- Email: [email protected]
Arbuscular mycorrhizal fungi decrease radiocesium accumulation in Medicago truncatula
Journal of Environmental Radioactivity 101 (2010) 591e596
Contents lists available at ScienceDirect
Journal of Environmental Radioactivity journal homepage: www.elsevier.com/locate/jenvrad
Arbuscular mycorrhizal fungi decrease radiocesium accumulation in Medicago truncatula Veronika Gyuricza, Stéphane Declerck, Hervé Dupré de Boulois* Université catholique de Louvain, Earth and Life Institute (ELI), Laboratoire de Mycologie, Croix du Sud 3, 1348 Louvain-la-Neuve, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 October 2009 Received in revised form 5 March 2010 Accepted 5 March 2010 Available online 7 April 2010
The role of arbuscular mycorrhizal fungi (AMF) in plant radiocesium uptake and accumulation remains ambiguous. This is probably due to the presence of other soil microorganisms, the variability of soil characteristics and plant nutritional status or the availability of its chemical analogue, potassium (K). Here, we used an in vitro culture system to study the impact of increased concentration of K on radiocesium accumulation in non K-starved mycorrhizal and non-mycorrhizal Medicago truncatula plants. In the presence of AMF radiocesium uptake decreased regardless of the concentration of K, and its translocation from root to shoot was also significantly lower. Potassium also reduced the accumulation of radiocesium in plants but to a lesser extent than mycorrhization, and without any effect on translocation. These results suggest that AMF in combination with K can play a key role in reducing radiocesium uptake and its subsequent translocation to plant shoots, thereby representing good potential for improved phytomanagement of contaminated areas. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Radiocesium Potassium Arbuscular mycorrhizal fungi In vitro Phytostabilisation
1. Introduction Radiocesium represents a major threat to terrestrial ecosystems as it can remain in the upper soil horizons for decades (Delvaux et al., 2001). Plant contamination results from root access to this pool of radiocesium and from the uptake mechanisms similar to that of potassium (White and Broadley, 2000; Zhu and Smolders, 2000). Physical countermeasures such as soil removal (Guillitte et al., 1994) and the application of soil amendments have been used to limit plant uptake of radiocesium (Ciuffo et al., 2003; Sandeep and Manjaiah, 2008; Shaw, 1993; Zhu and Shaw, 2000). However, these methods can be expensive and are often inadequate (Camps et al., 2003; Vidal et al., 2001; Zhu and Shaw, 2000). Therefore other methods, such as extraction of radiocesium by plants, were widely considered (e.g. Eapen et al., 2006; Fuhrmann et al., 2002). However, phytoextraction of radiocesium may be unsuitable for agricultural lands used for food production (White et al., 2003) and for these reasons the production of selected or genetically-engineered “safe” crops that do not accumulate radiocesium or could participate in its stabilisation was suggested by White et al. (2003) in combination with K-fertilisation. Several studies reported the potential of plant-associated microorganisms to restrict acquisition and/or accumulation of
* Corresponding author. Tel.: þ32 (0) 10 47 22 26; fax: þ32 (0) 10 45 15 01. E-mail address: [email protected] (H. Dupré de Boulois). 0265-931X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvrad.2010.03.004
radiocesium in plants (Dupré de Boulois et al., 2008). Among the soil microflora, arbuscular mycorrhizal fungi (AMF) have been considered in several studies on radiocesium, but an unequivocal conclusion on the behaviour of this element in mycorrhizal plants could not be reached. Authors either reported a reduced transfer of radiocesium from soil to plants (e.g. Berreck and Haselwandter, 2001), or an enhanced accumulation (e.g. Entry et al., 1999) or even no change (e.g. Rogers and Williams, 1986) in the presence of AMF. These apparently conflicting results could be attributed to plant and AMF species, to the presence of other microorganisms, to plant nutritional status and to the characteristics of the soil used in the different experiments (Dupré de Boulois et al., 2008) among other factors. Recently, Declerck et al. (2003); Dupré de Boulois et al. (2005, 2006) and Gyuricza et al. (2010) investigated the capacity of AMF to transport radiocesium under strict in vitro culture conditions. These authors demonstrated that AMF could take up, translocate and transfer radiocesium from a root-free labelled compartment to their host via their extraradical mycelial network. However, this transport was low and root to shoot translocation of radiocesium was limited. This suggested that even though AMF can transport radiocesium to their host plants, their participation in plant radiocesium accumulation is probably negligible. This corroborates earlier results by Joner et al. (2004) for pot experiments conducted with different substrates, host plants and AMF species. In parallel to these findings, Berreck and Haselwandter (2001) hypothesized that AMF could restrict direct root uptake through the accumulation of radiocesium within
592
V. Gyuricza et al. / Journal of Environmental Radioactivity 101 (2010) 591e596
extraradical structures. However, Dupré de Boulois et al. (2005, 2006) showed that this accumulation was limited. These authors therefore proposed another hypothesis according to which AMF could restrict radiocesium plant acquisition by modifying the expression or activity of transporters involved in plant radiocesium uptake. It was also suggested that AMF could play a role in radiocesium root to shoot translocation by restricting its transport towards or its loading within the xylem. Among the environmental factors affecting plant radiocesium acquisition, potassium (K) has been shown to decrease radiocesium root uptake under hydroponic conditions (see Zhu, 2001 and Zhu et al., 2000). Soil based studies have been less conclusive, which is understandable given the wide range of soils investigated and the various treatments applied (Nisbet, 1993) and possibly also the differences in plant K status (see Zhu et al., 2000). The studies on transporters responsible for radiocesium influx into root cells further supported the role of K in radiocesium uptake by plant roots (see White and Broadley, 2000 and Zhu and Smolders, 2000). For mycorrhizal plants the role of K was only considered in a soil experiment where the long-term effects of fertilization were investigated (Berreck and Haselwandter, 2001). Our study aims to examine the effects of the increased supply of K on radiocesium uptake and partitioning within plants in the presence or absence of AMF, under strict in vitro culture conditions. 2. Materials and methods 2.1. Biological material A strain of Glomus intraradices Schenck and Smith (MUCL 43194) grown in association with Ri T-DNA transformed carrot roots (Daucus carota L.) was purchased from GINCO (http://www.mbla.ucl.ac.be/ginco-bel). The AMF strain was delivered in a Petri plate (90 mm diameter) on the modified Strullu-Romand (MSR) medium (Declerck et al., 1998), solidified with 3 g l1 GelGroÔ (ICN, Biomedicals, Irvine, CA, USA). The mineral composition of the MSR medium is given in Table 1 (see mineral composition of MSR1 medium) but included sucrose and vitamins. The AM fungus was subsequently subcultured following the methodology of Cranenbrouck et al. (2005). Seeds of Medicago truncatula L., cv. Jemalong A17 (SARDI, Adelaide, Australia) were surface-sterilized by immersion in sodium hypochlorite (containing 5% active chloride) for 10 min, rinsed 3 times in sterile de-ionized water and germinated in groups of 10 in Petri plates (90 mm diameter) containing 40 ml of MSR1 medium (Table 1) solidified with 3 g l1 GelGroÔ. Germination was conducted in an incubator set at 27 C in the dark for 8 days.
2.2. Experimental design Eight-day old M. truncatula plantlets with shoots approximately 50 mm long and primary roots approximately 70 mm long, with two to five secondary roots, were selected for transplantation into in vitro culture systems. These systems consisted of two connected compartments: a shoot compartment (SC), where the shoot
Table 1 Mineral composition of the culture media used for germination and subsequent cultivation of plants before (MSR1) and during (MSR2) labelling.
Macroelements
Microelements
a
KNO3 MgSO4$7H2O K2PO4$2H2O NaH2PO4.2H2O Ca(NO3)2$4H2O KCl NaFeEDTA MnSO4$4H2O ZnSO4$7H2O H3BO3 CuSO4$5H2O (NH4)6Mo7O24$4H2O NaMoO4$2H2O
MSR1
MSR2
76 739 4.1 e 359 65 8 24.5 2.8 18.6 2.2 0.34 0.024
e 739 e 4.7 359 0, 74.4 or 744a 8
Amount varies according to treatmente see text for details.
developed, and a root compartment (RC), where roots developed, associated or not with an AMF (Dupré de Boulois et al., 2006). Shoots of M. truncatula plantlets were inserted through the opening between the RC and the SC. The roots were placed in the RC on the surface of 30 ml MSR1 medium (Table 1), solidified with 3 g l1 GelGroÔ. The initial concentration of K in the MSR1 medium was 1.65 mM (0.192 mg in 30 ml) added in the form of KH2PO4$2H2O, KNO3 and KCl. In half of the in vitro culture systems, the roots of the M. truncatula plantlets were inoculated with spores (100) of G. intraradices, while the other half was not inoculated. Inoculation was performed following the method described by Cranenbrouck et al. (2005). The in vitro culture systems were then sealed using cellophane film and the RCs were wrapped in black plastic bags to maintain roots and AMF in the dark. The culture systems were incubated in a controlled growth chamber set at 22/18 C (day/night) with a 16/8 h photoperiod. Daylight lamps provided an average photosynthetic photon flux at the level of the SCs of 225 mmol m2s1. To ensure adequate plant and fungal growth, 10 ml MSR1 medium, solidified with 3 g l1 GelGroÔ was added in the RCs of the in vitro culture systems 3 and 6 weeks after the start of the experiment. The medium was added at a temperature of approximately 35 C to avoid damage to the roots and AMF. The plants therefore received a total of 0.32 mg of K (in the form of KH2PO4$2H2O, KNO3 and KCl) before adding the medium that contained three different concentrations of K. 2.3.
