Journal of Experimental Botany, Vol. 63, No. 16,
2, pp.
2012
pp.695–709,
5815–5827,
2012
doi:10.1093/jxb/err313
doi:10.1093/jxb/ers230 Advance Access publication 4 November, 2011
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RESEARCH
Research PAPER
Paper
Ecophysiology
of nickel
phytoaccumulation:
a simplified
In
Posidonia oceanica
cadmium
induces changes
in DNA
biophysical approach
methylation
and chromatin patterning
1
1,
1
David Coinchelin
, François Bartoli
*, Christophe Robin
and Guillaume Echevarria
Maria
Greco, Adriana
Chiappetta, Leonardo
Bruno and 2Maria
Beatrice Bitonti*
1 Université de
Lorraine, University
Laboratoire
et Environnement
(ENSAIA)INPL-INRA,
BP 172,
54505
Vandoeuvre-les-Nancy
Department
of Ecology,
of Sols
Calabria,
Laboratory ofUMR
Plant1120
Cyto-physiology,
Ponte Pietro
Bucci,
I-87036
Arcavacata di Rende,
Cedex, France
Cosenza,
Italy
2 Université
de Lorraine, Laboratoire
Agronomie
et Environnement
UMR 1121 (ENSAIA)INPL-INRA, BP 172, 54505
* To
whom correspondence
should be
addressed.
E-mail: [email protected]
Vandoeuvre-les-Nancy Cedex, France
Received
29 correspondence
May 2011; Revised
8 Julybe
2011;
Accepted
18 August
2011
* To whom
should
addressed:
E-mail:
[email protected]
Received 25 May 2012; Revised 13 July 2012; Accepted 23 July 2012
Abstract
In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent
Abstract mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with
epigenetic
its
effect
on chromatin
reconfiguration
in Posidonia
oceanica.
methylation
level and pattern
were
analysed in
Solute
active
transport or
exclusion by plants
can be identified
byDNA
the values
of the Transpiration
Stream
Concentration
actively
growing
organs,
under
short(6
h)
and
long(2
d
or
4
d)
term
and
low
(10
mM)
and
high
(50
mM)
doses
Factor (TSCF=xylem:solution solute concentration ratio). The aim of this study was to estimate this parameter of
forCd,
Ni
through
a
Methylation-Sensitive
Amplification
Polymorphism
technique
and
an
immunocytological
approach,
uptake by the Ni-hyperaccumulator Leptoplax emarginata or the Ni-excluder Triticum aestivum cultivar ‘Fidel’. The
respectively.
The for
expression
of one )member
of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase,
Intact Plant TSCF
nickel (IPTSCF
Ni was calculated as the ratio between the nickel mass accumulation in the leaves
was
also
assessed
by
qRT-PCR.
Nuclear
chromatin
ultrastructure
investigatedNi by
transmission
electron
and the nickel concentration in solution per volume of water
transpired.was
Predominantly,
active
transport occurred
microscopy.
Cd
treatment
induced
a
DNA
hypermethylation,
as
well
as
an
up-regulation
of
CMT,
indicating
thatflow
de
for L. emarginata, with IPTSCFNi values of 4.7–7.2 and convective component proportions of the root Ni uptake
novo
methylation
did
indeed
occur.
Moreover,
a
high
dose
of
Cd
led
to
a
progressive
heterochromatinization
of
–1
of only 15–20% for a range of Ni concentrations in solutions of 2–16 µmol Ni l , regardless of the growth period and
interphase
nuclei
and
apoptotic
figures
were
also
observed
after
long-term
treatment.
The
data
demonstrate
that
Cd
the time of Ni uptake. Hyperaccumulator roots were permeable to both water and nickel (mean reflection coeffiperturbs
theσDNA
methylation status through the involvement of a specific methyltransferase. Such changes are
cient for Ni,
Ni, of 0.06), which was mainly attributed to an absence of exodermis. Results provide a new view of the
linked
to
nuclear
chromatin
reconfiguration
likely the
to establish
a new was
balance
of expressed/repressed
mechanisms of Ni hyperaccumulation.
By contrast,
wheat excluder
characterized
by an extremelychromatin.
low mean
Overall,
the
data
show
an
epigenetic
basis
to
the
mechanism
underlying
Cd
toxicity
in
plants.
IPTSCF value of 0.006, characterizing a predominantly Ni sequestration in roots. From a methodological viewpoint,
Ni
the ‘microscopic’ TSCFNi, measured directly on excised plants was 2.4 times larger than its recommended ‘macroKey words: 5-Methylcytosine-antibody,
cadmium-stress condition, chromatin reconfiguration, CHROMOMETHYLASE,
scopic’ IPTSCFNi counterpart. Overall, IPTSCF and σ determined on intact transpiring plants appeared to be very useDNA-methylation, Methylation- Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile.
ful biophysical parameters in the study of the mechanisms involved in metal uptake and accumulation by plants, and
in their modelling.
Key words: Active transport, convective transport, Intact Plant Transpiration Stream Concentration Factor, nickel concentration
Introduction
in solution, nickel phytoaccumulation, Ni-hyperaccumulator Leptoplax emarginata, reflection coefficient, transpiration, winter
wheat.
In
the Mediterranean coastal ecosystem, the endemic
seagrass Posidonia oceanica (L.) Delile plays a relevant role
by ensuring primary production, water oxygenation and
provides niches for some animals, besides counteracting
Introduction
coastal
erosion through its widespread meadows (Ott, 1980;
Piazzi et al., 1999; Alcoverro et al., 2001). There is also
Understanding and modelling the uptake and accumulation of
considerable evidence that P. oceanica plants are able to
metals by plants is crucial for reasons of food safety and for
absorb and accumulate metals from sediments (Sanchiz
the challenging perspectives of contaminated soil phytoremeet al., 1990; Pergent-Martini, 1998; Maserti et al., 2005) thus
diation and precious metal phytomining. Most plant species
influencing metal bioavailability in the marine ecosystem.
are recognized as excluder plants, limiting root-to-shoot metal
For this reason, this seagrass is widely considered to be
translocation via the xylem (Baker and Brooks, 1989). By cona metal bioindicator species (Maserti et al., 1988; Pergent
trast, a minority of plants growing on metalliferous soils accuet al., 1995; Lafabrie et al., 2007). Cd is one of most
mulate metals in their leaves to such high levels that they are
widespread heavy metals in both terrestrial and marine
environments.
© 2012 The Authors.
