Annals of Botany 85: 929±936, 2000
doi:10.1006/anbo.1999.1127, available online at http://www.idealibrary.com on
Cluster Roots in Casuarinaceae: Role and Relationship to Soil Nutrient Factors
H . G . D I E M{* , E . D U H O U X {, H . Z A I D} and M . A R A H O U}
{Institut FeÂdeÂratif d'Ecologie Fondamentale et AppliqueeÂ, Universite Paris b and Direction des Relations
Internationales, CNRS, 3 rue Michel-Ange, 75794 Paris, France, {IRD (GeneTrop/PCMA), BP 5045, 34032
Montpellier, France and }Departement de Biologie, Faculte des Sciences, Universite Mohammed V, Rabat, Maroc
Received: 8 August 1999 Returned for revision: 4 October 1999 Accepted: 3 November 1999
N2-®xing actinorhizal trees in the family Casuarinaceae are economically of great interest in tropical and sub-tropical
zones because they are used for many purposes including protection against wind, stabilization of sand dunes and the
production of ®rewood and charcoal. They are usually able to grow in sandy soils with low fertility by virtue of their
ability to ®x N2 . The objective of this review is to discuss brie¯y the role of mycorrhizas and, more extensively, that of
cluster ( proteoid) roots, developed by a number of species of Casuarinaceae to improve the absorption of nutrients
other than N from soil, especially those needed for N2 ®xation and growth. After evaluating the actual relationships
between mycorrhizas and the Casuarinaceae, we highlight the possible role of cluster roots as an e€ective alternative
to mycorrhizas, and as a means of improvement of growth of the trees in nutrient-de®cient soils. This raises the
question of what triggers the formation of cluster roots in the Casuarinaceae. In addition to phosphorus de®ciency,
iron de®ciency seems to be a major factor inducing the formation of cluster roots in Casuarina glauca and
C. cunninghamiana. The number of cluster roots and the precocity of their formation are directly related to plant
chlorosis due to Fe de®ciency, as expressed by the critical concentration of chlorophyll in the shoot (0.60 mg g ÿ1
shoot f.wt). The e€ect of the nitrogen source on cluster root formation is discussed in relation to pH values in the
plant culture solution. The number of cluster roots formed in nitrate-fed plants increases with pH in the range of
5 to 9. Experiments carried out with alkaline and acidic soils show that cluster roots are only produced when they are
needed to overcome soil nutrient de®ciency due to the immobilization of nutrient elements (P and Fe) by soil
alkalinity. The possible involvement of ethylene in the initiation and/or the morphogenesis of cluster roots is
# 2000 Annals of Botany Company
discussed.
Key words: Casuarinaceae, Casuarina cunninghamiana, Casuarina glauca, cluster roots, ethylene, iron de®ciency,
phosphorus de®ciency, proteoid roots.
I N T RO D U C T I O N
N2-®xing trees in the family Casuarinaceae are widely
planted in many tropical and subtropical countries for
various purposes such as protection against wind, ®rewood
production, stabilization of sand dunes and also land
reclamation. In addition, these trees are also considered
e€ective pioneer species capable of thriving in marginal soils
characterized by low fertility. The ability of these trees to
grow in poor soils is largely due to the N2-®xing activity in
the root nodules, through a mutualistic symbiosis which
they form with the actinomycete, Frankia. However nodules
of N2-®xing plants only function if the host plant is supplied
with sucient levels of nutrients other than N, which are
essential for the biochemical process of N2 ®xation. The
e€ectiveness of the Frankia-Casuarinaceae symbiosis is,
therefore, strongly dependent upon the potential of Casuarinaceae trees to acquire nutrient elements from soil,
particularly P (Sanginga et al., 1989; Yang, 1995) and also
Co, Mo (Hewitt and Bond, 1961, 1966) and Fe (Arahou
et al., 1996; Arahou and Diem, 1997).
