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. 2005 Feb 9;95(5):737–747. doi: 10.1093/aob/mci080

Structure and Development of Medicago truncatula Pod Wall and Seed Coat

HONG LI WANG 1,*, MICHAEL A GRUSAK 2
PMCID: PMC4246729  PMID: 15703184

Abstract

• Background and Aims Medicago truncatula has gained much attention as a genomic model species for legume biology, but little is known about the morphology of its pods and seeds. Structural and developmental characteristics of M. truncatula pod walls and seed coats are presented.

• Methods Plants of M. truncatula ecotype A17 were grown under controlled conditions in a greenhouse. Flowers were date-tagged at anthesis, so that pods of known age could be collected. Harvested pods were fixed and sectioned for light microscopy. Structural attributes of pod walls and seed coats were characterized at four time points throughout early to mid-stages of pod development (3, 6, 13 and 20 d post-pollination).

• Key Results Basic features of the pod wall are an exocarp comprised of a single epidermal layer, a mesocarp with seven to 14 layers of parenchyma cells, and an endocarp composed of an inner epidermal cell layer and three to five layers of sclerenchyma cells adjacent to it. Vascular bundles are abundant in the pod wall and include one lateral carpellary bundle, one median carpellary bundle and nine to 12 vascular bundles, all embedded within the mesocarp parenchyma. Seed coat features include an epidermal layer of macrosclereids, a sub-epidermal layer of osteosclereids, and two to five rows of internal parenchyma cells. The hilar region contains the tracheid bar and the chalazal vascular bundle, the latter of which expands to form only two short branches.

• Conclusions This characterization provides a needed understanding of pod structure and development in this model legume, and should facilitate various molecular investigations into legume fruit and seed biology.

Keywords: Development, structure, morphology, microscopy, pod, pod wall, seed, seed coat, Fabaceae, Medicago truncatula, legumes

INTRODUCTION

Medicago truncatula, a member of the Fabaceae, has garnered considerable attention recently as a genomic and molecular model system for legume biological studies (Cook, 1999; Oldroyd and Geurts, 2001), due to its small genome size and short life cycle. Researchers have generated an expressed sequence tag (EST) database of over 190 000 sequences as of November 2004 (http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html; Lamblin et al., 2003), genomic DNA sequencing is underway, with the gene-rich region of the genome scheduled to be completed by 2007 (http://www.medicago.org/genome/), and physical and genetic maps of the M. truncatula genome are being developed (Kulikova et al., 2001; Thoquet et al., 2002). Although much of the scientific effort with this species has focused on the biology of root–rhizobial interactions and the molecular regulation of nodule development (Cook et al., 1997), interest also exists to study seed biology in this plant (Gallardo et al., 2003). However, at present, there is little published on the structural attributes of M. truncatula pods and seeds. The ability of seed biologists to capitalize on the vast molecular and genomic resources of this model species will be limited until a thorough structural and developmental characterization of its pods and seeds is available.

Legume seeds develop within the confines of an ovary-derived pod whose walls provide numerous functions for the seeds. Pod walls serve to protect the seeds during development (Small and Brookes, 1982), they are part of the source–sink pathway that delivers nutrients to the seed coat (Harvey, 1973; Setia et al., 1987), and they can produce photosynthates (Willmer and Johnston, 1976; Atkins et al., 1977) and/or metabolize storage products (Thorne, 1979; Mounoury et al., 1984) for transfer to the seeds. The developing embryo is further surrounded by a seed coat, which itself is involved in the transit and/or metabolism of various nutrients (Murray, 1987; Van Dongen et al., 2003). Seed coat tissues can metabolize amino acids (Murray, 1987), accumulate polyphenolics (Islam et al., 2003) and store calcium oxalate crytstals (Barnabas and Arnott, 1990). The mature, dry seed coat provides a protective barrier that may also contribute to physical dormancy (Baskin and Baskin, 1998). More information is needed to understand how the pod wall or seed coat tissues in legumes provide these functions; a sound structural foundation should help in this effort.

Previous morphological studies with M. truncatula have documented gross features of the fruit reproductive tissues, such as the coiled nature of the pods (Small and Brookes, 1982; Small et al., 1991), the thickened pod walls (Lesins and Lesins, 1979) or the presence of macro- and osteosclereids in the seed coat (Jha and Pal, 1992). However, no detailed microscopic studies have been reported for this species' pods and seeds. In this paper, using light microscopy, a structural characterization of the developing pod wall and seed coat of M. truncatula ecotype A17 is provided, with the focus on pods in the age range 3–20 d post-pollination (DPP). The intention was to gain a better structural understanding of the tissues that serve both as a nutritional pathway and a protective enclosure for the developing embryos at early stages of pod and seed development.

