Annals of Botany 101: 491–499, 2008
doi:10.1093/aob/mcm322, available online at www.aob.oxfordjournals.org
Developmental Changes in Peanut Root Structure during Root Growth
and Root-structure Modification by Nodulation
RYO S U KE TAJ IM A 1, * , J U N A B E 2 , O NE W LEE 1 ,2 , S HI GEN OR I MORI TA 1 and AL E XA ND E R L UX 1,3
1
Field Production Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Nishi-tokyo, Tokyo 188-0002, Japan, 2AE-Bio, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Tokyo 113-8657, Japan and 3Department of Plant Physiology, Faculty of Natural Sciences,
Comenius University in Bratislava, Mlynská dolina B-2, SK-842 15 Bratislava, Slovak Republic
Received: 4 September 2007 Returned for revision: 26 October 2007 Accepted: 3 December 2007 Published electronically: 5 February 2008
† Background and Aims Basic information about the root and root nodule structure of leguminous crop plants is
incomplete, with many aspects remaining unresolved. Peanut (Arachis hypogaea) forms root nodules in a unique
process. Structures of various peanut root types were studied with emphasis on insufficiently characterized lateral
roots, changes in roots during their ontogenesis and root modification by nodule formation.
† Methods Peanut plants were grown in the field, in vermiculite or in filter paper. The taproot, first-order and secondorder lateral roots and root nodules were analysed using bright-field and fluorescence microscopy with hand sections
and resin sections.
† Key Results Three root categories were recognized. The primary seminal root was thick, exhibiting early and intensive secondary thickening mainly on its base. It was tetrarch and contained broad pith. First-order lateral roots were
long and thin, with limited secondary thickening; they contained no pith. Particularly different were second- and
higher-order lateral roots, which were anatomically simple and thin, with little or no secondary growth. Unusual
wall ingrowths were visible in the cells of the central part of the cortex in the first-order and second-order lateral
roots. The nodule body was formed at the junction of the primary and lateral roots by the activity of proliferating
cells derived originally from the pericycle.
† Conclusions Two morphologically and anatomically distinct types of lateral roots were recognized: long, firstorder lateral roots, forming the skeleton of the root system, and thin and short second- and higher-order lateral
roots, with an incomplete second state of endodermal development, which might be classified as peanut ‘feeder
roots’. Formation of root nodules at the base of the lateral roots was the result of proliferating cell divisions
derived originally from the pericycle.
Key words: Endodermis, lateral root structure, nodule structure, peanut, Arachis hypogaea, primary root structure.
IN TROD UCT IO N
The capability of legumes to fix atmospheric dinitrogen
(N2) using symbioses with specific bacteria has been a
research subject of agricultural and plant biology laboratories for a very long time (e.g. Fred et al., 1939;
Caetano-Anollés, 1997). As described in a review by
Sinclair (2004), this unique feature of legumes underscores
their high value as a component of many cropping systems,
and it might become increasingly important for alternative
cropping systems in both industrial and developing
countries. The capability is one reason for the recently
increasing number of studies, using molecular biology techniques, of the symbiotic association between legume roots
and their bacterial symbionts (e.g. Gleason et al., 2006;
Tirichine et al., 2006). However, in many cases basic information related to the root structure of a given species is
incomplete; numerous aspects of the relationship between
the host and its symbionts thereby remain unresolved.
One legume species that is studied intensively for its
agronomic and food value is the peanut (Arachis hypogaea). Yarbrough (1949) provided fundamental anatomical
observations of peanut vegetative organs, including data
* For correspondence. E-mail [email protected]
related to root primary and secondary structure, whilst
Allen and Allen (1940) described morphological development of peanut nodules. Structural and functional aspects
of legume root-nodule initiation and development were
documented in detail by Dart (1975). Important aspects
of the transport system in leguminous root nodules were
characterized by Pate et al. (1969) using transmission electron microscope, and Sen et al. (1986) and Bal et al. (1989)
later made detailed investigations of the ultrastructural
characteristics of the host– symbiont interaction of peanut.