In order to enhance the absorption of nutrients, especially insoluble elements, from soil, trees in the family
* For correspondence. Fax 33 1 44 96 49 10, e-mail diem.hoang@
cnrs-dir.fr
0305-7364/00/060929+08 $35.00/00
Casuarinaceae develop specialized structures of symbiotic
origin known as mycorrhizas. They may also form, in their
root system, many bottlebrush-like clusters of rootlets
(Diem et al., 1981; Gardner et al., 1982; Reddell et al., 1986)
now designated as cluster or proteoid roots with reference
to their unique root morphology (Lamont, 1981, 1982;
White and Robson, 1989; Racette et al., 1990; Crocker and
Schwintzer, 1993).
Until now, the variable distribution of mycorrhizas, and
their e€ects on the growth of Casuarinaceae, have not been
clearly understood, probably because mycorrhizal infection
depends on many factors including host-fungal compatibility and also adverse soil characteristics. Firstly, the
susceptibility of members in the genera Allocasuarina and
Casuarina to mycorrhizal infection is variable, depending
on the type of mycorrhizas involved. For example, there is
some indication that Allocasuarina spp. are more susceptible to ectomycorrhizal infection than Casuarina spp.
(Reddell et al., 1986), probably because trees in the genus
Casuarina show a true hypersensitive reaction of incompatibility against the ectomycorrhizal fungus, Pisolithus (Thoen
et al., 1990; Theodoru and Reddell, 1991; Dell et al., 1994).
Secondly, ®eld observations reported by many authors
(Trinick, 1977; Malajczuk et al., 1981; Berliner and Torrey,
1989; Louis et al., 1990; Crocker and Schwintzer, 1994),
# 2000 Annals of Botany Company
930
Diem et al.ÐCluster Roots in Casuarinaceae
show that a number of plant species, including N2-®xing
pioneer plants Comptonia peregrina and Myrica gale,
produce cluster roots rather than mycorrhizas to acquire
P from P-de®cient soils.
It is therefore assumed that Casuarinaceae trees spontaneously form cluster roots in response to soil nutrient
de®ciency when they are planted or when they grow as
pioneers in soil nutritionally poor or lacking mycorrhizas.
As discussed by Diem and Arahou (1996), cluster roots
improve nutrient uptake not only by means of an enlarged
absorptive root surface, but also through their capacity to
release relatively large amounts of hydrogen ions (H ‡ ),
reductants (organic acids and phenolics) and speci®c
chelators, thereby increasing the acquisition of poorlysoluble sources of P, Fe and other elements. For this reason,
cluster roots of Casuarinaceae are considered to be an
e€ective, adaptative mechanism and a reliable strategy
usually developed by plants of this family to substitute for
mycorrhizas in enhancing nutrient uptake.
O C C U R R E N C E O F C L U S T E R RO OT S I N
CA S U A R I N AC E A E
As expected, cluster roots of Casuarina glauca (Diem and
Arahou, 1996) are similar to those described for other plant
species, i.e. dense clusters of rootlets produced opposite
every protoxylem pole, at intervals along lateral secondary
roots. Each cluster root produces, on average, 20 rootlets,
which are initially white and become brown within 2 or
3 weeks, probably because of tannin deposition in the
rootlets (Fig. 1). Similar to cluster roots of other plant
species, cluster roots of C. glauca appear to be short-lived.
Cluster roots are found not only in the root system of species
in the genus Casuarina (Diem et al., 1981; Gardner et al.,
1982), but also in Allocasuarina campestris (Reddell et al.,
1986) and Gymnostoma papuanum (Racette et al., 1990).
Diem and Arahou (1996) did not ®nd any cluster roots on
Allocasuarina torulosa and A. verticillata grown in hydroponic conditions even in medium speci®cally designed to
induce cluster root formation. The exact reason is not
understood. Other families show a similar disjointed
occurrence of cluster roots. For example, not all species in
the genus Lupinus produce cluster roots (White and Robson,
1989). In our study, the percentage of plants producing
cluster roots and the number of cluster roots per plant are
signi®cantly higher in C. glauca than in C. cunninghamiana
and C. obesa. Since members in the family Casuarinaceae
may have di€erent requirements for P and Fe, it is possible
that di€erences among Casuarinaceae trees in forming
cluster roots are determined by their di€erent potentials to
grow under P and/or Fe-stressed conditions.