MATERIALS AND METHODS

Plant material

Plants of Medicago truncatula ecotype A17 were grown in a greenhouse with supplemental lighting (metal halide lamps) that provided a photoperiod of at least 15 h. Environmental conditions within the greenhouse were a temperature regime of 22 ± 3 °C day and 20 ± 3 °C night and relative humidity ranging from 45 to 65 % throughout the day/night cycle. Plants were grown in a medium composed of two parts synthetic soil (Metro-Mix 360; Scotts-Sierra Horticultural Products Co., Marysville, OH, USA) and one part medium-grade vermiculite (Strong-Lite Medium Vermiculite, Sun Gro Horticulture Co, Seneca, IL, USA). Plants were watered/fertilized by hand until approx. 2 weeks after planting; subsequently they were placed on an automatic drip line system. Plants were watered/fertilized twice daily, using a solution containing 1 mm K2SO4, 0·4 mm Ca(NO3)2, 0·15 mm KH2PO4, 0·1 mm MgSO4, 25 μm CaCl2, 25 μm H3BO3, 2 μm MnSO4, 2 μm ZnSO4, 0·5 μm CuSO4, 0·5 μm H2MoO4, 0·1 μm NiSO4 and 1 μm Fe(III)-EDDHA [N,N′-ethylenebis[2-(2-hydroxyphenyl)-glycine]].

Pod harvest and fresh weight analysis

Randomly selected flowers were date tagged (using small marking tags on strings) on the day of full bloom, which was 1 d prior to tripping; this date was designated as day −1 or −1 DPP. The tag was applied when the first flower at an axil was open; all other flowers at an axil with multiple flowers usually opened either on the same day as the first flower or 1 d later. Whole pods were collected at various DPPs (from 3 to 43 DPP) for the determination of pod wall and seed fresh weight; ten pods were harvested for each time point. Pods from 3 to 6 DPP (with peduncle and any attached sepals or petals removed) were weighed in their entirety within 5 min of harvest using a four-point electronic balance; developing ovules were very small during this time period and their weight was included with the pod walls. Pods aged 7–43 DPP were separated into wall and seed fractions within 5 min of harvest, and put into pre-weighed, capped microcentrifuge tubes. These also were weighed using a four-point electronic balance. Seed fresh weight was calculated as an average individual seed weight from each pod (total seed fresh weight divided by number of seeds).

Pods were collected at 3, 6, 13 and 20 DPP to be fixed and sectioned for light microscope observations. Pods also were collected at 1, 2, 3, 6, 13 and 20 DPP to photograph and record external characteristics (Coolpix 990 Digital Camera; Nikon Corp., Tokyo, Japan).

Tissue preparation and light microscopy observations

The developing pods of M. truncatula at 3, 6, 13 and 20 DPP were cut into small segments about 4·5 mm wide and 8 mm long. These pod segments were immediately fixed for 12 h in a 4 °C fixation solution containing 2·5 % glutaraldehyde, 2·5 % paraformaldehyde and 50 mm cacodylate buffer at pH 7·0. Following fixation, segments were processed through three 40-min washes of cacodylate buffer at 4 °C. Tissues were then dehydrated at 40-min intervals through a 10 %-step graded series of ethanol–water mixtures, ending at 100 % ethanol. Pod segments were subsequently infiltrated over 4 d with LR White (London Resin Company Ltd, Berkshire, UK) through a five-step graded series from 100 % ethanol to 100 % LR White, and then embedded in LR White resin. Blocks were polymerized by exposure to a temperature sequence of 37 °C for 12 h, 48 °C for 12 h and 60 °C for 24 h.

Semi-thin (1–2 µm thick) sections were cut with glass knives on an LKB 2088 Ultrotome V (LKB-Produkter AB, Bromma, Sweden), and dried onto superfrost/plus microscope slides (Fisher Scientific Co., Pittsburgh, PA, USA). Three blocks were sectioned for each time point, and a minimum of 80 sections were collected for each block. Sections were stained with toluidine blue O at pH 4·4 and observed with an Olympus-BH2 microscope (Olympus America Inc., Melville, NY, USA). Digital microscopic images were taken using a SPOT Insight Camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA).