Although not essential for plant growth, in terrestrial
plants, Cd is readily absorbed by roots and translocated into
aerial organs while, in acquatic plants, it is directly taken up
by leaves. In plants, Cd absorption induces complex changes
at the genetic, biochemical and physiological levels which
ultimately account for its toxicity (Valle and Ulmer, 1972;
called hyperaccumulators (Verbruggen et al., 2009). The latSanitz di Toppi and Gabrielli, 1999; Benavides et al., 2005;
ter are interesting plants for their potential use in phytoremeWeber et al., 2006; Liu et al., 2008). The most obvious
diation or phytomining (McGrath et al., 2002; Robinson et al.,
symptom of Cd toxicity is a reduction in plant growth due to
2003a; Chaney et al., 2007). The transport processes of metal
an inhibition of photosynthesis, respiration, and nitrogen
in excluders or hyperaccumulator plants are rather complex
metabolism, as well as a reduction in water and mineral
(Hopmans and Bristow, 2002; Seregin and Kozhevnikova, 2008;
uptake (Ouzonidou et al., 1997; Perfus-Barbeoch et al., 2000;
Verbruggen et al., 2009) and, consequently, it is not straightforShukla et al., 2003; Sobkowiak and Deckert, 2003).
ward to capture the successive combinations of metal transport
At the genetic level, in both animals and plants, Cd
can induce chromosomal aberrations, abnormalities in
This
is an
Open
Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
ª
2011
The
Author(s).
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
5816 | Coinchelin et al.
types from the rhizosphere to the xylem with relevant biophysical parameters. Our aims are concomitantly to determine
two of these biophysical parameters: the Transpiration Stream
Concentration Factor (TSCF) (Russel and Barber, 1960; Russel,
1977; Hopmans and Bristow, 2002) and the reflection coefficient
of the root membrane (Dalton et al., 1975; Steudle and Peterson,
1998; Hopmans and Bristow, 2002), during root Ni uptake and
leaf Ni accumulation by intact transpiring plants. The choice of
intact plants is significant as, to date, these biophysical parameters have mostly been estimated, with possible biases, on excised
stem root systems immersed in hydroponic solution.
TSCF is the xylem:solution solute concentration ratio at the
steady-state efflux, mainly estimated on excised stem root systems immersed in hydroponic solution. Its value reflects the net
outcome of the multiple steps and combined solute pathways.
When TSCF is lower than 1, this indicates that the solute has
moved from the solution to the shoot more slowly than water
and then accumulated onto or within the roots (excluders). When
TSCF is greater than 1, this indicates a predominantly active,
metabolically driven transport. Such predominantly active
transport occurs in metal hyperaccumulator plants, with a use
of photosynthetic energy by transporter proteins, allowing the
transport of metal against its concentration gradient in the xylem
(Hopmans and Bristow, 2002; Verbruggen et al., 2009). TSCF
(also referred to as ‘microscopic’ TSCF in the Results) was much
greater than 1 for a hyperaccumulator or an accumulator plant,
whereas it was lower for its non-accumulative plant counterpart
(see Supplementary Table S1 at JXB online).
The TSCF bioconcentration factor can also be determined
indirectly from a mass balance carried out on intact transpiring
plants. In this case, the bioconcentration factor was called the
Intact Plant Transpiration Stream Concentration Factor (IPTSCF)
by Bartoli et al. (2012) and this abbreviation has been used
throughout this paper in order clearly to differentiate IPTSCF
from the classical TSCF. IPTSCF has only been calculated for
metal uptake twice by (i) a Cd-excluder, durum wheat (Van der
Vliet et al., 2007) or (ii) the Ni-hyperacccumulator Leptoplax
emarginata (Bartoli et al., 2012). No comparison has been made
between TSCF and IPTSCF for a given solute/plant couple.
The second selected biophysical parameter is the dimensionless reflection coefficient of the root membrane, σ, ranging from
0 to 1. σ=0 indicates zero selectivity, meaning that all solutes
can pass through and that the membrane is as permeable to solutes as it is to water; σ=1 indicates total selectivity, i.e. no solute
can pass through and the membrane is only permeable to water,
1>σ>0 indicates a partial solute uptake. Over the past 30 years,
σ has mainly been estimated on excised stem root–hydroponic
solution systems placed in pressure chambers. σNaCl was 0.64
for maize (Steudle et al., 1987), 0.975 for barley (Dalton et al.,
1975), 0.32–0.64 for beech and 0.12–0.35 for oak (Steudle
and Peterson, 1998). The parallel compartment model called
the ‘composite transport model’ (Steudle et al., 1993; Steudle
and Peterson, 1998) may explain these results. However, there
is again serious doubt as to whether water flow through pressurized root systems is representative of what occurs in intact
transpiring plants. The latter is more appropriate when estimating σ according to the methodology described by Zimmermann
and co-workers (Zhu et al., 1995; Schneider et al., 1997a, b).
Intact transpiring plants were also used but this methodology
was partly adapted in this study. The purpose of the present
study is 4-fold.
(i) To evaluate that IPTSCFNi is much greater than 1 for the
Ni-hyperaccumulator L. emarginata and very low for the
Ni-excluder wheat (Triticum aestivum), regardless of the
growth period.
(ii) To compare IPTSCFNi and TSCFNi for the selected
Ni-hyperaccumulator plant cultivated in fertilized sand for
10 d, with Ni-contamination for the last 2 d of culture, and
excised 6 h thereafter. Our hypothesis was that TSCF values are overestimated compared with the IPTSCF values
because the sap flux is smaller than the transpiration flux.
(iii) To determine σNi for Ni uptake by the Ni-hyperaccumulator
and to discuss this new result for a better understanding of
metal hyperaccumulation.
(iv) To enter both the IPTSCFNi and σNi values in relevant mechanistic equations of coupled root water and solute uptake in
order to divide each Ni-hyperaccumulator root Ni uptake
flow into a convective Ni flow component and a combined
active and diffusive Ni flow component.
The paper is organized as follows. In the next section, the
coupled root water and solute uptake mechanistic models which
were used in this study are summarized. Then, after a description
of the materials and methods used, the results are presented and
interpreted. We conclude with the potential utility of the determination of the IPTSCF bioconcentration factor and the reflection
coefficient for better understanding and modelling of the uptake
and accumulation of trace metals by plants.
Coupled root water and solute uptake models and
their uses in this study
In the Dalton et al. (1975) model, a single semi-permeable
membrane separates the external solution from the root xylem.
The root solute uptake flow normalized by root area, Js (mol
cm–2 s–1), is assumed to be the sum of a convective component
(1–σ)CsJv, a diffusive component ωΔπ, and an active component
JS* (mol cm–2 s-–1):
J s = (1 −σ)Cs J v + ω ∆ π + J S* (1)
where σ is the dimensionless reflection coefficient of the root
membrane, ranging from 0 to 1, Cs (mol cm–3) is the solute concentration in solution, Jv (cm s–1) is the volume water flux of the
solution, ω (mol cm–2 s–1 bar–1) is the osmotic permeability of the
root membrane, and Δπ (bars) is the osmotic pressure difference
between the solution and the xylem.
