In situ hybridization of vicilin and legumin A mRNA accumulation in M. truncatula paraplast sections at two developmental stages. The anti-sense probe binding was evident as blue-dark signals in the cytoplasm (A, C, D, E, F). Negative control with sense probe hybridization did not yield any detectable signal (B, G). Positive control using a riboprobe is shown in (H). In 16 DAP seed sections, there is high density accumulation of vicilin (A) and legumin A (C); mRNA localization is mainly in the embryo parenchyma cells, to compare with the riboprobe pattern where the seed coat cells were also probed (H). Fewer signals were observed in the embryonic axis with the legumin A probe (D). Note the wave–like development of the embryo where different cell size population are observed (A, B). In 14 DAP seed sections, the labelling is significantly lighter in the cotyledon cells, and the mRNA accumulation is more abundant for vicilin (E) than legumin A (F). e, Embryo proper; ea, embryonic axis; ep, epidermis; c, cotyledon; sc, seed coat; vb, vascular bundle. Bars: (A, B, D, E, F) 40 μm; (C, G, H) 60 μm. Fig. 4. View largeDownload slide In situ hybridization of vicilin and legumin A mRNA accumulation in M. truncatula paraplast sections at two developmental stages. The anti-sense probe binding was evident as blue-dark signals in the cytoplasm (A, C, D, E, F). Negative control with sense probe hybridization did not yield any detectable signal (B, G). Positive control using a riboprobe is shown in (H). In 16 DAP seed sections, there is high density accumulation of vicilin (A) and legumin A (C); mRNA localization is mainly in the embryo parenchyma cells, to compare with the riboprobe pattern where the seed coat cells were also probed (H). Fewer signals were observed in the embryonic axis with the legumin A probe (D). Note the wave–like development of the embryo where different cell size population are observed (A, B). In 14 DAP seed sections, the labelling is significantly lighter in the cotyledon cells, and the mRNA accumulation is more abundant for vicilin (E) than legumin A (F). e, Embryo proper; ea, embryonic axis; ep, epidermis; c, cotyledon; sc, seed coat; vb, vascular bundle. Bars: (A, B, D, E, F) 40 μm; (C, G, H) 60 μm. In P. sativum 12 DAP seed sections, at the pre-storage stage, the legumin A mRNA which began to accumulate, showed a weak hybridization signal over the cotyledonary parenchyma cells (Fig. 5A, B, C). The vicilin mRNA anti-sense probe was detected in three or four layers beneath the embryo epidermis and decreased toward the core of the cotyledons. (Fig. 5D). Later, in 19 DAP pea seed sections, during seed filling, an intensive hybridization was observed with both the vicilin and the legumin A mRNA probe. The embryo epidermis, the plumule and the radicle tissues were never probed (Fig. 5E, F, G, H). Fig. 5. View largeDownload slide In situ hybridization of vicilin and legumin A mRNA in P. sativum paraplast sections at two developmental stages. (A, B, C, D) mRNA accumulation in 12 DAP seed sections after in situ hybridization with the anti-sense probe for legumin A (A, C), the sense probe (B), and the vicilin anti-sense probe (D). In 19 DAP seed sections (E, F, G, H) intensive mRNA accumulation is shown for the legumin A gene (E, F, G) and the vicilin gene (H). Note the difference in the expression level in (C) and (G). Arrows in (G) point on mRNA accumulation. Bars equal 40 μm. e, Embryo proper; ae, embryonic axis, ep, embryo epidermis; n, cell nucleus; s, starch. Fig. 5. View largeDownload slide In situ hybridization of vicilin and legumin A mRNA in P. sativum paraplast sections at two developmental stages. (A, B, C, D) mRNA accumulation in 12 DAP seed sections after in situ hybridization with the anti-sense probe for legumin A (A, C), the sense probe (B), and the vicilin anti-sense probe (D). In 19 DAP seed sections (E, F, G, H) intensive mRNA accumulation is shown for the legumin A gene (E, F, G) and the vicilin gene (H). Note the difference in the expression level in (C) and (G). Arrows in (G) point on mRNA accumulation. Bars equal 40 μm. e, Embryo proper; ae, embryonic axis, ep, embryo epidermis; n, cell nucleus; s, starch. Discussion The seed is a protein resource for seedling growth and also for food and feed. The immature seed is a heterogeneous, highly organized system, consisting of different organs where deviations from wild-type embryo morphology are frequently associated with a lack of storage protein accumulation, as shown by the study of Arabidopsis Emb mutants (Devic et al., 1996). The light microscopy analysis of M. truncatula immature seed sections allowed some morphological and histological features of the main seed organs to be defined and for these to be related to the expression of two storage protein genes by in situ hybridization. Earlier studies on interspecific Medicago seeds pointed to the importance of a co-ordinated regulation in both maternal and embryonic tissue (Sangduen et al., 1982). This study is the first histological report on seed development in the model legume M. truncatula. Morphological and histological characteristics of seed at mid-embryogenesis In M. truncatula, at the micropylar region, the seed coat highly implicated in the nutrient transport, displayed a prominent vascular system which was organized in characteristic tracheids like in most Fabaceae seeds (Esau, 1977; Van Dongen et al., 2003). Seed colour is derived from the accumulation of abundant anthocyanin pigments in the cell layer beneath the epidermis. These pigments may be involved in the regulation of the testa development as suggested by the study of the transparent testa mutants (tt) in Arabidopsis (Leon-Kloozsterziel et al., 1994). The determination of seed shape is dependent on macrosclereids which are well-developed in the palisade epidermis of M. truncatula seed coat. These observations identified an epidermal cell-type layer, which abuts the seed coat and encloses the endosperm vacuole, as a possible transfer cell layer. The ultrastructural characterization and the presence of cell wall ingrowths will allow its implication in exchange and nutrient uptake during seed filling to be ascertained. Similar observations in P. sativum, localized a transfer cell layer expressing an amino acid permease transporter gene (PsAAP1) at the inner surface of the seed coat (Tegeder et al., 2000). In Vicia faba, the transfer cell layer, differentiated from the epidermal embryo, was limited to the region where the cotyledon first contacts the seed coat (Borisjuk et al., 1995). Embryo development In the experimental conditions used here, 12 DAP M. truncatula seed corresponded to the early torpedo stage where the embryo has slowed cell division activity and initiated cellular expansion and storage accumulation. This coincided with a decrease in several proteins such as β-tubulin and annexin, which are associated with the cell division cycle (Gallardo et al., 2003). The pattern of distribution of the DAPI-stained embryo nuclei was region-dependent, indicating that cell division activity is not completely arrested in the abaxial region between 12 and 14 DAP. In grain legume seeds the maintenance of cell division activity is important because the final cell number in the cotyledons may determine the capacity of the storage organ to accumulate dry matter for feed (Munier-Jolain and Ney, 1998). The endosperm may also be a key player in the control of seed size through epigenetic controls as suggested by the analysis of haiku and titan Arabidopsis mutants (Garcia et al., 2003; Tzafir et al., 2002). At mid-embryogenesis, seed development in M. truncatula followed that of Arabidopsis. These data showed a well-developed endosperm, unlike faba bean and pea seeds where the coenocytic endosperm disappears before maturation (Borisjuk et al., 1995, 2002a). Even if the embryo proper in M. truncatula represents the major storage organ (Gallardo et al., 2003), it can be assumed that the endosperm supports embryogenesis by providing developmental signals (Berger, 1999) and by mediating metabolite transport as shown in M. sativa (Aivalakis et al., 2004). It may also protect the embryo from physical and osmotic stress. The developmental gradient in the embryo was visualized by differences in cell size. The cotyledon parenchyma showed a typical wave-like differentiation pattern similar to that reported in other legumes such as V. faba and P. sativum, where differentiation began in the adaxial region then spread to the abaxial surface. In M. truncatula storage accumulation started with the early transient accumulation of carbohydrate, mainly starch granules, in the seed coat from 8 DAP to 14–16 DAP. Transient starch accumulation in the seed coat characterized the early stages of Arabidopsis seed development (Baud et al., 2002). In P. sativum, the data revealed early starch granule accumulation in the outer integument and in the cotyledon cells at 12 DAP. The same layer underneath the outer epidermis was reported to express a sucrose transporter, indicating an increase in sucrose concentration (Rochat and Boutin, 1989; Borisjuk et al., 2002b). This increase in carbohydrate may be necessary to promote growth by cell division. Later, in 19 DAP pea seed sections the cotyledon parenchyma was filled with numerous dense storage protein bodies and starch granules. However, in M. truncatula, no starch granules were ever found in cotyledon cells even in 16–18 DAP seeds during the accumulation of the dense protein bodies. These dense bodies were immuno-globulin positive (Quillien and M Darmency, unpublished results) and their