134
Cs labelling
Eight weeks after the beginning of the experiment, a profuse mycelium had grown within and on the surface of the medium contained in the RCs. Ten ml of MSR2 medium (Table 1) solidified with 3 g l1 GelGroÔ was added to the RCs following the same method as above. This medium was either free of K (0 mM) or supplemented with KCl with a concentration of 1 or 10 mM of K. Therefore, the mycorrhizal and non-mycorrhizal plants received 0, 0.744 or 7.44 mg K. Six to eight replicates were used per treatment. After solidification of the MSR2 medium, filter-sterilized (AcrodiscÒ Syringe Filters, PALL Corporation, Ann Arbor, MI, USA) radiocesium (134Cs) was applied to the surface of the medium. Fifty ml 134Cs droplets (altogether 200 ml) were distributed with a micropipette on the whole surface of the medium with a total activity of 5050 50 Bq. After 48 h the 134Cs was uniformly distributed in the medium (based on a preliminary experiment where 134Cs was evenly distributed in the medium after 48 h e data not shown). The source of 134Cs was in the form of CsNO3 in water, supplied by the laboratory of nuclear chemistry (CMAT, UCL, Louvain-la-Neuve, Belgium). 2.4. Harvest and planteAMF analyses Before labelling, the total extraradical hyphal length and the number of spores in the RCs were estimated. The extraradical hyphal length was determined as detailed in Declerck et al. (2003), while spore count followed the methodology described in Declerck et al. (2001). One week after the addition of 134Cs (i.e. 9 weeks after inoculation), the M. truncatula shoots were collected. The roots were removed gently from the RCs and cleaned from the remaining solidified medium using citrate buffer (0.01 M). The medium was collected subsequently. All samples (i.e. shoots, mycorrhizal and nonmycorrhizal roots and medium with or without extraradical mycelium of AMF) were subjected to 134Cs counting on a Wallac 1480 Gamma Counter (Wallac, Turku, Finland). After counting, the roots and shoots were dried at 70 C to a constant weight and their dry weight was measured. Root colonisation of the mycorrhizal plants was estimated. The root subsamples were cleared in 10% KOH and stained with 0.2% Trypan blue following the method of Phillips and Hayman (1970). Roots were cut into 10 mm fragments and 30 randomly selected segments were examined under a compound microscope (Olympus BH2, Olympus Optical (Europa) GmbH, Hamburg, Germany) at 50e250 magnifications, to evaluate the frequency (%F) and intensity (%I) of AMF colonisation (Plenchette and Morel, 1996). 2.5. Potassium concentration in the plants Plant shoots and root subsamples were dried to a constant weight and digested by boiling aqua regia (concentrated nitric acid and hydrochloric acid in 1:3 ratio). Potassium concentrations in the extracts were determined with ICPOES (Perkin Elmer Optima 3300 DV, Waltham, MA, USA). Plant reference samples of known K concentration were also digested concomitantly with the samples as internal control. 2.6. Statistical analyses Data normally distributed and having homogeneous variances were subjected to analysis of variance (ANOVA). One-way ANOVA was used to compare plant and fungal variables in different groups and two-way ANOVA to compare mycorrhizal status and K levels.
V. Gyuricza et al. / Journal of Environmental Radioactivity 101 (2010) 591e596
593
Table 2 Plant and AMF growth variables in presence/absence of AMF and under increasing concentrations of K. Hyphal length and number of spores were estimated before labelling, i.e. eight weeks after inoculation while other variables (shoot dry weight (SDW), root dry weight (RDW), and Frequency (%F) and Intensity (%I) of root colonisation) were measured after labelling, i.e. nine weeks after inoculation. Mycorrhizal 0 mM K (N ¼ 8) SC RC
SDW (mg) Hyphal length (cm) Number of spores RDW (mg) %F %I
18a 1503a 1314a 70a 40a 15a
a
2b 353 251 6 9 2
Non-mycorrhizal
1 mM K (N ¼ 8)
10 mM K (N ¼ 8)
19a 1 1747a 244 1095a 235 63a 7 60a 26 14a 2
19a 1633a 1603a 56a 56a 13a
1 173 343 5 19 1
0 mM K (N ¼ 7)
1 mM K (N ¼ 7)
10 mM K (N ¼ 6)
20a 1 e e 77a 7 e e
22a 1 e e 60a 4 e e
20a 1 e e 58a 5 e e
SC Shoot compartment, RC Root compartment. a Number of replicates. b Values (standard errors) within a row followed by the same letter do not differ significantly at p 0.05. 134
3. Results
3.3.
3.1. Plant and AMF growth variables
3.3.1. Effect of AMF on 134Cs uptake One week after labelling, 134Cs was detected in the roots and shoots of M. truncatula plants in all the treatments. The total 134Cs activity measured either in the plants (i.e. shoots plus roots), or separately in the shoots and roots, was significantly higher in the nonmycorrhizal plants as compared to the mycorrhizal ones (Table 4). The activity measured in shoots of the non-mycorrhizal plants accounted for 3.9% 0.4 of the total 134Cs supplied to the RCs, while it was only 2.3% 0.5 in mycorrhizal plants. Non-mycorrhizal roots also contained a significantly higher percentage (6.9% 0.5) of the activity initially applied, as compared to mycorrhizal roots (5.7% 0.4) (Fig. 1). Identically, the total 134Cs activity concentration (measured in Bq mg1 dry weight) was significantly higher in the plants (i.e. shoots plus roots) and in the shoots of the non-mycorrhizal plants as compared to the mycorrhizal plants, while no significant differences were observed for this parameter in roots between the mycorrhizal and non-mycorrhizal plants (Table 4).
Data on plant and AMF growth variables are presented in Table 2. At the end of the experiment (i.e. nine weeks after inoculation), M. truncatula plants were still growing actively and new root apexes as well as emerging leaves were observed in all the treatments. Spore germination and appressoria were observed within the first ten days following inoculation. Following root colonisation, the extraradical mycelium of the AMF started to develop profusely on the surface and within the MSR1 medium of the RCs. This mycelium was characterized by runner hyphae and lower order hyphae bearing spores, branched absorbing structures (BAS) and spores associated with these structures (BAS-spores). At the end of the experiment, no significant differences were observed between the treatments (i.e. mycorrhizal and nonmycorrhizal plants receiving MSR2 supplemented with 0, 1 or 10 mM K) for any of the plant growth variables (i.e. shoot and root dry weight). Regarding the mycorrhizal plants, no significant differences were observed in hyphal length, number of spores produced, %F and %I between the three concentrations of K (Table 2). 3.2. Potassium concentration in the plants Potassium concentration was measured in the plant tissues (i.e. roots and shoots separately) at the end of the experiment (i.e. nine weeks after inoculation). Average K concentration ranged from 5.23 to 6.34 mg per 100 mg in roots and 2.93 to 4.15 mg per 100 mg in shoots (Table 3). No significant difference was noted in the K concentration of roots or shoots, even though K concentration in roots was always higher than in shoots. Identically, no difference in K concentration was found between the different K-treatments in either the mycorrhizal or the nonmycorrhizal plants.
Table 3 Potassium concentration in the plants (mg per 100 mg dry weight in roots and shoots) grown in presence/absence of mycorrhizal fungi and under increasing concentrations of K, at the end of the experiment i.e. nine weeks after inoculation. Inoculation treatment
K concentration (mM)
Roots (mg per 100 mg)
Mycorrhizal
0 1 10 0 1 10
5.23 5.99 5.66 5.61 6.34 5.67
Non-mycorrhizal
0.49 0.41 0.47 0.64 0.33 0.35
Shoots (mg per 100 mg) 3.21 4.15 3.25 3.51 2.93 3.02
0.14 0.67 0.17 0.16 0.17 0.20
Cs uptake and partitioning in plants
3.3.2. Effect of K on 134Cs uptake Increase in K supplied to the RCs (i.e. ten ml of medium containing either 0, 1 or 10 mM K at labelling) significantly lowered plant 134Cs activity measured in shoots and roots (separately and combined for the whole plant), as well as 134Cs activity concentration in shoots. Radiocesium activity concentrations in the whole plant (i.e. roots and shoots combined) and in the roots separately were however not significantly affected by K levels in the RCs (Table 4). 3.3.3. Interaction effect of AMF and K on 134Cs uptake The activity of 134Cs measured in the plants (i.e. shoots plus roots), or shoots and roots separately, was not significantly influenced by either in the presence of AMF or by the supply of K in the RCs (Table 4). Identically, no significant differences were observed in the total plant or root 134Cs activity concentration. However, for the 134Cs activity concentration of shoots, the interaction effect of inoculation and K proved to be significant (p < 0.05; Table 4). 3.3.4. Root/shoot ratio of 134Cs Root/shoot ratio was significantly lower in the non-mycorrhizal plants as compared to the mycorrhizal plants, both as regards activity and activity concentration (Table 4). The effect of different K concentrations was not significant on root/shoot ratio. There was no interaction effect between inoculation and K concentrations on root/shoot ratio. 4. Discussion The few studies examining the role of AMF on radiocesium uptake and plant accumulation suggested either an increase or
594
V. Gyuricza et al. / Journal of Environmental Radioactivity 101 (2010) 591e596
Table 4 Total activity (Bq per plant) and activity concentration (Bq mg1 dry weight) in plants grown in presence/absence of mycorrhizal fungi and under increasing concentrations of K measured by gamma-counting one week after labelling i.e. nine weeks after inoculation. Inoculation treatment
K concentration (mM)
Total plant activity (Bq)
Mycorrhizal
0 1 10 0 1 10
458 340 424 725 539 470
Inoculation Potassium Inoculation Potassium
Non-mycorrhizal
Significance ofb
Activity in shoots (Bq)
Activity in roots (Bq)
66a 26 43 86 43 26
127 32 96 16 135 22 210 20 174 25 173 19
332 245 289 436 366 297
p < 0.001c p < 0.01 NS
p < 0.001 p < 0.05 NS
p < 0.05 p < 0.05 NS
42 17 28 73 25 11
Total activity concentration (Bq mg1 dry weight) 5.23 4.55 5.72 7.00 6.66 6.15
0.70 0.55 0.71 0.63 0.62 0.3
p < 0.01 NS NS
Activity concentration in shoots (Bq mg1 dry weight)
Activity concentration in roots (Bq mg1 dry weight)
6.85 1.49 5.3 1.02 7.27 1.19 13.10 2.11 7.96 1.27 8.37 0.94
4.80 4.28 5.26 5.03 6.21 5.19
p < 0.001 p < 0.01 p < 0.05
NS NS NS
0.62 0.52 0.47 0.63 0.69 0.49
Root/shoot ratio (total activity) 3.59 3.01 2.41 1.64 2.69 1.87
0.63 0.43 0.32 0.38 0.73 0.30
p < 0.05 NS NS
Root/shoot ratio (activity conc.) 0.97 0.93 0.83 0.55 0.76 0.64
0.20 0.15 0.12 0.19 0.11 0.48
p < 0.05 NS NS
NS Not significant. a Values correspond to the means the standard errors (SE) of the replicates. b By analysis of variance. c P value obtained by the analysis of variance.
