•In a transcriptomic study of magnesium (Mg) starvation in Arabidopsis, we identified several genes that were differentially regulated which are involved in the detoxification process of nonessential heavy metals such as cadmium (Cd).
•We further tested the impact of low Mg status on Cd sensitivity in plants. Interestingly, a −Mg pretreatment of 7 d alleviated the bleaching of young leaves caused by Cd. No or little difference in Cd tissue concentration between the +Mg and −Mg plants was observed, suggesting that lower Cd toxicity was probably not attributable to modified root to shoot translocation.
•Mg deficiency also promoted an increase in the iron (Fe) concentration (up to one-fourth) in Cd-treated leaves. Because high Fe concentrations have previously been reported to prevent the harmful effects of Cd, we explored whether Fe homeostasis plays a role in the Mg–Cd interaction. A protective effect of −Mg pretreatment was also observed on Fe starvation. However, Fe foliar spray partially alleviated Cd-induced chloroses, while it almost completely restored chlorophyll content in Fe-deficient leaves.
•In conclusion, the protective effect of Mg against Cd toxicity could be attributable partly to the maintenance of Fe status but also to the increase in antioxidative capacity, detoxification and/or protection of the photosynthetic apparatus.
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Mineral interactions have been demonstrated to be important for the manifestation of deficiency and toxicity in plants (Suzuki, 2005; Ward et al., 2008). The role of high magnesium (Mg) supply in alleviating aluminium (Al) (Bose et al., 2011) and cadmium (Cd) (Kashem & Kawai, 2007) toxicity has already been described. Here we report a positive effect of low Mg status on the alleviation of toxicity symptoms caused by Cd exposure in Arabidopsis.
In our recent global transcriptomic studies of Mg deficiency in Arabidopsis (Hermans et al., 2010a,b), we identified a category of genes involved in the process of detoxification of drugs and heavy metals, such as Cd. In this report, we show that a Mg starvation pretreatment can alleviate Cd toxicity in plants.
Materials and Methods
Arabidopsis thaliana Heynh Columbia (Col-0) plants were grown hydroponically in short-day conditions (8 h light; 100 μmol photons m−2 s−1/16 h darkness). The growth conditions and the nutrient solution composition are described elsewhere in Hermans & Verbruggen (2005) and Hermans et al. (2010a).
Mineral content analysis
Plant tissues were harvested and dried at 60°C. Full mineral profiles were assayed by inductively coupled plasma mass spectrometry performed by Purdue Ionomics Information Management Systems (PIIMS, Purdue, IN, USA).
Chlorophyll content analysis
Pigments were extracted with 80% acetone and chlorophyll content was estimated using the method of Porra et al. (1989).
The behaviour of photosystem II was assessed using chlorophyll a (Chla) fast fluorescence kinetics (direct induction with 660-nm exposure), recorded with the Handy Plant Efficiency Analyser fluorometer (Hansatech Instruments, Norfolk, UK). The OJIP transients were analysed according to the JIP test procedure, as previously described by Hermans et al. (2003); Hermans & Verbruggen (2005).
RNA extraction, reverse transcription and quantitative PCR (qPCR)
Total RNA was extracted from 100 mg of ground frozen root and leaf samples with TRIzol reagent (RNeasy; Qiagen, Venlo, NL, USA). Reverse transcription was performed starting from 1 μg of total RNA using the RevertAid H Minus First Strand cDNA Synthesis kit (Fermentas, St. Leon-Rot, Germany). The Janus Automated Workstation (PerkinElmer, Waltham, MA, USA) handled all pipetting for PCRs in a final volume of 5 μl using the LightCycler 480 SYBR Green I Master mix (Roche). qPCR reactions were performed with the LightCycler 480 (Roche), with preincubation at 95°C for 10 min, 45 cycles at 95°C for 10 s, 60°C for 15 s and 72°C for 15 s, and melting curves according to the manufacturer’s recommendations. Primers designed using NCBI Primer-Blast (Rozen et al., 2000) are listed in Supporting Information Table S2.
Statistical analyses for biological data were carried out using the spss statistical software (SPSS, Chicago, IL, USA). One-way analysis of variance (ANOVA) and subsequent multiple comparison tests (LSDs) were performed to calculate the significance level of differences between means. Significance was set at the 5% level (P <0.05).
Five-week-old Araridopsis (Col-0) plants grown hydroponically were subjected to Mg starvation (complete omission of Mg from the nutrient solution) for a period of 1 wk. At day 7 of treatment, half of the Mg-fully supplied (+Mg) and Mg-starved (−Mg) plant populations were subjected to mild Cd exposure by transfer to a nutrient solution contaminated with 2 μM CdSO4 or were fed with an Fe-depleted solution (Fig. 1a). The experimental design included treatments in which plants were fully supplied with Mg and not subjected to Cd exposure (+Mg-Cd; the control), subjected to Cd exposure (+Mg+Cd) or subjected to Fe depletion (+Mg-Fe); and treatments in which plants were Mg-starved and not subjected to Cd exposure (-Mg-Cd), subjected to Cd exposure (-Mg+Cd) or subjected to Fe depletion (-Mg-Fe). We considered 1 wk of Mg depletion to be sufficient before applying the +Cd and −Fe treatments because, while no visible symptoms in leaves (Hermans et al., 2010b) or decrease in biomass (Fig. S1) was perceptible, the Mg content had already decreased considerably in plant organs. Indeed, the mineral analysis revealed that, after 7 d of Mg starvation treatment, the concentration had decreased significantly (P <0.05) by 74% in roots and by 56, 67 and 77%, respectively, in old mature leaves (OL), young mature/expanding leaves (YL) and immature leaves/rosette buds (IL) compared with Mg-fully supplied controls (Fig. 2a).
