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Influence of exposure solution composition and of plant cadmium content on root cadmium short-term uptake
Environmental and Experimental Botany 74 (2011) 131–139
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Influence of exposure solution composition and of plant cadmium content on root cadmium short-term uptake Thibault Sterckeman ∗ , Tanegmart Redjala, Jean Louis Morel Nancy Université, INRA, Laboratoire Sols et Environnement, BP 172, 54505 Vandoeuvre-lès-Nancy Cedex, France
a r t i c l e
i n f o
Article history: Received 8 June 2010 Received in revised form 12 May 2011 Accepted 15 May 2011 Keywords: Cadmium Root exposure Apoplast Symplast Ionic competition Uptake regulation
a b s t r a c t The aim was to evaluate the influence of (i) cations potentially present in soil solution and (ii) of plant Cd content on apoplastic and symplastic root uptake of Cd. Hydroponically grown maize (Zea mays) roots were exposed to two Cd concentrations together with a cation five times the strength of the Cd. The influence of three pH and four Ca levels was also assessed. In addition, maize and alpine penny-cress (Noccaea caerulescens) were grown so their tissues contained Cd at three levels when their roots were exposed for 1 h to three Cd concentrations. Maize Cd uptake in the 5 M Cd solution was reduced by the presence of Pb2+ , Cu2+ , Co2+ , Zn2+ , Mg2+ or Mn2+ . At a 1 M Cd concentration, Cu2+ , as well as Ca2+ and H+ reduced the Cd uptake. However, the Ca concentration had to be much higher than that of Cd to restrain its uptake. High plant Cd content was responsible for an increase in the apoplastic Cd uptake and a decrease in the symplastic absorption, for both species. Low plant Cd content neither affected the Cd apoplastic uptake whatever the exposure concentration, nor the symplastic uptake in 0.1 M Cd exposure solution. Moreover, the hyperaccumulator symplastic uptake increased when exposed to 10 M Cd, but not when exposed to 50 M Cd. Maize roots showed a decrease in membrane net flux when exposed to 10 M and 50 M Cd. Finally, the results suggest that for plants exposed to usual soil solution concentrations, it is mainly calcium and protons which interfere with Cd internalization. © 2011 Elsevier B.V. All rights reserved.
1. Introduction When present in the soil even at relatively low concentrations, Cd tends to accumulate in plant organs. The presence of this metal in consumable parts of crop plants is a threat to human health. On the other hand, it suggests a way to decontaminate Cd polluted soil through phytoextraction, using low accumulating plants with high biomass production, such as maize (Zea mays L.), sunflower (Helianthus annuus L.) and willow (Salix spp.) or hyperaccumulating species like alpine pennycress (Noccaea caerulescens (J. Presl & C. Presl) F.K. Mey). From a practical point of view, Cd phytoaccumulation should be reduced in the first case and enhanced in the second. To reach both these goals there is a need to better understand the mechanisms of soil to plant Cd transfer, in which root uptake is a key process. The Cd symplastic net flux (Fs , mol g−1 s−1 or mol m−2 s−1 ) is generally modelled by the classical Michaelis–Menten equation FS =
Imax ([Cd] − [Cd]min ) − E, Km + ([Cd] − [Cd]min )
∗ Corresponding author. Tel.: +33 03 83 59 58 66; fax: +33 03 83 59 57 91. E-mail addresses: [email protected] (T. Sterckeman), [email protected] (T. Redjala), [email protected] (J.L. Morel). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.05.010
where [Cd] represents the Cd concentration in the exposure solution (mol L−1 ). Imax (mol g−1 s−1 or mol m−2 s−1 ) and Km (mol L−1 ) are the maximum influx and the affinity coefficient, respectively. Considering that [Cd]min , the concentration under which there is no possible uptake and the efflux E (mol g−1 s−1 or mol m−2 s−1 ) are negligible, the equation becomes FS =
Imax ([Cd]) . Km + ([Cd])
This supposes the existence of a transport system with a high affinity to Cd (HATS) in the plasma membrane (Cataldo et al., 1983; Hart et al., 1998; Lombi et al., 2001). It is not clear if this HATS is specific to Cd or if the metal enters the root cell using a Zn, Cu or Mn carrier, even a Ca or Mg cation channel (Welch and Norvell, 1999). More recently, it has been shown that the uptake kinetics model could be completed with a linear component that might correspond to the flow controlled by a low affinity transport system (LATS) acting at higher concentrations (Redjala et al., 2009, 2010a): FS =
Fmax [Cd] + a[Cd], Km + [Cd]
where a is the slope of the linear component. One can put forward the hypothesis that the Michaelis–Menten parameters (Fmax , Km and a) poorly reflect the root uptake in the
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field, at least for two reasons. The first one is that the plant used in the published works had not been exposed to Cd during cultivation before their roots were briefly exposed (generally for 20 min) to a Cd solution. In the field the Cd root uptake is that of a plant which contains Cd at varying concentrations. Knowing the numerous reactions that control the Cd homeostasis (Welch and Norvell, 1999; Clemens, 2001), it cannot be excluded that the Cd content in the plant tissue would have a feedback effect on the root uptake which is not taken into account in the classically measured Michaelis–Menten parameters. The second reason is that the Cd uptake kinetics were carried out in quasi-standard solutions containing predominantly 0.2–0.5 mM Ca and 2–5 mM MES to buffer the pH around 6 (Cataldo et al., 1983; Hart et al., 1998, 2002, 2006; Lombi et al., 2001; Zhao et al., 2002, 2006). This matrix is different in composition from the soil solution, in which various cations are present, at relatively high levels such as Ca2+ , or at lower or trace levels, such as H+ , NH4+ , K+ , Zn2+ or Cu2+ (Wolt, 1994). The composition of the soil solution varies according to factors such as soil type, horizon, use and contamination. It can therefore be hypothesized that the Michaelis–Menten parameters measured in vitro do not take into account any possible competition between Cd and other cations for the sorption on the plasmalemma and also on the cell wall sites. As already mentioned by Hart et al. (2002), it has been demonstrated that Cd phytoaccumulation can be modulated by the presence of Zn. However, as most of the results are based on analysis of plants grown in soil or in nutrient solution, it is not possible to know to which of the soil chemistry, root uptake or translocation processes the interaction between Cd and Zn must be attributed. There are few studies of the interaction between Cd and other cations for the uptake at the root level sensu stricto. Competition between Cd and Zn for root uptake has been shown in bread and durum wheat (Triticum aestivum L. and T. turgidum L.) (Smeyers-Verbeke et al., 1978; Hart et al., 2002), a low Cd accumulating population (Prayon) of alpine pennycress (Lombi et al., 2001; Zhao et al., 2002) or Arabidopsis halleri (L) O’Kane & Al-Shehbaz. This Cd/Zn competition was not seen for the Ganges population of alpine pennycress which accumulates more Cd than the Prayon population (Lombi et al., 2001; Zhao et al., 2002). The Mn also depressed the Cd uptake of Cd by the Prayon population of alpine pennycress but not by the Ganges one (Zhao et al., 2002). Copper was found to interfere with Cd uptake by wheat roots (Smeyers-Verbeke et al., 1978), while Ca at high concentrations (5 mM) depressed the Cd uptake by the Prayon population of alpine pennycress but not the Ganges one (Zhao et al., 2002). In the latter work, Co, Fe(II) and Ni were shown not to interfere with Cd uptake by either the Prayon and Ganges populations. However, we found no results on the effect of other cations present in soil solutions, such as H+ , K+ , Na+ , NH4+ and Pb2+ . It must added that in all the above-mentioned research, only symplastic uptake was considered, any apoplastic sorption being neglected. However, this component can represent 30–90% of the root Cd uptake after short term exposure (Redjala et al., 2009). To our knowledge, there is no information on the effect of both plant Cd content and ionic composition of the exposure solution on apoplastic uptake. To summarize, it can be hypothesized that the plant regulates Cd internalization according to the metal concentration in its tissues and that cations in the soil solution compete with Cd for sorption both on the cell membrane transporters and on cell wall sites. The aim of the work presented here was therefore (i) to evaluate the interference of the various cations potentially present in soil solution (H+ , K+ , Na+ , NH4 + , Ca2+ , Co2+ , Cu2+ , Fe2+ , Mg2+ , Mn2+ , Ni2+ , Pb2+ or Zn2+ ) on both the apoplastic and symplastic root uptake of Cd, and (ii) to test the hypothesis of a feedback effect of plant Cd content on the short term root uptake of the metal.