a decrease and even no change at all in plant radiocesium accumulation (see Dupré de Boulois et al., 2008 for review). These divergent results could be attributed to several factors such as internal and external concentration of K, a chemical analogue of radiocesium. For instance in K-starved wheat plants the radiocesium influx rates increased by a factor of 10 compared with nonstarved plants (Zhu et al., 2000). Potassium is therefore considered to be a key-element affecting plant radiocesium accumulation (Zhu and Shaw, 2000), but its role in mycorrhizal plants remains to be elucidated. Here we observed that increased concentrations of K reduced the accumulation of radiocesium both in mycorrhizal and non-mycorrhizal M. truncatula plants under controlled in vitro culture conditions. Our results also suggest that inoculation with AMF in combination with increased K supply could play a key role in the reduction of plant radiocesium acquisition and accumulation and that inoculation could restrict its translocation to plant shoots. Even though we observed an uptake of radiocesium and translocation from roots to shoots both in the non-mycorrhizal and mycorrhizal plants, the latter had a significantly lower total activity and activity concentration of radiocesium. This tends to
Percentage of radiocaesium in different plant parts 8
% of total supplied radiocesium
7
b a
Mycorrhizal Non-mycorrhizal
6
b
5 4
a
3 2 1 0 Roots
Shoots
Fig. 1. Percentage of radiocesium of the total amount supplied in roots and shoots of mycorrhizal and non-mycorrhizal plants (regardless of K supply) measured by gammacounting one week after labelling i.e. nine weeks after inoculation. Significant differences at p < 0.05 are identified with a different letter in each plant part.
support the limited contribution of the extraradical mycelium to radiocesium uptake (Dupré de Boulois et al., 2008). However, the mechanisms preventing the uptake and accumulation of radiocesium within the mycorrhizal M. truncatula plants remain unclear. It has been suggested that the extraradical mycelium might be able to accumulate radiocesium (Berreck and Haselwandter, 2001) by the same mechanisms described for the adsorption (Joner et al., 2000) and sequestration (Cornejo et al., 2008; González-Chávez et al., 2004) of several heavy metals. However, Dupré de Boulois et al. (2006) showed that radiocesium accumulation by the extraradical phase of AMF is limited. Another hypothesis is a mechanism of down-regulation of gene expression of K-transporters in the presence of AMF, as it was already observed for other genes encoding P and N transporters (Javot et al., 2007). For instance, Burleigh et al., (2003) demonstrated a down-regulation of the Zn expression in roots of M. truncatula colonised by AMF. This resulted in a reduced level of Zn in both roots and shoots, even though AMF probably participated in plant Zn acquisition. Although our research does not investigate these mechanisms, the results suggest a tendency whereby AMF might be more likely to influence plant radiocesium uptake at low K concentrations (Table 4). This might be through an influence on the expression of genes encoding for the high affinity K (HAK) transporters, which also mediate radiocesium uptake in plant roots (White and Broadley, 2000; Zhu and Smolders, 2000). Earlier studies demonstrated that, while the voltage-insensitive monovalent-cation (VIC) channels are not affected by K concentration, at low concentration of Kþ (<0.25 mM), the uptake of radiocesium is mediated by HAK transporters and at a higher concentration (>1 mM) by K inwards rectifying (KIR) channels (White and Broadley, 2000; Zhu and Smolders, 2000). In this study, the higher root/shoot ratios (both for total activity and activity concentration) indicate reduced root to shoot translocation of radiocesium in mycorrhizal plants as compared to the non-mycorrhizal controls. As suggested by Dupré de Boulois et al. (2005) this reduced translocation could be caused by abscisic acid (ABA), a hormone that has been reported in higher concentrations in mycorrhizal roots (Bothe et al., 1994; Danneberg et al., 1993; Ludwig-Müller, 2000) and might hinder radiocesium loading into the xylem by influencing the expression or activity of K outwards rectifying (KOR) channels which mediate the majority of K and radiocesium efflux from the root cells (White and Broadley, 2000; Zhu and Smolders, 2000). Indeed, Roberts (1998) and Roberts and Snowman (2000)
V. Gyuricza et al. / Journal of Environmental Radioactivity 101 (2010) 591e596
demonstrated that ABA significantly reduced root to shoot K translocation, explained later by the reduced expression of gene encoding for KOR channels (Ache et al., 2000; Becker et al., 2003; Gaymard et al., 1998; Pilot et al., 2003). It is also worth noticing that the HAK transporters and KIR channels previously mentioned both appear insensitive to increasing root ABA levels (Roberts, 1998; Pilot et al., 2003). The availability of K in soil affects the accumulation of radiocesium in plants (see Zhu and Smolders, 2000 for a review). We found that additional K resulted in a lower accumulation of radiocesium in M. truncatula plants, but that root to shoot radiocesium translocation was not affected. Previous studies have shown (e.g. White and Broadley, 2000; Zhu and Smolders, 2000) that K can significantly influence radiocesium root to shoot translocation, contrary to our findings. It has been observed that at low K availability, the efflux of K/Cs towards the xylem is limited to prevent K release from root cells. This is probably due to the KOR channels, which have a probability of openness close to zero when internal K concentration is low (Maathuis and Sanders, 1997). In addition, Pilot et al. (2003) observed that in K-starved plants, the expression of genes encoding for KOR channels in Arabidopsis thaliana was reduced. Therefore, when low K availability results in a low internal K concentration, the limited efflux of radiocesium to the xylem would lead to the local accumulation of radiocesium in the roots (Zhu et al., 1999). This also corroborates the work of Buysse et al. (1996) and Smolders et al. (1996), showing that root to shoot radiocesium concentration ratio increases with decreasing K supply. As it was mentioned, we did not detect any effect of K on the root to shoot translocation of radiocesium. This can be explained by the identical and satisfactory supply of K in all treatments until 134 Cs labelling, with the result that all the plants were non-Kdeficient by the end of the experiment (based on Fink, 1991). Therefore, it can be suggested that radiocesium root to shoot translocation was not affected by the K concentration of the medium, due to the high internal K concentration of all the plants in the different treatments. To conclude, we demonstrated that AMF root colonisation could reduce radiocesium accumulation in the model legume M. truncatula, and restrict root to shoot radiocesium translocation (by a factor of 2 under low external K concentration). In these non-Kdeficient plants, increasing external K concentration affected the accumulation of radiocesium both in mycorrhizal and non-mycorrhizal plants but did not affect radiocesium root to shoot translocation. Moreover, root colonisation by AMF had a more pronounced effect on plant radiocesium accumulation than an increase in K supply. Combining K application and AMF inoculation would thus lead to the reduced uptake of radiocesium (effect of AMF and K) and radiocesium root to shoot translocation (effect of AMF). AMF could thus participate in the improvement of phytomanagement of radiocesium contaminated areas. Finally, the in vitro system used proved to be an efficient tool to investigate the effects of AMF on radiocesium accumulation and to study the mechanisms underlying this process. Acknowledgements This research project was sponsored by a Marie Curie Early stage Research Training Fellowship of the European Community’s Sixth Framework Programme under contract number MEST-CT-2005020387. HDdB acknowledges the FRS-FNRS grant as a Research Fellow (Chargé de Recherches). We would like to thank Eric Smolders for the potassium analysis and Jurgen Buekers for his practical help. We also thank Anne Bol for her assistance with gamma-radiation measurements.