At high CsJv values, Js is linear in JvCs with slope (1–σ)
(Fiscus, 1986). Moreover, the determination of σNi allows the
calculation of the convective component of the root Ni uptake
flow occurring during either a 1 h period of symplastic 63Ni root
uptake:
J s (convective) = (1 − σ Ni )Cs Jv (2)
Ecophysiology of nickel phytoaccumulation | 5817
or during successive weekly periods of leaf Ni accumulation:
dQconvective = (1 − σ Ni )CS dVT (3)
where dQconvective (µg Ni week–1) is the convective component
of the weekly root Ni uptake flow occurring between two successive plant and solution sampling times t2 and t1, CS (µg Ni l–1)
is the mean concentration of nickel in solution calculated from
the two measured Cs(t2) and Cs(t1) Ni concentration values in the
solutions, and dVT (l–1) is the volume of water transpired during
the considered weekly period of plant growth.
The assumption that the flow is steady implies that the solute
flux (Js) by continuity is equal to the product of the solute concentration in the xylem (Cx) by the volumetric water flux (Jv):
J s = Cx J v (4)
IPTSCFNi, was calculated by substituting Cx by Cs IPTSCFNi
into Equation (4), assuming an equality between the symplastic
Ni flow and the xylem Ni flow:
JS
CS − J V
IPTSCFNi =
(5)
Similarly, the mean weekly nickel mass flow from the roots
to the leaves occurring between two successive sampling times
t2 and t1, dQL (µg Ni) (dQL=mean measured QL(t2)–mean measured QL(t1)), by continuity is equal to the product of the mean
concentration of nickel in the xylem for the considered weekly
period, CX (µg l–1) by the volume of water transpired during the
considered period, dVT (l–1):
dQL = dVT CX (6)
Substituting CX by CS IPTSCFNi into Equation (6) gives:
IPTSCFNi =
dQL
(7)
CS dVT
The convective:total root Ni uptake ratio was calculated either
at the hour Ni uptake time-scale by combining Equations (2)
and (5):
J S( convective )
JS
=
1 −σ Ni
(8)
IPTSCFNi
or at the week Ni uptake time-scale, by combining Equations
(3) and (7):
dQ( convective )
dQL
=
1 −σ Ni
(9)
IPTSCFNi
Finally, Combining Equations (1) and (5) also leads to:
IPTSCFNi = 1 −σ +
J*
ω∆π
+ S (10)
CS J V CS J V
Materials and methods
Plants, porous media, and solutions
The selected C3 plants were the Ni-excluder winter wheat (Triticum
aestivum L.) cultivar ‘Fidel’ and the Ni-hyperaccumulator Leptoplax
emarginata (Boiss.) O. E. Schulz (Brassicaceae), endemic to serpentine soils in Greece (Reeves et al., 1980; Cecchi et al., 2010). The L.
emarginata seeds were sampled in July 2006 in the Trigona village,
Pindus Mountains, Central Greece (830 m a.s.l.) and removed from their
siliques before use. The Ni content of the seeds without their outer shells
was 16.5 µg Ni seed–1.
The porous media were a sedimentary sand (HN 0.4–0.8 mm, Sibelco,
France) from Hostun, South-Eastern France and a sandy topsoil from a
cultivated podzol (WRB, 2001), Cestas, South-Western France. Their
main characteristics are listed in Supplementary Table S2 at JXB online.
Quartz was the dominant phase in both materials, with iron and aluminium coatings for the sand and organic coatings for the sandy topsoil
(X-ray photoelectron spectroscopy compared with bulk analyses, not
shown). Ni was not detectable in the sand. The initial DTPA-extractable
Ni content (a good estimate of labile Ni in soils: Echevarria et al., 1998)
was negligible for the sand (<0.02 mg kg–1), very low for the sandy topsoil (0.11 mg kg–1) (Table 1), and negligible compared with the supplied
Ni content (14.6 mg kg–1).
Both porous media were supplied with a nutritive solution containing
998 µM Ca(NO3)2.4H2O; 823 µM MgSO4.7H2O; 3570 µM NH4NO3;
2580 µM KH2PO4; 71.6 µM FeSO4.7H2O; 9.1 µM MnSO4.H2O;
4.59 µM ZnSO4.7H2O; 9.25 µM H3BO3.4H2O; 0.157 µM CuSO4.5H2O;
0.104 µM Na2MoO4.2H2O. This unique nutritive solution is a compromise between nutritive solutions used for crop cereals and those used
for hyperaccumulator plants. The three levels of Ni contamination
(NiSO4.7H2O) in the initial fertilizing solution were: 1 mg Ni l–1 for both
L. emarginata (L1) and wheat (W1) cultivated in the sand, 10 mg Ni l–1
or 100 mg Ni l–1 for L. emarginata cultivated in the sand (L10) or in
the sandy topsoil (L100), respectively. Each pot (1.5 l) was lined with
a polyethylene bag to prevent leakage and filled with either 1.40 kg of
sand or 1.37 kg of sandy topsoil, gently mixed with 200 ml of the fertilized and Ni contaminated solution in order to obtain a homogeneous
volumetric solution of 0.20 cm3 cm-3. A white polyethylene film covered
the top of each pot, with a single 1 cm diameter hole. For the sand, the
cultures (and the reference sand R1) started just after the fertilization
and Ni-contaminations of the sand. In contrast for the soil (L100 and its
reference R100), the mixture between the soil and the Ni-contaminated
fertilizing solution was made one week before the culture.
Growth/transpiration kinetics, plant and solution samplings
Before being cultured in sand, seeds of L. emarginata and wheat were
germinated on moistened filter paper in a Petri dish at 25 °C for 5 d in
the dark. Seeds of L.emarginata were then set in the fertilized sand for
a pre-culture of 3 weeks whereas wheat seeds were directly sown after
their germination. Before being cultured in soil, seeds of L. emarginata
were directly sown in the fertilized sand for a pre-culture of 1 week.
The pots were distributed into four blocks in the growth chamber: one
planted pot replicate per sampling period for each block, one unplanted
pot per sampling period for the two blocks. Two permanent unplanted
pots per block were used for daily evaporation measurements. The
growth conditions were as follows: 16 h photoperiod, photon flux density of 325 µmol photons m–2 s–1 in the PAR range, 20/18 °C day/night
temperatures, and 50% relative humidity. The total culture time was 6
weeks for plants cultivated in sand (first series of experiments) and 5
weeks in soil (second series of experiments).
The volume of daily transpired water, T, was calculated by subtracting
the mean daily water volume lost by the two unplanted pots of a block
5818 | Coinchelin et al.
and the mean calculated daily fresh biomass from the water volume
daily lost by each planted pot of the block considered. The mean daily
total fresh biomass was calculated from the non-linear total fresh biomass versus time relationships. After these daily weighings, the plants
cultivated in the sand were irrigated daily with a fertilizing solution
containing 50% or 5% of hte initial Ni concentration for L. emarginata
or wheat, respectively. This was done in order to maintain the volumetric solution of 0.20 cm3 cm–3 constant throughout the culture and
the Ni concentrations in solution nearly constant, thanks to preliminary
experiments. By contrast, the hyperaccumulator plants cultivated in the
soil were irrigated with deionized water only, assuming a sufficient soil
buffer power favouring a constant Ni concentration in the solution.
