Annals of Botany 95: 737–747, 2005

doi:10.1093/aob/mci080, available online at www.aob.oupjournals.org

Structure and Development of Medicago truncatula Pod Wall and Seed Coat
H O N G L I W A N G 1, * and M I C H A E L A . G R U S A K 2
1
Department of Biology, University of Arkansas-Little Rock, Little Rock, AR, USA and 2USDA/ARS Children’s
Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA

Received: 1 September 2004 Returned for revision: 1 November 2004 Accepted: 3 December 2004 Published electronically: 9 February 2005

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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 osteo-
sclereids, 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.

Key words: Development, structure, morphology, microscopy, pod, pod wall, seed, seed coat, Fabaceae, Medicago
truncatula, legumes.

INTRODUCTION seeds. Pod walls serve to protect the seeds during develop-
ment (Small and Brookes, 1982), they are part of the
Medicago truncatula, a member of the Fabaceae, has gar-
source–sink pathway that delivers nutrients to the seed
nered considerable attention recently as a genomic and
coat (Harvey, 1973; Setia et al., 1987), and they can produce
molecular model system for legume biological studies
photosynthates (Willmer and Johnston, 1976; Atkins et al.,
(Cook, 1999; Oldroyd and Geurts, 2001), due to its small
1977) and/or metabolize storage products (Thorne, 1979;
genome size and short life cycle. Researchers have gener-
Mounoury et al., 1984) for transfer to the seeds. The
ated an expressed sequence tag (EST) database of over
developing embryo is further surrounded by a seed coat,
190 000 sequences as of November 2004 (http://www.ncbi.
which itself is involved in the transit and/or metabolism of
nlm.nih.gov/dbEST/dbEST_summary.html; Lamblin et al.,
various nutrients (Murray, 1987; Van Dongen et al., 2003).
2003), genomic DNA sequencing is underway, with the
Seed coat tissues can metabolize amino acids (Murray,
gene-rich region of the genome scheduled to be completed
1987), accumulate polyphenolics (Islam et al., 2003) and
by 2007 (http://www.medicago.org/genome/), and physical
store calcium oxalate crytstals (Barnabas and Arnott, 1990).
and genetic maps of the M. truncatula genome are being
The mature, dry seed coat provides a protective barrier that
developed (Kulikova et al., 2001; Thoquet et al., 2002).
may also contribute to physical dormancy (Baskin and
Although much of the scientific effort with this species
Baskin, 1998). More information is needed to understand
has focused on the biology of root–rhizobial interactions
how the pod wall or seed coat tissues in legumes provide
and the molecular regulation of nodule development (Cook
these functions; a sound structural foundation should help
et al., 1997), interest also exists to study seed biology in
in this effort.
this plant (Gallardo et al., 2003). However, at present, there
Previous morphological studies with M. truncatula have
is little published on the structural attributes of M. truncatula
documented gross features of the fruit reproductive tissues,
pods and seeds. The ability of seed biologists to capitalize
such as the coiled nature of the pods (Small and Brookes,
on the vast molecular and genomic resources of this model
1982; Small et al., 1991), the thickened pod walls (Lesins
species will be limited until a thorough structural and
and Lesins, 1979) or the presence of macro- and osteo-
developmental characterization of its pods and seeds is
sclereids in the seed coat (Jha and Pal, 1992). However, no
available.
detailed microscopic studies have been reported for this
Legume seeds develop within the confines of an ovary-
species’ pods and seeds. In this paper, using light micro-
derived pod whose walls provide numerous functions for the
scopy, a structural characterization of the developing
* For correspondence. E-mail hxwang@ualr.edu pod wall and seed coat of M. truncatula ecotype A17 is
Published by Oxford University Press 2005
738 Wang and Grusak — Medicago Pod Wall and Seed Coat Structure
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 6 3  C day and 20 6 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 plant-
ing; subsequently they were placed on an automatic drip line
system. Plants were watered/fertilized twice daily, using
a solution containing 1 mM K2SO4, 04 mM Ca(NO3)2,
015 mM KH2PO4, 01 mM MgSO4, 25 mM CaCl2, 25 mM
H3BO3, 2 mM MnSO4, 2 mM ZnSO4, 05 mM CuSO4, 05 mM
H2MoO4, 01 mM NiSO4 and 1 mM Fe(III)-EDDHA [N,N0 -
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 bal-
ance; 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 45 mm wide
and 8 mm long. These pod segments were immediately fixed
for 12 h in a 4  C fixation solution containing 25 % glut-
araldehyde, 25 % paraformaldehyde and 50 mM cacodylate
buffer at pH 70. Following fixation, segments were pro-
cessed 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 mix-
tures, ending at 100 % ethanol. Pod segments were sub-
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sequently 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 mm thick) sections were cut with glass
knives on an LKB 2088 Ultrotome V (LKB-Produkter AB,
Bromma, Sweden), and dried onto superfrost/plus micro-
scope 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 44
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.
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), 35 coils by
 Wang and Grusak — Medicago Pod Wall and Seed Coat Structure 739
250 ovule, the inner of the two integuments disappears, while
Pod wall fresh weight (mg pod–1)