595
References Ache, P., Becker, D., Ivashikina, N., Dietrich, P., Roelfsema, M.R.G., Hedrich, R., 2000. GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a Kþ-selective, Kþ-sensing ion channel. FEBS Letters 486, 93e98. Becker, D., Hoth, S., Ache, P., Wenkel, S., Roelfsema, M.R., Meyerhoff, O., et al., 2003. Regulation of the ABA-sensitive Arabidopsis potassium channel gene GORK in response to water stress. FEBS Letters 554, 119e126. Berreck, M., Haselwandter, K., 2001. Effect of the arbuscular mycorrhizal symbiosis upon uptake of cesium and other cations by plants. Mycorrhiza 10, 275e280. Bothe, H., Klingner, A., Kaldorf, M., Schmitz, O., Esch, H., Hundeshagen, B., Kernebeck, H., 1994. Biochemical approaches to the study of plant-fungal interactions in arbuscular mycorrhiza. Experientia 50, 919e925. Burleigh, S.H., Kristensen, B.K., Bechmann, I.E., 2003. A plasma membrane zinc transporter from Medicago truncatula is up-regulated in roots by Zn fertilization, yet down-regulated by arbuscular mycorrhizal colonisation. Plant Molecular Biology 52, 1077e1088. Buysse, J., VandenBrande, K., Merckx, R., 1996. Genotypic differences in the uptake and distribution of radiocaesium in plant. Plant and Soil 178, 265e271. Camps, M., Rigol, A., Vidal, M., Rauret, G., 2003. Assessment of the suitability of soil amendments to reduce Cs-137 and Sr-90 root uptake in meadows. Environmental Science and Technology 37, 2820e2828. Ciuffo, L., Velasco, H., Belli, M., Sansone, U., 2003. Cs-137 soil-to-plant transfer for individual species in a semi-natural grassland. Influence of potassium soil content. Journal of Radiation Research 44, 277e283. Cornejo, P., Meiera, S., Borie, G., Rillig, M.C., Borie, F., 2008. Glomalin-related soil protein in a Mediterranean ecosystem affected by a copper smelter and its contribution to Cu and Zn sequestration. Science of the Total Environment 406, 154e160. Cranenbrouck, S., Voets, L., Bivort, C., Renard, L., Strullu, D.G., Declerck, S., 2005. Methodologies for in vitro cultivation of arbuscular mycorrhizal fungi with root organs. In: Declerck, S., Strullu, D.G., Fortin, J.A. (Eds.), In Vitro Culture of Mycorrhizas. Springer, Berlin, pp. 341e375. Danneberg, G., Latus, C., Zimmer, W., Hundeshagen, B., Schneiderpoetsch, H., Bothe, H., 1993. Influence of vesicular-arbuscular mycorrhiza on phytohormone balances in maize (Zea mays L). Journal of Plant Physiology 141, 33e39. Declerck, S., Strullu, D.G., Plenchette, C., 1998. Monoxenic culture of the intraradical forms of Glomus sp. isolated from a tropical ecosystem: a proposed methodology for germplasm collection. Mycologia 90, 579e585. Declerck, S., D’Or, D., Cranenbrouck, S., Le Boulengé, E., 2001. Modelling the sporulation dynamics of arbuscular mycorrhizal fungi in monoxenic culture. Mycorrhiza 11, 225e230. Declerck, S., Dupré de Boulois, H., Bivort, C., Delvaux, B., 2003. Extraradical mycelium of the arbuscular mycorrhizal fungus Glomus lamellosum can take up, accumulate and translocate radiocaesium under root-organ culture conditions. Environmental Microbiology 5, 510e516. Delvaux, B., Kruyts, N., Maes, E., Smolders, E., 2001. Fate of radioceasium in soil and rhizopshere. In: Gobran, G.R., Wenzel, W.W., Lombi, E. (Eds.), Trace Elements in the Rhizosphere. CRC Press, Boca Raton, pp. 61e91. Dupré de Boulois, H., Delvaux, B., Declerck, S., 2005. Effects of arbuscular mycorrhizal fungi on the root uptake and translocation of radiocaesium. Environmental Pollution 134, 515e524. Dupré de Boulois, H., Joner, E.J., Leyval, C., Jakobsen, I., Chen, B.D., Roos, P., et al., 2008. Role and influence of mycorrhizal fungi on radiocesium accumulation by plants. Journal of Environmental Radioactivity 99, 775e784. Dupré de Boulois, H., Voets, L., Delvaux, B., Jakobsen, I., Declerck, S., 2006. Transport of radiocaesium by arbuscular mycorrhizal fungi to Medicago truncatula under in vitro conditions. Environmental Microbiology 8, 1926e1934. Eapen, S., Singh, S., Thorat, V., Kaushik, C.P., Raj, K., D’Souza, S.F., 2006. Phytoremediation of radiostrontium (Sr-90) and radiocesium (Cs-137) using giant milky weed (Calotropis gigantea R.Br.) plants. Chemosphere 65, 2071e2073. Entry, J.A., Watrud, N.S., Reeves, M., 1999. Accumulation of Cs-137 and Sr-90 from contaminated soil by three grass species inoculated with mycorrhizal fungi. Environmental Pollution 104, 449e457. Fink, A., 1991. Pflanzenernehrung in Stichworten. Hirt, Berlin. Fuhrmann, M., Lasat, M.M., Ebbs, S.D., Kochian, L.V., Cornish, J., 2002. Uptake of cesium-137 and strontium-90 from contaminated soil by three plant species; application to phytoremediation. Journal of Environmental Quality 31, 904e909. Gaymard, F., Pilot, G., Lacombe, B., Bouchez, D., Bruneau, D., Boucherez, J., et al., 1998. Identification and disruption of a plant shaker-like outward channel involved in Kþ release into the xylem sap. Cell 94, 647e655. González-Chávez, M.C., Carrillo-Gonzalez, R., Wright, S.F., Nichols, K.A., 2004. The role of glomalin, a protein produced by arbuscular mycorrhizal fungi, in sequestering potentially toxic elements. Environmental Pollution 130, 317e323. Guillitte, O., Tikhomirov, F.A., Shaw, G., Vetrov, V., 1994. Principles and practices of countermeasures to be carried out following radioactive contamination of forest areas. The Science of the Total Environment 157, 399e406. Gyuricza, V., Dupré de Boulois, H., Declerck, S., 2010. Effect of potassium and phosphorus on the transport of radiocesium by arbuscular mycorrhizal fungi. Journal of Environmental Radioactivity 101 (6), 482e487. Javot, H., Pumplin, N., Harrison, M.J., 2007. Phosphate in the arbuscular mycorrhizal symbiosis: transport properties and regulatory roles. Plant Cell and Environment 30, 310e322.
596
V. Gyuricza et al. / Journal of Environmental Radioactivity 101 (2010) 591e596
Joner, E.J., Briones, R., Leyval, C., 2000. Metal-binding capacity of arbuscular mycorrhizal mycelium. Plant and Soil 226, 227e234. Joner, E.J., Roos, P., Jansa, J., Frossard, E., Leyval, C., Jakobsen, I., 2004. No significant contribution of arbuscular mycorrhizal fungi to transfer of radiocesium from soil to plants. Applied and Environmental Microbiology 70, 6512e6517. Ludwig-Müller, J., 2000. Hormonal balance in plants during colonisation by mycorrhizal fungi. In: Douds, D.D., Kapulnik, Y. (Eds.), Arbuscular Mycorrhizas: Molecular Biology and Physiology. Kluwer Academic Publishers, Dordrecht, pp. 263e285. Maathuis, F.J.M., Sanders, D., 1997. Regulation of Kþ absorption in plant root cells by external Kþ: interplay of different plasma membrane Kþ transporters. Journal of Experimental Botany 48, 451e458. Nisbet, A.F., 1993. Effects of soil-based contermeasures on solid-liquid equilibria in agricultural soils contaminated with radiocaesium and radiostrontium. The Science of the Total Environment 137, 99e118. Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society 55, 158e161. Pilot, G., Gaymard, F., Mouline, K., Chérel, I., Sentenac, H., 2003. Regulated expression of Arabidopsis Shaker Kþ channel genes involved in Kþ uptake and distribution in the plant. Plant Molecular Biolology 51, 773e787. Plenchette, C., Morel, C., 1996. External phosphorus requirements of mycorrhizal and non-mycorrhizal barley and soybean plants. Biology and Fertility of Soils 21, 303e308. Roberts, S.K., 1998. Regulation of Kþ channels in maize roots by water stress and abscisic acid. Plant Physiology 116, 145e153. Roberts, S.K., Snowman, B.N., 2000. The effects of ABA on channel-mediated Kþ transport across higher plant roots. Journal of Experimental Botany 51, 1585e1594. Rogers, R.D., Williams, S.E., 1986. Vesicular-arbuscular mycorrhiza: influence on plant uptake of cesium and cobalt. Soil Biology and Biochemistry 18, 371e376.
Sandeep, S., Manjaiah, K.M., 2008. Transfer factors of 134Cs to crops from Typic Haupslept under tropical region as influenced by potassium application. Journal of Environmental Radioactivity 99, 349e358. Shaw, G.,1993. Blockade by fertilizers of caesium and strontium uptake in crops: effects on the root uptake process. The Science of the Total Environment 137, 119e133. Smolders, E., Kiebooms, L., Buysse, J., Merckx, R., 1996. Cs-167 uptake in spring wheat (Trticum aestivum L. cv. Tonic) at varying K supply: I. The effect in soil solution culture. Plant and Soil 181, 205e209. Vidal, M., Camps, M., Grebenshikova, N., Sanzharova, N., Ivanov, Y., Vandecasteele, C., Shand, C., Rigol, A., Firsakova, S., Fesenki, S., Levchuk, S., Cheshire, S., Sauras, T., Rauget, G., 2001. Soil- and plant-based countermeasures to reduce Cs-137 and Sr-90 uptake by grasses in natural meadows: the REDUP project. Journal of Environmental Radioactivity 56, 139e156. White, P.J., Broadley, M.R., 2000. Mechanisms of caesium uptake by plants. New Phytologist 147, 241e256. White, P.J., Swarup, K., Escobar-Gutierrez, A.J., Bowen, H.C., Willey, N.J., Broadley, M. R., 2003. Selecting plants to minimise radiocaesium in the food chain. Plant and Soil 249, 177e186. Zhu, Y.G., 2001. Effect of external potassium (K) supply on the uptake of Cs-137 by spring wheat (Triticum aestivum cv. Tonic): a large-scale hydroponic study. Journal of Environmental Radioactivity 55, 303e314. Zhu, Y.G., Shaw, G., 2000. Soil contamination with radionuclides and potential remediation. Chemosphere 41, 121e128. Zhu, Y.G., Shaw, G., Nisbet, A.F., Wilkins, B.T., 1999. Effects of external potassium supply on compartmentation and flux characteristics of radiocaesium in spring wheat roots. Annals of Botany 84, 639e644. Zhu, Y.G., Shaw, G., Nisbet, A.F., Wilkins, B.T., 2000. Effect of potassium starvation on the uptake of radiocesium by spring wheat (Triticum aestivum cv. Tonic). Plant and Soil 220, 27e34. Zhu, Y.G., Smolders, E., 2000. Plant uptake of radiocaesium: a review of mechanisms, regulation and application. Journal of Experimental Botany 51, 1635e1645.