N U T R I T I O N A L FAC TO R S A F F E C T I N G T H E
FO R M AT I O N O F C L U S T E R RO OT S I N
CA S UA R I N AC E A E
Environmental factors in¯uencing the formation of cluster
roots have been extensively investigated (see review by
F I G . 1. Cluster roots of C. glauca. Plants were grown in a plant culture medium lacking exogenous P and Fe, and supplemented with the strong
Fe-binding chelator, EDDHA. A, General view with 1±2 cm long cluster roots; B, cluster root with three rows of rootlets. Bar ˆ 1 cm.
Diem et al.ÐCluster Roots in Casuarinaceae
E€ect of P de®ciency
In the study of the relationship between mineral nutrient
availability and growth of Gymnostoma papuanum, Racette
et al. (1990) showed that only P de®ciency induced a
signi®cant increase in cluster root formation, as previously
reported for other actinorhizal plants (Louis et al., 1990;
Crocker and Schwintzer, 1993). This observation is consistent with the general ®nding that low P status in soil is a
major cause of cluster root formation.
Interestingly, Arahou and Diem (1997) found that P
de®ciency alone did not induce any cluster root formation
in C. glauca provided the plant culture medium contained
available Fe. The stimulating e€ect of P de®ciency on
cluster root formation was only signi®cant when synergistically associated with Fe de®ciency, as evidenced by the
highest number of cluster roots in Fe- and P-stressed plants
grown in media lacking both P and Fe and supplemented
with the strong Fe-binding chelator, EDDHA (ethylenediamine di (o-hydroxyphenyl) acetate). C. glauca probably
does not form cluster roots in media lacking only P because
it does not require large amounts of available P for growth,
at least during the early growth stage, and especially when it
is fed with mineral N. By that time, plants continue to grow
well and do not show any apparent symptoms of P
de®ciency. This is in agreement with the suggestion that
Casuarina has a great requirement for P only for the
symbiotic N ®xation process, not for plant growth per se
(Sanginga et al., 1989). It may be concluded that cluster
roots do not necessarily occur in Casuarina as long as plant
growth is not severely a€ected by nutrient de®ciency. In
such a case there would be no physiological/biochemical
signal for inducing cluster roots.
E€ect of Fe de®ciency
Typical morphological and physiological changes were
observed in iron-de®cient cultures of plants. In Helianthus
annuus L., additional cell divisions in the rhizodermis layer
and enhanced formation of root hairs and swollen root tips
have been observed (RoÈmheld and Marschner, 1981). In
Fe-de®cient roots, the position of emerging hairs di€ers
from control plants whereas initiation of lateral root
primordia occurs in the swollen zone (Landsberg, 1996).
According to Arahou and Diem (1997), Fe de®ciency in
the plant culture medium, rather than P de®ciency,
appeared to be determinative in inducing cluster root
formation in C. glauca. The number of cluster roots formed
by this plant species and also the number of plants producing cluster roots were inversely correlated with the
concentration of FeCl3 in the plant culture medium. The
presence of FeCl3 at a concentration 100 mM completely
inhibited cluster root formation even in a medium lacking P
and containing 50 mM EDDHA to remove contaminate Fe
in the medium.
It should be mentioned that there is no cluster root
formation in a medium that is not supplied with exogenous
Fe. Cluster roots only occurred if NaHCO3 or Fe-binding
chelators were used to remove all traces of contaminate Fe
in the medium, resulting in an actual Fe-stressed condition
(Arahou and Diem, 1997). Rosen®eld et al. (1991) also
reported that Ficus benjamina only produced cluster roots
when all contaminate Fe in the medium was previously
eliminated by using Na2EDTA (ethylenediamine tetraacetate). These results suggest that cluster roots of C. glauca
and F. benjamina might be considered as good indicators of
total Fe de®ciency status in the root environment.
As reported by Diem and Arahou (1996), the number of
cluster roots increased when C. glauca plants were grown
continuously in Fe-deprived medium (medium lacking both
P and Fe and supplemented with 50 mM EDDHA). By
contrast, plants ceased to produce cluster roots as soon as
100 mM FeCl3 was temporarily supplied, even for a short
time i.e. 24 h (Fig. 2). This ®nding suggests that cluster root
formation is inhibited by the increased Fe content in plant
tissue due to the temporary Fe supply. Plants only produce
cluster roots again 7 weeks after being transferred into
Fe-deprived medium, probably when the amount of Fe in
the plant tissue is exhausted.