Low Mg status alleviates Cd toxicity symptoms in leaves
Cd exposure resulted in chlorotic symptoms primarily in IL and YL (Fig. 1a). Interestingly, the Mg starvation pretreatment alleviated these chloroses upon Cd exposure (Fig. 1a). After 7 d of Cd exposure, a decrease was observed in the total chlorophyll content in YL of −58% between the +Mg−Cd and +Mg+Cd treatments and of −20% between the −Mg−Cd and −Mg+Cd treatments (Fig. 1b). Also, the photosynthetic performance index (PI), characterizing overall photochemical processes in photosystem II (Hermans et al., 2003), decreased, respectively, by −94% between +Mg−Cd and +Mg+Cd and by −46% between −Mg−Cd and −Mg+Cd in YL (Fig. 1b). Note that, because IL were too small, no photosynthetic analysis could be performed on these leaves. After (7 d)+7 of toxic metal exposure, Cd accumulation was greater in roots than in leaves, in accordance with previous studies with the same species (Gong et al., 2003; Smeets et al., 2008; Li et al., 2010), and Cd concentrations were equal among the three leaf groups (Fig. 2b). Cd concentrations in roots and leaves (except for OL) did not differ significantly (P <0.05) between Mg-deficient and Mg-fully supplied backgrounds (Fig. 2b). The impact of Cd exposure was also assessed on the whole ionic profile. The addition of Cd decreased manganese (Mn) concentrations in roots (−61%) and leaves, with the highest difference observed in IL (−55%), compared with the +Mg–Cd treatment (Figs 2d, S2). The decrease in Mn concentration was similar in roots of +Mg and –Mg backgrounds but smaller in leaves of the −Mg background (Figs 2d, S1). Upon Cd exposure, the sulphur (S) concentration was increased in leaves of both backgrounds but to a greater extent in the −Mg background (up to +79% in OL between the −Mg+Cd and +Mg−Cd treatments) and Zn concentration was increased (+92%) in −Mg roots only (Fig. S2). Furthermore, ratios of mineral elements are reported to be crucial for chloroplast stability and chlorophyll contents and often considered as diagnostic criteria for chlorosis (Nenova & Stoyanov, 1999; Fernández-Falcón et al., 2006). We found that Cd treatment markedly increased the Mg : Mn ratio in YL and IL, and the Fe : Mn ratio in the same organs and in roots (Fig. S3). Mg deficiency pretreatment led to a more modest increase in the Fe : Mn ratio in leaves (Fig. S3).
Low Mg status improves Fe deficiency symptoms in leaves
Fe starvation applied for 1 wk also resulted in chlorotic symptoms but affected the very young leaves less and the mature leaves more, compared with Cd exposure (Fig. 1a). The Fe starvation treatment depleted the Fe content by −55% in roots and in leaves (up to −28% in IL) after (7 d)+7 in +Mg conditions (Fig. 2c). The Mg deficiency pretreatment was also able to prevent the chlorotic symptoms produced by Fe deficiency and to improve photosynthetic performance (Fig. 1b). Under those conditions, there was less of a reduction in the Fe concentrations of Mg-deficient leaves (up to −12% in IL between −Mg−Fe and +Mg−Fe) compared with Mg-sufficient conditions (Fig. 2c). The Fe depletion treatment had much more pronounced effects on the ionic profile than Cd exposure: copper (Cu), Mn and Zn (+553%) concentrations increased in roots and Cu (up to +197% in IL), Mn, S and Zn concentrations increased in leaves (Fig. S1). These differences were similar in the +Mg and −Mg backgrounds, with the exception of Cu, for which lower concentrations were found in −Mg leaves (up to +110% in IL between −Mg−Fe and +Mg−Fe).