Contents lists available at ScienceDirect
Journal of Environmental Radioactivity journal homepage: www.elsevier.com/locate/jenvrad
Arbuscular mycorrhizal fungi decrease radiocesium accumulation in Medicago truncatula Veronika Gyuricza, Stéphane Declerck, Hervé Dupré de Boulois* Université catholique de Louvain, Earth and Life Institute (ELI), Laboratoire de Mycologie, Croix du Sud 3, 1348 Louvain-la-Neuve, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 October 2009 Received in revised form 5 March 2010 Accepted 5 March 2010 Available online 7 April 2010
The role of arbuscular mycorrhizal fungi (AMF) in plant radiocesium uptake and accumulation remains ambiguous. This is probably due to the presence of other soil microorganisms, the variability of soil characteristics and plant nutritional status or the availability of its chemical analogue, potassium (K). Here, we used an in vitro culture system to study the impact of increased concentration of K on radiocesium accumulation in non K-starved mycorrhizal and non-mycorrhizal Medicago truncatula plants. In the presence of AMF radiocesium uptake decreased regardless of the concentration of K, and its translocation from root to shoot was also significantly lower. Potassium also reduced the accumulation of radiocesium in plants but to a lesser extent than mycorrhization, and without any effect on translocation. These results suggest that AMF in combination with K can play a key role in reducing radiocesium uptake and its subsequent translocation to plant shoots, thereby representing good potential for improved phytomanagement of contaminated areas. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Radiocesium Potassium Arbuscular mycorrhizal fungi In vitro Phytostabilisation
1. Introduction Radiocesium represents a major threat to terrestrial ecosystems as it can remain in the upper soil horizons for decades (Delvaux et al., 2001). Plant contamination results from root access to this pool of radiocesium and from the uptake mechanisms similar to that of potassium (White and Broadley, 2000; Zhu and Smolders, 2000). Physical countermeasures such as soil removal (Guillitte et al., 1994) and the application of soil amendments have been used to limit plant uptake of radiocesium (Ciuffo et al., 2003; Sandeep and Manjaiah, 2008; Shaw, 1993; Zhu and Shaw, 2000). However, these methods can be expensive and are often inadequate (Camps et al., 2003; Vidal et al., 2001; Zhu and Shaw, 2000). Therefore other methods, such as extraction of radiocesium by plants, were widely considered (e.g. Eapen et al., 2006; Fuhrmann et al., 2002). However, phytoextraction of radiocesium may be unsuitable for agricultural lands used for food production (White et al., 2003) and for these reasons the production of selected or genetically-engineered “safe” crops that do not accumulate radiocesium or could participate in its stabilisation was suggested by White et al. (2003) in combination with K-fertilisation. Several studies reported the potential of plant-associated microorganisms to restrict acquisition and/or accumulation of
* Corresponding author. Tel.: þ32 (0) 10 47 22 26; fax: þ32 (0) 10 45 15 01. E-mail address: [email protected] (H. Dupré de Boulois). 0265-931X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvrad.2010.03.004
radiocesium in plants (Dupré de Boulois et al., 2008). Among the soil microflora, arbuscular mycorrhizal fungi (AMF) have been considered in several studies on radiocesium, but an unequivocal conclusion on the behaviour of this element in mycorrhizal plants could not be reached. Authors either reported a reduced transfer of radiocesium from soil to plants (e.g. Berreck and Haselwandter, 2001), or an enhanced accumulation (e.g. Entry et al., 1999) or even no change (e.g. Rogers and Williams, 1986) in the presence of AMF. These apparently conflicting results could be attributed to plant and AMF species, to the presence of other microorganisms, to plant nutritional status and to the characteristics of the soil used in the different experiments (Dupré de Boulois et al., 2008) among other factors. Recently, Declerck et al. (2003); Dupré de Boulois et al. (2005, 2006) and Gyuricza et al. (2010) investigated the capacity of AMF to transport radiocesium under strict in vitro culture conditions. These authors demonstrated that AMF could take up, translocate and transfer radiocesium from a root-free labelled compartment to their host via their extraradical mycelial network. However, this transport was low and root to shoot translocation of radiocesium was limited. This suggested that even though AMF can transport radiocesium to their host plants, their participation in plant radiocesium accumulation is probably negligible. This corroborates earlier results by Joner et al. (2004) for pot experiments conducted with different substrates, host plants and AMF species. In parallel to these findings, Berreck and Haselwandter (2001) hypothesized that AMF could restrict direct root uptake through the accumulation of radiocesium within
592
V. Gyuricza et al. / Journal of Environmental Radioactivity 101 (2010) 591e596
extraradical structures. However, Dupré de Boulois et al. (2005, 2006) showed that this accumulation was limited. These authors therefore proposed another hypothesis according to which AMF could restrict radiocesium plant acquisition by modifying the expression or activity of transporters involved in plant radiocesium uptake. It was also suggested that AMF could play a role in radiocesium root to shoot translocation by restricting its transport towards or its loading within the xylem. Among the environmental factors affecting plant radiocesium acquisition, potassium (K) has been shown to decrease radiocesium root uptake under hydroponic conditions (see Zhu, 2001 and Zhu et al., 2000). Soil based studies have been less conclusive, which is understandable given the wide range of soils investigated and the various treatments applied (Nisbet, 1993) and possibly also the differences in plant K status (see Zhu et al., 2000). The studies on transporters responsible for radiocesium influx into root cells further supported the role of K in radiocesium uptake by plant roots (see White and Broadley, 2000 and Zhu and Smolders, 2000). For mycorrhizal plants the role of K was only considered in a soil experiment where the long-term effects of fertilization were investigated (Berreck and Haselwandter, 2001). Our study aims to examine the effects of the increased supply of K on radiocesium uptake and partitioning within plants in the presence or absence of AMF, under strict in vitro culture conditions. 2. Materials and methods 2.1. Biological material A strain of Glomus intraradices Schenck and Smith (MUCL 43194) grown in association with Ri T-DNA transformed carrot roots (Daucus carota L.) was purchased from GINCO (http://www.mbla.ucl.ac.be/ginco-bel). The AMF strain was delivered in a Petri plate (90 mm diameter) on the modified Strullu-Romand (MSR) medium (Declerck et al., 1998), solidified with 3 g l1 GelGroÔ (ICN, Biomedicals, Irvine, CA, USA). The mineral composition of the MSR medium is given in Table 1 (see mineral composition of MSR1 medium) but included sucrose and vitamins. The AM fungus was subsequently subcultured following the methodology of Cranenbrouck et al. (2005). Seeds of Medicago truncatula L., cv. Jemalong A17 (SARDI, Adelaide, Australia) were surface-sterilized by immersion in sodium hypochlorite (containing 5% active chloride) for 10 min, rinsed 3 times in sterile de-ionized water and germinated in groups of 10 in Petri plates (90 mm diameter) containing 40 ml of MSR1 medium (Table 1) solidified with 3 g l1 GelGroÔ. Germination was conducted in an incubator set at 27 C in the dark for 8 days.
2.2. Experimental design Eight-day old M. truncatula plantlets with shoots approximately 50 mm long and primary roots approximately 70 mm long, with two to five secondary roots, were selected for transplantation into in vitro culture systems. These systems consisted of two connected compartments: a shoot compartment (SC), where the shoot
Table 1 Mineral composition of the culture media used for germination and subsequent cultivation of plants before (MSR1) and during (MSR2) labelling.
Macroelements
Microelements
a
KNO3 MgSO4$7H2O K2PO4$2H2O NaH2PO4.2H2O Ca(NO3)2$4H2O KCl NaFeEDTA MnSO4$4H2O ZnSO4$7H2O H3BO3 CuSO4$5H2O (NH4)6Mo7O24$4H2O NaMoO4$2H2O
MSR1
MSR2
76 739 4.1 e 359 65 8 24.5 2.8 18.6 2.2 0.34 0.024
e 739 e 4.7 359 0, 74.4 or 744a 8
Amount varies according to treatmente see text for details.
developed, and a root compartment (RC), where roots developed, associated or not with an AMF (Dupré de Boulois et al., 2006). Shoots of M. truncatula plantlets were inserted through the opening between the RC and the SC. The roots were placed in the RC on the surface of 30 ml MSR1 medium (Table 1), solidified with 3 g l1 GelGroÔ. The initial concentration of K in the MSR1 medium was 1.65 mM (0.192 mg in 30 ml) added in the form of KH2PO4$2H2O, KNO3 and KCl. In half of the in vitro culture systems, the roots of the M. truncatula plantlets were inoculated with spores (100) of G. intraradices, while the other half was not inoculated. Inoculation was performed following the method described by Cranenbrouck et al. (2005). The in vitro culture systems were then sealed using cellophane film and the RCs were wrapped in black plastic bags to maintain roots and AMF in the dark. The culture systems were incubated in a controlled growth chamber set at 22/18 C (day/night) with a 16/8 h photoperiod. Daylight lamps provided an average photosynthetic photon flux at the level of the SCs of 225 mmol m2s1. To ensure adequate plant and fungal growth, 10 ml MSR1 medium, solidified with 3 g l1 GelGroÔ was added in the RCs of the in vitro culture systems 3 and 6 weeks after the start of the experiment. The medium was added at a temperature of approximately 35 C to avoid damage to the roots and AMF. The plants therefore received a total of 0.32 mg of K (in the form of KH2PO4$2H2O, KNO3 and KCl) before adding the medium that contained three different concentrations of K. 2.3.