The relationship between cluster root formation in
C. glauca and Fe deprivation was also con®rmed by the
following experiment. Twelve-week old plants of C. glauca
were ®rst grown in the complete culture medium of
Broughton and Dilworth (Arahou and Diem, 1997). They
were then deprived of Fe for periods varying from 1 to 8
weeks by transferring into a plant culture medium lacking
30
No. of cluster roots/plant
Dinkelaker et al., 1995; Skene, 1998). Soil organic matter
(Lamont, 1993), aeration (Crocker and Schwintzer, 1993)
and rhizosphere microorganisms (Lamont and McComb,
1974) may be involved in the production of cluster roots.
However most attention has been directed towards the
e€ect of soil nutrient de®ciencies. There is now evidence
that cluster roots are generally formed by plants in response
to P and Fe de®ciencies in the plant culture medium or in soil
(Lamont, 1981, 1982; Gardner et al., 1982; Keerthisinghe
et al., 1998; White and Robson, 1989; Rosen®eld et al.,
1991).
931
25
20
15
10
5
0
0
5
10
15
20
Weeks
F I G . 2. Kinetics of cluster root production in Casuarina glauca and
e€ect of temporary Fe supply (Figure from Diem and Arahou, 1996).
(h), Plants continuously grown in a medium lacking both Fe and P
and containing 50 mM of EDDHA to remove contaminate Fe; (s),
plants grown as above, but with 100 mM FeCl3 for 24 h in the seventh
week. Note that the production of cluster roots was interrupted until
the fourteenth week.
932
Diem et al.ÐCluster Roots in Casuarinaceae
T A B L E 1. E€ect of Fe deprivation (temporary interruption of Fe supply) on cluster root formation and chlorophyll synthesis in
hydroponically-grown Casuarina glauca
Fe deprivation*time
(weeks)
Time (weeks) after transfer
to Fe-containing medium{
No. of cluster
roots per plant
Chlorophyll content in shoot
(mg g ÿ1 f.wt)
0
1
2
3
4
5
6
7
8
8
7
6
5
4
3
2
1
0
0a
0a
0a
0a
2ab
5b
9c
13d
15d
1.59e
1.37d
0.98c
0.79b
0.63a
0.61a
0.60a
0.58a
0.58a
For each parameter, values followed by a di€erent superscript are signi®cantly di€erent according to the Newman and Keuls test at P ˆ 0.001
with ten replicates. *Medium lacking Fe and containing 50 mM EDDHA. {Medium containing 100 mM FeCl3 .
exogenous Fe and supplemented with 50 mM EDDHA to
remove contaminate Fe. After growing under Fe-stressed
conditions, plants were returned to the culture medium
containing 100 mM FeCl3 for the remainder of the 8-week
experiment. Table 1 shows that the number of cluster roots
per plant markedly increased with increasing time of Fe
deprivation. As expected, chlorophyll content in shoots
decreased progressively and C. glauca produced cluster
roots after 4 weeks of Fe deprivation when plants began to
exhibit chlorotic symptoms and when the chlorophyll
concentration in the shoot was reduced to 0.60 mg g ÿ1
shoot f.wt. It is generally admitted that green plants require
a continuous supply of Fe for growth. As soon as this
supply is interrupted, growth is hampered by Fe de®ciency
resulting in leaf chlorosis (Brown, 1978; Mengel, 1994).
Similarly, C. glauca plants which were Fe-stressed due to
the interruption of Fe supply, exhibited chlorotic symptoms
which were more, or less, severe according to the duration
of the interruption. As a consequence, the number of cluster
roots and the precocity of their formation were directly
related to plant chlorosis severity.