RESULTS

Growth of M. truncatula pod walls and seeds

The fresh weight of the developing pod walls and seeds of M. truncatula ecotype A17 increased until 39 DPP, at which time a net desiccation of the pod walls and seeds was observed (Fig. 1). The growth dynamics of the pod walls displayed four phases (Fig. 1A): a rapid fresh weight gain was seen from 3 to 8 DPP; a delay in growth followed by a gradual weight gain was observed from 8 to 20 DPP; a plateau in fresh weight gain was found from 20 to 39 DPP; and fresh weight eventually declined sharply from 39 to 43 DPP. For M. truncatula developing seeds, the fresh weight dynamics exhibited three phases (Fig. 1B). These included a period during which the rate of fresh weight gain increased with time (7–15 DPP), a period during which fresh weight continued to increase with time, but at a declining rate (15–39 DPP) and, finally, a period of fresh weight loss (39–43 DPP). Based on this characterization, four time points were chosen to represent the early to mid-stages of pod development: 3, 6, 13 and 20 DPP.

Fig. 1.

Fig. 1.

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Growth dynamics of developing pod walls (A) and seeds (B) of Medicago truncatula. Data are mean fresh weight values (± s.e.) based on ten pods collected at each time point.

Development and structure of the pod wall and seed coat

General morphology

The pod of M. truncatula is derived from a superior ovary that consists of a single carpel. After pollination, the carpel grows from a short, curved form into a spiral structure (Fig. 2). Pods of ecotype A17 develop nearly two full coils by 1 DPP (Fig. 2A), 3·5 coils by 2 DPP (Fig. 2B) and their full five to six coils by 3 DPP (Fig. 2C). Viewed from the apical end of the developing pod, coil growth occurs in a clock-wise direction. Spines, which develop along the edges of the dorsal suture, are visible as spine initials at 3 DPP; these insert at approx. 135° to the face of the pod wall (Fig. 2C–F). The diameter of the pod ranges from about 2 mm at 1 DPP to 8 mm by 20 DPP.

Fig. 2.

Fig. 2.

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External morphology of developing Medicago truncatula pods: (A) developing pod at 1 DPP with remaining petals removed; (B) developing pod at 2 DPP with remaining petals removed; (C) pod at 3 DPP; (D) pod at 6 DPP; (E) pod at 13 DPP; (F) pod at 20 DPP. Scale bars: A–C = 0·5 cm; D–F = 1·0 cm.

Pod age: 3 DPP

A transverse section cut 90° to the face of the coils shows the major features of the pod wall and developing ovules at 3 DPP (Fig. 3A). The pod wall (or pericarp) is composed of an exocarp layer (the outer epidermis), seven to 15 layers of mesocarp parenchyma cells, and inner endocarp layers. The endocarp is comprised of three to five layers of small dividing sclerenchyma precursor cells and a single layer of large inner epidermal cells (Fig. 3B and D). Most cells within different layers of the pod wall are still undergoing cell division, as demonstrated by their alignments at various orientations.

Fig. 3.

Fig. 3.

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Light micrographs of developing Medicago truncatula pods at 3 d post-pollination: (A) longitudinal section of the pods (×36); (B) the median carpellary bundle and the pod wall (×184); (C) cellular structure of the ovule (×460); (D) cellular structure of the pod wall (×460). Endo, Endocarp; Ep, epidermis; Exo, exocarp; Hy, Hypodermis; IE, inner epidermis; LCB, lateral carpellary bundle; MCB, median carpellary bundle; Meso, mesocarp; Nu, nucellus; Ov, Ovule; Pa, parenchyma; Ph, phloem; PW, pod wall; SP, sclerenchyma precursor cell; VB, vascular bundle; Xy, xylem.

The vasculature of the pod wall at this stage includes one lateral carpellary bundle (Fig. 3A), one median carpellary bundle (Fig. 3A and B) and 9–12 vascular bundles that are embedded among the mesocarp parenchyma cells on each face of the pod wall (Fig. 3A, B and D). Although most of the vascular cells appear to be still dividing, some of the protoxylem cells have differentiated and show thickened walls, as indicated by the light blue staining with toluidine blue O (Fig. 3B and D).