2. Materials and methods 2.1. Plant species Maize (cv INRA MB 682) and alpine pennycress of the Viviez population from the south of France were chosen because of their contrasting capacities to tolerate and accumulate Cd. Maize plants accumulate the metal in their roots (Jarvis et al., 1976; Florijn and Van Beusichem, 1993). In contrast, alpine pennycress has been reported to accumulate more than 1400 mg kg−1 DW of Cd in its shoots (Reeves et al., 2001; Sirguey et al., 2006). Only maize was used for the study of the influence of the composition of the exposure solution on the Cd short term uptake, because the reactivity of its apoplastic adsorption and symplastic absorption sites could be regarded as similar to those of the hyperaccumulating plant (Redjala et al., 2009, 2010a). 2.2. Plant cultivation Maize seeds were put on filter paper moistened with distilled water and placed in an incubator at 25 ◦ C for 4 days. After germination, the seedlings were transferred into a growth chamber with a photon flux density of 300 mol s−1 m−2 , a photoperiod of 16 h and day/night temperatures of 25/20 ◦ C. They were placed in hydroponics, onto a sheet of polystyrene floating on 40 L of the following nutrient solution (in M): 3000 Ca(NO3 )2 , 250 Ca(H2 PO4 )2 , 500 K2 SO4 , 1000 MgSO4 , 2000 NH4 NO3 , 46 H3 BO3 , 9 MnSO4 , 0.3 CuSO4 , 0.8 Na2 MoO4 , 0.8 ZnSO4 , 7.5 FeSO4 . CHESS software (van der Lee, 1998) was used to check the availability of each type of nutrient. The nutrient solution was continuously aerated thanks to air bubbling from capillary tubes. pH was adjusted to 5.7 through KOH or NaOH addition when the solution was renewed twice a week. Cultivation of the plants used for the solution composition study lasted 12 days. For the study of the influence Cd content, the maize plants were grown for 20 days and during the last 10 days, Cd was added to the nutrient solution to maintain three different exposure levels: 0 M (control), 0.1 M and 10 M Cd. Alpine pennycress seeds were sown onto a filter paper lying on cotton wool soaked with distilled water. Germination lasted 10 days in the dark, at 20 ◦ C. Seedlings were then transferred into a growth chamber for cultivation in hydroponics, on floating polystyrene sheets. Plants were fed with an aerated solution containing (in M): 3500 Ca(NO3 )2 , 1500 MgSO4 , 1200 KNO3 , 100 K2 HPO4 , 10 KCl, 10 H3 BO3 , 10 MnCl2 , 7.5 FeSO4 , 5 ZnSO4 , 0.7 NiSO4 , 0.2 CuSO4 , 0.2 Na2 MoO4 . The pH was adjusted to 5.7 by adding KOH or HNO3 . The growth chamber conditions were the same as for maize cultivation. The cultivation lasted 6 weeks, with weekly renewals of the nutrient solution. During the last 3 weeks, Cd was added to the nutrient solution to maintain an exposure concentration of 0 M (control), 0.1 M and 10 M Cd. Half of the control and of the plants exposed to Cd during cultivations were harvested just after the growth period in order to be analyzed for quantification of the accumulated stable Cd. 2.3. Root exposure to Cd Roots of the plants cultivated in Cd enriched nutrient solutions were rinsed and exposed to a desorption treatment in order both to minimize contamination of the radio-labelled solution with Cd leakage from the cell walls, and to liberate all exchange sites able to adsorb Cd. For that, each root system was immersed for 2 h in 80 mL of buffered solution (pH = 5.7) containing 5 mM of Ca(NO3 )2 and 2 mM MES buffer, then for two further hours in 80 mL of buffered solution (pH = 5.7) containing 0.5 mM of Ca(NO3 )2 and 2 mM MES buffer.
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Table 1 Ionic composition of the exposure solutions used in the different experiments. Experiment Influence of solution composition on Cd uptake
Influence of Cd plant content on Cd uptake a
Influence of Ca Influence of pH Influence of other cations (K+ , Na+ , NH4 + , Ca2+ , Co2+ , Cu2+ , Fe2+ , Mg2+ , Mn2+ , Ni2+ , Pb2+ or Zn2+ )
[CaCl2 ] mM
[Cd] M
pH
Other cation M
0, 0.001, 0.5 and 2 0.5 0.5 0.5
1 (CdCl2 ) 1 (CdCl2 ) 1 (CdNO3 ) 30 (CdNO3 )
5.7 4, 5 and 6 5.7 5.7
No No 5a 150a
0.5
0.1, 10 and 50 (CdCl2 )
5.7
No
As nitrate, except for Fe2+ and Mn2+ added as sulphate
Before exposure to Cd solution, all seedling roots were first rinsed in tap water, then in distilled water. They were then immersed in 650 mL of a solution containing 2 mM MES buffer (pH 5.7) with CaCl2 , Cd2+ and eventually another cation in concentrations varying according to the experiment, as presented in Table 1. Except in the study of the influence of Ca on Cd uptake, CaCl2 concentration was 0.5 mM. pH was adjusted by adding KOH or HNO3 . Cadmium was used with the same accompanying anion as the potentially interfering cation i.e. chloride or nitrate form. Only Fe2+ and Mn2+ , which could not be found as nitrate were used as sulphates. Cadmium concentrations for short term exposure were chosen according to a previous study (Redjala et al., 2009), in which it was shown that for maize, the HATS prevailed at Cd exposure concentrations below 1 M, while it prevailed up to 30 M for alpine pennycress. Thus, for maize the short-term exposure to 0.1 M Cd solution concerns the HATS activity, while the 10 M, 30 M and 50 M exposure concentrations concern that of the LATS. In the case of alpine pennycress, the short-term exposure to 0.1 M and 10 M Cd solutions theoretically concern the HATS, while the exposure with 50 M Cd solution should give information about the LATS activity. Cadmium was labelled with 109 Cd (GE Healthcare, Chalfont St. Giles, United Kingdom) at activity levels adapted to the exposure concentrations, so that the counting results were far above the quantification limit. Root exposure to Cd lasted 1 h, as in previous works (Redjala et al., 2009, 2010a). This duration enabled the apoplastic adsorption equilibrium to be approached without significant symplastic efflux. Indeed, Han et al. (2006) showed that Cd entry into the intracellular compartment is linear over the first 60 min, which suggests that possible efflux or regulation processes are still negligible during the first hour of exposure. During the whole duration of absorption, the exposure concentrations could be considered constant, as their decrease due to root uptake was always lower than 10% of the initial concentration. 2.4. Root cadmium fractionation For most of the experiments, the fractionation method was the same as the one previously used and detailed elsewhere (Redjala et al., 2010a). After exposure, roots were rinsed in distilled water for about ten seconds, and gently blotted using paper towels. They were then excised from shoots before the desorption kinetics procedure. The desorption solution contained 5 mM CaCl2 , 2 mM MES buffer (pH 5.7) and 2 mM unlabelled CdCl2 . The desorption kinetics procedure was carried out using a series of pots containing 80 mL of ice-cold desorption solution. Maize roots were carefully transferred into five successive pots, at the following times (t): 0, 5, 15, 30, 60 min. At t = 90 min, roots were frozen by dipping them into liquid nitrogen for 2 min. At t = 92 min, they were placed and agitated in another 80 mL desorption solution, at room temperature to help thawing. Ice-cold desorption kinetics continued thereafter, but with broken roots retained by a synthetic tissue mesh to facilitate the renewal of the desorption solution. At the end of the desorption (t = 270 min), the roots were dried by placing them in
an oven at 72 ◦ C for 3 days and then weighed before 109 Cd determination. For alpine pennycress plants, the same procedure was applied, but with freezing/thawing occurring 180 min after desorption kinetics started. Freezing was performed when the desorption kinetics started to reach a quasi-plateau. The freezing time (90 min for maize and 180 min for alpine pennycress) was chosen thanks to preliminary experiments carried out to describe the desorption kinetics. For each exposure solution and each root system replicate, samples of 20 mL were taken from the desorption pots and 0.22 m filtered before 109 Cd determination. Four replicates were carried out for maize and five for alpine pennycress. In the experiments carried out to evaluate the effect of Ca2+ and H+ , apoplastic Cd was estimated through a 30 min desorption of the roots. These were soaked in three successive desorption baths of 10 min each. The desorption solution had the same composition as the one previously described. Samples of 20 mL were taken from the desorption pots and 0.22 m filtered before 109 Cd determination. The roots were then dried at 72 ◦ C for 3 days and then weighed before 109 Cd determination (symplastic Cd). 2.5. Cadmium determination For the determination of stable Cd content in the plant tissues, shoots and root systems were separated, dried in an oven (3 days at 72 ◦ C), ground in an agate mill (Retsch, Germany) and digested in a microwave oven (Mars 5, CEM Corporation, Matthews, Caroline du Nord, USA). Cadmium in the digested material was then quantified through inductively coupled plasma atomic emission spectrometry (ICP-AES, Liberty II, Varian, Inc, Palo Alto, California, USA). The radioisotope 109 Cd was quantified by gamma counting (Wallac 1480 Wizard®3, Perkin Elmer Life Sciences Wallac Oy, Turku, Finland). The total Cd in the samples was calculated from the initial isotopic ratio in the exposure solution. In roots and shoots, 109 Cd was determined directly in the dry matter. To relate Cd content found in the root dry matter to that in solution, close linear correlations (DR = 0.7935 UR, R2 = 0.99) found between gamma-counting in un-ground (UR, cpm) and ground and digested root material (DR, cpm) were used. 2.6. Data processing The 109 Cd content in the shoots being negligible, the amounts of Cd taken up were calculated using the data produced by the root Cd fractionation. A Student test was used to compare mean values of Cd taken up by plants exposed to a factor to unexposed (control) plants. 3. Results 3.1. Influence of pH and calcium in the exposure solution Both Cd apoplastic adsorption and symplastic absorption clearly decreased with the pH (Fig. 1). Total Cd uptake at pH = 4 was about
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Fig. 1. Mean Cd root uptake by maize plants exposed for 60 min to a 1 M CdCl2 solution at three different pH levels. The error segment represents two standard deviations. For one root compartment (symplast or apoplast), means with different letters are significantly different (p = 0.05).
one fourth of the uptake at pH = 6. The presence of 1 M CaCl2 in the exposure solution did not seem to affect the Cd uptake (Fig. 2). On the contrary, the Cd uptake of the roots exposed to a solution containing 500 M CaCl2 was far below the uptake of the control roots, without calcium chloride in the exposure solution. The apoplastically adsorbed Cd was reduced approximately 2.5 times more than the symplastically absorbed metal. Indeed, the addition of 500 M CaCl2 to the exposure solution decreased the apoplastic adsorption by a factor close to 5 and the symplastic absorption by a factor close to 2. The addition of 2000 M CaCl2 did not significantly reduce the uptake compared to the 500 M CaCl2 . The presence of Ca in Ca/Cd ratio of 5 did not affect the apoplastic and symplastic Cd uptake, when the concentration of this metal was 1 M (Fig. 3) as well as when it was 30 M (Fig. 4).