Plant and solution samplings were carried out nearly every week (4
replicates). Gas exchange was recorded on the 4th leaf of each plant
the day before each sampling period, using a CIRAS-1 portable photosynthesis system (PP SYSTEMS Inc., USA). Photon flux density and
temperature inside the leaf chamber delivered at the measured leaf area
were normalized using a lamp (PAR of 260 µmol photons m–2 s–1 and
temperature of 24.3 °C).
Plant and solution analyses
Stem, root, and leaf biomass were determined after oven-drying at 70 °C
for 48 h. Leaf area was determined using the WinFOLIA® Software,
specific leaf area being calculated thereafter. Roots were collected and
washed with deionized water in order to remove all the root-adhering
particles. Roots were then air-dried for 10 min, washed for 1 min with
150 ml of 0.01 M Na2H2EDTA to remove root-adsorbed Ni, washed with
deionized water, and oven-dried at 70 °C for 48 h (Kalis et al., 2006).
The oven-dried roots and leaves were carefully crushed using an agate
mortar and then 0.5 g aliquots were dissolved by a HNO3:H2O2 solution (8:2 v/v). The mixture was then heated in a microwave oven (Mars
5, CEM Corporation Inc., USA). Nickel content in the resulting solutions was determined by inductively coupled plasma optical emission
spectrometry (ICP-OES, Liberty RL, Varian, Inc., USA), except for the
EDTA-desorbed Ni from roots at harvest, which was determined by
ICP-mass spectrometry.
Solutions were extracted and filtered at 0.22 µm at a matric pressure
potential of –30 kPa by vacuum pump depression from 200 ml of wetted sand or by centrifugation from 12 ml of wetted sandy topsoil. Their
pH was measured with a microelectrode, and major ion concentrations
determined by ionic chromatography (IC25 Ion Chromatograph, Dionex
Corporation, USA). Ni concentrations were measured by ICP-OES after
acidification.
Ni phytoaccumulation kinetics
A homogeneous plant population for a considered plant and treatment
was postulated. This was validated for all the plant samples thanks to
the ANOVA analysis of the transpired water volumes occurring on the
7th day of cultivation. The exceptions were the hyperaccumulator plants
cultivated on soil and sampled on the 14th day of culture, the data of
which are therefore not being used. Thus, the weekly rates of root and
leaf biomass production and the weekly transpiration and nickel flows
(total Ni uptake, root-adsorbed Ni, root-sequestrated Ni, and root-toshoot translocated Ni flows) were calculated. Each rate or flow was
calculated as the difference of the mean values of the considered parameter between two sampling periods, normalized to a weekly period if
necessary.
Comparison between IPTSCFNi and TSCFNi
Ten hyperaccumulator plant replicates were cultivated in sand mixed
with 150 ml of the fertilizing solution for 8 d and irrigated daily with the
fertilizing solution, maintaining the volumetric solution of 0.15 cm3 cm–3
constant throughout the culture. For the last two days corresponding to
Ni contamination (the addition of 50 ml of a Ni solution at 68 µmol l–1
during the 8th day of culture), the volumetric solution was maintained
at 0.20 cm3 cm–3. At the end of the culture, sand solutions were extracted
and filtered at 0.22 µm at a matric pressure potential of –30 kPa and
analysed thereafter. The ‘macroscopic’ IPTSCFNi was determined and
compared with the ‘microscopic’ TSCFNi (Cx:Cs ratio) estimated after
stem excision and 6 h of xylem sampling.
Symplastic 63Ni uptake by L.emarginata (data from Redjala
et al., 2010)
After 45 d of culture in hydroponic nutrient solution, the Nihyperaccumulator L. emarginata plants were transferred into a radioelement room with a Vapour Pressure Deficit value of 2.12 kPa. The roots
were rinsed in deionized water and then immersed for 1 h in a solution
containing 0.5 mM Ca(NO3)2, 2 mM MES buffer (pH 5.7), and Ni(NO3)2
at one the following concentrations: 5, 15, 30, 50, 75, 100, 130, 160,
200, and 250 µM. Nickel was labelled with 267 kBq of 63Ni as NiCl2.
After exposure, the roots were rinsed with distilled water before starting
the Ni fractionation step: exchangeable apoplastic Ni after a desorption
step, and symplastic Ni after root immersion in a methanol–chloroform
mixture (2:1, v/v) for 3 d at room temperature, then into two successive
baths of deionized water for 24 h each and, finally, a final desorption
step (Redjala et al., 2010).
A constant transpiration flow was assumed because of the very short
63
Ni uptake time and the fact that low or high concentrations of Ni had
no effect on transpiration for two hyperaccumulator plants (Whiting
et al., 2003). The transpiration rate per root area (Jv of 10–6 cm s–1) was
estimated from the mean daily transpiration to aerial biomass ratio of
20 ± 1 ml g–1 d–1 of the hyperaccumulator plants cultivated on the fertilized and Ni-contaminated sand, the different Vapour Pressure Deficit
values of 2.12 kPa (Redjala et al., 2010, final experiments) and 1.24 kPa
(this study), a root/shoot ratio of 0.47 (this study), a root area/root biomass ratio of 1844 cm2 g–1 (Redjala et al., 2010) and the fact that transpiration of Thlaspi caerulescens was 1.3 times greater when cultivated
in hydroponic solution than in soil (Whiting et al., 2003).
Statistical analysis
Statistical analysis and curve-fitting were carried out using the XLSTAT
2010 Excel package software or the KaleidaGraph™ 3.52 package software. The effect of treatments and plant types on the parameters studied
were tested by performing a one-way ANOVA and the Bonferroni post
hoc test with α=0.05 and P <0.05. The Michaelis–Menten or Dalton
models were applied to the revisited Redjala et al. (2010) results using
the KaleidaGraph™ 3.52 package.
Results
Sand and soil solutions
From the 14th day of culture, the Ni concentration in the solutions
of the reference R1 sand was quite constant over time (Fig. 1A)
and was 14 times lower than the Ni concentration in the initial
contaminating solution. For the unplanted soil, physico-chemical
equilibrium was reached after 9 d (2nd day of culture) (Fig. 1A)
and Ni concentration in solution was 165 times lower than the Ni
concentration in the initial contaminating solution.
From the 14th day of culture, the pH was significantly higher in
the rhizosphere of the L1 hyperaccumulator plant (pH 7.67 ± 0.04)
than in its reference soil R1 (pH 7.46 ± 0.07) (Fig. 1B). As a complement, the Ni concentration in solution was significantly lower in
the rhizosphere of the L1 hyperaccumulator plant (1.19 ± 0.07 µM
Ni) than in its reference soil (1.38 ± 0.03 µM Ni) (Fig. 1B). In contrast, for wheat, a significantly strong acidification from pH 8.07 to
5.93 occurred from 14–21 to 35–42 d of culture, leading to a significantly strong increase in Cs from 1.12 to 3.71 µM Ni (Fig. 1A, 1B).