More recently, structural features of peanut nodule cells
have been studied from the aspect of lipid storage and
metabolism by Bal (1990), Jayaram and Bal (1991) and
Khetmalas and Bal (1997).
Two routes of Rhizobium infection have been described
for root-nodule formation in legume roots: entry via roothairs and via cracks. Root-hair entry occurs in most
legumes, e.g. soybean and common bean (Phaseolus vulgaris). Crack entry occurs in few legumes: peanut and
Sesbania. In peanut, root nodules develop only at the sites
of lateral-root emergence (Uheda et al., 2001), where the
epidermis and cortex of the parent root are broken by emergence of the lateral root (reviewed in Boogerd and van
Rossum, 1997). Using gus-A-marked Bradyrhizobium,
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Tajima et al. — Peanut Root Structure and Nodule Development
Uheda et al. (2001) visually demonstrated Rhizobium
infection into the root through the intercellular gap
created by lateral-root emergence.
The nitrogen-fixing activity of individual nodules is
affected by oxygen diffusion into the inner part of the
nodule and by the partition of photosynthate from the
plant to the nodule through the root. These physiological
traits are probably affected by the anatomy of the nodule
and by the junction between the root and nodule (Brown
and Walsh, 1994). The diffusion barrier, which restricts
O2 flux into the nodule, is of vital importance because it
protects nitrogenase from inactivation by oxygen (Sinclair
and Goudriaan, 1981). The mechanism of changes in the
gas permeability of this barrier remain largely unexplained,
but some models present the possible role of structural features such as opened or closed pores in the nodule cortex
(Denison, 1992). The function and structure of the nodule
surface are important subjects that need to be examined.
In this study, structural differences of various peanut root
types and their changes during growth and ontogenesis were
characterized. Furthermore, the question was addressed of,
in structural terms, the incompletely defined root-nodule
development, with special reference to the presence and
changes of the root apoplasmic barrier, the endodermis,
and to the formation and development of peridermal
tissues of the root and of the nodule.
M AT E R I A L S A N D M E T H O D S
Plant materials
Field cultivation of peanut. For this study, plants of
‘Chibahandachi’, a leading cultivar of peanut (Arachis
hypogaea L.) in Japan, were grown using conventional
Japanese methods in a Kanto loam (humic Andosol) field
at the Field Production Science Center of the University
of Tokyo, Tokyo, Japan (358430 N, 1398320 E) in 2004 and
2006. Peanut seeds were inoculated with Bradyrhizobium
spp. (J2P21; Tokachi Nokyoren, Hokkaido, Japan) and
planted in the field by hand at 5 cm depth in May. The
row direction was east– west, with row spacing of 0.7 m
and plant spacing of 0.3 m. Chemical fertilizer including
N, P, and K was applied as a basal dressing before planting
at rates of 15, 50 and 50 kg ha21, respectively. The plants
were harvested at 71 d after seeding (DAS, pod-filling
stage) in 2004 and 49 DAS (flowering stage) in 2006.
After removing the above-ground parts, the root systems
were extracted using a shovel and the roots in the topsoil
layer (approx. 30 cm depth) were washed out carefully.
Vermiculite culture of peanut. Peanuts were grown in vermiculite (Burnpiece S; Technon) using 1-L plastic pots in a growth
chamber at 30/25 8C (day/night) with 12 h daily light of
308 mmol m22 s21. Peanut seeds were sown directly in the
vermiculite, with one seed per pot. The individual pots were
irrigated every fifth day with 100 mL of nitrogen-free
solution
(NaH2PO4.2H2O,
64 mmol m23;
K2SO4,
23
86 mmol m ;
KCl,
0.20 mmol m23;
CaCl2.2H2O,
0.34 mmol m23; MgSO4.7H2O, 81 mmol m23; FeSO4.
7H2O, 7.2 mmol m23; MnSO4.4H2O, 2.2 mmol m23;
H3BO3, 8.1 mmol m23; ZnSO4.7H2O, 0.70 mmolm23;
CuSO4.5H2O,
0.040 mmol m23;
Na2MoO4.2H2O,
23
0.021 mmol m ; pH 5.2). Peanut plants were inoculated
with Bradyrhizobium spp. at 5 DAS. Peanut root systems
were sampled at 15 DAS.