Fig. 3. Mean Cd root uptake by maize plants exposed for 60 min to a 1 M Cd solution with 5 M of another cation. The error segment represents two standard deviations. The mean marked with * is significantly different from the control mean with p = 0.05. The effect of NH4 + could not be assessed in this case because of the accidental loss of replicates during the experiment.
80%, respectively, when roots were exposed to a 30 M Cd solution (Fig. 4a). Also Zn, Ni, Mn, Co and Ca reduced the average apoplastic adsorption by about 20%, although these decreases were not statistically significant. Manganese, Mg, Co, Zn, Cu and Pb at 150 M significantly decreased the Cd symplastic absorption by approximately 30–70% (Fig. 4b). Nickel and Ca also had a relatively strong depressive effect on the symplastic which was however, not significant.
3.2. Influence of minor cations in the exposure solution The influence of K could not be assessed because of the addition of KOH to buffer the pH. The presence of another cation (Co, Cu, Fe, Mg, Mn, Na, Ni, Pb or Zn) at 5 M did not significantly affect the Cd apoplastic adsorption of roots exposed to a 1 M Cd solution (Fig. 3). Only the presence of Cu reduced the Cd symplastic absorption, by about 30% (Fig. 3). In contrast, the presence of 150 M of Mg, Cu and Pb significantly reduced the apoplastic Cd uptake by nearly 20%, 60% and
Fig. 2. Mean Cd root uptake by maize plants exposed for 60 min to a 1 M CdCl2 solution with increasing concentrations of CaCl2 . The error segment represents two standard deviations. For one root compartment (symplast or apoplast), means with different letters are significantly different (p = 0.05).
Fig. 4. Mean apoplastic (a) and symplastic (b) Cd root uptake by maize plants exposed for 60 min to a 30 M Cd solution with 150 M of another cation. The error segment represents two standard deviations. Means marked with *, ** and *** are significantly different from the control mean with p = 0.05, p = 0.01 and p = 0.001, respectively.
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Table 2 Cadmium concentrations in nutrient solutions and in the plant organs grown in these solutions (mean values with standard deviation between brackets). Zea mays Nutrient solution Roots Shoots
mol L−1 mg kg−1 DW
0.1 295 (31) 24 (6.1)
3.3. Influence of plant Cd content Root dry mass was not affected by the contamination level during growth. On the other hand, some interveinal chlorosis was observed in the highly contaminated alpine pennycress plants. Alpine pennycress accumulated more Cd in shoots than in roots, while it was the reverse for maize (Table 2). Moreover, Cd accumulated in roots and shoots was positively correlated with Cd contamination level, which showed that the contamination levels were efficient in generating contrasting internal Cd accumulations. Apoplastic and symplastic sorption increased with external concentration during the short-term exposure in both species, regardless of the amount of Cd internally accumulated during cultivation (Figs. 5 and 6). In both plant species, low internal accumulation of Cd (after cultivation in 0.1 M Cd) did not affect the short-term Cd symplastic uptake in 0.1 M Cd exposure solution. On the other hand, the contrasted plant species showed different behaviors when exposed to 10 M Cd or 50 M Cd after cultivation in 0.1 M Cd solution. Indeed, roots of the hyperaccumulating plant showed a significant increase in Cd membrane net influx when exposed to 10 M, but no variation when exposed to a 50 M solution. On the contrary, maize roots showed a more or less significant decrease in membrane influx (Fig. 6). After high internal accumulation of Cd (after cultivation in 10 M Cd), root symplastic absorption was reduced for both plants, except for alpine pennycress when exposed to a 50 M Cd solution. This decrease in the absorption rate is more significant in maize than in alpine pennycress. Nevertheless, when considering the lowest short-term exposure concentrations (0.1 M Cd and 10 M Cd), both plants showed a greater drop in symplastic influx than when exposed to 50 M Cd. As regards Cd binding to cell walls in both plant species, low internal accumulation of Cd (after cultivation in 0.1 M Cd) did not affect the short-term Cd apoplastic uptake whatever the Cd concentration in the exposure solution. Cultivation in 10 M Cd was responsible for a more or less significant increase in the adsorption rate, for both species and the three exposure concentrations (Fig. 5). 4. Discussion 4.1. Influence of calcium and protons in the exposure solution on Cd root uptake The depressive effect of Ca on the Cd uptake can be explained by the competition for the adsorption sites in the cell walls and for the absorption sites on the plasmalemma. It is well known that a high proportion of Ca in plant tissues is located in the cell walls because of the abundance of more or less specific binding sites in the apoplasm. Divalent cations such as Ca can be bound to the sites of the so-called Donnan free space of the root apoplasm (Marschner, 1995). Cadmium can also be sorbed in root cell wall, as was shown in a previous works (Redjala et al., 2009): 30–90% of Cd was adsorbed on the apoplasm of root exposed for 1 h to solutions containing 0.05–50 mol L−1 M Cd. One proposed mechanism to explain the reduction of Cd symplastic absorption by Ca is the displacement of cell-surface Cd2+ by
Noccaea caerulescens 10 849 (1.2) 128 (18.4)
0.1 16 (1.2) 471 (55)
10 3361 (9.8) 3377 (86)
Ca2+ , as suggested by Kinraide (1998). Increased concentration of Ca2+ reduces the negativity of the electrical potential at the plasma membrane exterior surface. This reduction decreases the activity of Cd2+ at the membrane surface, and therefore its symplastic uptake (Wang et al., 2011). Another mechanism is that Cd is taken up through Ca channels and that both ions compete for this absorption site (Perfus-Barbeoch et al., 2002; Lindberg et al., 2004). However, since several transport systems might be involved in the symplastic uptake of Cd (Welch and Norvell, 1999), the precise role of Ca in decreasing Cd absorption still has to be elucidated. The competition with Ca most probably explains the absence of a saturable component in the Cd absorption isotherms found for maize by Perriguey et al. (2008), who carried out the uptake experiment in a nutrient solution containing, among other cations, 3250 M Ca. It must be noted that when Ca concentration is the same as that of Cd, the former element does not reduce the uptake of the latter, in the apoplastic as well as in the symplastic compartment. Calcium must be at much higher concentration than Cd to restrain its uptake. This could be related to the reactivity of Cd2+ which seems much higher than that of Ca2+ , as reflected by the Misono softness parameter (0.309 and 0.163 for Cd2+ and Ca2+ , respectively) (Misono et al., 1967). The depressive effect of Ca is relatively higher on apoplastic adsorption than on symplastic absorption, suggesting that the reaction of Cd with absorption sites is stronger than that with adsorption sites. Calcium is most frequently the dominant cation in soil solution, 800 M being its representative concentration. However, the latter can vary greatly, from around 10 M to more than 100 mM (Wolt, 1994). In our results, the effect of Ca on Cd uptake is evidenced when a 1 M Ca concentration is compared to a 500 M concentration. There is a need to more finely describe the relationship between Ca concentration (or Ca/Cd) and Cd uptake, for Ca concentrations varying between 10 M and 1000 M approximately. This could be useful in the mechanistic modelling of Cd uptake (Sterckeman et al., 2004), by representing the Cd influx as a function of the Ca concentration. Acidity when concerning soil or rhizosphere is often shown to increase Cd phytoaccumulation, mainly because of the increase in Cd solubility with proton concentration (Alloway, 1995). However, in solution, protons seem to compete with Cd for the binding site on the cell wall and the plasma membrane. This is not surprising, as most of the sites in the apoplasm are weak acids, with pK values similar to that of polygalacturonic acid (Grignon and Sentenac, 1991). The depressive effect of acidity on symplastic absorption could also be explained by an alteration of the root functioning. It is well established that the major driving force for the absorption of cations and anions is the extrusion of H+ , catalysed by an ATPase (Haynes, 1990; Marschner, 1995). On the other hand, it is thermodynamically consistent that increasing the external proton concentrations would reduce the extrusion of protons by the root (Yan et al., 1992) and at the same time the Cd symplastic absorption, as has been shown for various nutrients (Schubert et al., 1990). The effect of pH can also be explained by the fact that increased concentration of H+ reduces the negativity of the electrical potential at the plasma membrane exterior surface. This decreases the activity of Cd2+ at the membrane surface and, as a consequence, the symplastic uptake of the metal (Wang et al., 2011).
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Fig. 5. Mean apoplastic Cd root uptake by maize (a) and alpine penny-cress (b) cultivated in three differently Cd contaminated nutrient solutions and subsequently exposed for 60 min to three Cd concentrations. The error segment represents two standard deviations. Means marked with *, ** and *** are significantly different from the control mean with p = 0.05, p = 0.01 and p = 0.001, respectively.
4.2. Influence of minor cations on Cd symplastic absorption The Cd symplastic absorption for an exposure to a 1 M Cd solution is dominated by HATS activity (Redjala et al., 2009). Here, the only competitor to Cd for symplastic absorption is Cu, suggesting that both metals could use the same transport system to cross the root cell plasmalemma. Copper reduces the Cd flux by about 30%
on average, although it is five times more concentrated. This is consistent with the Misono softness parameter of Cu (0.284) which is lower than that of Cd (0.309). However, Pb has a much higher Misono softness parameter (0.396) and could therefore interfere in the Cd absorption, principally because ion reactivity would control its reaction with the absorption site. This suggests that the Cd transporter has a high affinity to Cd and could be specific to this element.