For the L1 and L10 plant–sand systems, the factor of ten occurring
Ecophysiology of nickel phytoaccumulation | 5819
Fig. 1. Kinetics of mean Ni concentration, Cs (A) or pH (B) in the sand or soil solutions of the planted (W1, L1, L10, and L100) or
unplanted pots (R1 and R100). For the soil (L100 and R100), the mixture between the soil and the Ni-contaminated fertilizing solution
5820 | Coinchelin et al.
between the two Ni concentrations in the initial contaminating solutions remained nearly the same after the observed strong Ni adsorption on the solid phases (Fig. 1A). However, for the L10 plant–sand
systems, the nickel concentration in solution decreased during the
culture (Fig. 1A) and hte pH was variable (Fig. 1B).
From the second day of culture, Cs and pH were statistically
the same and quite constant over time for the L100 plant–soil
systems and their reference soil without plants R100, with a
slight tendency to a pH increase over time (Fig. 1A, 1B).
The dynamics of nutrients in solutions are listed in
Supplementary Table S3 and Fig. S2 at JXB online. Concentrations
of K+, Mg2+, Ca2+, and NO–3 were constant over time in both
the sand solutions and the soil solutions of the reference pots.
A strong nutrient uptake by the L1, L10 or L100 hyperaccumulator plants was shown by the strong depletion of K+, Ca2+, SO2–4,
PO3–4, NH+4, and NO–3 concentrations from the 7th day (L100)
or 14th day (L1 and L10) until the end of the culture. A similarly
strong decrease in the NO–3 concentrations occurred in the wheat
rhizosphere solutions, whereas the corresponding decrease in
Ca2+ and SO2–4 concentrations was moderate.
Plant growth and ecophysiological characteristics
Plant growth was three times faster for wheat than for hyperaccumulator plants (Fig. 1C, 1D; see Supplementary Table S4 at
JXB onlione). The root/shoot ratio, relatively constant over time,
was significantly greater for wheat (R:S ratio of 0.66 ± 0.04 g
g–1) than for L1 and L10 hyperaccumulator plants (R:S ratio
of 0.47 ± 0.04 g g–1 and 0.48 ± 0.05 g g–1, respectively). SLA
was twice as large for L100 as for both L1 and L10, and it was
almost time-constant for each hyperaccumulator plant (see
Supplementary Fig. S1A at JXB online). By contrast, a dramatic
SLA decrease occurred from 14–21 d to the end of the wheat
culture (see Supplementary Fig. S1A at JXB online).
The cumulative volume of transpired water per plant (VT) was
much greater for wheat than for the hyperaccumulator plants
which were characterized by an increasing L1<L10<L100 VT
gradient (see Supplementary Table S4 at JXB online). The daily
transpiration flow was significantly greater for wheat than for the
L1, L10, and L100 hyperaccumulator plants, and always with
T:LA decreases from the 21st or 28th day to the end of culture
(see Supplementary Fig. S1B at JXB online).
Water use efficiency (WUE) for shoot biomass production was
significantly better for L1 hyperaccumulator plants than for both
L10 hyperaccumulator plants and wheat (see Supplementary
Fig. S1C at JXB online). The values for L100 hyperaccumulator plants cultivated in sandy topsoil fell between these. The
L1 and L10 WUE kinetics curves were parallel, with WUE
decreases followed by relatively constant WUE values thereafter
(see Supplementary Fig. S1C at JXB online). By contrast, WUE
slightly increased or was almost constant for L100 hyperaccumulator plants and for wheat. Similar, but much more pronounced,
trends were observed on the weekly WUE for shoot biomass production kinetics (see Supplementary Fig. S1D at JXB online).
Leaf transpiration flux (E) was significantly greater for L100
hyperaccumulator plants (E of 3.51 ± 0.25 mmol H2O m–2 s–1) than
for L1 hyperaccumulator plants (E of 2.08 ± 0.18 mmol H2O m–2
s–1). By contrast, statistically similar CO2 assimilation flux (A)
values were recorded for L1 (A of 5.01 ± 0.47 µmol CO2 m–2 s–1)
and for L100 (A of 5.61 ± 0.69 µmol CO2 m–2 s–1). This explains
why two statistically different groups were identified for the
‘microscopic’ water use efficiency (µWUE) for photosynthesis
(A:E ratio), with a decreasing L1>L100 µWUE gradient (µWUE
of 2.49 ± 0.26 µmol CO2 mmol–1 H2O and 1.64 ± 0.21 µmol CO2
mmol–1 H2O, respectively).
Ni concentrations in plant organs
Ni concentrations in L10 leaves and in L100 leaves were relatively constant during the culture and were greater than the Ni
hyperaccumulation CL threshold of 1 mg Ni g–1 given by Brooks
et al. (1977) (Fig. 1E). CL was nearly ten times less for L1 leaves
than for L10 leaves (Fig. 1A), as was the case for Ni concentration in solutions (Fig. 1A). By contrast, Ni concentrations in
wheat leaves were extremely low. Ni quantities in leaves (QL)
increased as a non-linear function of culture time (Fig. 1E) as
leaf biomass (Fig. 1C).
Ni concentrations strongly decreased in roots (CR) (Fig. 1F),
CR data being unavailable for the L100 hyperaccumulator plants.
For L1 and L10, CR decreased as a function of root biomass production, leading to a slight decrease in the Ni quantity in roots
(QR) (Fig. 1H), which did not include root-adsorbed Ni. By contrast, QR strongly increased for wheat (Fig. 1H).
For each plant and treatment, Ni concentration in leaves did
not decrease as a function of leaf biomass (‘dilution’ curves),
except slightly for wheat, and CL was relatively constant when
CR increased (results not shown). The root-to-leaf Ni translocation factor, CL:CR, was always greater than 1 for the hyperaccumulator plants, increasing during the culture: e.g. for L10, it was
at 3.3 at the beginning of the culture, 18.7–44.6 from the 21st
day to the 35th day and 143.3 at the end of the culture period.
The proportion of Ni translocated to the leaves increased from
the 14th day to the 28th day (L1) or from the 14th day to the 21st
day (L10) of culture and was constant thereafter (92.7 ± 0.8%
and 97.3 ± 0.6%, respectively). As a complement, the proportions
of root-adsorbed Ni and of root-sequestrated Ni were very low
was made one week before the culture, whereas cultures started just after fertilization and Ni-contamination of the sand (W1, L1, L10,
and R1). Plant growth: kinetics of mean leaf biomass (C) and mean root biomass (D). Kinetics of mean concentrations (E, F) or mean
quantities (G, H) of nickel in leaves (E, G) or in roots (F, H, without taking into account the nickel adsorbed on the roots). Error bars show
standard errors of mean values (n=4). Open triangles and fine dotted line, winter wheat W1; circles, the Ni-hyperaccumulator Leptoplax
emarginata: with open circles and fine full line, L1; closed circles and fine full line, L10; grey circles and thick full line, L100. Only for (A)
and (B): small open lozenges and fine full line, reference soil R1 without plants (reference for both W1 and L1), small grey lozenges and
thick full line, reference soil R100 without plants (reference for L100). Inlets of (E) and (G): the same figures for winter wheat W1 with
specific y-axis scales.