134
Cs labelling
Eight weeks after the beginning of the experiment, a profuse mycelium had grown within and on the surface of the medium contained in the RCs. Ten ml of MSR2 medium (Table 1) solidified with 3 g l1 GelGroÔ was added to the RCs following the same method as above. This medium was either free of K (0 mM) or supplemented with KCl with a concentration of 1 or 10 mM of K. Therefore, the mycorrhizal and non-mycorrhizal plants received 0, 0.744 or 7.44 mg K. Six to eight replicates were used per treatment. After solidification of the MSR2 medium, filter-sterilized (AcrodiscÒ Syringe Filters, PALL Corporation, Ann Arbor, MI, USA) radiocesium (134Cs) was applied to the surface of the medium. Fifty ml 134Cs droplets (altogether 200 ml) were distributed with a micropipette on the whole surface of the medium with a total activity of 5050 50 Bq. After 48 h the 134Cs was uniformly distributed in the medium (based on a preliminary experiment where 134Cs was evenly distributed in the medium after 48 h e data not shown). The source of 134Cs was in the form of CsNO3 in water, supplied by the laboratory of nuclear chemistry (CMAT, UCL, Louvain-la-Neuve, Belgium). 2.4. Harvest and planteAMF analyses Before labelling, the total extraradical hyphal length and the number of spores in the RCs were estimated. The extraradical hyphal length was determined as detailed in Declerck et al. (2003), while spore count followed the methodology described in Declerck et al. (2001). One week after the addition of 134Cs (i.e. 9 weeks after inoculation), the M. truncatula shoots were collected. The roots were removed gently from the RCs and cleaned from the remaining solidified medium using citrate buffer (0.01 M). The medium was collected subsequently. All samples (i.e. shoots, mycorrhizal and nonmycorrhizal roots and medium with or without extraradical mycelium of AMF) were subjected to 134Cs counting on a Wallac 1480 Gamma Counter (Wallac, Turku, Finland). After counting, the roots and shoots were dried at 70 C to a constant weight and their dry weight was measured. Root colonisation of the mycorrhizal plants was estimated. The root subsamples were cleared in 10% KOH and stained with 0.2% Trypan blue following the method of Phillips and Hayman (1970). Roots were cut into 10 mm fragments and 30 randomly selected segments were examined under a compound microscope (Olympus BH2, Olympus Optical (Europa) GmbH, Hamburg, Germany) at 50e250 magnifications, to evaluate the frequency (%F) and intensity (%I) of AMF colonisation (Plenchette and Morel, 1996). 2.5. Potassium concentration in the plants Plant shoots and root subsamples were dried to a constant weight and digested by boiling aqua regia (concentrated nitric acid and hydrochloric acid in 1:3 ratio). Potassium concentrations in the extracts were determined with ICPOES (Perkin Elmer Optima 3300 DV, Waltham, MA, USA). Plant reference samples of known K concentration were also digested concomitantly with the samples as internal control. 2.6. Statistical analyses Data normally distributed and having homogeneous variances were subjected to analysis of variance (ANOVA). One-way ANOVA was used to compare plant and fungal variables in different groups and two-way ANOVA to compare mycorrhizal status and K levels.
V. Gyuricza et al. / Journal of Environmental Radioactivity 101 (2010) 591e596
593
Table 2 Plant and AMF growth variables in presence/absence of AMF and under increasing concentrations of K. Hyphal length and number of spores were estimated before labelling, i.e. eight weeks after inoculation while other variables (shoot dry weight (SDW), root dry weight (RDW), and Frequency (%F) and Intensity (%I) of root colonisation) were measured after labelling, i.e. nine weeks after inoculation. Mycorrhizal 0 mM K (N ¼ 8) SC RC
SDW (mg) Hyphal length (cm) Number of spores RDW (mg) %F %I
18a 1503a 1314a 70a 40a 15a
a
2b 353 251 6 9 2
Non-mycorrhizal
1 mM K (N ¼ 8)
10 mM K (N ¼ 8)
19a 1 1747a 244 1095a 235 63a 7 60a 26 14a 2
19a 1633a 1603a 56a 56a 13a
1 173 343 5 19 1
0 mM K (N ¼ 7)
1 mM K (N ¼ 7)
10 mM K (N ¼ 6)
20a 1 e e 77a 7 e e
22a 1 e e 60a 4 e e
20a 1 e e 58a 5 e e
SC Shoot compartment, RC Root compartment. a Number of replicates. b Values (standard errors) within a row followed by the same letter do not differ significantly at p 0.05. 134
3. Results
3.3.
3.1. Plant and AMF growth variables
3.3.1. Effect of AMF on 134Cs uptake One week after labelling, 134Cs was detected in the roots and shoots of M. truncatula plants in all the treatments. The total 134Cs activity measured either in the plants (i.e. shoots plus roots), or separately in the shoots and roots, was significantly higher in the nonmycorrhizal plants as compared to the mycorrhizal ones (Table 4). The activity measured in shoots of the non-mycorrhizal plants accounted for 3.9% 0.4 of the total 134Cs supplied to the RCs, while it was only 2.3% 0.5 in mycorrhizal plants. Non-mycorrhizal roots also contained a significantly higher percentage (6.9% 0.5) of the activity initially applied, as compared to mycorrhizal roots (5.7% 0.4) (Fig. 1). Identically, the total 134Cs activity concentration (measured in Bq mg1 dry weight) was significantly higher in the plants (i.e. shoots plus roots) and in the shoots of the non-mycorrhizal plants as compared to the mycorrhizal plants, while no significant differences were observed for this parameter in roots between the mycorrhizal and non-mycorrhizal plants (Table 4).
Data on plant and AMF growth variables are presented in Table 2. At the end of the experiment (i.e. nine weeks after inoculation), M. truncatula plants were still growing actively and new root apexes as well as emerging leaves were observed in all the treatments. Spore germination and appressoria were observed within the first ten days following inoculation. Following root colonisation, the extraradical mycelium of the AMF started to develop profusely on the surface and within the MSR1 medium of the RCs. This mycelium was characterized by runner hyphae and lower order hyphae bearing spores, branched absorbing structures (BAS) and spores associated with these structures (BAS-spores). At the end of the experiment, no significant differences were observed between the treatments (i.e. mycorrhizal and nonmycorrhizal plants receiving MSR2 supplemented with 0, 1 or 10 mM K) for any of the plant growth variables (i.e. shoot and root dry weight). Regarding the mycorrhizal plants, no significant differences were observed in hyphal length, number of spores produced, %F and %I between the three concentrations of K (Table 2). 3.2. Potassium concentration in the plants Potassium concentration was measured in the plant tissues (i.e. roots and shoots separately) at the end of the experiment (i.e. nine weeks after inoculation). Average K concentration ranged from 5.23 to 6.34 mg per 100 mg in roots and 2.93 to 4.15 mg per 100 mg in shoots (Table 3). No significant difference was noted in the K concentration of roots or shoots, even though K concentration in roots was always higher than in shoots. Identically, no difference in K concentration was found between the different K-treatments in either the mycorrhizal or the nonmycorrhizal plants.
Table 3 Potassium concentration in the plants (mg per 100 mg dry weight in roots and shoots) grown in presence/absence of mycorrhizal fungi and under increasing concentrations of K, at the end of the experiment i.e. nine weeks after inoculation. Inoculation treatment
K concentration (mM)
Roots (mg per 100 mg)
Mycorrhizal
0 1 10 0 1 10
5.23 5.99 5.66 5.61 6.34 5.67
Non-mycorrhizal
0.49 0.41 0.47 0.64 0.33 0.35
Shoots (mg per 100 mg) 3.21 4.15 3.25 3.51 2.93 3.02
0.14 0.67 0.17 0.16 0.17 0.20
Cs uptake and partitioning in plants
3.3.2. Effect of K on 134Cs uptake Increase in K supplied to the RCs (i.e. ten ml of medium containing either 0, 1 or 10 mM K at labelling) significantly lowered plant 134Cs activity measured in shoots and roots (separately and combined for the whole plant), as well as 134Cs activity concentration in shoots. Radiocesium activity concentrations in the whole plant (i.e. roots and shoots combined) and in the roots separately were however not significantly affected by K levels in the RCs (Table 4). 3.3.3. Interaction effect of AMF and K on 134Cs uptake The activity of 134Cs measured in the plants (i.e. shoots plus roots), or shoots and roots separately, was not significantly influenced by either in the presence of AMF or by the supply of K in the RCs (Table 4). Identically, no significant differences were observed in the total plant or root 134Cs activity concentration. However, for the 134Cs activity concentration of shoots, the interaction effect of inoculation and K proved to be significant (p < 0.05; Table 4). 3.3.4. Root/shoot ratio of 134Cs Root/shoot ratio was significantly lower in the non-mycorrhizal plants as compared to the mycorrhizal plants, both as regards activity and activity concentration (Table 4). The effect of different K concentrations was not significant on root/shoot ratio. There was no interaction effect between inoculation and K concentrations on root/shoot ratio. 4. Discussion The few studies examining the role of AMF on radiocesium uptake and plant accumulation suggested either an increase or
594
V. Gyuricza et al. / Journal of Environmental Radioactivity 101 (2010) 591e596
Table 4 Total activity (Bq per plant) and activity concentration (Bq mg1 dry weight) in plants grown in presence/absence of mycorrhizal fungi and under increasing concentrations of K measured by gamma-counting one week after labelling i.e. nine weeks after inoculation. Inoculation treatment
K concentration (mM)
Total plant activity (Bq)
Mycorrhizal
0 1 10 0 1 10
458 340 424 725 539 470
Inoculation Potassium Inoculation Potassium
Non-mycorrhizal
Significance ofb
Activity in shoots (Bq)
Activity in roots (Bq)
66a 26 43 86 43 26
127 32 96 16 135 22 210 20 174 25 173 19
332 245 289 436 366 297
p < 0.001c p < 0.01 NS
p < 0.001 p < 0.05 NS
p < 0.05 p < 0.05 NS
42 17 28 73 25 11
Total activity concentration (Bq mg1 dry weight) 5.23 4.55 5.72 7.00 6.66 6.15
0.70 0.55 0.71 0.63 0.62 0.3
p < 0.01 NS NS
Activity concentration in shoots (Bq mg1 dry weight)
Activity concentration in roots (Bq mg1 dry weight)
6.85 1.49 5.3 1.02 7.27 1.19 13.10 2.11 7.96 1.27 8.37 0.94
4.80 4.28 5.26 5.03 6.21 5.19
p < 0.001 p < 0.01 p < 0.05
NS NS NS
0.62 0.52 0.47 0.63 0.69 0.49
Root/shoot ratio (total activity) 3.59 3.01 2.41 1.64 2.69 1.87
0.63 0.43 0.32 0.38 0.73 0.30
p < 0.05 NS NS
Root/shoot ratio (activity conc.) 0.97 0.93 0.83 0.55 0.76 0.64
0.20 0.15 0.12 0.19 0.11 0.48
p < 0.05 NS NS
NS Not significant. a Values correspond to the means the standard errors (SE) of the replicates. b By analysis of variance. c P value obtained by the analysis of variance.