Fig. 6. Mean symplastic Cd root uptake by maize (a) and alpine penny-cress (b) cultivated in three differently Cd contaminated nutrient solutions and subsequently exposed for 60 min to three Cd concentrations. The error segment represents two standard deviations. Means marked with *, ** and *** are significantly different from the control mean with p = 0.05, p = 0.01 and p = 0.001, respectively.
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However, this seems very unlikely as Cd is considered to be a nonessential and toxic element to maize and most other plants and it is difficult to understand why such a transporter would have been selected during plant evolution. The results obtained in this study are only partially consistent with those available in published articles. The exposure to a 1 M Cd solution did not evidence the competition with Zn found in previous more or less comparable works for wheat (Hart et al., 2002), alpine pennycress (Prayon population) (Lombi et al., 2001; Zhao et al., 2002) and A. halleri (Zhao et al., 2006). The interference of Mn found on Cd absorption by alpine pennycress (Prayon population) (Zhao et al., 2002) was not evidenced here. Using 5 M concentrations for Cd and competing cations, Zhao et al. (2002) did not find any interference of Co, Cu, Mn, Ni and Zn with Cd absorption by the Ganges population of alpine pennycress. These differences are difficult to explain. One remark that can be made is that in the previous works, the uptake period was shorter than the one used here (20 min vs. 60 min). It is not sure that the pseudoequilibrium between cell membranes and solution cations reached after a 20 min exposure is the same as that after a 60 min exposure. Moreover, in the previous studies, the desorption step lasted only 15 min, which might be insufficient to remove all the trace metals adsorbed on the apoplasm. This could have created a bias in the measurement of the symplastic uptake of Cd (Redjala et al., 2009, 2010a,b), a bias which could vary according to the presence of competing ions for the apoplastic adsorption. On the other hand, for an exposure to a 30 M Cd solution, several cations seem to interfere more or less significantly. This supports the hypothesis that in this range of exposure concentrations, the Cd symplastic uptake is mainly the consequence of a LATS activity or a passive absorption (Redjala et al., 2009). The significantly interfering cations can be ranked according to the decrease in the average Cd uptake: Pb > Cu > Co > Zn > Mg > Mn. This ranking is close to that of the Misono softness parameters of the same elements: Pb(0.396) > Cu(0.284) > Co(0.268) > Zn(0.237) > Mn(0.151) > Mg(0.096). The coincidence of these rankings confirms that the adsorption is governed by the chemical properties of the elements rather than by those of the transport system. 4.3. Influence of minor cations on Cd apoplastic adsorption The Cd adsorption on root cell walls does not seem to be interfered with by the other cations when roots are exposed to a 1 M Cd solution. On the contrary, competition with three cations (Pb, Cu and Mg) appears when the exposure concentration is higher (30 M Cd solution). Moreover, it must be noted that the variability of the measurement of the apoplastic uptake is relatively high, and could hide the competition with Cd adsorption of other cations such as Ca, Co, Mn, Ni and Zn. This suggests that sites with different affinities to Cd are responsible for the adsorption of the metal in the cell walls. This hypothesis is also supported by previous results. Indeed, it was shown that after an exposure to a 1 M Cd solution, up to 33% of the Cd in the maize apoplasm remained unexchangeable, while at higher exposure concentrations (35 M) this proportion was around 5% or even less (Redjala et al., 2009, 2010a). This tightly bound Cd could be the consequence of covalent bonds with sulphhydryl and other functional groups of components such as hemicelluloses and proteins, but also of the complexation with the pectin chains (Liners et al., 1989; Cohen et al., 1998).
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Therefore, lower Cd affinity adsorption sites dominate the Cd adsorption of root apoplasm exposed to a 30 M solution, which make the competition of Cd with other cations more possible than when the metal is sorbed to higher affinity sites, as in the case of a 1 M Cd exposure. 4.4. Influence of plant Cd content on Cd uptake According to our results, chronic exposure to Cd does not exert the same influence on the different components of root uptake and depends on the level of exposure. In a previous study (Redjala et al., 2009), we showed that Cd transport through root cell membranes was ensured by at least two different transport mechanisms, one of them acting predominantly at low external concentrations (HATS) and the other one prevailing at higher concentrations. The latter could be a LATS or simple diffusion through the membrane. For maize, the HATS prevailed at external concentrations below 1 M, while it prevailed up to 30 M for alpine pennycress. Thus for maize, the short-term exposure to 0.1 M will concern HATS activity, while the 10 and 50 M exposure concentrations will preponderantly concern LATS. In alpine pennycress, the short-term exposure to 0.1 M and 10 M theoretically concerns HATS, while the 50 M exposure concentrations will principally concern LATS (Redjala et al., 2009). The HATS activity of maize and alpine pennycress is not affected at all by the low internal accumulation of Cd during growth. The withdrawal of free Cd ion from the cytosol by the complexation with phytochelatins (PCs) and possible transport to the vacuole or to the shoots may depress other mechanisms (active efflux, carrier mediated uptake) regulating the cytosolic Cd concentrations (Larsson et al., 2002). On the other hand, the plants do not show the same behavior for the high short-term exposure concentrations: the 0.1 M Cd contamination of the growth solution would down-regulate the LATS of maize (at 10 M and 50 M short term exposure), while stimulating the HATS of alpine pennycress after short-term exposure to a 10 M Cd solution and having no effect on its LATS (at 50 M short-term exposure). After high Cd accumulation during growth (cultivation in a 10 M Cd nutrient solution), the Cd symplastic influx falls significantly for both plants, except for the hyperaccumulating plant when exposed to 50 M Cd. Such a diminution in the intracellular uptake of Cd had already been observed on wheat root protoplasts (Lindberg et al., 2007). This may come from the down-regulation of the short-term Cd absorption by both high- and low-affinity transport systems for maize. In the case of alpine pennycress, it may come from the down-regulation of the short-term Cd absorption by HATS only, as short-term absorption at a 50 M exposure was not affected by the highest plant Cd content. This suggests that the down-regulation affects the HATS rather than the LATS in alpine pennycress. The decrease in Cd net influx can also result from upregulation of the Cd extrusion from intracellular to extracellular space, through Cd2+ /H+ antiport (Salt and Wagner, 1993) or vesicle excretion (Seregin and Kozhevnikova, 2008). In both species, there might be a regulation controlled by a threshold value for Cd plant content. Long-term contamination with 0.1 M would be below this threshold. On the other hand, long-term contamination with 10 M would exceed this limit and result in down-regulation of the absorption, possibly because of an excess of Cd in the roots or because of some signal from the shoots. For both plants, cell-wall sorption efficiency appears to be improved by the highest Cd concentration in the growth solution (10 M), whereas the lower concentration generally had no significant impact. The increase in the cell wall binding efficiency after high internal Cd accumulation may be related to the downregulation of symplastic absorption. However, the reduction in symplastic influx is minor compared to the increase in the apoplas-
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tic adsorption. Indeed, the up-regulation of the adsorption rate may well account for the decrease in the symplastic uptake. The apparent up-regulation of Cd binding properties may be due to modifications of the root cell-wall sorption characteristics by long term cadmium exposure. Cadmium stress is known to affect cellwall composition (Douchiche et al., 2007, 2010). First, Cd increases the proportion of acidic pectins (compared to neutral pectins). Secondly, the cell-wall CEC may be increased through regulation of enzymes. For instance, pectinmethylesterase has been suggested as being stimulated in the outer cell-wall domains of Cd-stressed plants, resulting in a strong decrease in the methylesterification of the acidic pectins. Thus, Cd strongly increases the acid pectins/esterified pectins ratio, particularly in the middle lamellae (Douchiche et al., 2007). This low degree of esterification enhances the apoplastic adsorption of all metallic trace elements (Kupchik et al., 2006). These results are supported by those previously found for Cu (Konno et al., 2005), Al (Eticha et al., 2005) or Pb (Khotimchenko et al., 2004). As dry mass did not vary with the level of contamination, there may be no significant difference in the proportion of young roots and then no decrease in the root CEC due to the age of roots. Therefore, the insignificant effect of low Cd contamination on the apoplastic adsorption rate could be accounted for by the existence of some Cd-stress threshold below which there is no regulation mechanism.
5. Conclusions The competition between Cd and other cations found in this work support the hypothesis of the existence of at least two transport systems for Cd symplastic uptake by maize, e.g. a HATS acting at low concentrations and a LATS or membrane diffusion at higher concentrations. They also show that two cations, Ca2+ and H+ are principally responsible for interference with Cd for uptake in the Cd concentration range that is frequently found in soil solution (≤1 M). However, a more detailed description of the relationships between Ca, proton concentrations and Cd uptake is necessary to ameliorate the modelling of the latter. The plant Cd content also influences the root uptake of the metal, both in the apoplastic and symplastic compartments. The direction (up- or down-regulation) and the intensity of this influence depend on the plant species, its Cd content and the Cd concentration in the exposure solution. However, for plants cultivated in and exposed to usual soil Cd concentrations, a plant regulation of the root uptake flow could not be evidenced.
Acknowledgements The authors wish to thank Cyril Bauland, from “Unité de génétique végétale du Moulon” Gif-sur-Yvette and Jacques Laborde of “Unité Expérimentale de Saint Martin de Hinx” for providing the maize seeds. We are very grateful to ADEME, for funding, and to Stéphane Colin for his helpful management of the radioisotope laboratory.