Most legume seeds differentiate from an ovule with two integuments (Esau, 1977). During development of the ovule, the inner of the two integuments disappears, while the outer integument differentiates into different layers of the seed coat. The seed coat at 3 DPP consists of a layer of epidermal cells which contain dense cytoplasm and are still undergoing division (Fig. 3A and C). Beneath the epidermis there are one to two layers of dividing hypodermal cells, and one to three layers of large parenchyma cells (Fig. 3A and C). Just inside the seed coat, several layers of nucellar cells are observed that vary in shape and size, and surround the embryo sac (Fig. 3A and C). Although a number of seeds were sectioned and viewed, the embryo was not yet identifiable in any of the 3 DPP sections.

Pod age: 6 DPP

At this stage, most cells in the pod are expanding, and the width of the pod has increased to about 5 mm (Figs 2D and 4A); however, the external morphology of the pod (cf. Figs 2C and 2D) and many cellular features of the pod wall are similar to those at 3 DPP (cf. Figs 3A and 4A). The walls of cells within the exocarp are thickening, especially at the outside surface (Fig. 4C and D). The vascular bundles within the pod walls are further differentiated, with more xylem elements developing thickened walls (Fig. 4D).

Fig. 4.

Fig. 4.

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Light micrographs of developing Medicago truncatula pods at 6 d post-pollination: (A) longitudinal section of pods (×36); (B) cross-section of the pod wall and ovule (×45); (C) longitudinal section of pod wall and ovule (×178); (D) cellular structure of the pod wall (×490); (E) cellular structure of the seed coat (×490). Endo, Endocarp; Ep, epidermis; ES, embryo sac; Exo, exocarp; Hy, hypodermis; IE, inner epidermis; LCB, lateral carpellary bundle; MCB, median carpellary bundle; Meso, mesocarp; Nu, nucellus; Ov, ovule; Pa, parenchyma; Ph, phloem; PW, pod wall; SC, seed coat; SP, sclerenchyma precursor cell; VB, vascular bundle; Xy, xylem.

Each growing ovule is seen attached to the pod wall via a funiculus, and the ovule has initiated a curved and bent orientation (Fig. 4B). A multinuclear endosperm appears within the embryo sac (Fig. 4C). The embryo consists of a number of suspensor cells and a globular embryo (data not shown). The epidermal cells of the seed coat have elongated and their vacuoles stain blue-green with toluidine blue O (Fig. 4E). Numerous granular bodies are found in the parenchyma cells throughout the seed coat (Fig. 4E).

Pod age: 13 DPP

By 13 DPP, the cells of the exocarp and mesocarp in the pod walls have further expanded (Fig. 5A and B), as the pod width has increased to about 7 mm (Fig. 2E). Sclerenchyma cells with thickened cell walls, that have differentiated from vascular parenchyma cells, appear in the vascular bundles that are close to the dorsal suture (Fig. 5A). Moreover, sclerenchyma cells have also differentiated in the endocarp and display thickened cell walls (Fig. 5B). Vascular bundles in the pod wall are fully differentiated, consisting of mature phloem and xylem (Fig. 5A and B). The large lateral carpellary bundle extends from the funiculus into the chalazal region and branches into the nucellus of the seed (Fig. 5A and C).

Fig. 5.

Fig. 5.

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Light micrographs of developing Medicago truncatula pods at 13 d post-pollination: (A) longitudinal section of pods (×37); (B) longitudinal section of pod wall and seed coat (×186); (C) hilum region of the seed coat (×186); (D) macrosclereid and osteosclereid precursor cells of the seed coat, and the inner epidermis of the pod wall (×585); (E) parenchyma cells of the seed coat (×585). CP, Chalazal phloem; Cu, cuticle; CV, chalazal vascular bundle; En, endosperm; Endo, endocarp; ES, embryo sac; Exo, exocarp; Fun, funicular tissue; IE, inner epidermis; Ma, macrosclereid; Meso, mesocarp; Nu, nucellus; OsP, osteosclereid precursor; Pa, parenchyma; PL, palisade layer of macrosclereids; PW, pod wall; SC, seed coat; Scl, sclerenchyma; TB, tracheid bar; VB, vascular bundle.