number increased during seed filling. They are assumed to be storage protein bodies which will integrate the mature protein storage vacuoles (PSV). The origin of PSV in maturating legume seeds is a matter of controversy regarding the presence of one or two distinct vacuole populations (reviewed in Muntz, 1998; Marty, 1999). The dense vesicle (DV)-mediated pathway for the transport of storage proteins into the PSV was identified in pea (Hillmer et al., 2001). In soybean, in addition to the mature PSV, 1–2 μm diameter dense globules (DG) were identified in both the cytoplasm and vacuoles (Elmer et al., 2003). It is assumed that dense globules are similar to the protein bodies in this study. They might correspond to the coalescence of several dense vesicles. Using immunogold labelling and electron microscopy will determine how the dense protein bodies observed in this study can be related to (DV), but also to know if the mechanism underlying their formation is the same in M. truncatula and P. sativum. mRNA accumulation pattern of two storage protein genes: vicilin and legumin A The two storage proteins genes for vicilin and legumin A are highly expressed and tightly regulated both spatially and temporally during the cell expansion phase. In this study, the transcripts were detected in the embryo cells specifically at mid-embryogenesis. In situ hybridization of mRNA accumulation has shown that transcription occurred mainly in the embryo storage parenchyma. In M. truncatula seed at 16 DAP, legumin A mRNA was also weakly detected in the embryonic axis cells, as reported in Arabidopsis where the 2S1 storage protein gene was expressed in the embryonic axis (Devic et al., 1996). The in situ hybridization pattern of mRNA accumulation indicated that vicilin and legumin A gene expression was just beginning in 12–14 DAP seeds, which corroborated the 2-D protein electrophoresis data (Gallardo et al., 2003). Protein studies also showed that vicilin was accumulated earlier than legumin, which may explain the lower expression level of the legumin gene at this stage, furthermore the legumin A transcripts represent only a part of the whole legumin mRNA production. Later during seed filling the seed sections studied, 16 DAP in M. truncatula and 19 DAP in pea, displayed an intensive labelling with a gradient towards the inner enlarged cells for both antisense probes. In this study, the legumin A probe never showed signals in the embryo epidermis, the cotyledon provascular region, the endosperm, or the seed coat. A different pattern was reported in V. faba for the legumin B gene which was transcribed in the seed coat at mid-embryogenesis, and earlier in the globular embryo, the suspensor, and the endosperm (Panitz et al., 1995). As the legumin A probe does not cross-hybridize to RNA encoded by the legumin B subfamily genes, it can be assumed that the expression pattern of legumin A may be different from that of legumin B. This suggests differences in the promoter activity regulation of the two legumin genes. Finally, legumin A and vicilin expression patterns appeared as suitable embryo-specific markers during histo-differentiation at mid-embryogenesis in M. truncatula and P. sativum. The importance of morphogenesis on the induction of gene storage expression programmes has been explored in Arabidopsis through the analysis of embryo-defective mutants (McElver et al., 2001). Nevertheless, how this regulation is achieved is still poorly understood. Amino acid transporters and transcription factors are candidate genes for being involved in the regulation of seed protein composition (Tegeder et al., 2000; Miranda et al., 2001; Stone et al., 2001; Kwong et al., 2003). Many genes such as ABI3, FUS3, and LEC2 were identified to act in concert in the control of SSP expression (Kroj et al., 2003). Their corresponding mutant alleles in M. truncatula will be sought in Tilling populations. Embryo morphogenesis, and the nature of interactions between the main seed organs will be approached by cytology, genetic, and molecular analyses of embryonic lethal, defectives and pattern mutants. Molecular cytology is fundamental for defining the expression pattern of genes, and also for studying the cellular mechanism of protein accumulation. This work was funded by the Institut National de la Recherche Agronomique (INRA). 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TI - In situ expression of two storage protein genes in relation to histo-differentiation at mid-embryogenesis in Medicago truncatula and Pisum sativum seeds
JF - Journal of Experimental Botany
DO - 10.1093/jxb/eri200
DA - 2005-06-27
UR - https://www.deepdyve.com/lp/oxford-university-press/in-situ-expression-of-two-storage-protein-genes-in-relation-to-histo-EmYGgcmV5o
SP - 2019
EP - 2028
VL - 56
IS - 418
DP - DeepDyve
ER -