Paper culture of peanut. For observation of the seedling
roots and their primary structure, peanuts were grown in
rolls of filter paper in a growth chamber at 28 8C with
12 h daily light of 228 mmol m22 s21. Rolled filter papers
(10 cm high) holding a seed near the top were placed
upright in a beaker and wetted every other day. Roots
were sampled at 5 DAS.
Observation of root nodules
The taproot, first-, second- and third-order lateral roots,
and root nodules were observed. Root nodules and roots
that exhibited nodule formation were collected.
For hand sections, all samples were washed and sectioned fresh without prior fixation. The cells were cleared
using lactic acid (Lux et al., 2005). Sections were stained
with either toluidine blue O, fluorol yellow (detecting
lamellar suberin), phloroglucinol and HCl (detecting
lignin), or berberine (detecting amorphous suberin) (see
Lux et al., 2004, 2005). Cells were then observed using
optical and fluorescence microscopy.
For resin sections, the samples were fixed using two
methods. (1) Samples were fixed using formalin – acetic
acid – ethanol solution (FAA), and roots and root nodules
were embeded in Technovit 7100 (Heraeus Kulzer GmbH).
Sections were stained with toluidine blue O and observed
using optical microscopy. (2) Samples were fixed using glutaraldehyde and post-fixed using osmium tetroxide in cacodylate buffer, then embedded in Spurr resin. For both
methods, sections were cut to 1-mm thickness using an ultramicrotome with glass knives. They were stained with toluidine blue and basic fuchsin (Lux, 1981).
R E S U LT S
Peanut root structure
Peanut plants have a typical dicotyledonous root system
with a single taproot branched by the formation of first-,
second- and third-order lateral roots (Fig. 1).
Primary root (taproot) structure. After germination, the
seminal root is thick and covered on its surface by a thin
peripheral zone (Fig. 2A, B). Yarbrough (1949) has characterized the anatomical structure previously; thus, only a
brief summary with some added amendments will be
given here. This paper does not specifically address the
root apical meristem organization as it is outside the
scope of our study.
The basic primary structure is typical of leguminous crop
plants, with an epidermis formed by small cells, a broad
cortical zone of parenchyma cells arranged radially in its
inner part and alternately in its peripheral region, and a
broad central cylinder with tetrarch organization of the vascular system (Fig. 2A – C). The exodermis, as defined by
Peterson et al. (1981), is not developed: no Casparian
Tajima et al. — Peanut Root Structure and Nodule Development
493
F I G . 1. Peanut taproot. (A) Taproot root system of a field-grown peanut at the pod-filling stage. (B) Taproot of a field-grown peanut plant at the flowering
stage. (C) Peanut seedling (5 DAS). PR, primary root; 1stb, first-order lateral root formed on the basal part of the primary root; 1std, first-order lateral root
formed on the distal part of the primary root; 2nd, second-order lateral root; arrowhead, root nodule. Scale bars: (A, B) ¼ 50 mm; (C) ¼ 10 mm.
F I G . 2. Primary root structures. (A–F) Peanut grown in filter paper, (G, H) field-grown plants. (A– H) Hand sections. (A, B, D, E, G) Observed under
white light, (C, F, H) observed under UV light. (A) Cross-section 2 cm from the root apex of a seedling root. (B) Magnified image of (A). (C) Primary
structure of a central cylinder of the seedling root with fluorol yellow and toluidine blue O staining. (D) Pericycle adjacent to the phloem pole. (E, F)
Pericycle adjacent to the xylem pole with several cell layers. (G) Taproot secondary structure of a field-grown plant in the basal part after toluidine
blue staining. (H) Taproot secondary structure of a field-grown plant in the distal part. CB, cork cambium; CO, cortex; EN, endodermis; EP, epidermis;
LP, lateral root primordium; PC, pericycle; PHF, phloem fibers; PH, phloem; PP, parenchyma pith; SPH, secondary phloem; SXY, secondary xylem;
VCB, vascular cambium; XY, xylem; arrowhead, proto-xylem. Scale bars: (A, B, G, H) ¼ 100 mm; (C– F) ¼ 50 mm.