a decrease and even no change at all in plant radiocesium accumulation (see Dupré de Boulois et al., 2008 for review). These divergent results could be attributed to several factors such as internal and external concentration of K, a chemical analogue of radiocesium. For instance in K-starved wheat plants the radiocesium influx rates increased by a factor of 10 compared with nonstarved plants (Zhu et al., 2000). Potassium is therefore considered to be a key-element affecting plant radiocesium accumulation (Zhu and Shaw, 2000), but its role in mycorrhizal plants remains to be elucidated. Here we observed that increased concentrations of K reduced the accumulation of radiocesium both in mycorrhizal and non-mycorrhizal M. truncatula plants under controlled in vitro culture conditions. Our results also suggest that inoculation with AMF in combination with increased K supply could play a key role in the reduction of plant radiocesium acquisition and accumulation and that inoculation could restrict its translocation to plant shoots. Even though we observed an uptake of radiocesium and translocation from roots to shoots both in the non-mycorrhizal and mycorrhizal plants, the latter had a significantly lower total activity and activity concentration of radiocesium. This tends to
Percentage of radiocaesium in different plant parts 8
% of total supplied radiocesium
7
b a
Mycorrhizal Non-mycorrhizal
6
b
5 4
a
3 2 1 0 Roots
Shoots
Fig. 1. Percentage of radiocesium of the total amount supplied in roots and shoots of mycorrhizal and non-mycorrhizal plants (regardless of K supply) measured by gammacounting one week after labelling i.e. nine weeks after inoculation. Significant differences at p < 0.05 are identified with a different letter in each plant part.
support the limited contribution of the extraradical mycelium to radiocesium uptake (Dupré de Boulois et al., 2008). However, the mechanisms preventing the uptake and accumulation of radiocesium within the mycorrhizal M. truncatula plants remain unclear. It has been suggested that the extraradical mycelium might be able to accumulate radiocesium (Berreck and Haselwandter, 2001) by the same mechanisms described for the adsorption (Joner et al., 2000) and sequestration (Cornejo et al., 2008; González-Chávez et al., 2004) of several heavy metals. However, Dupré de Boulois et al. (2006) showed that radiocesium accumulation by the extraradical phase of AMF is limited. Another hypothesis is a mechanism of down-regulation of gene expression of K-transporters in the presence of AMF, as it was already observed for other genes encoding P and N transporters (Javot et al., 2007). For instance, Burleigh et al., (2003) demonstrated a down-regulation of the Zn expression in roots of M. truncatula colonised by AMF. This resulted in a reduced level of Zn in both roots and shoots, even though AMF probably participated in plant Zn acquisition. Although our research does not investigate these mechanisms, the results suggest a tendency whereby AMF might be more likely to influence plant radiocesium uptake at low K concentrations (Table 4). This might be through an influence on the expression of genes encoding for the high affinity K (HAK) transporters, which also mediate radiocesium uptake in plant roots (White and Broadley, 2000; Zhu and Smolders, 2000). Earlier studies demonstrated that, while the voltage-insensitive monovalent-cation (VIC) channels are not affected by K concentration, at low concentration of Kþ (<0.25 mM), the uptake of radiocesium is mediated by HAK transporters and at a higher concentration (>1 mM) by K inwards rectifying (KIR) channels (White and Broadley, 2000; Zhu and Smolders, 2000). In this study, the higher root/shoot ratios (both for total activity and activity concentration) indicate reduced root to shoot translocation of radiocesium in mycorrhizal plants as compared to the non-mycorrhizal controls. As suggested by Dupré de Boulois et al. (2005) this reduced translocation could be caused by abscisic acid (ABA), a hormone that has been reported in higher concentrations in mycorrhizal roots (Bothe et al., 1994; Danneberg et al., 1993; Ludwig-Müller, 2000) and might hinder radiocesium loading into the xylem by influencing the expression or activity of K outwards rectifying (KOR) channels which mediate the majority of K and radiocesium efflux from the root cells (White and Broadley, 2000; Zhu and Smolders, 2000). Indeed, Roberts (1998) and Roberts and Snowman (2000)
V. Gyuricza et al. / Journal of Environmental Radioactivity 101 (2010) 591e596
demonstrated that ABA significantly reduced root to shoot K translocation, explained later by the reduced expression of gene encoding for KOR channels (Ache et al., 2000; Becker et al., 2003; Gaymard et al., 1998; Pilot et al., 2003). It is also worth noticing that the HAK transporters and KIR channels previously mentioned both appear insensitive to increasing root ABA levels (Roberts, 1998; Pilot et al., 2003). The availability of K in soil affects the accumulation of radiocesium in plants (see Zhu and Smolders, 2000 for a review). We found that additional K resulted in a lower accumulation of radiocesium in M. truncatula plants, but that root to shoot radiocesium translocation was not affected. Previous studies have shown (e.g. White and Broadley, 2000; Zhu and Smolders, 2000) that K can significantly influence radiocesium root to shoot translocation, contrary to our findings. It has been observed that at low K availability, the efflux of K/Cs towards the xylem is limited to prevent K release from root cells. This is probably due to the KOR channels, which have a probability of openness close to zero when internal K concentration is low (Maathuis and Sanders, 1997). In addition, Pilot et al. (2003) observed that in K-starved plants, the expression of genes encoding for KOR channels in Arabidopsis thaliana was reduced. Therefore, when low K availability results in a low internal K concentration, the limited efflux of radiocesium to the xylem would lead to the local accumulation of radiocesium in the roots (Zhu et al., 1999). This also corroborates the work of Buysse et al. (1996) and Smolders et al. (1996), showing that root to shoot radiocesium concentration ratio increases with decreasing K supply. As it was mentioned, we did not detect any effect of K on the root to shoot translocation of radiocesium. This can be explained by the identical and satisfactory supply of K in all treatments until 134 Cs labelling, with the result that all the plants were non-Kdeficient by the end of the experiment (based on Fink, 1991). Therefore, it can be suggested that radiocesium root to shoot translocation was not affected by the K concentration of the medium, due to the high internal K concentration of all the plants in the different treatments. To conclude, we demonstrated that AMF root colonisation could reduce radiocesium accumulation in the model legume M. truncatula, and restrict root to shoot radiocesium translocation (by a factor of 2 under low external K concentration). In these non-Kdeficient plants, increasing external K concentration affected the accumulation of radiocesium both in mycorrhizal and non-mycorrhizal plants but did not affect radiocesium root to shoot translocation. Moreover, root colonisation by AMF had a more pronounced effect on plant radiocesium accumulation than an increase in K supply. Combining K application and AMF inoculation would thus lead to the reduced uptake of radiocesium (effect of AMF and K) and radiocesium root to shoot translocation (effect of AMF). AMF could thus participate in the improvement of phytomanagement of radiocesium contaminated areas. Finally, the in vitro system used proved to be an efficient tool to investigate the effects of AMF on radiocesium accumulation and to study the mechanisms underlying this process. Acknowledgements This research project was sponsored by a Marie Curie Early stage Research Training Fellowship of the European Community’s Sixth Framework Programme under contract number MEST-CT-2005020387. HDdB acknowledges the FRS-FNRS grant as a Research Fellow (Chargé de Recherches). We would like to thank Eric Smolders for the potassium analysis and Jurgen Buekers for his practical help. We also thank Anne Bol for her assistance with gamma-radiation measurements.