From 6 to 13 DPP, the seed coat has undergone a dramatic developmental change. The epidermis has differentiated into a uniform palisade layer of macrosclereids (or Malpighian layer; Fig. 5A). These macrosclereids are radially elongated, and are covered by a thick cuticle layer on the outer surface (Fig. 5B and D). Abundant vacuoles, stained blue-green by toluidine blue O, are found in the cytoplasm of these macrosclereids (Fig. 5B and D). A hilar groove is located at the centre of the seed hilum area, and consists of more elongated macrosclereids (Fig. 5A and C). Other cells in the hilar region are round shaped and also contain numerous vacuoles that are stained blue-green by toluidine blue O. Adjacent to the macrosclereids, towards the interior of the seed coat, a layer of precursor cells are found that will subsequently differentiate into osteosclereids (Fig. 5D). Large granules (apparently starch grains as indicated by IKI staining; data not shown) appear in the nucellar cells (Fig. 5C) especially the inner parenchyma cells that are adjacent to the embryo sac (Fig. 5E).

The endosperm has begun to cellularize, forming one or two cell layers that encircle the embryo sac (Fig. 5A, C and E). By 13 DPP, the embryo has now become heart shaped and is still attached to the suspensor cells (data not shown).

Pod age: 20 DPP

By 20 DPP, the exocarp layer exhibits only limited connections with the mesocarp, as cells of the outer mesocarp have become more elongated and irregularly shaped (Fig. 6A and B). Exocarp cells also have become further elongated, especially along regions of the pod wall where intercellular air spaces have enlarged within the outer mesocarp (Fig. 6A, B and E). Many of the mesocarp cells contain small starch granules (identified by IKI staining; data not shown) (Fig. 6E). Sclerenchyma cells in the vascular bundles and in the pod wall endocarp have developed heavily thickened cell walls (Fig. 6B, D and E). The inner epidermal cells contain dense cytoplasm and cell wall ingrowths are observed on the side facing the seed coat (Fig. 6E). Within the pod wall vasculature, phloem companion cells also appear to differentiate transfer cell-like wall ingrowths (Fig. 6D).

Fig. 6.

Fig. 6.

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Light micrographs of developing Medicago truncatula pods at 20 d post-pollination: (A) longitudinal section of the developing pod, including pod wall, seed coat and cotyledons (×35); (B) longitudinal section of pod wall, seed coat and cotyledon (×88); (C) hilum region of seed coat (×175); (D) vascular bundles in the pod wall (×438); (E) longitudinal section of pod wall and seed coat (×350) [insert: inner epidermal cells with wall ingrowths (×525)]; (F) macrosclereid, osteosclereid and parenchyma cells of the seed coat (×438). Cot, Cotyledon; CP, chalazal phloem; CV, chalazal vascular bundle; En, endosperm; Endo, endocarp; ES, embryo sac; Exo, exocarp; Fun, funicular tissue; IE, inner epidermis; Ma, macrosclereid; Meso, mesocarp; Nu, nucellus; Os, osteosclereid; Pa, parenchyma; Ph, phloem; PL, palisade layer of macrosclereids; PW, pod wall; SC, seed coat; Scl, sclerenchyma; TB, tracheid bar; Tr, transfer cell; VB, vascular bundles; WI, wall ingrowths; Xy, xylem.

Seed size has increased significantly by 20 DPP. Cell division within the embryo has enabled the cotyledons to fill most of the embryo sac cavity (Fig. 6A), which itself has expanded in size since 13 DPP. Additionally, many seed coat cells have elongated, apparently in concert with the increase in seed size and volume. The cells in the different layers of the seed coat have undergone dramatic structural changes. In the hilum region, the tracheid bar has expanded and its cells have differentiated into mature vessels with thickened walls (Fig. 6C). The palisade macrosclereids, which comprise the seed coat epidermis, display dense cytoplasm and a large vacuole that stains green-blue with toluidine blue O (Fig. 6B, E and F). Moreover, the light line, which is seen because of refraction within a restricted region of the epidermal cell wall, is oriented tangentially along the central third of the epidermal cell layer (Fig. 6B and F). The sub-epidermal layer (hypodermis) has differentiated into osteosclereids (hourglass cells); these cells exhibit a high density of starch grains (identified by IKI staining; data not shown) (Fig. 6F). Situated below the osteosclereid layer are an inner layer of two to five rows of parenchyma cells, which also contain numerous starch grains at this developmental stage (Fig. 6F). Interior to the seed coat, two to four rows of endosperm cells are visible at the funicular end of the embryo sac, while many of the endosperm cells at the other end of the embryo sac appear to have degraded and become collapsed (Fig. 6A, C and F).