494
Tajima et al. — Peanut Root Structure and Nodule Development
bands are formed in the tissue internally adjacent to the epidermis. In the young, fast-growing primary root of the seedling, the innermost cortical layer, the endodermis, starts its
differentiation approximately 10– 15 mm from the root-cap
junction. Casparian bands (the first state of endodermal
development) are identifiable at this distance. The second
state, suberin lamellae formation, starts in these roots at a
distance of approx. 50 mm from the root tip, by the deposition of suberin lamellae in the cells adjacent to the
phloem poles (Fig. 2C). At 100 mm from the root tip,
almost all endodermal cells are in the second stage of
their development. This is the final stage of endodermal
development in roots of peanut plants.
The stele exterior tissue, the pericycle, is not uniform
(Fig. 2D –F). The pericycle adjacent to the xylem poles is
3 –5 layered (Fig. 2E, F), whilst the zones adjacent to the
phloem poles are 1 (22) layered (Fig. 2D). The xylem
elements of the fast-growing seminal root of the seedling
mature close to the apex, at about the same distance where
the formation of endodermis starts (approx. 10 mm from
the root cap boundary). The first narrow protoxylem elements
are followed quickly by lignification of metaxylem elements
as the process of xylem development proceeds centripetally
(Fig. 2E, F). Phloem poles are extensive, and are
accompanied peripherally by groups of sclerified primary
phloem fibres that give a positive reaction for lignin (Fig. 2F).
Secondary growth of the taproot starts very early. The
first tangential divisions of vascular parenchyma cells
that start the formation of cambium cells adjacent to the
phloem poles were observed approx. 20 mm from the
root tip in young seedlings (Fig. 2D – F). The activity of
the vascular cambium later fosters formation of extensive
secondary vascular tissues. Close to the root base in particular, the taproot becomes considerably thickened – it is
apparent at the flowering stage of the plant (Fig. 1B, G).
However, the distal part of the same root remains much
thinner with a limited secondary growth (Fig. 2H). The
secondary xylem is formed by broad vessel elements:
libriform and parenchyma cells (Fig. 2G, H). The original
tetrarch organization of the taproot primary structure can
be identified clearly from the persistence of four groups
of prominent primary phloem fibres (Fig. 2C, H). Cork
cambium is initiated soon after formation of the vascular
cambium, by the activity of pericycle cells (Fig. 2G, H).
Peripheral root layers, epidermis and cortical tissues are
subject to dilatation growth for some time. This involves
cell extension and limited additional cell divisions of
some cortical cells, including those of the endodermis.
Epidermal and hypodermal cell walls become slightly lignified and suberized, as indicated by phloroglucinol staining (see Fig. 3G). However, in later stages of secondary
growth, all peripheral tissues are destroyed and are gradually shed. Lignified and suberized peridermal tissues
formed by the cork cambium protect the root surface.
The broad parenchyma pith, which is present during root
primary growth, might be preserved even during the flowering stage of the plant (Fig. 2G).
First-order lateral roots. The formation of the first lateral
roots starts soon after germination. Well-developed lateral
root primordia emerging from the primary root are
already present 20 mm from the root tip in young seedlings
(Figs 1C and 2A, B). The structure of these roots differs
from that of the taproot (Fig. 3A– D); they are thinner,
and consequently the cortex and stele are also less extensive
than in the primary root (Figs 2B and 3A). These lateral
roots are diarch or triarch, with two-to-three xylem and
phloem poles; however, their structure differs along the
root axis. At the base of the taproot, the first-emerging laterals are thick, but later-emerging ones become thinner and
their structure is simpler, with a less extensive cortex
(Fig. 1A, ‘1std’). First-order lateral roots formed from the
distal part of the taproot exhibit a similar structure and
probably serve a similar function to that of higher-order
lateral roots (see below).
In the central area of the cortex, some very specific cells are
developed with wall ingrowths in the form of regularly
shaped, semi-globular structures (Fig. 3C, arrowhead).