Many workers have estimated Fe content of plant tissue
with the objective of establishing the critical levels of Fe
below which de®ciency symptoms occur. However, according to the reviews by Chen and Barak (1982) and Mengel
(1994) there is evidence that the determination of chlorophyll content in shoot rather than Fe content is the most
suitable approach to quantify Fe de®ciency. The decreased
chlorophyll content observed in N-fertilized Casuarina
plants (Table 1) is undoubtedly associated with Fe de®ciency in their shoot tissues. As soon as plants become
chlorotic, with a chlorophyll concentration lower than
0.60 mg g ÿ1 shoot f.wt, cluster roots were formed to
facilitate Fe uptake. This indicates that plant chlorosis, as
expressed by the critical concentration of chlorophyll (as
indicated above), is possibly a visible signal for cluster root
initiation.
E€ect of KNO3 vs. NH4Cl
In several plant species, cluster root formation has been
related to N nutrition (Dinkelaker et al., 1995). Lamont
(1993) reported that cluster root production was inversely
correlated with the increase of N content in shoots of
Hakea (Proteaceae). Crocker and Schwintzer (1993),
however, stated that N de®ciency in the culture medium
did not induce any cluster root formation in three
actinorhizal plant species, Gymnostoma papuanum, Myrica
cerifera and M. gale. There was no di€erence in cluster root
formation between plants receiving KNO3 and those grown
solely with N derived from N2 ®xation.
The relationship between N source and cluster root
formation is not yet clear and varies greatly according to
plant species. For example, M. gale produced more cluster
roots when it was supplied with urea than when it was given
KNO3 (Crocker and Schwintzer, 1993). However, in the case
of G. papuanum (Casuarinaceae), the number of cluster
roots was higher in plants grown in nitrate-containing
medium than in plants grown in the presence of ammonium
(Racette et al., 1990). Interestingly, C. glauca, also belonging
to the Casuarinaceae, only showed chlorotic symptoms, and
subsequently produced cluster roots, when it was fed with
nitrate. This suggests that cluster root formation in
Casuarinaceae is signi®cantly in¯uenced by N source
(Arahou and Diem, 1997). In contrast, there were neither
cluster roots nor chlorotic symptoms in C. glauca grown in
medium supplemented with NH4Cl. This study provides
additional evidence that cluster roots of C. glauca are an
adaptation and remediation mechanism that is only developed by plants su€ering from severe nutrient de®ciencies.
To explain the absence of chlorosis in C. glauca given
NH4Cl, it is suggested that the low pH of the NH4Clcontaining medium (Table 2) may have allowed mobilization of ferric iron, which often precipitates in the root cell
apoplast, thus forming a pool of available ferric iron
(Bienfait et al., 1985).
E€ect of pH
Since plants given NH4Cl release considerable amounts
of H ‡ ions leading to a decrease in rhizosphere pH and a
better absorption of insoluble nutrients (P and Fe particularly), this raises the question as to which of the two factors
(i.e. pH or N source), actually controls cluster root
Diem et al.ÐCluster Roots in Casuarinaceae
933
T A B L E 2. E€ect of nitrogen sources (KNO3 and NH4Cl) and NaHCO3 supply (5 mM) on cluster root formation by
Casuarina glauca and Casuarina cunninghamiana hydroponically grown for 10 weeks in Fe-free medium
C. glauca
Nitrogen source (0.5 mM)
KNO3
NH4Cl
C. cunninghamiana
‡NaHCO3
ÿNaHCO3
‡NaHCO3
ÿNaHCO3
33.2d
( pH 8.2)
4.1b
( pH 7.6)
23.4
( pH 6.8)
30.4c
( pH 8.2)
13.2b
( pH 7.60)
28.7c
( pH 6.8)
0a
( pH 4.7)
0a
( pH 4.7)
Results are expressed as number of cluster roots per plant. pH of the medium is indicated. For each plant species, values followed by a di€erent
superscript are signi®cantly di€erent according to the Newman and Keuls test at P ˆ 0.01 with 10 replicates.
T A B L E 3. E€ect of pH on cluster root (CR) formation by Casuarina glauca and Casuarina cunninghamiana grown for
10 weeks in Fe-free medium containing 0.5 mM KNO3
C. glauca
pH of the medium
3
5
7
9
C. cunninghamiana
No. of plants with CR (%)
No. of CR per plant
No. of plants with CR (%)
No. of CR per plant
0
40
100
100
0.0a
8.2b
19.3c
31.9d
0
50
100
90
0.0a
6.6b
17.3c
18.7c
For each plant species, values followed by a di€erent superscript are signi®cantly di€erent according to the Newman and Keuls test at P ˆ 0.01
with 10 replicates.