Ecophysiology of nickel phytoaccumulation | 5821
(Fig. 2A, 2B). By contrast, the wheat CL:CR was always very
low and rather constant (CL:CR ratio of 0.005) with very low proportions of Ni translocated to the leaves (0.7 ± 0.1%), significant
proportions of root-adsorbed Ni (9.0 ± 1.6%) and very dominant
proportions of root-sequestered Ni (90.3 ± 1.7%,) (Fig. 2C).
During the first 14 d of culture, the root-adsorbed Ni content was
eight times higher for the wheat (16.9 ± 1.0 µg Ni g–1) than for
the L1 Ni-hyperaccumulator (2.0 ± 0.2 µg Ni g–1), the pH and
Cs values being the same in their sand solutions. The strong Cs
increase recorded from the 21st day to the 35th day of the wheat
culture (Fig. 1A) was also positively correlated to a decrease in
root-adsorbed Ni from 13.8 ± 0.7 µg Ni g–1 to 5.6 ± 0.9 µg Ni g–1.
Finally, the mean cumulative phytoextracted Ni mass (Fig. 2),
correspond to 29, 32, and 17% of the total Ni input for wheat, L1,
and L10 hyperaccumulator plants, respectively.
Intact Plant Transpiration Stream Concentration Factor
for nickel
The kinetics of cumulative leaf Ni mass (Fig. 1G), cumulative
volume of transpired water (see Supplementary Table S4 at JXB
online), and nickel concentration in solution (Fig. 1A) allowed us
to calculate the nickel mass flow from the roots to the leaves, dQL
(Fig. 3A), the corresponding transpiration flow, dVT (Fig. 3B), and
the mean Ni concentration in solution, CS (Fig. 3C). Then for each
weekly period considered, IPTSCFNi was calculated from these
three parameters using Equation (7). Two statistically different plant
groups were identified: L1, L10, and L100 Ni-hyperaccumulator
plants characterized by IPTSCFNi values much greater than 1
(7.2 ± 1.8, 5.8 ± 0.7, and 4.7 ± 0.5, respectively) and the Ni-excluder,
wheat, characterized by an extremely low IPTSCFNi value
(0.006 ± 0.004) (Fig. 3D). For the Ni-hyperaccumulator plants,
these IPTSCFNi values and those independently estimated from
Equation (5) applied to the revisited Redjala et al. (2010) results
(Fig. 4A) were plotted as a function of Cs (Fig. 4B), showing (i)
the same order of magnitude for the IPTSCFNi belonging to the
same Cs range of 2–16 µmol Ni l–1, and (ii) a non-linear IPTSCFNi
decrease as a function of Cs, down to a IPTSCFNi value of nearly
1.5 for Cs values larger than 100 µmol Ni l–1.
Equation (6) was validated as follows. Two linear but scattered regression lines forced to the origin occurred between dQL
and dVT, with a slope ( Cx ) of 2.75 µg Ni ml–1 for the L1 and
L100 Ni-hyperaccumulator plants (except for a couple of data
points), and of 0.38 µg Ni ml–1 for the L1 Ni-hyperaccumulator
plants (Fig. 3E). These two groups correspond to two groups of
CS values (Fig. 3C).
Finally, for all the L1, L10, and L100 Ni-hyperaccumulator
plants, a linear regression line forced to the origin occurred
between dQL and the dVT CS product (Fig. 3F), validating
Equation (7). The slope of this regression line gives a IPTSCFNi
value of 5.0 ± 0.1.
Comparison between IPTSCFNi and TSCFNi
The ‘microscopic’ TSCFNi (TSCFNi of 21.7 ± 4.4, with mean
Cx and Cs of 4.65 and 0.22 mg Ni l–1, respectively), directly
measured on the excised Ni-hyperaccumulator plants, was
2.4 times larger than its ‘macroscopic’ IPTSCFNi counterpart
(IPTSCFNi of 9.0 ± 2.3), estimated on the intact transpiring
Ni-hyperaccumulator plants just before stem excision at 6 h.
Fig. 2. Proportions of total plant Ni content in leaves (grey), in
roots (white), and adsorbed on the root surfaces (black) for the Ni
hyperaccumulator L. emarginata L1 (A) and L10 (B), and for wheat
W1 (C).
Reflection coefficient for Ni and symplastic Ni flow
modelling
For the highest JvCs values of the symplastic 63Ni uptakes by the
Ni-hyperaccumulator L. emarginata, the slope of the Js versus
5822 | Coinchelin et al.
Fig. 3. For each weekly period considered, mean kinetics of nickel mass flow from the roots to the leaves, dQL (A), transpiration rate,
dVT (B), mean Ni concentration in the sand or soil solutions of the planted pots, CS (C), and Ni Intact Plant Transpiration Stream
Concentraton Factor, IPTSCFNi calculated from these three parameters using Equation (7) (D, with an inset for winter wheat W1 with
specific y-axis scales). The mean culture time corresponds to the centred culture time for each weekly period: 7, 18, 25, 32, and 39 d
for the plants cultivated in the fertilized and Ni-contaminated sand (W1, L1, and L10), and 4, 14, and 28 d for the plants cultivated in the
fertilized and Ni-contaminatd soil (L100). Ni concentrations in the reference unplanted sand (R1) or soil (R100) at the very beginning of
the cultures were used for the calculation of the CS values for W1/L1 and L100 at the centred culture time of 7 d and 4 d, respectively.
Linear regression lines forced to the origin between the weekly nickel mass flow from the roots to the leaves, dQL, and either the weekly
transpiration rate, dVT (E) or the product between dVT and the corresponding mean Ni concentration in solution (F). A couple of L10 data
points were not taken into account for the linear relationship occurring between dQL and dVT (L10 and L100 data). Open triangles and
fine dotted line, winter wheat W1; circles, the Ni-hyperaccumulator Leptoplax emarginata: with open circles and fine full line, L1; closed
circles and fine full line, L10; grey circles and thick full line, L100.
Ecophysiology of nickel phytoaccumulation | 5823
JvCs regression line (Js=0.939JvCs+1.009 × 10–13, r=0.928, P
<0.001, Fig. 4A) is equal to (1–σNi) (Equation (1); Fiscus, 1986).
This leads to a σNi value of 0.06 ± 0.15.
Both the Michaelis–Menten model (Redjala et al., 2010) and
the Dalton et al. (1975) model fitted well the data plotted on
Fig. 4A (r of 0.959 and 0.946, respectively, with P <0.001 for
both). However, the first model was only non-linear, whereas
the second model was non-linear for the lowest JvCs values and
linear thereafter (nearly the regression line plotted on Fig. 4A).