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Influence of exposure solution composition and of plant cadmium content on root cadmium short-term uptake Thibault Sterckeman ∗ , Tanegmart Redjala, Jean Louis Morel Nancy Université, INRA, Laboratoire Sols et Environnement, BP 172, 54505 Vandoeuvre-lès-Nancy Cedex, France
a r t i c l e
i n f o
Article history: Received 8 June 2010 Received in revised form 12 May 2011 Accepted 15 May 2011 Keywords: Cadmium Root exposure Apoplast Symplast Ionic competition Uptake regulation
a b s t r a c t The aim was to evaluate the influence of (i) cations potentially present in soil solution and (ii) of plant Cd content on apoplastic and symplastic root uptake of Cd. Hydroponically grown maize (Zea mays) roots were exposed to two Cd concentrations together with a cation five times the strength of the Cd. The influence of three pH and four Ca levels was also assessed. In addition, maize and alpine penny-cress (Noccaea caerulescens) were grown so their tissues contained Cd at three levels when their roots were exposed for 1 h to three Cd concentrations. Maize Cd uptake in the 5 M Cd solution was reduced by the presence of Pb2+ , Cu2+ , Co2+ , Zn2+ , Mg2+ or Mn2+ . At a 1 M Cd concentration, Cu2+ , as well as Ca2+ and H+ reduced the Cd uptake. However, the Ca concentration had to be much higher than that of Cd to restrain its uptake. High plant Cd content was responsible for an increase in the apoplastic Cd uptake and a decrease in the symplastic absorption, for both species. Low plant Cd content neither affected the Cd apoplastic uptake whatever the exposure concentration, nor the symplastic uptake in 0.1 M Cd exposure solution. Moreover, the hyperaccumulator symplastic uptake increased when exposed to 10 M Cd, but not when exposed to 50 M Cd. Maize roots showed a decrease in membrane net flux when exposed to 10 M and 50 M Cd. Finally, the results suggest that for plants exposed to usual soil solution concentrations, it is mainly calcium and protons which interfere with Cd internalization. © 2011 Elsevier B.V. All rights reserved.
1. Introduction When present in the soil even at relatively low concentrations, Cd tends to accumulate in plant organs. The presence of this metal in consumable parts of crop plants is a threat to human health. On the other hand, it suggests a way to decontaminate Cd polluted soil through phytoextraction, using low accumulating plants with high biomass production, such as maize (Zea mays L.), sunflower (Helianthus annuus L.) and willow (Salix spp.) or hyperaccumulating species like alpine pennycress (Noccaea caerulescens (J. Presl & C. Presl) F.K. Mey). From a practical point of view, Cd phytoaccumulation should be reduced in the first case and enhanced in the second. To reach both these goals there is a need to better understand the mechanisms of soil to plant Cd transfer, in which root uptake is a key process. The Cd symplastic net flux (Fs , mol g−1 s−1 or mol m−2 s−1 ) is generally modelled by the classical Michaelis–Menten equation FS =
Imax ([Cd] − [Cd]min ) − E, Km + ([Cd] − [Cd]min )
∗ Corresponding author. Tel.: +33 03 83 59 58 66; fax: +33 03 83 59 57 91. E-mail addresses: [email protected] (T. Sterckeman), [email protected] (T. Redjala), [email protected] (J.L. Morel). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.05.010
where [Cd] represents the Cd concentration in the exposure solution (mol L−1 ). Imax (mol g−1 s−1 or mol m−2 s−1 ) and Km (mol L−1 ) are the maximum influx and the affinity coefficient, respectively. Considering that [Cd]min , the concentration under which there is no possible uptake and the efflux E (mol g−1 s−1 or mol m−2 s−1 ) are negligible, the equation becomes FS =
Imax ([Cd]) . Km + ([Cd])
This supposes the existence of a transport system with a high affinity to Cd (HATS) in the plasma membrane (Cataldo et al., 1983; Hart et al., 1998; Lombi et al., 2001). It is not clear if this HATS is specific to Cd or if the metal enters the root cell using a Zn, Cu or Mn carrier, even a Ca or Mg cation channel (Welch and Norvell, 1999). More recently, it has been shown that the uptake kinetics model could be completed with a linear component that might correspond to the flow controlled by a low affinity transport system (LATS) acting at higher concentrations (Redjala et al., 2009, 2010a): FS =
Fmax [Cd] + a[Cd], Km + [Cd]
where a is the slope of the linear component. One can put forward the hypothesis that the Michaelis–Menten parameters (Fmax , Km and a) poorly reflect the root uptake in the
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field, at least for two reasons. The first one is that the plant used in the published works had not been exposed to Cd during cultivation before their roots were briefly exposed (generally for 20 min) to a Cd solution. In the field the Cd root uptake is that of a plant which contains Cd at varying concentrations. Knowing the numerous reactions that control the Cd homeostasis (Welch and Norvell, 1999; Clemens, 2001), it cannot be excluded that the Cd content in the plant tissue would have a feedback effect on the root uptake which is not taken into account in the classically measured Michaelis–Menten parameters. The second reason is that the Cd uptake kinetics were carried out in quasi-standard solutions containing predominantly 0.2–0.5 mM Ca and 2–5 mM MES to buffer the pH around 6 (Cataldo et al., 1983; Hart et al., 1998, 2002, 2006; Lombi et al., 2001; Zhao et al., 2002, 2006). This matrix is different in composition from the soil solution, in which various cations are present, at relatively high levels such as Ca2+ , or at lower or trace levels, such as H+ , NH4+ , K+ , Zn2+ or Cu2+ (Wolt, 1994). The composition of the soil solution varies according to factors such as soil type, horizon, use and contamination. It can therefore be hypothesized that the Michaelis–Menten parameters measured in vitro do not take into account any possible competition between Cd and other cations for the sorption on the plasmalemma and also on the cell wall sites. As already mentioned by Hart et al. (2002), it has been demonstrated that Cd phytoaccumulation can be modulated by the presence of Zn. However, as most of the results are based on analysis of plants grown in soil or in nutrient solution, it is not possible to know to which of the soil chemistry, root uptake or translocation processes the interaction between Cd and Zn must be attributed. There are few studies of the interaction between Cd and other cations for the uptake at the root level sensu stricto. Competition between Cd and Zn for root uptake has been shown in bread and durum wheat (Triticum aestivum L. and T. turgidum L.) (Smeyers-Verbeke et al., 1978; Hart et al., 2002), a low Cd accumulating population (Prayon) of alpine pennycress (Lombi et al., 2001; Zhao et al., 2002) or Arabidopsis halleri (L) O’Kane & Al-Shehbaz. This Cd/Zn competition was not seen for the Ganges population of alpine pennycress which accumulates more Cd than the Prayon population (Lombi et al., 2001; Zhao et al., 2002). The Mn also depressed the Cd uptake of Cd by the Prayon population of alpine pennycress but not by the Ganges one (Zhao et al., 2002). Copper was found to interfere with Cd uptake by wheat roots (Smeyers-Verbeke et al., 1978), while Ca at high concentrations (5 mM) depressed the Cd uptake by the Prayon population of alpine pennycress but not the Ganges one (Zhao et al., 2002). In the latter work, Co, Fe(II) and Ni were shown not to interfere with Cd uptake by either the Prayon and Ganges populations. However, we found no results on the effect of other cations present in soil solutions, such as H+ , K+ , Na+ , NH4+ and Pb2+ . It must added that in all the above-mentioned research, only symplastic uptake was considered, any apoplastic sorption being neglected. However, this component can represent 30–90% of the root Cd uptake after short term exposure (Redjala et al., 2009). To our knowledge, there is no information on the effect of both plant Cd content and ionic composition of the exposure solution on apoplastic uptake. To summarize, it can be hypothesized that the plant regulates Cd internalization according to the metal concentration in its tissues and that cations in the soil solution compete with Cd for sorption both on the cell membrane transporters and on cell wall sites. The aim of the work presented here was therefore (i) to evaluate the interference of the various cations potentially present in soil solution (H+ , K+ , Na+ , NH4 + , Ca2+ , Co2+ , Cu2+ , Fe2+ , Mg2+ , Mn2+ , Ni2+ , Pb2+ or Zn2+ ) on both the apoplastic and symplastic root uptake of Cd, and (ii) to test the hypothesis of a feedback effect of plant Cd content on the short term root uptake of the metal.