Arahou and Diem (1997) reported that in acid soil ( pH 5.8)
from the Rabat (Morocco) area, C. glauca plants did not
show any chlorotic symptoms and did not produce any
cluster roots because nutrients needed for plant growth
were readily available in this soil (Fig. 3). By contrast, in
calcareous Moroccan soil from Meknes ( pH 8.3), numerous
plants exhibited symptoms of severe Fe de®ciency during
the ®rst 4 weeks of growth as shown by the decreasing
chlorophyll content in shoot tissue. In this soil plants began
to produce cluster roots when chlorophyll content in the
shoot was as low as 0.50 mg g ÿ1 shoot f.wt, con®rming the
observation stated above for hydroponically-grown plants.
The increment of chlorophyll content in shoots of cluster
root-producing plants (green plants) grown in calcareous
Meknes soil compared to that of non-cluster root-producing
2
60
1.5
40
1
20
0.5
0
0
1
2
3 4
5
6
7
8
0
9 10 11 12 13 14
No. of cluster roots per plant
I M P ROV E D G ROW T H O F CA S U A R I N A
G L A U CA B Y C L U S T E R RO OT FO R M AT I O N
IN SOIL
plants (chlorotic plants) grown in the same calcareous
Meknes soil suggests that plant chlorosis alleviation is
closely related to cluster root production (Table 4). It is
therefore assumed that C. glauca produces cluster roots to
scavenge Fe from calcareous soils as previously reported for
Lupinus consentinii (White and Robson, 1989), L. albus
(Gardner et al., 1982; Dinkelaker et al., 1989) and Ficus
benjamina (Rosen®eld et al., 1991).
El-Lakany and Luard (1982) and Gill and Abrol (cited
by Batra, 1991) reported that Casuarina spp. can be
Chlorophyll content (mg g−1 shoot f. wt)
formation. Table 2 shows that the addition of 5 mM
NaHCO3 to increase the pH of NH4Cl-containing medium
up to 7.6 only slightly stimulates cluster root formation in
C. glauca and C. cunninghamiana. The number of cluster
roots in this culture medium is still signi®cantly lower than
that obtained in KNO3-containing medium at the same
pH (7.6). This con®rms that cluster root formation is
a€ected by the form of N supplied. However we should
mention that increasing pH of the KNO3-containing
medium also induces a higher number of cluster roots
(Table 3). This suggests that cluster root formation is
additionally in¯uenced by the pH of the root environment.
Weeks
F I G . 3. Relationship between chlorophyll content in shoot and cluster
root formation in C. glauca grown in alkaline and acid soils for
12 weeks (Figure from Arahou and Diem, 1997). (h), Chlorophyll
content in plants grown in acid soil; ($), chlorophyll content in plants
grown in alkaline soil; (s), number of cluster roots produced by
plants grown in acid soil; (n), number of cluster roots produced by
plants grown in alkaline soil.
934
Diem et al.ÐCluster Roots in Casuarinaceae
successfully grown and nodulate in saline and alkaline soils.
High pH in the culture medium inhibits in vitro nodulation
of most Casuarina species, but Batra (1991) reported that
in soil-grown C. glauca and C. equisetifolia the nodule
development, as expressed by nodule weight, was signi®cantly higher at pH 8.5 than at lower pH values. This ®nding
is consistent with our observation that C. glauca is able to
grow and nodulate in an alkaline medium, provided that
cluster roots are formed.
The most impressive example of a successful plantation
trial with Casuarina in alkaline soil is the story of the
rehabilitation of a limestone quarry at Baobab Farm, a
renowned recreational resort near Mombasa in Kenya.