Convective root Ni flow versus overall root Ni flow for
the Ni-hyperaccumulator
The convective component proportion of the root Ni uptake flow
(Equation 9) was of 17.6 ± 5.1, 17.7 ± 2.8, and 15.0 ± 2.8% for
the L1, L10, and L100 Ni-hyperaccumulator plants, respectively,
assuming a constant σNi value of 0.06. Adding these results to
those obtained during a 1 h period of symplastic 63Ni root uptake
(Equation 8) leads to a significative power-law relationship
(P <0.001) being identified between the convective component
proportion of the root Ni uptake flow and Cs (Fig. 4C).
Discussion
Nickel concentration in solution and methodology
Fig. 4. Relationship between the mean 63Ni symplastic flux by
L. emarginata root area unit across the root membrane, Js, or
its convective Ni flow counterpart (dotted line with the same
slope (1–σNi) as that determined on the regression line, full line)
and the mean product between water flux by root area unit
across the membrane, Jw, and Ni concentration in solution,
Cs (A). Relationships between either the mean Ni Intact Plant
Transpiration Stream Concentraton Factor, IPTSCFNi (B) or the
passive convective component proportion of the root Ni uptake
flow (C) and the Ni concentration in solution, Cs. Error bars show
standard errors of mean values (n=4). Open squares, data from
the revisited Redjala et al. (2010) results; circles, data from the
results of this study: with open circles, L1 Ni-hyperaccumulator
plants; closed circles, L10 Ni-hyperaccumulator plants; grey
circles, L100 Ni-hyperaccumulator plants.
For the sand, the very strong decrease in Ni concentration from
the initial solution is attributed to iron coatings on the quartz particles and to Fe hydroxide precipitation from FeSO4. For the sandy
topsoil, the huge drop in the Ni concentration is attributable to the
pH-dependent cationic exchange capacity of the organic coatings
and to the Fe oxyhydroxides. In both cases, Ni and SO4 should
coadsorb jointly on the surface OH groups, greatly decreasing Ni
solubility (Swedlund et al., 2003). Physico-chemical equilibrium
was only obtained from the 14th day of culture for the plants cultivated in the sand but was never obtained for the L10 plant–sand
system. This had crucial consequences on the dQL kinetics which
were rather chaotic for the very unstable L10 plant–sand system
but continuously non-linear for the other Ni-hyperaccumulator
plant–sand or plant–soil systems and characterized by a predominant equilibrium. For future studies, we therefore recommend
(i) to wait a long time, for example, six months after the addition of the fertilizing Ni-contaminated solution to the sand in
order to have a good physico-chemical equilibrium and (ii) not
to add nickel or nutrients to the irrigation solution so as not to
disturb this equilibrium. We have already successfully used the
experience from these methodological problems for the second
series of experiments where the mixture between the soil and the
Ni-contaminated fertilizing solution was made one week before
the culture, which led to an equilibrium after 9 d, and irrigation
was only made with demineralized water.
Rhizosphere pH
The alkalinization occurring in the rhizosphere of the L1
Ni-hyperaccumulator is attributed to the fact that the plants
absorbed more anions, mostly nitrates (see Supplementary Fig.
S2A at JXB online), than cations (the relative deficit of anions in
5824 | Coinchelin et al.
order of magnitude as the IPTSCFNi value of 0.006 ± 0.004 found
in this study for the winter wheat cultivar ‘Fidel’ but much less
variable because of the use of radio-labelling.
the L1 solution against rather more electroneutrality for the R1
solution: see Supplementary Fig. S2D at JXB online) leading to
anionic exudates in order to maintain the electroneutrality of the
solution. This alkalinization should explain the decrease in Ni
solubility from the reference soil to the planted soil. Similarly,
alkalinization in the rhizosphere of the Zn-Cd hyperaccumulator
Noccea caerulescens grown in a Cd-contaminated soil has also
been reported (Luo et al., 2000; Monsant et al., 2008; Blossfeld
et al., 2010). Hedley et al. (1982) also demonstrated that alkalinization in the rhizosphere of Brassica napus is related to an
anionic depletion in the solution because the plants absorb more
anions than cations. In the Ni-contaminated sandy topsoil, the
pH of the rhizosphere was similar to that of its reference soil.
Unfortunately, NH+4 and PO3–4 were not determined in the soil
solution, rendering impossible a calculation of the anion–cation balance for discussion. Finally, the significant acidification
occurring in the wheat rhizosphere has been widely reported,
being attributed to root exudates (Cieslinski et al., 1998; Bertin
et al., 2003).
The ‘microscopic’ TSCFNi, directly measured on the excised
Ni-hyperaccumulator plants, was 2.4 times larger than its ‘macroscopic’ IPTSCFNi counterpart, indirectly measured on intact
transpiring plants. Similarly, a greater sap ion concentration in
excised stem roots than in intact transpiring plants for maize was
reported by Goodger et al. (2005). ‘Microscopic’ TSCF values
might be overestimated because the mass flux should be smaller
than the transpiration flux, a non-linear decrease in the sap element concentration versus mass flux being reported (Schurr,
1998). It is therefore recommended to measure TSCF either on
excised plants in a pressure chamber, adjusting the sap flux to the
transpiration flux, as previously recommended by Schurr (1998),
or on intact transpiring plants, as was carried out in this study.
Intact Plant Transpiration Stream Concentration Factor
for nickel
Reflection coefficient for Ni and symplastic Ni flow
modelling
It has been widely reported that metals are highly translocated
from hyperaccumulator roots to leaves, predominantly because
of their active transport (see Supplementary Table S1 at JXB
online; Robinson et al., 2003b; Seregin and Kozhevnikova,
2008; Zhao et al., 2002, 2006; Xing et al., 2008). Our results
validated these previous findings for the Ni-hyperaccumulator L.
emarginata, namely the high translocation of Ni from the roots to
the leaves (93–97% of the overall root Ni uptake), and IPTSCFNi
values ranging from 7.2 to 4.7 for a range of Ni concentrations in
solution of 2–16 µmol Ni l–1, regardless of the growth period, the
time of Ni uptake, and the method used. These large IPTSCFNi
values were logically associated with low convective component proportions of the root Ni uptake flow (15–20% of the total
transport). The mean IPTSCFNi value of 5.0 ± 0.1 is very near to
the IPTSCFNi value of 5.2 ± 0.9 recently found by Bartoli et al.
(2012) for Ni uptake by the same Ni-hyperaccumulator, with a
similar Cs range of 0.8–10.2 µmol Ni l–1.