DISCUSSION

In this report, a detailed structural and developmental characterization of M. truncatula pod walls and seed coats are presented at the four time points (3, 6, 13 and 20 DPP), which encompass early to mid-stages of pod development. Pods studied at 3 and 6 DPP reflect rapidly growing pod walls (Fig. 1A) and the early emergence of developing ovules (Fig. 1B). Pods studied at 13 DPP reflect pods at the end of an early pause in fresh weight growth (Fig. 1A) and seeds in a rapid period of fresh weight gain (Fig. 1B). Pods studied at 20 DPP represent pod walls that have reached a plateau in fresh weight mass (Fig. 1A) and seeds that are in a mid-stage of growth (Fig. 1B). A detailed structural and developmental analysis of M. truncatula pods and seeds in late stages of development will be reported in a subsequent paper.

Pods of ecotype A17 reach full maturity (i.e. seed and pod desiccation) in about 43 d (Fig. 1), but the complete coiled nature of the pod (five to six coils) is already achieved by 3 DPP (Fig. 2). From this early age, the pod tissues undergo continued cell division, differentiation and growth, as the pod expands from 3 mm to 8 mm in diameter by 20 DPP. Although the intention was to characterize the structural attributes of the maternal reproductive tissues, which serve to protect and nourish the developing embryos, it was fortunate that the time points of 3, 6, 13 and 20 DPP were selected for this study, which covered a range of embryo stages. As noted in the results, embryos were found to be within the proembryonic stage at 3 DPP, the globular stage at 6 DPP, the heart-shaped stage at 13 DPP and the cotyledon filling stage at 20 DPP.

The general features of the M. truncatula pod wall are an exocarp comprising a single epidermal layer, a mesocarp with seven to 14 layers of parenchyma cells, and an endocarp composed of three to five layers of sclerenchyma cells plus an inner epidermal cell layer (Figs 3–6). Vascular bundles are abundant in the pod wall; these include one lateral carpellary bundle, one median carpellary bundle and nine to 12 vascular bundles, all embedded in the mesocarp parenchyma cells (Figs 3A, 4A and B, 5A and 6A). Differentiation was evident in various endocarp cells during pod wall development; the deposition of cell wall materials occurred from 6 to 20 DPP as the cell walls of the sclerenchyma precursors thickened (Figs 4D and 5B). Moreover, large strands of sclerenchyma cells differentiated by 20 DPP at the outer boundary of the phloem in the pod wall (Fig. 6A, B and D). These sclerenchyma cells are long (Fig. 6A and B) and contain thickened cell walls (Fig. 6D). Intriguingly, the inner epidermal cells of the endocarp are also undergoing structural changes; transfer cell-like wall ingrowths arise at the side of the cell wall adjacent to the developing seed by 20 DPP (Fig. 6E). The role of these wall ingrowths is unclear, although it is possible that the inner epidermal cells of the M. truncatula endocarp are scavenging nutrients released within the pod cavity. In various legumes, developing pod walls can act as a temporary reservoir of assimilates and other nutrients (although usually coming from the leaves) that are destined for later transport to the developing seeds (Setia et al., 1987). Alternatively, with the endocarp inner epidermis situated next to the seed surface, the possibility cannot be ruled out that this expanded surface area facilitates nutrient exchange towards the seed. This could be similar to the transfer cell-facilitated exchange of solutes found at the inner seed coat/cotyledon interface in developing Vicia faba seeds (Offler and Patrick, 1993). The absorption of some nutrients may be possible in developing seed coats, as has been suggested for the movement of Ca2+ from pod walls into Phaseolus vulgaris seeds (Mix and Marschner, 1976). More research is needed to define the role of this interesting pod wall structure.

Relative to agronomic legume species, the structure and development of M. truncatula pod walls are similar to those in Glycine max and Pisum sativum, but some differences exist with Phaseolus vulgaris. In pods of both Glycine max (Esau, 1977) and Pisum sativum (Vercher et al., 1984), their mesocarp regions consist of numerous layers of large parenchyma cells, and their endocarp regions contain several layers of sclerenchyma cells with thick walls, along with a single-layered inner epidermis (cf. Figs 5B and D and 6B and E for M. truncatula). By contrast, the pod walls of Phaseolus vulgaris also are composed of an exocarp, mesocarp and endocarp, but these pod walls are quite thick and fleshy, due to a prolonged period of cell division in the endocarp (Reeve and Brown, 1968). This prolonged cell division occurs in the inner region of the pod wall, such that a zone of parenchyma tissue, consisting of 30–40 cell layers, arises adjacent to the region of endocarp sclereid cells (Reeve and Brown, 1968). As noted, the inner endocarp region in M. truncatula consists of only a single cell layer (Figs 5B and D and 6B and E).