These structures are also abundant in the second-order and
third-order lateral roots (Fig. 3E – G; third-order lateral
roots are not shown) and exhibit strong autofluorescence
and a positive reaction to lignin (Fig. 3F, G). They are
present more-or-less regularly in the central cortex, but
never in the layer immediately adjecent to the endodermis
(Fig. 3C, F). The wall ingrowths develop in the cell
‘corners’ opposite to the intercellular spaces of internally
adjacent cells. X-ray analyses coupled with environmental
scanning electron microscopy revealed no specific elemental
composition of these ingrowths (data not shown). Therefore,
we might conclude that these structures are formed purely by
organic materials of the cell walls. The endodermis of the
first-order laterals, similar to the primary root, matures
close to the root apex at a distance of several millimeters by
the formation of Casparian bands. Later, the second stage
of endodermal development occurs; at 50 mm from the root
tip, almost all endodermal cells are covered by a prominent
layer of lamellar suberin (Fig. 3B). The pericycle is two- or
three-layered (Fig. 3A).
First-order lateral roots exhibit secondary growth that
resembles that of the primary roots. Activity of the vascular
cambium is followed by formation of cork cambium of
pericycle origin (Fig. 3C). At the initial stage of this
process, two layers of lignified and suberized tissues are
located centripetally to the primary cortical layers: the peripheral one is the endodermis, the internal one is the first
layer derived from the cork cambium (Fig. 3C). The
number of layers increases gradually; however, the peripheral zones, including the former endodermal layer, are
gradually destroyed and detached. Outer peridermal cells
become lignified and suberized, as shown by histochemical
reactions. Other events occurring in the cortical layers as a
reaction to the secondary thickening closely resemble those
of the primary roots.
The epidermis is occasionally double-layered and the
size of epidermal cells varies (Fig. 3A, G, H, black
arrow). Abundant root hairs, some forked or branched
(Fig. 3H, I), were formed when the peanut seedlings were
grown in wet filter paper. No root hairs were observed
when peanut plants were grown in field cultivation or in
vermiculite culture.
Tajima et al. — Peanut Root Structure and Nodule Development
495
F I G . 3. Peanut lateral root. (A, B, H, I) Peanut grown in filter paper, (C–G) plants grown in vermiculite. (A– D, H, I) First-order lateral root, (E –G)
second-order lateral root. (A, B, E– H) Hand sections, (C, D) Spurr-embedded semi-thin sections stained with toluidine blue and basic fuchsin. (A,
C–E, G, H, I) Observed in white light, (B, F) observed under UV light. (A, B) Primary structure. (B) Primary structure at 50 mm from the root tip
with endodermis in second state of development, shown by fluorol yellow staining. (C) Secondary structure. (D) Secondary structure of a central cylinder.
(E) Simple structure of a second-order lateral root. (F) Second state of the endodermis against phloem poles, detected with fluorol yellow staining and
autofluorescence of the cell-wall ingrowths in cortex. (G) Cell-wall ingrowths in cortex detected with phloroglucinol staining. (H, I) Epidermis with root
hairs. CCB, cork cambium; CO, cortex; EN, endodermis; EP, epidermis; PC, pericycle; PH, phloem; RH, root hair; VCB, vascular cambium; XY, xylem;
white arrow, Casparian band; black arrow, double-layered epidermis; arrowhead, cell-wall ingrowths. Scale bars: (A– F, H, I) ¼ 100 mm; (G) ¼ 50 mm.
Second- and third-order lateral roots. These roots differ from
both the previously described types. Differences in their
structure, and probably their function, are greater when
compared to the difference between the primary and firstorder laterals. These roots are, and remain, thin with a
diameter of 200 mm. They exhibit no secondary growth at
the flowering stage. However, in older plants of 90 DAS,
some second-order lateral roots were detected with
limited secondary thickening. The second- and third-order
lateral roots have simple primary structures. Under an epidermis formed by small cells, 5 – 6 (even fewer in the
case of third-order laterals) layers of alternately arranged
cortical cells exist (Fig. 3E). In most of the sections of
the second- and third-order lateral roots, special cells with
lignified wall ingrowths were found (Fig. 3F, G, arrowhead). Their presence was usually restricted to the third cortical layer from the root surface. The innermost layer of the
cortex, the endodermis, is formed by 16– 22 small cells in
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Tajima et al. — Peanut Root Structure and Nodule Development
the second-order laterals (Fig. 3E). The endodermis also
matures via two states in these roots; however, the second
state (suberin lamellae deposition) is restricted to the cells
that are adjacent to the phloem poles (Fig. 3F). No endodermis was found with all cells in the second state in the
second-order lateral roots of plants at the flowering stage.