A the outer integument differentiates into different layers of

200 the seed coat. The seed coat at 3 DPP consists of a layer of
epidermal cells which contain dense cytoplasm and are still
150 undergoing division (Fig. 3A and C). Beneath the epidermis
there are one to two layers of dividing hypodermal cells, and
100 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
50
the embryo sac (Fig. 3A and C). Although a number of
seeds were sectioned and viewed, the embryo was not
0 yet identifiable in any of the 3 DPP sections.

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9
B
Seed fresh weight (mg pod–1)

8 Pod age: 6 DPP. At this stage, most cells in the pod are
7 expanding, and the width of the pod has increased to about
6 5 mm (Figs 2D and 4A); however, the external morphology
5
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
4
4A). The walls of cells within the exocarp are thickening,
3 especially at the outside surface (Fig. 4C and D). The vas-
2 cular bundles within the pod walls are further differentiated,
1 with more xylem elements developing thickened walls
0 (Fig. 4D).
0 5 10 15 20 25 30 35 40 45 Each growing ovule is seen attached to the pod wall via a
Pod age (days post-pollination) funiculus, and the ovule has initiated a curved and bent
orientation (Fig. 4B). A multinuclear endosperm appears
F I G . 1. Growth dynamics of developing pod walls (A) and seeds (B) of within the embryo sac (Fig. 4C). The embryo consists of
Medicago truncatula. Data are mean fresh weight values (6 s.e.) based on
ten pods collected at each time point. 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
2 DPP (Fig. 2B) and their full five to six coils by 3 DPP (Fig. 4E). Numerous granular bodies are found in the par-
(Fig. 2C). Viewed from the apical end of the developing pod, enchyma cells throughout the seed coat (Fig. 4E).
coil growth occurs in a clock-wise direction. Spines, which
develop along the edges of the dorsal suture, are visible as Pod age: 13 DPP. By 13 DPP, the cells of the exocarp
spine initials at 3 DPP; these insert at approx. 135 to the and mesocarp in the pod walls have further expanded
face of the pod wall (Fig. 2C–F). The diameter of the pod (Fig. 5A and B), as the pod width has increased to about
ranges from about 2 mm at 1 DPP to 8 mm by 20 DPP. 7 mm (Fig. 2E). Sclerenchyma cells with thickened cell
walls, that have differentiated from vascular parenchyma
Pod age: 3 DPP. A transverse section cut 90 to the face cells, appear in the vascular bundles that are close to the
of the coils shows the major features of the pod wall and dorsal suture (Fig. 5A). Moreover, sclerenchyma cells have
developing ovules at 3 DPP (Fig. 3A). The pod wall (or also differentiated in the endocarp and display thickened
pericarp) is composed of an exocarp layer (the outer epi- cell walls (Fig. 5B). Vascular bundles in the pod wall are
dermis), seven to 15 layers of mesocarp parenchyma cells, fully differentiated, consisting of mature phloem and xylem
and inner endocarp layers. The endocarp is comprised of (Fig. 5A and B). The large lateral carpellary bundle extends
three to five layers of small dividing sclerenchyma from the funiculus into the chalazal region and branches into
precursor cells and a single layer of large inner epidermal the nucellus of the seed (Fig. 5A and C).
cells (Fig. 3B and D). Most cells within different layers of From 6 to 13 DPP, the seed coat has undergone a dramatic
the pod wall are still undergoing cell division, as demon- developmental change. The epidermis has differentiated
strated by their alignments at various orientations. into a uniform palisade layer of macrosclereids (or
The vasculature of the pod wall at this stage includes one Malpighian layer; Fig. 5A). These macrosclereids are radi-
lateral carpellary bundle (Fig. 3A), one median carpellary ally elongated, and are covered by a thick cuticle layer on
bundle (Fig. 3A and B) and 9–12 vascular bundles that are the outer surface (Fig. 5B and D). Abundant vacuoles,
embedded among the mesocarp parenchyma cells on each stained blue-green by toluidine blue O, are found in the
face of the pod wall (Fig. 3A, B and D). Although most of cytoplasm of these macrosclereids (Fig. 5B and D). A
the vascular cells appear to be still dividing, some of the hilar groove is located at the centre of the seed hilum
protoxylem cells have differentiated and show thickened area, and consists of more elongated macrosclereids
walls, as indicated by the light blue staining with toluidine (Fig. 5A and C). Other cells in the hilar region are round
blue O (Fig. 3B and D). shaped and also contain numerous vacuoles that are stained
Most legume seeds differentiate from an ovule with two blue-green by toluidine blue O. Adjacent to the macro-
integuments (Esau, 1977). During development of the sclereids, towards the interior of the seed coat, a layer of
740 Wang and Grusak — Medicago Pod Wall and Seed Coat Structure