The non-linear decrease of IPTSCFNi from 7.2 to 1.2 when
Cs increased from 2 µmol Ni l–1 to 200 µmol Ni l–1 validates
the theory, IPTSCFNi being a complex inverse function of Cs,
assuming constant values for ω, Jv, and JS* (Equation 10). The
lowest IPTSCFNi values of 1.2–1.7 occurred for the highest Cs
range of 130–250 µmol Ni l–1. It should be of the same order of
magnitude as the (1–σNi) value of 0.94. This should be attributed
to the numerical tendency for IPTSCFNi to approach (1–σNi) for
extremely high CsJv values (Equation 10).
Our results also validated that wheat is a metal excluder: shown
by the predominant root-adsorbed and root-storage Ni (99.3% of
the overall root Ni uptake), the extremely low CL:CR ratio of 0.005,
and the extremely low mean IPTSCFNi value of 0.006. Gajewska
and Sklodowska (2008) found similar (CR+CR-ads) values of 191 µg
Ni g–1, a similar CL value of nearly 2 µg Ni g–1 and a similar CL:CR
ratio of 0.01 when Ni concentration in solution was of 10 mmol l–1.
For the durum wheat cultivar ‘Kyle’ 106Cd system, Van der Vliet
et al. (2007) found a IPTSCFCd value of 0.070 ± 0.001 of the same
σNi was very close to zero for the Ni-hyperaccumulator L. emarginata (σNi value of 0.06 ± 0.15). This was mainly attributed to
the fact that the L. emarginata root structure was characterized
by a hypodermis without Casparian bands (I Zelko, unpublished
data), which is extremely rare among angiosperms (Perumalla
et al., 1990; Enstone et al., 2003). As with root structures of some
other hyperaccumulating and non-accumulating Brassicaceae
(Enstone et al., 2003; Seregin and Kozhevnikova, 2008; Zelko
et al., 2008), the root structure of L. emarginata has other specific features: one or two layers of large cortical cells, and endodermic phi-thickenings (I Zelko, unpublished data).
The use of the Dalton et al. (1975) model on the Ni symplastic influx in the Ni-hyperaccumulator roots showed that the
fitted curve was non-linear for the lowest JvCs values and linear thereafter. Such a linear component, often associated with
a Michaelis–Menten non-linear component, for the lowest Cs
values, has been interpreted as the non-desorbed metal from the
cell walls (Hart et al., 1998, 2006; Zhao et al., 2002; Lu et al.,
2008) or a fingerprint of low-affinity metal transport (Redjala
et al., 2009). The Dalton et al. (1975) transpiration-based model
should, therefore, also be considered in further work in this area.
Comparison between IPTSCFNi and TSCFNi
Nickel root uptake and leaf accumulation modelling
The results showed that the IPTSCF and σ determined on intact
transpiring plants are very useful biophysical parameters in the
study of the mechanisms involved in metal uptake and accumulation by plants. The question now arises as to applying the
results of this study to process-based simplified models. This is a
crucial challenge because most process-based simplified models
do not sufficiently take into account ecophysiological processes,
for example, as discussed by Hopmans and Bristow (2002).
Biophysical Equation (7) has often been used in reactive
transport or root solute uptake models, TSCF being used as a
variable to be parameterized (Schoups and Hopmans, 2002;
Ecophysiology of nickel phytoaccumulation | 5825
Manzoni et al., 2011), fixed with an assumption (Ingwersen and
Streck, 2005) or independently calculated (Burken and Schnoor,
1997; Goktas and Aral, 2011). In the Robinson et al. (2003a)
model, the combined measurements of TSCFmetal, the metal concentration in solution, the volume of transpirated water, the soil
bulk density, and the vertical distribution of the root density fraction potentially allow the prediction of the shoot metal mass of
metal-hyperaccumulator plants and then the depth change of the
soil metal concentration. However, this model was not simulated
nor calibrated nor validated.
We have usefully taken into account forces and weaknesses
of these previous biophysical-based models and of some others in order to predict the kinetics of both Ni concentration in
soil solution and leaf Ni mass for the Ni-hyperaccumulator L.
emarginata cultivated on a fertilized and Ni-contaminated sandy
topsoil. For this, the plant sink term of the model was approximated by the above recommended biophysical equation. This
differential equation was coupled with a one-site rate-limited
desorption model. The model calibrations have been improved
and the model was finally succesfully validated for both Ni concentration in soil solution and leaf Ni mass (D Coinchelin, D
Stemmelen, and F Bartoli, unpublished data).
work (Redjala et al., 2010), Ivan Zelko, Bratislava University,
Slovakia, for kindly providing us with his results on the structure of the Leptoplax emarginata roots we prepared for him,
Mathilde Royer for her participation in the hyperaccumulator/
soil experiment, Stéphane Colin for designing the culture pots,
and Aurélien Renard, LCPME Nancy University-CNRS and the
INRA Arras laboratory in providing the XPS spectrometry and
ICP-MS Ni data, respectively. Trust and financial support from
INPL, ADEME, and Lorraine Regional Council given to the first
author for his PhD grant were greatly appreciated. We finally
thank Helen Selliez for improving the English, and the Associate
Editor and the two reviewers for their very helpful comments
that have greatly improved the former versions of this paper.
Supplementary data
Bertin C, Xiaohan Yang X, Weston LA. 2003. The role of root
exudates and allelochemicals in the rhizosphere. Plant and Soil , 256,
67–83.
Supplementary data can be found at JXB online.
Supplementary Fig. S1. Kinetics of Specific Leaf Area, SLA
(A), daily transpiration rate normalized to the leaf area, T/LA
(B), Water Use Efficiency (WUE) for shoot biomass production
(leaf biomass/cumulated volume of transpired water ratio) (C),
and weekly WUE for shoot biomass production (leaf biomass
production/transpiration ratio) (D).
Supplementary Fig. S2. Kinetics of mean composition of the
sand solutions for the fertilized and Ni-contaminated sand (1 mg
l–1 of Ni) planted with the Ni-hyperaccumulator Leptoplax
emarginata (L1) or unplanted (reference R1): NH+4 and NO–3
(A), K+, Mg2+, and Ca2+ (B), SO2–4 and PO3–4 (C), and total
cations and total anions (D).
Supplementary Table S1. Review of the Metal Transpiration
Stream Concentration Factor (TSCFM) values calculated as the
root xylem/hydroponic solution metal concentration ratio from
the results found in the series of papers listed in the table.
Supplementary Table S2. Main characteristics of the sand and
the sandy topsoil used in the pot experiments.
Supplementary Table S3. The nutrient concentration in porous
media solutions at the beginning and end of the cultures of the
Ni-hyperaccumulators Leptoplax emarginata L1, L10, and L100
and for wheat W1, with the references without plants R1 (reference for both W1 and L1) and R100 (reference for L100).
Supplementary Table S4. Kinetics of plant growth and transpiration for the Ni-hyperaccumulators Leptoplax emarginata L1,
L10, and L100 and for wheat W1.
Acknowledgements
We thank our colleagues Samia Skiker, Tanegmart Redjala, and
Thibault Sterckeman for useful discussions on their published
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