The stele of these roots is narrow, with a diarch xylem.
The pericycle is uniseriate: xylem and phloem poles are
formed by a few conductive elements (Fig. 3E, F). For
the third-order laterals, only a single protoxylem and a
single metaxylem element exist in each xylem pole (data
not shown). A group of phloem fibres is formed even in
these roots; the pith is absent in both second- and
third-order lateral roots.
Nodule structure and development
Peanut root nodules are of a determinate type. In the root
systems both of flowering and pod-filling plants, their development is restricted mostly to first-order lateral roots (Tajima
et al., 2006). Occasionally, they occur also on the taproot, or
on the second-order laterals. Nodules change their size and
structure as they mature. Young nodules are white in crosssection, whereas mature nodules are intensively pink-reddish
in colour in their centre due to the presence of leghemoglobin
(Fig. 4A, B). Even macroscopically, it is possible to distinguish central, coloured, homogeneous infected areas and
a white, semi-transparent peripheral zone (Fig. 4B).
Light-brown peridermal tissue is formed on the nodule
surface. In later stages, senescing nodules turn greenish in
their interior (not shown here).
The development of nodules in this species is connected
with the development of lateral roots. The nodule body is
formed at the junction of the main and lateral roots by the
activity of proliferating cells derived originally from the
pericycle, which is most clearly apparent in Fig. 4(C, D).
In the cross-section of the first-order lateral root, it is possible
to distinguish a dark-stained layer of cork cells derived peripherally from the cork cambium. The remainder of the
primary cortical and epidermal tissues, including collapsed
endodermal cells, is present on the root surface. These
layers are shed later, and the nodule surface is then protected
by a thin peridermal layer that is impregnated with lignin
and suberin, as demonstrated by histochemical reactions
(Fig. 4B).
Detailed observations made at higher magnification
support the connection of a nodule vascular bundle with
vascular bundles of both parent and lateral roots at their
junction (Fig. 4E). The connecting vascular bundle is
branched to form nodule vascular bundles (Fig. 4E).
Individual nodule bundles are covered and surrounded by
one endodermal layer with prominent Casparian bands
(Fig. 4F– H, arrow).
DISCUSSION
Structures of various peanut root types
The root structures of crops have been studied and
documented intensively by earlier plant anatomists.
However, most of our knowledge is based on structural
analyses of primary (seminal, pole or prop) roots in dicotyledons, and of both primary seminal and adventitious roots
in monocotyledons. Very little is known about the structure
of lateral roots, their different types and categories, or their
different structure-related functions. Lateral root structures
have been analysed in detail, to the best of our knowledge,
only for a few cereals. Cereal plants form L-type and S-type
lateral roots: the former being thick and long whilst the
latter are thin and short (Yamauchi et al., 1987a, b) In
rice, the L-type are approx. 150 mm in diameter at the
base and the S-type are 50– 80 mm. Only L-type lateral
roots form higher-order lateral roots. Kawata et al. (1977)
observed first-order through to fifth-order lateral roots of
rice and reported that thick lateral roots (i.e. L-type) have
an anatomical structure resembling that of the primary
root, whereas thin lateral roots (i.e. S-type) lack the midcortex (i.e. aerenchyma in the case of rice) and late
meta-xylems in the central cylinder. Consequently, the epidermis, exodermis, aerenchyma, endodermis and central
cylinder, with phloem and protoxylem, are commonly
present in all primary lateral roots, thick and thin. In
other monocotyledons, the disparities between the lateral
roots might be different. For maize, they have been classified into four different types based on their thickness and
anatomy (Varney et al., 1991).