Originally this site was a bare area covered by a calcareous
layer (CaO: 51%; pH of the ground water: 7.4; Wood, 1987)
resulting from the exploitation of fossil coral limestone for
cement production. To restore the landscape and bring back
some life, the cement company decided, in 1971, to set up
some tree plantation trials with 26 tree species. Surprisingly,
the Casuarinas were the most promising of them all (Wood,
1987). The rehabilitation project has proven to be successful
since, over a few years, Casuarinas have established a
luxuriant vegetation. Falling leaves from Casuarina trees
were progressively decomposed into a layer of humus on the
top of the limestone and this allowed other introduced plant
species to grow.
The fact that Casuarinas can grow and survive in the
harsh conditions of the quarry, with minimal management,
is striking. This raises the question of what determines the
ability of Casuarinas to take up nutrients, especially
insoluble P and Fe, from limestone.
As indicated in Table 4, shoot re-greening in cluster rootproducing plants is accompanied by an increased shoot dry
weight. This obviously gives evidence that improved growth
of C. glauca is due to an enhancement of nutrient uptake by
cluster roots. In the absence of cluster roots, the trees do not
regreen and probably have poorly developed N2-®xing root
nodules, because of low carbohydrate supply from chlorotic
shoots. Decreased nodule formation associated with Fede®ciency has been observed in C. glauca (Arahou et al.,
1996) and a number of legume species (Tang et al., 1993).
We believe that growth performance, via N2 ®xation, of
®eld-grown Casuarinaceae trees essentially depends on their
ability to spontaneously produce cluster roots as a major
mechanism to overcome internal P and/or Fe de®ciencies,
thus making nodule development and activity possible.
CO N C L U S I O N S A N D R E S E A R C H N E E D S
Cluster roots were described 40 years ago (Purnell, 1960)
but our knowledge of their distribution, their relationship
to soil nutrients and their mode of action is only increased
thanks to a number of subsequent studies since 1970.
Despite this progress, there are still many gaps in our
knowledge of cluster roots. The ®rst is the lack of ecological
research into cluster roots, as discussed by Skene (1998).
The second major challenge is to elucidate the physiological/biochemical mechanisms involved in the initiation
and the morphogenesis of cluster roots. What is the
signalling pathway that induces the di€erentiation of
some pericycle cells into rootlet primordia? What are the
factors that determine the distinctive morphology of
rootlets in the clusters?
Before we can address these questions, we ®rst must
elucidate the biochemical events that occur in plants grown
under P and Fe de®ciency. Interestingly many authors
(Romera and Alcantara, 1994; Lynch and Brown, 1997;
Romera et al., 1999) recently reported that ethylene (C2H4)
is involved in the regulation of plant responses to P and Fe
de®ciency stress. Lynch and Brown (1997) suggested that
low P increases C2H4 production which in turn results in
changes in root architecture that enhance the plant's ability
to acquire P.
Along with Lynch and Brown's observation on P (Lynch
and Brown, 1997), Romera et al. (1999) reported that the
increased C2H4 production in Fe-de®cient plants promoted
the ferric-reducing capacity of roots of Strategy I plant
species ( plants capable of acquiring Fe through Fe(III)
reduction and proton release mechanisms). According
to these authors, C2H4 is a key compound that acts as a
signal to trigger the expression of genes responsible for
physiological and morphological changes, for instance, the
formation of root epidermal transfer cells in root tissues, in
order to improve Fe uptake, as described by Landsberg
(1996).
Taking into account the data stated above and considering that cluster roots are induced by Fe de®ciency to assist
plants in taking up Fe, Diem, Duhoux, Arahou and Zaid,
at the Symposium on cluster roots (XVIth International
Congress of Botany in Saint Louis, August 1±7, 1999)
hypothesized that C2H4 may also be implicated in the
signalling process leading to cluster root formation.
Simultaneously, but independently, Watt and Evans in
T A B L E 4. Relationship between iron chlorosis, growth and cluster root (CR) formation in Casuarina glauca grown for
12 weeks in acid and alkaline soils
Soil type
Meknes soil ( pH 8.3)
Rabat soil ( pH 5.8)
Plant status
Shoot dry weight
(g per plant)
Chlorophyll content in
shoot (mg g ÿ1 f.wt)
No. of CR per plant
Chlorotic (25%)
Green (75%)
0.24a
0.72b
0.68a
1.77b
0a
19b
Chlorotic (0%)
Green (100%)
±
1.08c
±
1.86c
±
0a
Data from Arahou and Diem (1997). Plant observation and measurements were carried out at harvest. Values in parentheses indicate the
percentage of plants showing chlorotic symptoms or green colouration. Values within columns followed by a di€erent superscript are signi®cantly
di€erent according to the Newman and Keuls test at P ˆ 0.01 with 24 replicates.