The present observations of pod wall vasculature, being localized within several layers of mesocarp, are consistent with a taxonomical character that is used to discern this species from other medics. The lateral veins in the pod wall are not readily visible in some Medicago species (e.g. M. truncatula), whereas they are clearly defined and visible in the thinner walls of others (e.g. M. polymorpha) (Lesins and Lesins, 1979). Apparently, there is species-dependent variation in the development of mesocarp layers within the Medicago genus.

The general features of the M. truncatula seed coat are an epidermal layer of macrosclereids (epidermis), a sub-epidermal layer of osteosclereids (hypodermis) and two to five rows of internal parenchyma cells (Figs 3–6), similar to other Medicago species (Small and Brookes, 1990; Jha and Pal, 1992). The parenchyma layer is thinnest at the end of the seed coat opposite the hilum (Fig. 6A). The hilar region contains the chalazal vascular bundle and the tracheid bar (Fig. 6A and C). Maturation of these cell types varies; the epidermal macrosclereids and the chalazal vasculature are well developed by 13 DPP, while the tracheid bar and sub-epidermal osteosclereids do not fully differentiate until sometime between 13 and 20 DPP.

Legume seed coats play a critical role in the lateral transfer of assimilates and other nutrients, prior to their release to the developing embryo (Lush and Evans, 1980; Offler and Patrick, 1984, 1993; Offler et al., 1989). Seed coat vascular systems vary structurally amongst legumes; some species possess extensive vascular systems that anastomose to form reticulated networks throughout the entire seed coat (e.g. Phaseolus vulgaris, Offler and Patrick, 1984; Glycine max, Thorne, 1979), while other species have relatively simple vascular systems, with only a single chalazal vascular bundle and two lateral branches extending into the seed coats (e.g. Pisum sativum, Hardham, 1976; Vicia faba, Offler et al., 1989). The present study shows that the vascular system in the seed coat of M. truncatula also is simple, consisting of a single chalazal vascular bundle with two short branches (Figs 5A and 6A). It is worth noting that the maturation of the chalazal vasculature in M. truncatula precedes the extensive cellularization and growth of the developing embryo (and presumably the extensive import of nutrients) that occurs between 13 and 20 DPP.

Legume seed coats also serve to protect the mature embryo, and can create a physical barrier to water absorption that delays germination (Baskin and Baskin, 1998). Medicago truncatula seed coats are relatively thin, in comparison to agronomic legumes such as Phaseolus vulgaris and Pisum sativum, whose seed coats possess more than 12 cell layers at full maturity (Esau, 1977; Offler and Patrick, 1984; Van Dongen et al., 2003). However, seeds of these agronomic legumes are dispersed from dehiscent pods, while Medicago seeds are retained within indehiscent pods in the seed bank (Heida and Jones, 1988). Embryo protection in M. truncatula can thus be provided both by the seed coat and the pod walls; there may be no selective advantage for thick seed coats in this species. As to physical dormancy, several Medicago species (including M. truncatula) are known to have hard-seededness that delays germination (Crawford et al., 1989). Because only a few cell layers exist in M. truncatula seed coats, the double layer of macrosclereids and osteosclereids in this seed coat (Fig. 6) may be sufficient to inhibit water absorption; however, this requires verification.

In summary, the structural characterization of M. truncatula pod walls and seed coats described in this paper has identified the specific cell types found in these tissues and has revealed the developmental timing of their differentiation, during early to mid-stages of pod development. This basic understanding can now be combined with the molecular resources available for M. truncatula, to study various aspects of fruit and seed biology pertinent to legumes.

Acknowledgments

We thank Dr M. Kleeve for his technical assistance on the microscope and digital imaging system. This work was funded in part by grant No. 01-B-29 to H.L.W. from Arkansas Science & Technology Authority, by a start-up fund to H.L.W. from the University of Arkansas-LR, and in part by the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement Number 58-6250-1-001. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

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