2. Materials and methods 2.1. Plant species Maize (cv INRA MB 682) and alpine pennycress of the Viviez population from the south of France were chosen because of their contrasting capacities to tolerate and accumulate Cd. Maize plants accumulate the metal in their roots (Jarvis et al., 1976; Florijn and Van Beusichem, 1993). In contrast, alpine pennycress has been reported to accumulate more than 1400 mg kg−1 DW of Cd in its shoots (Reeves et al., 2001; Sirguey et al., 2006). Only maize was used for the study of the influence of the composition of the exposure solution on the Cd short term uptake, because the reactivity of its apoplastic adsorption and symplastic absorption sites could be regarded as similar to those of the hyperaccumulating plant (Redjala et al., 2009, 2010a). 2.2. Plant cultivation Maize seeds were put on filter paper moistened with distilled water and placed in an incubator at 25 ◦ C for 4 days. After germination, the seedlings were transferred into a growth chamber with a photon flux density of 300 mol s−1 m−2 , a photoperiod of 16 h and day/night temperatures of 25/20 ◦ C. They were placed in hydroponics, onto a sheet of polystyrene floating on 40 L of the following nutrient solution (in M): 3000 Ca(NO3 )2 , 250 Ca(H2 PO4 )2 , 500 K2 SO4 , 1000 MgSO4 , 2000 NH4 NO3 , 46 H3 BO3 , 9 MnSO4 , 0.3 CuSO4 , 0.8 Na2 MoO4 , 0.8 ZnSO4 , 7.5 FeSO4 . CHESS software (van der Lee, 1998) was used to check the availability of each type of nutrient. The nutrient solution was continuously aerated thanks to air bubbling from capillary tubes. pH was adjusted to 5.7 through KOH or NaOH addition when the solution was renewed twice a week. Cultivation of the plants used for the solution composition study lasted 12 days. For the study of the influence Cd content, the maize plants were grown for 20 days and during the last 10 days, Cd was added to the nutrient solution to maintain three different exposure levels: 0 M (control), 0.1 M and 10 M Cd. Alpine pennycress seeds were sown onto a filter paper lying on cotton wool soaked with distilled water. Germination lasted 10 days in the dark, at 20 ◦ C. Seedlings were then transferred into a growth chamber for cultivation in hydroponics, on floating polystyrene sheets. Plants were fed with an aerated solution containing (in M): 3500 Ca(NO3 )2 , 1500 MgSO4 , 1200 KNO3 , 100 K2 HPO4 , 10 KCl, 10 H3 BO3 , 10 MnCl2 , 7.5 FeSO4 , 5 ZnSO4 , 0.7 NiSO4 , 0.2 CuSO4 , 0.2 Na2 MoO4 . The pH was adjusted to 5.7 by adding KOH or HNO3 . The growth chamber conditions were the same as for maize cultivation. The cultivation lasted 6 weeks, with weekly renewals of the nutrient solution. During the last 3 weeks, Cd was added to the nutrient solution to maintain an exposure concentration of 0 M (control), 0.1 M and 10 M Cd. Half of the control and of the plants exposed to Cd during cultivations were harvested just after the growth period in order to be analyzed for quantification of the accumulated stable Cd. 2.3. Root exposure to Cd Roots of the plants cultivated in Cd enriched nutrient solutions were rinsed and exposed to a desorption treatment in order both to minimize contamination of the radio-labelled solution with Cd leakage from the cell walls, and to liberate all exchange sites able to adsorb Cd. For that, each root system was immersed for 2 h in 80 mL of buffered solution (pH = 5.7) containing 5 mM of Ca(NO3 )2 and 2 mM MES buffer, then for two further hours in 80 mL of buffered solution (pH = 5.7) containing 0.5 mM of Ca(NO3 )2 and 2 mM MES buffer.
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Table 1 Ionic composition of the exposure solutions used in the different experiments. Experiment Influence of solution composition on Cd uptake
Influence of Cd plant content on Cd uptake a
Influence of Ca Influence of pH Influence of other cations (K+ , Na+ , NH4 + , Ca2+ , Co2+ , Cu2+ , Fe2+ , Mg2+ , Mn2+ , Ni2+ , Pb2+ or Zn2+ )
[CaCl2 ] mM
[Cd] M
pH
Other cation M
0, 0.001, 0.5 and 2 0.5 0.5 0.5
1 (CdCl2 ) 1 (CdCl2 ) 1 (CdNO3 ) 30 (CdNO3 )
5.7 4, 5 and 6 5.7 5.7
No No 5a 150a
0.5
0.1, 10 and 50 (CdCl2 )
5.7
No
As nitrate, except for Fe2+ and Mn2+ added as sulphate
Before exposure to Cd solution, all seedling roots were first rinsed in tap water, then in distilled water. They were then immersed in 650 mL of a solution containing 2 mM MES buffer (pH 5.7) with CaCl2 , Cd2+ and eventually another cation in concentrations varying according to the experiment, as presented in Table 1. Except in the study of the influence of Ca on Cd uptake, CaCl2 concentration was 0.5 mM. pH was adjusted by adding KOH or HNO3 . Cadmium was used with the same accompanying anion as the potentially interfering cation i.e. chloride or nitrate form. Only Fe2+ and Mn2+ , which could not be found as nitrate were used as sulphates. Cadmium concentrations for short term exposure were chosen according to a previous study (Redjala et al., 2009), in which it was shown that for maize, the HATS prevailed at Cd exposure concentrations below 1 M, while it prevailed up to 30 M for alpine pennycress. Thus, for maize the short-term exposure to 0.1 M Cd solution concerns the HATS activity, while the 10 M, 30 M and 50 M exposure concentrations concern that of the LATS. In the case of alpine pennycress, the short-term exposure to 0.1 M and 10 M Cd solutions theoretically concern the HATS, while the exposure with 50 M Cd solution should give information about the LATS activity. Cadmium was labelled with 109 Cd (GE Healthcare, Chalfont St. Giles, United Kingdom) at activity levels adapted to the exposure concentrations, so that the counting results were far above the quantification limit. Root exposure to Cd lasted 1 h, as in previous works (Redjala et al., 2009, 2010a). This duration enabled the apoplastic adsorption equilibrium to be approached without significant symplastic efflux. Indeed, Han et al. (2006) showed that Cd entry into the intracellular compartment is linear over the first 60 min, which suggests that possible efflux or regulation processes are still negligible during the first hour of exposure. During the whole duration of absorption, the exposure concentrations could be considered constant, as their decrease due to root uptake was always lower than 10% of the initial concentration. 2.4. Root cadmium fractionation For most of the experiments, the fractionation method was the same as the one previously used and detailed elsewhere (Redjala et al., 2010a). After exposure, roots were rinsed in distilled water for about ten seconds, and gently blotted using paper towels. They were then excised from shoots before the desorption kinetics procedure. The desorption solution contained 5 mM CaCl2 , 2 mM MES buffer (pH 5.7) and 2 mM unlabelled CdCl2 . The desorption kinetics procedure was carried out using a series of pots containing 80 mL of ice-cold desorption solution. Maize roots were carefully transferred into five successive pots, at the following times (t): 0, 5, 15, 30, 60 min. At t = 90 min, roots were frozen by dipping them into liquid nitrogen for 2 min. At t = 92 min, they were placed and agitated in another 80 mL desorption solution, at room temperature to help thawing. Ice-cold desorption kinetics continued thereafter, but with broken roots retained by a synthetic tissue mesh to facilitate the renewal of the desorption solution. At the end of the desorption (t = 270 min), the roots were dried by placing them in
an oven at 72 ◦ C for 3 days and then weighed before 109 Cd determination. For alpine pennycress plants, the same procedure was applied, but with freezing/thawing occurring 180 min after desorption kinetics started. Freezing was performed when the desorption kinetics started to reach a quasi-plateau. The freezing time (90 min for maize and 180 min for alpine pennycress) was chosen thanks to preliminary experiments carried out to describe the desorption kinetics. For each exposure solution and each root system replicate, samples of 20 mL were taken from the desorption pots and 0.22 m filtered before 109 Cd determination. Four replicates were carried out for maize and five for alpine pennycress. In the experiments carried out to evaluate the effect of Ca2+ and H+ , apoplastic Cd was estimated through a 30 min desorption of the roots. These were soaked in three successive desorption baths of 10 min each. The desorption solution had the same composition as the one previously described. Samples of 20 mL were taken from the desorption pots and 0.22 m filtered before 109 Cd determination. The roots were then dried at 72 ◦ C for 3 days and then weighed before 109 Cd determination (symplastic Cd). 2.5. Cadmium determination For the determination of stable Cd content in the plant tissues, shoots and root systems were separated, dried in an oven (3 days at 72 ◦ C), ground in an agate mill (Retsch, Germany) and digested in a microwave oven (Mars 5, CEM Corporation, Matthews, Caroline du Nord, USA). Cadmium in the digested material was then quantified through inductively coupled plasma atomic emission spectrometry (ICP-AES, Liberty II, Varian, Inc, Palo Alto, California, USA). The radioisotope 109 Cd was quantified by gamma counting (Wallac 1480 Wizard®3, Perkin Elmer Life Sciences Wallac Oy, Turku, Finland). The total Cd in the samples was calculated from the initial isotopic ratio in the exposure solution. In roots and shoots, 109 Cd was determined directly in the dry matter. To relate Cd content found in the root dry matter to that in solution, close linear correlations (DR = 0.7935 UR, R2 = 0.99) found between gamma-counting in un-ground (UR, cpm) and ground and digested root material (DR, cpm) were used. 2.6. Data processing The 109 Cd content in the shoots being negligible, the amounts of Cd taken up were calculated using the data produced by the root Cd fractionation. A Student test was used to compare mean values of Cd taken up by plants exposed to a factor to unexposed (control) plants. 3. Results 3.1. Influence of pH and calcium in the exposure solution Both Cd apoplastic adsorption and symplastic absorption clearly decreased with the pH (Fig. 1). Total Cd uptake at pH = 4 was about
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Fig. 1. Mean Cd root uptake by maize plants exposed for 60 min to a 1 M CdCl2 solution at three different pH levels. The error segment represents two standard deviations. For one root compartment (symplast or apoplast), means with different letters are significantly different (p = 0.05).
one fourth of the uptake at pH = 6. The presence of 1 M CaCl2 in the exposure solution did not seem to affect the Cd uptake (Fig. 2). On the contrary, the Cd uptake of the roots exposed to a solution containing 500 M CaCl2 was far below the uptake of the control roots, without calcium chloride in the exposure solution. The apoplastically adsorbed Cd was reduced approximately 2.5 times more than the symplastically absorbed metal. Indeed, the addition of 500 M CaCl2 to the exposure solution decreased the apoplastic adsorption by a factor close to 5 and the symplastic absorption by a factor close to 2. The addition of 2000 M CaCl2 did not significantly reduce the uptake compared to the 500 M CaCl2 . The presence of Ca in Ca/Cd ratio of 5 did not affect the apoplastic and symplastic Cd uptake, when the concentration of this metal was 1 M (Fig. 3) as well as when it was 30 M (Fig. 4).
