Impact of phosphorus mineral source Al P

Plant Soil (2008) 304:169–178
DOI 10.1007/s11104-007-9535-7

REGULAR ARTICLE

Impact of phosphorus mineral source (Al–P or Fe–P)
and pH on cluster-root formation and carboxylate exudation
in Lupinus albus L.
M. W. Shane & H. Lambers & G. R. Cawthray &
A. J. Kuhn & U. Schurr

Received: 28 September 2007 / Accepted: 20 December 2007 / Published online: 10 January 2008
# Springer Science + Business Media B.V. 2007

Abstract Lupinus albus L. were grown in rhizoboxes
containing a soil amended with sparingly available
Fe–P or Al–P (100 μg P g−1 soil/resin mixture). Root
halves of individual plants were supplied with
nutrient solution (minus P) buffered at either pH 5.5
or 7.5, to assess whether the source of mineral-bound
P and/or pH influence cluster-root growth and

carboxylate exudation. The P-amended soil was
mixed 3:1 (w/w) with anion-exchange resins to allow
rapid fixation of carboxylates. Treatments lasted
10 weeks. Forty percent and 30% of the root mass
developed as cluster roots in plants grown on Fe–P
and Al–P respectively, but cluster-root growth was the
same on root-halves grown at pH 5.5 or 7.5. Mineralbound P source (Al– or Fe–P) had no influence on the
types of carboxylates measured in soil associated with
cluster roots—citrate (and trace amounts of malate

Responsible Editor: P. Randall.
M. W. Shane (*) : H. Lambers : G. R. Cawthray
School of Plant Biology,
Faculty of Natural and Agricultural Sciences,
The University of Western Australia,
35 Stirling Highway,
Crawley, WA 6009, Australia
e-mail: mshane@cyllene.uwa.edu.au
M. W. Shane : A. J. Kuhn : U. Schurr
ICG-3 (Phytosphere), Research Centre Jülich,

52425 Jülich, Germany

and fumarate) was the only major carboxylate
detected. The [citrate] in the rhizosphere of cluster
roots decreased with increased shoot P status (suggesting a systemic effect) and also, only for plants
grown on Al–P, with decreased pH in the root
environment (suggesting a local effect). In a separate
experiment using anion exchange resins pre-loaded
with malate or citrate, we measured malate (50%) and
citrate (79%) recovery after 30 days in soil. We
therefore, also conclude that measurements of [citrate]
and [malate] at the root surface may be underestimated and would be greater than the 40- and 1.6μmol g−1 root DM, respectively estimated by us and
others because of decomposition of carboxylates
around roots prior to sampling.
Keywords Citrate . P-deficiency . Proteoid roots .
Split-root design . Systemic signal . White lupin

Introduction
Phosphorus (P) deficiency induces formation of dense
numbers of closely spaced, short-lived, determinate

lateral roots (rootlets) termed ‘cluster’ or ‘cluster-like’
roots (e.g., L. cosentinii, Trinick 1977; L. albus,
Gardner et al. 1981; Clements et al. 1993; Skene
1998, 2000) in two-thirds of the ‘old-world’ Lupinus
species (8 of the 11 species; Fabaceae, Longnecker et
al. 1998). In L. albus, cluster roots can represent a
significant proportion of the plant’s investment in

170

biomass (i.e. 30% to 80% of the total root mass,
Keerthisinghe et al. 1998; Skene 2000; Hocking and
Jeffery 2004). Cluster roots acquire highly immobile
phosphate and micronutrients that need to be chemically extracted from soils (Braum and Helmke 1995),
and cluster-root producing Lupinus species are well
known for their capacity to grow on soils where P is
highly unavailable (Trinick 1977; Neumann and
Martinoia 2002).
The P status of shoots has a strong systemic effect on
cluster-root formation and the amount of citrate released

from cluster roots in L. albus (Marschner et al. 1986,
1987; Gilbert et al. 1997; Shane et al. 2003a) and in
other cluster-root forming species (e.g., Fabaceae,
Viminaria juncea, Walker and Pate 1986; Proteaceae,
Hakea prostrata and Grevillea crithmifolia; Lamont
1972; Shane and Lambers 2006). There is also some
evidence that a low supply of N, Fe, K or Zn (Lamont
1972; Hagström et al. 2001; Liang and Li 2003;
McCluskey et al. 2004) can increase cluster-root
formation in L. albus. Furthermore, Peiter et al.
(2001) reported that pH of the root environment can
influence cluster-root development. They found that
cluster-root numbers, at a constant P supply, doubled
in L. albus grown in nutrient solution buffered at
pH 7.7 as compared with that at pH 5.5.
The release of exudates (e.g., carboxylates such as
citrate, Jones 1998) from cluster roots of L. albus
mobilises sparingly available mineral-bound inorganic and organic P as well as micronutrients (e.g., Mn
and Zn, Gardner et al. 1982a, b; Dinkelaker et al.
1989; Vance et al. 2003). In the case of Lupinus