For dicotyledons, available information on this topic is
scarce. In dicotyledonous plants, large lateral roots
develop secondary thickening and form a permanent root
system structure, although small feeder lateral roots do
not develop secondary thickening (Zobel, 1986).
Differences between large lateral roots and small feeder
lateral roots are insufficient to elucidate their anatomical
structures and functions, as inferred already by Zobel
(1986). For instance, in a recently published monograph
related to soybeans, only five lines of text were given to
present knowledge about lateral (branch) roots (Lersten
and Carlson, 2004). Similarly, in a structural study of
peanut seedlings published by Yarbrough (1949), the
characteristics attributed to the lateral roots referred to the
fact that their vascular pattern does not correspond to that
of the primary root: laterals are diarch and without pith.
Fundamentally identical information is included in a monograph about peanuts (Ramanatha Rao and Murty, 1994).
Our study revealed a principal difference between firstorder and higher-order lateral roots of peanut plants. The
first-order lateral roots, at least most of them, resemble
the primary seminal root but have simpler organization
because of their smaller diameter and lack of pith. They
become secondarily thickened and form the skeleton of
the entire root system. In contrast, higher-order laterals
are very thin and have a simple structure; we might classify
them as ‘feeder roots’ (Zobel, 1986).
An interesting phenomenon is the incomplete development of the second state of the endodermis, suberin lamellae deposition, in these roots. Sections of the endodermis
opposite the xylem poles remain with Casparian bands
only, even during the flowering stage of the plant. Similar
incomplete development of the second state of the endodermis was also observed in thin lateral roots of the shrub
Tajima et al. — Peanut Root Structure and Nodule Development
497
F I G . 4. Root nodule and peanut root modified by nodule formation for plants grown in vermiculite. (A) Cross-section of a nodule and first-order lateral
root at low magnification without staining. (B) Cross-section of nodules and first-order lateral root at low magnification with red phloroglucinol staining,
showing lignification of the root xylem and peridermal layers of the nodules. (C, D) Spurr-embedded semi-thin sections stained with toluidine blue and
basic fuchsin. (C) Cross-section of a first-order lateral root modified by a second-order lateral root and nodule formation. (D) Longitudinal section of a
first-order lateral root and nodule formation. (E– G) Technovit-embedded sections stained with toluidine blue. (E) Magnified view of a vascular bundle
connection. (F) Peripheral structure of a root nodule. (G) Vascular bundle in peripheral tissue of the root nodule. (H) UV-fluorescent view of a handsection of a nodule vascular bundle stained by berberin, showing Casparian bands. 1st, first-order lateral root; CCB, cork cambium; CO, cortex; EN,
endodermis; IA, Rhizobia-infected area; ND, root nodule; PC, pericycle; PD, peridermal layers of a nodule impregnated with lignin and suberin; PH,
phloem; ST, stele; VB1, vascular bundle of the first-order lateral root; VB2, vascular bundle of the second-order lateral root; VBN, vascular bundle
of the root nodule; VE, vessel; XY, xylem; arrow, Casparian band. Scale bars: (A, B) ¼ 1 mm; (C– F) ¼ 100 mm; (G, H) ¼ 10 mm.
Karwinskia (Lux et al., 2004). This might be an important
feature for uptake and transport processes in these ‘feeder’
roots.
An unusual development of the epidermis in peanut was
addressed long ago by Yarbrough (1949), who noted a
meristem-like hypodermal region producing a multilayered
‘sloughing zone’ and commonly missing root hairs, except
at the root base where ‘tuft hairs’ occur. Exceptional shedding of the epidermal and even outer cortical cells from the
root surface in this species was examined recently by Uheda
et al. (1997). Apparently, root hairs are formed in this
species only under specific conditions in first-order lateral
roots, in our case in wet filter paper but not in the soil;
they might become branched.