Diem et al.ÐCluster Roots in Casuarinaceae
their review (1999) also mentioned the possible involvement
of C2H4 in the auxin signal for cluster root formation. If
this hypothesis is correct, C2H4 may act as a regulating
agent of plant responses to Fe de®ciency in two ways: it
may induce changes in the chemical properties of roots of
non-cluster root-producing plants as reported by Romera
et al. (1999), or it may interact with auxins to initiate cluster
roots in species that are able to produce them.
Borch et al. (1999) reported that, under P-de®ciency
conditions, C2H4 induced the reduction of lateral root
density. This is apparently in contradiction with the fact
that P-de®ciency, which also induces high production of
endogenous C2H4 (Lynch and Brown, 1997), is usually
considered a major cause of production of cluster roots on
lateral roots. The observation reported by Borch et al.
(1999) is essentially due to the e€ect of C2H4 on the
promotion of main root extension without any signi®cant
change in lateral root number, thus leading to an increased
lateral root spacing and a reduced lateral root density.
It should be noted that the literature regarding the
involvement of C2H4 in root formation is con¯icting.
Although Liu et al. (1990) found that wound-induced
increase in C2H4 was a key stimulatory factor of adventitious
rooting from seedlings, there is no clear evidence that C2H4
is involved in lateral root initiation. Some authors ( for
example NordstroÈm and Eliasson, 1984) even showed an
inhibitory e€ect of C2H4 on root formation. Further investigations are needed to determine whether, and to what
extent, activation and subsequent proliferation of speci®c
root pericycle cells are induced by auxin-C2H4 interaction.
The mechanism by which auxin triggers initiation of new
roots from pericycle cells of the parent root is still unknown
(Malamy and Benfey, 1997). Gilbert et al. (1997, 2000) and
Skene and James (2000) were able to induce the formation
of cluster roots in L. albus by using auxin even when P was
supplied at a level that normally inhibits their development.
Laskowski et al. (1995) also succeeded in inducing the
formation of a series of contiguous lateral root primordia,
structurally similar to cluster roots, in radish treated with
90 mM IAA. Strikingly, cluster roots are only observed on
lateral roots of the secondary order in the root system,
never on primary roots. It is possible that pericycle cells
perceive the C2H4-controlled auxin signal di€erently at a
parent root level, depending on the order of their parent
root in the root system. C2H4 may also interfere with the
determination of the particular morphology of cluster
roots. This assumption is jointly supported by the following
considerations.
According to many authors, at high concentrations C2H4
a€ects root morphogenesis by inhibiting root elongation
(Goodlass and Smith, 1979; Whalen and Feldman, 1988;
Lee et al., 1990), probably because it causes a reduction in
the number of dividing cells in the root meristem and
controls root growth by modulating the degree and
orientation of cell growth behind the meristem (Barlow,
1976). C2H4 is therefore able to modify root architecture by
a€ecting root gravitropism (Lee et al., 1990; Lynch and
Brown, 1997). Of particular interest with regard to the
modi®cation of root architecture is the induction, by C2H4 ,
of root growth in a horizontal direction i.e. perpendicular to
935
the normal root axis (Goodlass and Smith, 1979). From
this account, C2H4 seems to play a signi®cant role in the
regulation of root growth pattern.
The putative involvement of C2H4 , with its speci®c
e€ects, may explain why all rootlets in cluster roots are
identically bent in a horizontal position and have a
determinate development (Skene et al., 1998) resulting in
a short and similar rootlet length and a limited longevity
(see Dinkelaker et al., 1995).
AC K N OW L E D G E M E N T S
We thank Dr Y. Dommergues for reading the manuscript
and for helpful comments.
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