A D

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0.5 cm

1.0 cm

B
E

C
F

F I G . 2. 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 = 05 cm;
D–F =10 cm.
 Wang and Grusak — Medicago Pod Wall and Seed Coat Structure 741

MCB LCB

VB
MCB

Exo
Ov

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Ov
PW
VB

Meso

Endo

MCB B
A
Exo
Ph
Ep
Nu Xy
Meso
Hy

Pa SP
IE
Endo

C D
F I G . 3. 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.

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).
742 Wang and Grusak — Medicago Pod Wall and Seed Coat Structure

PW

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MCB VB
LCB

PW
PW
MCB
SC

Nu
Ov ES
ES
LCB

B C VB

Exo
Ep
Meso ES Pa
Endo
Ph Hy
Xy
SP
IE

VB

D E
F I G . 4. 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.
 Wang and Grusak — Medicago Pod Wall and Seed Coat Structure 743

Fun VB
CV
Pa
Scl
OsP IE Exo
SC Meso
Ma
ES Endo

PW

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PW
B SC

VB
Scl
Scl
OsP Ma IE Scl
Cu
A

D
PL

CP

TB

Nu Pa
En

En
C E
ES
F I G . 5. 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.

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 com-
panion cells also appear to differentiate transfer cell-like
wall ingrowths (Fig. 6D).
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
744 Wang and Grusak — Medicago Pod Wall and Seed Coat Structure

Fun

SC Scl

CV Scl
Cot
SC PW

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Scl
B VB

Cot

PW

Tr
Xy Scl
Scl
A
Ph

D
PL

TB
CP IE
IE Scl

Exo
Nu Endo
WI
Meso

En E

Ma

Os
Cot Pa
C F
En
F I G . 6. 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.
 Wang and Grusak — Medicago Pod Wall and Seed Coat Structure
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 develop-
mental 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 char-
acterization 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 endo-
carp composed of three to five layers of sclerenchyma cells
plus an inner epidermal cell layer (Figs 3 –6). Vascular
745
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 meso-
carp 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 scler-
enchyma 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,
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the inner epidermal cells of the endocarp are also under-
going 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, devel-
oping pod walls can act as a temporary reservoir of assim-
ilates 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 pos-
sibility 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 devel-
oping 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 par-
enchyma 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, meso-
carp 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 endo-
carp 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
746 Wang and Grusak — Medicago Pod Wall and Seed Coat Structure
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 trans-
fer 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 vas-
cular 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 absorp-
tion that delays germination (Baskin and Baskin, 1998).
Medicago truncatula seed coats are relatively thin, in com-
parison 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 develop-
ment. 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.
ACKNOWLEDGEMENTS
We thank Dr M. Kleeve for his technical assistance on the
Downloaded from https://academic.oup.com/aob/article-abstract/95/5/737/201067 by guest on 23 May 2019
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 Depart-
ment of Agriculture, nor does mention of trade names,
commercial products or organizations imply endorsement
by the US Government.
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