albus, leaf Mn accumulation are frequently observed
as a consequence of Mn co-mobilization associated
with rhizosphere-chemical changes involved in P
acquisition (Shane and Lambers 2005a; Dinkelaker et
al. 1995). Citrate is almost always observed to be the
most abundant carboxylate found in cluster-root
exudates (Jones 1998). Similar findings have been
made for specialised root clusters developed in species
from other families [e.g., Proteaceae (Dinkelaker et al.
1997; Shane et al. 2004), Cyperaceae (Playsted et al.
2006; Shane et al. 2006)]. Root growth and turnover
of new cluster roots provides further P mobilisation by
the root system (e.g., proteoid roots in Banksia, Pate
and Dell 1984).
The carboxylate composition (e.g., citrate, malate
etc.) and concentrations in root exudates have been
found to vary amongst some species as related to soil

Plant Soil (2008) 304:169–178

factors (Jones 1998). Investigations by Ström et al.
(1994) discovered that nine species adapted to
calcareous soils released more citrate and oxalate
compared to nine species adapted to acidic soils.
Furthermore, cluster roots of Proteaceae (i.e. Banksia
grandis native to soils of extremely low fertility) have
been shown to release mainly di- and tri-carboxylates
when supplied with Al–P but released additional
mono-carboxylates when Fe–P was supplied (Lambers
et al. 2002). Although it is not known if carboxylate
exudation by cluster roots of L. albus varies with the
type ‘form’ of P (e.g., Al–P or Fe–P) supplies in the
soil, Veneklaas et al. (2003) found that the proportion
of malate to citrate in the rhizosphere varied as a
function of soil pH: more malate at pH6.5.
The aim of the present study was to assess whether
cluster-root development, and net accumulation of
individual carboxylates in the rhizosphere of L. albus
are affected by the ‘form’ of P supplied and/or pH of
the root environment.

Materials and methods
Experimental design and split-root culture
A nutrient-deficient soil (pH 6 in water, Jülich,
Germany) (acid-washed with 0.5 M HCl and rinsed
several times in DI water, and dried) was mixed with
anion exchange resin (Amberlite IRA-400, Amberjet
4200, Sigma-Aldrich, Deisenhofen, Germany) in the
Cl− form (using 0.5 M NaCl) in a 3:1 ratio (soil/
resin). The soil/resin mixture was amended with 100
mg P kg−1 supplied as either Al2O3P2O5 or FePO4
(both AR grade). The P-amended soil/resin mixture
was added to each root compartment (9.5 cm wide
and 33 cm in height) of the split-root rhizoboxes
(inner thickness 1 cm) until half-filled. The remaining
upper halves of the rhizoboxes were filled with the
unamended, acid-washed soil, without added P and
without anion exchange resin.
Seven day-old seedlings of L. albus L. were
transplanted into the rhizoboxes (one plant in each

split root container (Fig. 1a). The root system of each
seedling was carefully placed so that equal numbers
of lateral roots were growing into each root compartment, and the clear backing was screwed into position
and covered with aluminium foil to exclude light.

Plant Soil (2008) 304:169–178

Fig. 1 Lupinus albus L. in split-root rhizoboxes grown on Al–
P or Fe–P. a Individual plants were transferred to split-root
rhizoboxes 7 days after germination. Roots grow-out into
separate root compartments. b L. albus after 6 weeks treatment.
Root halves of individual plants were fertilised daily and
automatically by drip irrigation with nutrient solution. One root

171

half received nutrient solution buffered at pH 5.5 while the
other half received nutrient solution buffered at pH 7.5. c The
cluster roots were closely associated with soil/resin. d Scanning
electron micrograph of root hairs in contact with anionexchange resin bead

172

Plants were positioned in special trays so that each
rhizobox was at approximately 30° relative to the
vertical axis. For the first 2 weeks the plants were
grown in a controlled environment chamber at
400 μmol quanta m 2 s −1 (PAR) and received
approximately 25 ml nutrient solution (to each root
half every 2nd day) of the following composition (in
μM): 1,200 NO3 , 600, Ca2+, 600 K+, 678 SO24 , 378
Mg2+, 1.9 Mn2+, 0.8 Zn2+, 0.14 Cu2+, 19.2 H3BO3,
0.24 Mo, 25 Fe2+ and 20 Cl− (pH 5.8). Subsequently,
and for the remaining 10 weeks of the experiment, the
plants were grown in a glasshouse, where they
received natural daylight supplemented by growth
lamps (Son-T-Agro 400W (Philips, Köln, Germany)
and HQI-Lamps (Osram, München, Germany); approximately 400–500 μmol m2 s−1 (PAR) at the level
of the leaves). An automated watering system was
used (Fig. 1b) so that each root-half received 15 ml