Fig. 3. Mean Cd root uptake by maize plants exposed for 60 min to a 1 M Cd solution with 5 M of another cation. The error segment represents two standard deviations. The mean marked with * is significantly different from the control mean with p = 0.05. The effect of NH4 + could not be assessed in this case because of the accidental loss of replicates during the experiment.
80%, respectively, when roots were exposed to a 30 M Cd solution (Fig. 4a). Also Zn, Ni, Mn, Co and Ca reduced the average apoplastic adsorption by about 20%, although these decreases were not statistically significant. Manganese, Mg, Co, Zn, Cu and Pb at 150 M significantly decreased the Cd symplastic absorption by approximately 30–70% (Fig. 4b). Nickel and Ca also had a relatively strong depressive effect on the symplastic which was however, not significant.
3.2. Influence of minor cations in the exposure solution The influence of K could not be assessed because of the addition of KOH to buffer the pH. The presence of another cation (Co, Cu, Fe, Mg, Mn, Na, Ni, Pb or Zn) at 5 M did not significantly affect the Cd apoplastic adsorption of roots exposed to a 1 M Cd solution (Fig. 3). Only the presence of Cu reduced the Cd symplastic absorption, by about 30% (Fig. 3). In contrast, the presence of 150 M of Mg, Cu and Pb significantly reduced the apoplastic Cd uptake by nearly 20%, 60% and
Fig. 2. Mean Cd root uptake by maize plants exposed for 60 min to a 1 M CdCl2 solution with increasing concentrations of CaCl2 . The error segment represents two standard deviations. For one root compartment (symplast or apoplast), means with different letters are significantly different (p = 0.05).
Fig. 4. Mean apoplastic (a) and symplastic (b) Cd root uptake by maize plants exposed for 60 min to a 30 M Cd solution with 150 M of another cation. The error segment represents two standard deviations. Means marked with *, ** and *** are significantly different from the control mean with p = 0.05, p = 0.01 and p = 0.001, respectively.
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Table 2 Cadmium concentrations in nutrient solutions and in the plant organs grown in these solutions (mean values with standard deviation between brackets). Zea mays Nutrient solution Roots Shoots
mol L−1 mg kg−1 DW
0.1 295 (31) 24 (6.1)
3.3. Influence of plant Cd content Root dry mass was not affected by the contamination level during growth. On the other hand, some interveinal chlorosis was observed in the highly contaminated alpine pennycress plants. Alpine pennycress accumulated more Cd in shoots than in roots, while it was the reverse for maize (Table 2). Moreover, Cd accumulated in roots and shoots was positively correlated with Cd contamination level, which showed that the contamination levels were efficient in generating contrasting internal Cd accumulations. Apoplastic and symplastic sorption increased with external concentration during the short-term exposure in both species, regardless of the amount of Cd internally accumulated during cultivation (Figs. 5 and 6). In both plant species, low internal accumulation of Cd (after cultivation in 0.1 M Cd) did not affect the short-term Cd symplastic uptake in 0.1 M Cd exposure solution. On the other hand, the contrasted plant species showed different behaviors when exposed to 10 M Cd or 50 M Cd after cultivation in 0.1 M Cd solution. Indeed, roots of the hyperaccumulating plant showed a significant increase in Cd membrane net influx when exposed to 10 M, but no variation when exposed to a 50 M solution. On the contrary, maize roots showed a more or less significant decrease in membrane influx (Fig. 6). After high internal accumulation of Cd (after cultivation in 10 M Cd), root symplastic absorption was reduced for both plants, except for alpine pennycress when exposed to a 50 M Cd solution. This decrease in the absorption rate is more significant in maize than in alpine pennycress. Nevertheless, when considering the lowest short-term exposure concentrations (0.1 M Cd and 10 M Cd), both plants showed a greater drop in symplastic influx than when exposed to 50 M Cd. As regards Cd binding to cell walls in both plant species, low internal accumulation of Cd (after cultivation in 0.1 M Cd) did not affect the short-term Cd apoplastic uptake whatever the Cd concentration in the exposure solution. Cultivation in 10 M Cd was responsible for a more or less significant increase in the adsorption rate, for both species and the three exposure concentrations (Fig. 5). 4. Discussion 4.1. Influence of calcium and protons in the exposure solution on Cd root uptake The depressive effect of Ca on the Cd uptake can be explained by the competition for the adsorption sites in the cell walls and for the absorption sites on the plasmalemma. It is well known that a high proportion of Ca in plant tissues is located in the cell walls because of the abundance of more or less specific binding sites in the apoplasm. Divalent cations such as Ca can be bound to the sites of the so-called Donnan free space of the root apoplasm (Marschner, 1995). Cadmium can also be sorbed in root cell wall, as was shown in a previous works (Redjala et al., 2009): 30–90% of Cd was adsorbed on the apoplasm of root exposed for 1 h to solutions containing 0.05–50 mol L−1 M Cd. One proposed mechanism to explain the reduction of Cd symplastic absorption by Ca is the displacement of cell-surface Cd2+ by
Noccaea caerulescens 10 849 (1.2) 128 (18.4)
0.1 16 (1.2) 471 (55)
10 3361 (9.8) 3377 (86)
Ca2+ , as suggested by Kinraide (1998). Increased concentration of Ca2+ reduces the negativity of the electrical potential at the plasma membrane exterior surface. This reduction decreases the activity of Cd2+ at the membrane surface, and therefore its symplastic uptake (Wang et al., 2011). Another mechanism is that Cd is taken up through Ca channels and that both ions compete for this absorption site (Perfus-Barbeoch et al., 2002; Lindberg et al., 2004). However, since several transport systems might be involved in the symplastic uptake of Cd (Welch and Norvell, 1999), the precise role of Ca in decreasing Cd absorption still has to be elucidated. The competition with Ca most probably explains the absence of a saturable component in the Cd absorption isotherms found for maize by Perriguey et al. (2008), who carried out the uptake experiment in a nutrient solution containing, among other cations, 3250 M Ca. It must be noted that when Ca concentration is the same as that of Cd, the former element does not reduce the uptake of the latter, in the apoplastic as well as in the symplastic compartment. Calcium must be at much higher concentration than Cd to restrain its uptake. This could be related to the reactivity of Cd2+ which seems much higher than that of Ca2+ , as reflected by the Misono softness parameter (0.309 and 0.163 for Cd2+ and Ca2+ , respectively) (Misono et al., 1967). The depressive effect of Ca is relatively higher on apoplastic adsorption than on symplastic absorption, suggesting that the reaction of Cd with absorption sites is stronger than that with adsorption sites. Calcium is most frequently the dominant cation in soil solution, 800 M being its representative concentration. However, the latter can vary greatly, from around 10 M to more than 100 mM (Wolt, 1994). In our results, the effect of Ca on Cd uptake is evidenced when a 1 M Ca concentration is compared to a 500 M concentration. There is a need to more finely describe the relationship between Ca concentration (or Ca/Cd) and Cd uptake, for Ca concentrations varying between 10 M and 1000 M approximately. This could be useful in the mechanistic modelling of Cd uptake (Sterckeman et al., 2004), by representing the Cd influx as a function of the Ca concentration. Acidity when concerning soil or rhizosphere is often shown to increase Cd phytoaccumulation, mainly because of the increase in Cd solubility with proton concentration (Alloway, 1995). However, in solution, protons seem to compete with Cd for the binding site on the cell wall and the plasma membrane. This is not surprising, as most of the sites in the apoplasm are weak acids, with pK values similar to that of polygalacturonic acid (Grignon and Sentenac, 1991). The depressive effect of acidity on symplastic absorption could also be explained by an alteration of the root functioning. It is well established that the major driving force for the absorption of cations and anions is the extrusion of H+ , catalysed by an ATPase (Haynes, 1990; Marschner, 1995). On the other hand, it is thermodynamically consistent that increasing the external proton concentrations would reduce the extrusion of protons by the root (Yan et al., 1992) and at the same time the Cd symplastic absorption, as has been shown for various nutrients (Schubert et al., 1990). The effect of pH can also be explained by the fact that increased concentration of H+ reduces the negativity of the electrical potential at the plasma membrane exterior surface. This decreases the activity of Cd2+ at the membrane surface and, as a consequence, the symplastic uptake of the metal (Wang et al., 2011).
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Fig. 5. Mean apoplastic Cd root uptake by maize (a) and alpine penny-cress (b) cultivated in three differently Cd contaminated nutrient solutions and subsequently exposed for 60 min to three Cd concentrations. The error segment represents two standard deviations. Means marked with *, ** and *** are significantly different from the control mean with p = 0.05, p = 0.01 and p = 0.001, respectively.
4.2. Influence of minor cations on Cd symplastic absorption The Cd symplastic absorption for an exposure to a 1 M Cd solution is dominated by HATS activity (Redjala et al., 2009). Here, the only competitor to Cd for symplastic absorption is Cu, suggesting that both metals could use the same transport system to cross the root cell plasmalemma. Copper reduces the Cd flux by about 30%
on average, although it is five times more concentrated. This is consistent with the Misono softness parameter of Cu (0.284) which is lower than that of Cd (0.309). However, Pb has a much higher Misono softness parameter (0.396) and could therefore interfere in the Cd absorption, principally because ion reactivity would control its reaction with the absorption site. This suggests that the Cd transporter has a high affinity to Cd and could be specific to this element.