595
References Ache, P., Becker, D., Ivashikina, N., Dietrich, P., Roelfsema, M.R.G., Hedrich, R., 2000. GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a Kþ-selective, Kþ-sensing ion channel. FEBS Letters 486, 93e98. Becker, D., Hoth, S., Ache, P., Wenkel, S., Roelfsema, M.R., Meyerhoff, O., et al., 2003. Regulation of the ABA-sensitive Arabidopsis potassium channel gene GORK in response to water stress. FEBS Letters 554, 119e126. Berreck, M., Haselwandter, K., 2001. Effect of the arbuscular mycorrhizal symbiosis upon uptake of cesium and other cations by plants. Mycorrhiza 10, 275e280. Bothe, H., Klingner, A., Kaldorf, M., Schmitz, O., Esch, H., Hundeshagen, B., Kernebeck, H., 1994. Biochemical approaches to the study of plant-fungal interactions in arbuscular mycorrhiza. Experientia 50, 919e925. Burleigh, S.H., Kristensen, B.K., Bechmann, I.E., 2003. A plasma membrane zinc transporter from Medicago truncatula is up-regulated in roots by Zn fertilization, yet down-regulated by arbuscular mycorrhizal colonisation. Plant Molecular Biology 52, 1077e1088. Buysse, J., VandenBrande, K., Merckx, R., 1996. Genotypic differences in the uptake and distribution of radiocaesium in plant. Plant and Soil 178, 265e271. Camps, M., Rigol, A., Vidal, M., Rauret, G., 2003. Assessment of the suitability of soil amendments to reduce Cs-137 and Sr-90 root uptake in meadows. Environmental Science and Technology 37, 2820e2828. Ciuffo, L., Velasco, H., Belli, M., Sansone, U., 2003. Cs-137 soil-to-plant transfer for individual species in a semi-natural grassland. Influence of potassium soil content. Journal of Radiation Research 44, 277e283. Cornejo, P., Meiera, S., Borie, G., Rillig, M.C., Borie, F., 2008. Glomalin-related soil protein in a Mediterranean ecosystem affected by a copper smelter and its contribution to Cu and Zn sequestration. Science of the Total Environment 406, 154e160. Cranenbrouck, S., Voets, L., Bivort, C., Renard, L., Strullu, D.G., Declerck, S., 2005. Methodologies for in vitro cultivation of arbuscular mycorrhizal fungi with root organs. In: Declerck, S., Strullu, D.G., Fortin, J.A. (Eds.), In Vitro Culture of Mycorrhizas. Springer, Berlin, pp. 341e375. Danneberg, G., Latus, C., Zimmer, W., Hundeshagen, B., Schneiderpoetsch, H., Bothe, H., 1993. Influence of vesicular-arbuscular mycorrhiza on phytohormone balances in maize (Zea mays L). Journal of Plant Physiology 141, 33e39. Declerck, S., Strullu, D.G., Plenchette, C., 1998. Monoxenic culture of the intraradical forms of Glomus sp. isolated from a tropical ecosystem: a proposed methodology for germplasm collection. Mycologia 90, 579e585. Declerck, S., D’Or, D., Cranenbrouck, S., Le Boulengé, E., 2001. Modelling the sporulation dynamics of arbuscular mycorrhizal fungi in monoxenic culture. Mycorrhiza 11, 225e230. Declerck, S., Dupré de Boulois, H., Bivort, C., Delvaux, B., 2003. Extraradical mycelium of the arbuscular mycorrhizal fungus Glomus lamellosum can take up, accumulate and translocate radiocaesium under root-organ culture conditions. Environmental Microbiology 5, 510e516. Delvaux, B., Kruyts, N., Maes, E., Smolders, E., 2001. Fate of radioceasium in soil and rhizopshere. In: Gobran, G.R., Wenzel, W.W., Lombi, E. (Eds.), Trace Elements in the Rhizosphere. CRC Press, Boca Raton, pp. 61e91. Dupré de Boulois, H., Delvaux, B., Declerck, S., 2005. Effects of arbuscular mycorrhizal fungi on the root uptake and translocation of radiocaesium. Environmental Pollution 134, 515e524. Dupré de Boulois, H., Joner, E.J., Leyval, C., Jakobsen, I., Chen, B.D., Roos, P., et al., 2008. Role and influence of mycorrhizal fungi on radiocesium accumulation by plants. Journal of Environmental Radioactivity 99, 775e784. Dupré de Boulois, H., Voets, L., Delvaux, B., Jakobsen, I., Declerck, S., 2006. Transport of radiocaesium by arbuscular mycorrhizal fungi to Medicago truncatula under in vitro conditions. Environmental Microbiology 8, 1926e1934. Eapen, S., Singh, S., Thorat, V., Kaushik, C.P., Raj, K., D’Souza, S.F., 2006. Phytoremediation of radiostrontium (Sr-90) and radiocesium (Cs-137) using giant milky weed (Calotropis gigantea R.Br.) plants. Chemosphere 65, 2071e2073. Entry, J.A., Watrud, N.S., Reeves, M., 1999. Accumulation of Cs-137 and Sr-90 from contaminated soil by three grass species inoculated with mycorrhizal fungi. Environmental Pollution 104, 449e457. Fink, A., 1991. Pflanzenernehrung in Stichworten. Hirt, Berlin. Fuhrmann, M., Lasat, M.M., Ebbs, S.D., Kochian, L.V., Cornish, J., 2002. Uptake of cesium-137 and strontium-90 from contaminated soil by three plant species; application to phytoremediation. Journal of Environmental Quality 31, 904e909. Gaymard, F., Pilot, G., Lacombe, B., Bouchez, D., Bruneau, D., Boucherez, J., et al., 1998. Identification and disruption of a plant shaker-like outward channel involved in Kþ release into the xylem sap. Cell 94, 647e655. González-Chávez, M.C., Carrillo-Gonzalez, R., Wright, S.F., Nichols, K.A., 2004. The role of glomalin, a protein produced by arbuscular mycorrhizal fungi, in sequestering potentially toxic elements. Environmental Pollution 130, 317e323. Guillitte, O., Tikhomirov, F.A., Shaw, G., Vetrov, V., 1994. Principles and practices of countermeasures to be carried out following radioactive contamination of forest areas. The Science of the Total Environment 157, 399e406. Gyuricza, V., Dupré de Boulois, H., Declerck, S., 2010. Effect of potassium and phosphorus on the transport of radiocesium by arbuscular mycorrhizal fungi. Journal of Environmental Radioactivity 101 (6), 482e487. Javot, H., Pumplin, N., Harrison, M.J., 2007. Phosphate in the arbuscular mycorrhizal symbiosis: transport properties and regulatory roles. Plant Cell and Environment 30, 310e322.
596
V. Gyuricza et al. / Journal of Environmental Radioactivity 101 (2010) 591e596
Joner, E.J., Briones, R., Leyval, C., 2000. Metal-binding capacity of arbuscular mycorrhizal mycelium. Plant and Soil 226, 227e234. Joner, E.J., Roos, P., Jansa, J., Frossard, E., Leyval, C., Jakobsen, I., 2004. No significant contribution of arbuscular mycorrhizal fungi to transfer of radiocesium from soil to plants. Applied and Environmental Microbiology 70, 6512e6517. Ludwig-Müller, J., 2000. Hormonal balance in plants during colonisation by mycorrhizal fungi. In: Douds, D.D., Kapulnik, Y. (Eds.), Arbuscular Mycorrhizas: Molecular Biology and Physiology. Kluwer Academic Publishers, Dordrecht, pp. 263e285. Maathuis, F.J.M., Sanders, D., 1997. Regulation of Kþ absorption in plant root cells by external Kþ: interplay of different plasma membrane Kþ transporters. Journal of Experimental Botany 48, 451e458. Nisbet, A.F., 1993. Effects of soil-based contermeasures on solid-liquid equilibria in agricultural soils contaminated with radiocaesium and radiostrontium. The Science of the Total Environment 137, 99e118. Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society 55, 158e161. Pilot, G., Gaymard, F., Mouline, K., Chérel, I., Sentenac, H., 2003. Regulated expression of Arabidopsis Shaker Kþ channel genes involved in Kþ uptake and distribution in the plant. Plant Molecular Biolology 51, 773e787. Plenchette, C., Morel, C., 1996. External phosphorus requirements of mycorrhizal and non-mycorrhizal barley and soybean plants. Biology and Fertility of Soils 21, 303e308. Roberts, S.K., 1998. Regulation of Kþ channels in maize roots by water stress and abscisic acid. Plant Physiology 116, 145e153. Roberts, S.K., Snowman, B.N., 2000. The effects of ABA on channel-mediated Kþ transport across higher plant roots. Journal of Experimental Botany 51, 1585e1594. Rogers, R.D., Williams, S.E., 1986. Vesicular-arbuscular mycorrhiza: influence on plant uptake of cesium and cobalt. Soil Biology and Biochemistry 18, 371e376.
Sandeep, S., Manjaiah, K.M., 2008. Transfer factors of 134Cs to crops from Typic Haupslept under tropical region as influenced by potassium application. Journal of Environmental Radioactivity 99, 349e358. Shaw, G.,1993. Blockade by fertilizers of caesium and strontium uptake in crops: effects on the root uptake process. The Science of the Total Environment 137, 119e133. Smolders, E., Kiebooms, L., Buysse, J., Merckx, R., 1996. Cs-167 uptake in spring wheat (Trticum aestivum L. cv. Tonic) at varying K supply: I. The effect in soil solution culture. Plant and Soil 181, 205e209. Vidal, M., Camps, M., Grebenshikova, N., Sanzharova, N., Ivanov, Y., Vandecasteele, C., Shand, C., Rigol, A., Firsakova, S., Fesenki, S., Levchuk, S., Cheshire, S., Sauras, T., Rauget, G., 2001. Soil- and plant-based countermeasures to reduce Cs-137 and Sr-90 uptake by grasses in natural meadows: the REDUP project. Journal of Environmental Radioactivity 56, 139e156. White, P.J., Broadley, M.R., 2000. Mechanisms of caesium uptake by plants. New Phytologist 147, 241e256. White, P.J., Swarup, K., Escobar-Gutierrez, A.J., Bowen, H.C., Willey, N.J., Broadley, M. R., 2003. Selecting plants to minimise radiocaesium in the food chain. Plant and Soil 249, 177e186. Zhu, Y.G., 2001. Effect of external potassium (K) supply on the uptake of Cs-137 by spring wheat (Triticum aestivum cv. Tonic): a large-scale hydroponic study. Journal of Environmental Radioactivity 55, 303e314. Zhu, Y.G., Shaw, G., 2000. Soil contamination with radionuclides and potential remediation. Chemosphere 41, 121e128. Zhu, Y.G., Shaw, G., Nisbet, A.F., Wilkins, B.T., 1999. Effects of external potassium supply on compartmentation and flux characteristics of radiocaesium in spring wheat roots. Annals of Botany 84, 639e644. Zhu, Y.G., Shaw, G., Nisbet, A.F., Wilkins, B.T., 2000. Effect of potassium starvation on the uptake of radiocesium by spring wheat (Triticum aestivum cv. Tonic). Plant and Soil 220, 27e34. Zhu, Y.G., Smolders, E., 2000. Plant uptake of radiocaesium: a review of mechanisms, regulation and application. Journal of Experimental Botany 51, 1635e1645.