nutrient solution at 6, 12, 18 and 24 h (as above
without P) that had been buffered (using 1 M MES) to
pH 5.5 or buffered to pH 7.5.
Plant harvest and chemical analyses of tissues
After 10 weeks of treatment (13 weeks growth), the
plants were harvested and fresh mass determined for
stems, and young, mature and older leaves. The
rhizoboxes were carefully opened, soil samples taken
(see below), and the roots gently removed and washed
with DI water to remove soil. The fresh mass was
determined for cluster and non-cluster roots on each
root half.
Tissues were dried (to constant mass) at 70°C for
1 week, and ground in a ball-mill. Samples of ground
tissues were acid-digested in HNO3/H2O2 (3:1 v/v).
Concentrations of macro-nutrients (P, Mg, K, Ca), and
micronutrients (Mn, Fe, Zn and Cu) were determined
by inductively coupled plasma with mass spectroscopy (ICP-MS, ELAN 6100 Perkin Elmer). Concentrations of N and S were determined after combustion
of dried, homogenised, plant material at 1,000°C in
flowing oxygen using a LECO CHNS-932 (Leco

Corporation, St Joseph, MI, USA).
Solubilities of Al–P and Fe–P
Samples of Al–P or Fe–P were shaken for 24 h in nutrient
solutions (as above) buffered at pH 5.5 or 7.5, and then
filtered (0.45 μm). Total P concentration was deter-

Plant Soil (2008) 304:169–178

mined using the Malachite green method (Motomizu
et al. 1983).
Carboxylate analysis by HPLC
Samples of soil/resin were taken from the bulk soil in
each rhizobox compartment where no roots were
growing and within 3 mm around cluster roots.
Carboxylates were eluted from sub-samples by
tumbling for 3 h in 0.3 ml of 1 M HCl. Samples
were filtered to 0.45 μm and stored at 4°C until
analysis. Carboxylates were separated on an Alltima
C-18 column (250 mm long × 4.6 mm internal
diameter with 5 μm diameter packing), and identified
using Waters® HPLC (600E pump, 717 auto injector
and 996 photodiode-array detector, Milford, MA,
USA). The mobile phase was a mixture of 25 mM
KH 2 PO 4 (pH 2.50) and MeOH (i.e. 93%:7%)
(pH 2.50) at a flow rate of 1 ml min−1. Detection
was at 210 nm, but data from 195 to 400 nm were
collected and used for spectrum matching and peak
purity analysis according to Cawthray (2003). The
sample injection volume was generally 100 μl, but
was reduced for samples with very high citrate
concentrations. The column was completely flushed
(gradient elution using 60% (v/v) methanol) after
every 10 samples to eliminate the transfer of highly
nonpolar compounds. Data acquisition and processing
was with Millennium© software (Waters, Milford
MA, USA). Retention times of organic acid standards
including malic, iso-citric, malonic, lactic, acetic,
maleic, citric, succinic, fumaric, cis-aconitic and
trans-aconitic acids were used to identify carboxylates
in rhizosphere extracts.
Measurements of malate and citrate decomposition
In a separate experiment (at the University of Western
Australia), white lupin (n=12) were grown from seed
in acid-washed quartz sand mixed 3:1 (as above) with
anion-exchange resin in the Cl− from. Small nylon
bags, 38 μm mesh (containing 15 g resin pre-loaded
with either malate or citrate) were buried in the top
4 cm of soil (malate, n=6, or citrate, n=5, resin bags).
Plants were grown in a glasshouse (Jan and Feb,
2006), and watered daily as required with nutrient
solution (minus P) as above. After 4 weeks, carboxylates were recovered from a 1-g sub-sample of anion
exchange resin from each resin bag. Three subsequent

Plant Soil (2008) 304:169–178

Total plant fresh mass (g)

b

15
10
5
0
5
a
4

b

pH 7.5

a
a

3
2
1
0

% cluster roots

Data were analysed with two-way analysis of variance
(GenStat 7.1, Lawes Agricultural Trust; Rothamsted
Experimental Station). Tukey’s pair-wise multiple
comparison tests were used to determine which levels
differed significantly (α=0.05).

pH 5.5

a

50

Statistics

a

a

20

Total root fresh mass (g)

extractions were carried out on each sub-sample as
follows, (1) in 5 ml of DI water for 1.5 h on a shaker,
(2) in 5 ml 0.5 M HCl for 1 h, and (3) again in 5 ml
0.5 M HCl for 1 h. After each extraction a 1-ml
sample was filtered for HPLC analysis. Between
extractions the remaining extraction solution was
removed by filtration using a syringe and syringe
filter (0.45 μ). The plants were also harvested and
resin/soil that was not tightly bound to the cluster
roots (Fig. 1c) was removed by gentle shaking. Only
resin beads strongly attached to cluster rootlets by
root hairs (Fig. 1d) were collected and extracted twice
in 1.5 ml of 0.5 M HCl for 1 h on a shaker, filtered
and a sample taken for HPLC analysis. The dry mass
for each cluster root associated with resin beads
collected for carboxylate extraction (24 roots from
12 plants) was determined. All samples were analysed
for carboxylates as described above.

173

40

pH 5.5
a

pH 7.5

b

b

c

a

30
20
10
0
Al-P
Fe-P
Source of phosphorus

Results
Solubility of Al–P and Fe–P
The solubility of both phosphorus sources was very
low (i.e.