Fig. 6. Mean symplastic Cd root uptake by maize (a) and alpine penny-cress (b) cultivated in three differently Cd contaminated nutrient solutions and subsequently exposed for 60 min to three Cd concentrations. The error segment represents two standard deviations. Means marked with *, ** and *** are significantly different from the control mean with p = 0.05, p = 0.01 and p = 0.001, respectively.
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However, this seems very unlikely as Cd is considered to be a nonessential and toxic element to maize and most other plants and it is difficult to understand why such a transporter would have been selected during plant evolution. The results obtained in this study are only partially consistent with those available in published articles. The exposure to a 1 M Cd solution did not evidence the competition with Zn found in previous more or less comparable works for wheat (Hart et al., 2002), alpine pennycress (Prayon population) (Lombi et al., 2001; Zhao et al., 2002) and A. halleri (Zhao et al., 2006). The interference of Mn found on Cd absorption by alpine pennycress (Prayon population) (Zhao et al., 2002) was not evidenced here. Using 5 M concentrations for Cd and competing cations, Zhao et al. (2002) did not find any interference of Co, Cu, Mn, Ni and Zn with Cd absorption by the Ganges population of alpine pennycress. These differences are difficult to explain. One remark that can be made is that in the previous works, the uptake period was shorter than the one used here (20 min vs. 60 min). It is not sure that the pseudoequilibrium between cell membranes and solution cations reached after a 20 min exposure is the same as that after a 60 min exposure. Moreover, in the previous studies, the desorption step lasted only 15 min, which might be insufficient to remove all the trace metals adsorbed on the apoplasm. This could have created a bias in the measurement of the symplastic uptake of Cd (Redjala et al., 2009, 2010a,b), a bias which could vary according to the presence of competing ions for the apoplastic adsorption. On the other hand, for an exposure to a 30 M Cd solution, several cations seem to interfere more or less significantly. This supports the hypothesis that in this range of exposure concentrations, the Cd symplastic uptake is mainly the consequence of a LATS activity or a passive absorption (Redjala et al., 2009). The significantly interfering cations can be ranked according to the decrease in the average Cd uptake: Pb > Cu > Co > Zn > Mg > Mn. This ranking is close to that of the Misono softness parameters of the same elements: Pb(0.396) > Cu(0.284) > Co(0.268) > Zn(0.237) > Mn(0.151) > Mg(0.096). The coincidence of these rankings confirms that the adsorption is governed by the chemical properties of the elements rather than by those of the transport system. 4.3. Influence of minor cations on Cd apoplastic adsorption The Cd adsorption on root cell walls does not seem to be interfered with by the other cations when roots are exposed to a 1 M Cd solution. On the contrary, competition with three cations (Pb, Cu and Mg) appears when the exposure concentration is higher (30 M Cd solution). Moreover, it must be noted that the variability of the measurement of the apoplastic uptake is relatively high, and could hide the competition with Cd adsorption of other cations such as Ca, Co, Mn, Ni and Zn. This suggests that sites with different affinities to Cd are responsible for the adsorption of the metal in the cell walls. This hypothesis is also supported by previous results. Indeed, it was shown that after an exposure to a 1 M Cd solution, up to 33% of the Cd in the maize apoplasm remained unexchangeable, while at higher exposure concentrations (35 M) this proportion was around 5% or even less (Redjala et al., 2009, 2010a). This tightly bound Cd could be the consequence of covalent bonds with sulphhydryl and other functional groups of components such as hemicelluloses and proteins, but also of the complexation with the pectin chains (Liners et al., 1989; Cohen et al., 1998).
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Therefore, lower Cd affinity adsorption sites dominate the Cd adsorption of root apoplasm exposed to a 30 M solution, which make the competition of Cd with other cations more possible than when the metal is sorbed to higher affinity sites, as in the case of a 1 M Cd exposure. 4.4. Influence of plant Cd content on Cd uptake According to our results, chronic exposure to Cd does not exert the same influence on the different components of root uptake and depends on the level of exposure. In a previous study (Redjala et al., 2009), we showed that Cd transport through root cell membranes was ensured by at least two different transport mechanisms, one of them acting predominantly at low external concentrations (HATS) and the other one prevailing at higher concentrations. The latter could be a LATS or simple diffusion through the membrane. For maize, the HATS prevailed at external concentrations below 1 M, while it prevailed up to 30 M for alpine pennycress. Thus for maize, the short-term exposure to 0.1 M will concern HATS activity, while the 10 and 50 M exposure concentrations will preponderantly concern LATS. In alpine pennycress, the short-term exposure to 0.1 M and 10 M theoretically concerns HATS, while the 50 M exposure concentrations will principally concern LATS (Redjala et al., 2009). The HATS activity of maize and alpine pennycress is not affected at all by the low internal accumulation of Cd during growth. The withdrawal of free Cd ion from the cytosol by the complexation with phytochelatins (PCs) and possible transport to the vacuole or to the shoots may depress other mechanisms (active efflux, carrier mediated uptake) regulating the cytosolic Cd concentrations (Larsson et al., 2002). On the other hand, the plants do not show the same behavior for the high short-term exposure concentrations: the 0.1 M Cd contamination of the growth solution would down-regulate the LATS of maize (at 10 M and 50 M short term exposure), while stimulating the HATS of alpine pennycress after short-term exposure to a 10 M Cd solution and having no effect on its LATS (at 50 M short-term exposure). After high Cd accumulation during growth (cultivation in a 10 M Cd nutrient solution), the Cd symplastic influx falls significantly for both plants, except for the hyperaccumulating plant when exposed to 50 M Cd. Such a diminution in the intracellular uptake of Cd had already been observed on wheat root protoplasts (Lindberg et al., 2007). This may come from the down-regulation of the short-term Cd absorption by both high- and low-affinity transport systems for maize. In the case of alpine pennycress, it may come from the down-regulation of the short-term Cd absorption by HATS only, as short-term absorption at a 50 M exposure was not affected by the highest plant Cd content. This suggests that the down-regulation affects the HATS rather than the LATS in alpine pennycress. The decrease in Cd net influx can also result from upregulation of the Cd extrusion from intracellular to extracellular space, through Cd2+ /H+ antiport (Salt and Wagner, 1993) or vesicle excretion (Seregin and Kozhevnikova, 2008). In both species, there might be a regulation controlled by a threshold value for Cd plant content. Long-term contamination with 0.1 M would be below this threshold. On the other hand, long-term contamination with 10 M would exceed this limit and result in down-regulation of the absorption, possibly because of an excess of Cd in the roots or because of some signal from the shoots. For both plants, cell-wall sorption efficiency appears to be improved by the highest Cd concentration in the growth solution (10 M), whereas the lower concentration generally had no significant impact. The increase in the cell wall binding efficiency after high internal Cd accumulation may be related to the downregulation of symplastic absorption. However, the reduction in symplastic influx is minor compared to the increase in the apoplas-
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tic adsorption. Indeed, the up-regulation of the adsorption rate may well account for the decrease in the symplastic uptake. The apparent up-regulation of Cd binding properties may be due to modifications of the root cell-wall sorption characteristics by long term cadmium exposure. Cadmium stress is known to affect cellwall composition (Douchiche et al., 2007, 2010). First, Cd increases the proportion of acidic pectins (compared to neutral pectins). Secondly, the cell-wall CEC may be increased through regulation of enzymes. For instance, pectinmethylesterase has been suggested as being stimulated in the outer cell-wall domains of Cd-stressed plants, resulting in a strong decrease in the methylesterification of the acidic pectins. Thus, Cd strongly increases the acid pectins/esterified pectins ratio, particularly in the middle lamellae (Douchiche et al., 2007). This low degree of esterification enhances the apoplastic adsorption of all metallic trace elements (Kupchik et al., 2006). These results are supported by those previously found for Cu (Konno et al., 2005), Al (Eticha et al., 2005) or Pb (Khotimchenko et al., 2004). As dry mass did not vary with the level of contamination, there may be no significant difference in the proportion of young roots and then no decrease in the root CEC due to the age of roots. Therefore, the insignificant effect of low Cd contamination on the apoplastic adsorption rate could be accounted for by the existence of some Cd-stress threshold below which there is no regulation mechanism.
5. Conclusions The competition between Cd and other cations found in this work support the hypothesis of the existence of at least two transport systems for Cd symplastic uptake by maize, e.g. a HATS acting at low concentrations and a LATS or membrane diffusion at higher concentrations. They also show that two cations, Ca2+ and H+ are principally responsible for interference with Cd for uptake in the Cd concentration range that is frequently found in soil solution (≤1 M). However, a more detailed description of the relationships between Ca, proton concentrations and Cd uptake is necessary to ameliorate the modelling of the latter. The plant Cd content also influences the root uptake of the metal, both in the apoplastic and symplastic compartments. The direction (up- or down-regulation) and the intensity of this influence depend on the plant species, its Cd content and the Cd concentration in the exposure solution. However, for plants cultivated in and exposed to usual soil Cd concentrations, a plant regulation of the root uptake flow could not be evidenced.
Acknowledgements The authors wish to thank Cyril Bauland, from “Unité de génétique végétale du Moulon” Gif-sur-Yvette and Jacques Laborde of “Unité Expérimentale de Saint Martin de Hinx” for providing the maize seeds. We are very grateful to ADEME, for funding, and to Stéphane Colin for his helpful management of the radioisotope laboratory.
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