498
Tajima et al. — Peanut Root Structure and Nodule Development
The presence of cell-wall ingrowths, which we detected
in the cortical cells, is another uncommon structural
feature of the peanut root. Several types of wall ingrowths
that are present in the root cortex were well documented
by von Guttenberg (1968). The best known are so-called
phi-thickenings, which are found in hypodermal or innercortical layers in some species. These cells are widely
believed to have a mechanical function in the root, but
other opinions have also been advanced (Peterson et al.,
1981; Weerdenburg and Peterson, 1983; Soukup et al.,
2004; Gerrath et al., 2005). Other types of cell-wall
thickening were identified recently in the metal hyperaccumulator species Thlaspi caerulescens, but not in a closely
related non-accumulator species, T. arvense (Broadley
et al., 2007; Zelko et al., in press). The layer of these
cells, localized adjacent to the endodermis, was designated
as the peri-endodermal layer. Although the precise function
of these cells remains unclear, we speculate that they are
somehow related to transport processes in the roots of
these exceptional plants. Similarly, in the case of the
peanut, the regular development of massive, lignified wall
ingrowths opposite the intercellular spaces in some peanut
cortical cells might have some other function for the root,
aside from its purely mechanical function.
Connection of the vascular bundle between the nodule
and roots
Both the vascular bundles of the nodule and the lateral
root were connected at the same site of the parent root.
Grant and Trese (1996) reported that the peanut lateral
roots that formed nodules at the base were shorter than
lateral roots without nodules, which was inferred to result
from competition for photosynthate products between
nodules and the lateral roots through the junction of the vascular bundles. On the other hand, the vascular bundles of
nodules were found to be connected with vascular
bundles of parent roots, separately from the vascular
bundle of the lateral root in soybean (Ikeda, 1955).
Moreover, the branching site of the nodule vascular
bundle was closer to the junction with the parent root in
peanut than in soybean. This interspecific difference
might arise from the different positions of the primary
infection of Rhizobia in their respective roots: the outer
cortex of the root in soybean, and the pericycle in peanut
(Kamata, 1958). Our results confirm that this type of
nodule formation originates from the pericycle in peanut.
The frequently discussed problem of a ‘common endodermis’ of the nodule and the root (Dart, 1975) might be
resolved in this species as follows. The early formed peridermal tissue, resulting from periclinal division of pericycle
cells (forming the cork cambium) covers both the root and
the nodule surface. The first derivative of the cork cambium
forms a layer of suberized cork cells. This layer separates
the outer root layers, consisting of the epidermal and
primary cortical layers. Their cells die and are gradually
shed. A network of thin vascular bundles is formed
within the peripheral part of the nodule, each one surrounded by endodermis. The root and nodule surface is
covered and protected by a thin peridermal layer.
The peanut nodule is formed nearer to the centre of the
parent root than the soybean nodule, with larger overlapping of nodule and root tissues. Consequently, the vascular
bundle of the peanut nodule branches near the junction with
the root vascular bundle. The short distance between the
root and nodule in peanut might engender close ecophysiological interactions between the plant and Rhizobia.
Anatomical structures of non-infected peripheral zones
in nodules
Legume nodules are of two types – determinate and
indeterminate – viewed in terms of the growing periods
of the individual nodules. The determinate nodule is oval,
whilst the indeterminate nodule has an axis and is elongated
by the meristem at the apical part of the nodule (reviewed in
Puppo et al., 2005). The peanut nodules are of determinate
type (Stalker, 1997). The non-infected layers regulate
oxygen diffusion into the inner part of the nodule, which
is a very important feature for nitrogen fixation (Serraj
et al., 1999). The thin and simple structure of the noninfected peripheral zones in peanut nodules raises the possibility that oxygen diffusion might easily be affected by
environmental stresses, thereby decreasing the nitrogenfixing capability of peanut plants. However, Sinclair and
Serraj (1995) compared several legume crops and determined that the suppression of nitrogen fixation by drought
stress is less in peanut than in soybean. Although soybean
has three peripheral zones, the peanut nodule, with a thin
and simple peripheral zone, might have a specific mechanism for the maintenance of nitrogen fixation, even under
external stresses.
ACKN OW LED GEMEN T
This research was supported in part by a Grant-in-Aid for
Scientific Research (No.17380009) from the Japan
Society for the Promotion of Science and a grant from
Slovak Research and Development Agency COST000406. A. Lux would like to express his gratitude for the hospitality provided during his stay as visiting professor at the
Field Production Science Center, the University of Tokyo.
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