Chloroses of Cd-treated plants are partially recovered by application of Fe foliar spray
Cd exposure and Fe depletion both induced chlorotic symptoms but on somewhat different leaves (Fig. 1a). The two treatments resulted in similar decreases in Fe concentrations in leaves but only Fe starvation resulted in a decrease in roots (Fig. 2c). To determine whether chloroses on Cd-treated leaves are related to an induced Fe deficiency or are attributable to other types of damage, we monitored the expression of markers related to Fe homeostasis in leaves such as FERRETIN 1 (FER1) (Thimm et al., 2001; Murgia et al., 2007) and three isoforms of ACONITASE (ACO1, ACO2 and ACO3) (Arnaud et al., 2007). Upon Fe depletion, we observed the down-regulation of FER1 and ACO2 in leaves at (7d)+3 and (7d)+7. ACO1 and ACO3 turned out not to be reliable markers for Fe deficiency at most of the time-points (data not shown). In Cd-treated leaves, FER1 and ACO2 were also repressed (Fig. 3b). Those results tend to indicate that leaves treated with Cd were subjected to an induced Fe deficiency. However, application of an Fe spray to rosette leaves exposed to Cd only partially alleviated chlorotic symptoms, while it almost completely reversed these symptoms in Fe-deficient plants (Fig. S4).
In this study, as demonstrated in Fig. 1, we found a clear protective effect of a low Mg concentration in the plant against the bleaching of young leaves typically caused by Cd exposure (Collin et al., 2008; Morel et al., 2009). No impact of Mg starvation on Cd translocation from the root to the shoot was found (Fig. 2b). This observation is in contrast to the finding that Cd toxicity is alleviated by a high supply of calcium in Arabidopsis (Suzuki, 2005) or of Mg in Brassica (Kashem & Kawai, 2007), where Cd uptake and accumulation have been reported to be reduced. However, in our case, the cellular localization of Cd could have been different and/or protection mechanisms against this heavy metal may have been enhanced by the low-Mg conditions. In recent genome-wide transcriptomic studies of the response to Mg starvation in Arabidopsis (Hermans et al., 2010a,b), we identified several Mg-responsive genes involved in the response to oxidative stress, the photoprotection of the photosynthetic apparatus, the detoxification process for drugs and heavy metals and the homeostasis of other essential elements (Table S1). We discuss here the possibilities that Mg deficiency (1) increases the antioxidative capacity, (2) protects chloroplasts against Cd-induced injuries, (3) alters Fe homeostasis to prevent Cd toxicity and (4) favours cytosolic efflux transport or vacuolar storage of Cd.
Low Mg status may enhance defence mechanisms against oxidative stress and protection of the photosynthetic apparatus against Cd
Low Mg status may indirectly alter mineral homeostasis to prevent Cd toxicity
An antagonistic interaction between Mg and Fe is widely recognized to occur in plants (Agarwala & Mehrotra, 1984; Ward et al., 2008). Particularly in Arabidopsis, Mg deficiency promotes a slight increase in the Fe content in leaves (Fig. 2c; Hermans et al., 2010b) and elevated Fe concentrations have been reported to prevent the harmful effects of Cd (Solti et al., 2008). Before Cd exposure at (d7)+0, we already observed a higher Fe concentration in Mg-deficient leaves and higher expression of FER1 (Fig. 4, Table S1), which encodes a well-characterized chloroplastic Fe-storage protein that accumulates under conditions of Fe excess (Murgia et al., 2007). The major physiological role of ferritins in plants is to fine-tune the quantity of metal available to meet metabolic requirements by sequestering Fe (Briat et al., 2009). In addition to Fe detoxification, another function of ferritins may be to prevent oxidative stress (Ravet et al., 2009) and to detoxify heavy metals, as these proteins are also capable of binding Cd2+ (Sczekan & Joshi, 1989; Rama Kumar & Prasad, 1999). Therefore, ferritins could also play an important role in controlling the interaction between Fe homeostasis, Cd excess and oxidative stress generated by Cd.
An influence of modified Fe and Cd supply on the uptake and distribution of both metals and photosynthesis has been reported (Siedlecka & Krupa, 1999; Sávári et al., 2008). Upon mild Cd concentration exposure, we did not find differences in Fe concentration (Fig. 2c) and in the expression of the Fe transporter IRT1 (Fig. 3a) in roots of the +Mg+Cd treatment in comparison with the control. This is in contrast to the findings of other studies which have reported a transient induction of IRT1 expression at higher Cd concentrations in different species (Hodoshima et al., 2007; Besson-Bard et al., 2009). Also, we found here that chloroses on Cd-treated leaves were not directly related to Fe deficiency but may be in part attributable to interactions with other elements such as Mn (Figs 2d, S2) or other types of Cd-induced damage. Overall, an Fe foliar spray had a positive influence on the chlorophyll content and photosynthetic activity of Cd-treated plants (Fig. S4). Incomplete restoration of Cd-induced chloroses by application of the Fe foliar spray may be a result of the direct inhibitory effect of Cd on the transcription of genes encoding apoLHC proteins (causing incomplete recovery of the light-harvesting complex of photosystem II), as previously suggested by Solti et al. (2008).
Finally, we note that the Mg deficiency pretreatment resulted in other ionic profile variations (Figs 2, S2); however, it is difficult to ascertain whether such variations account for the protective effect against Cd toxicity. Reference mineral concentrations in tissues, below which there is deficiency and above which there is toxicity, are available for some land and crop species (Bennett, 1993; Marschner, 1995; Krämer, 2010) but are not necessarily relevant for Arabidopsis. In addition, these thresholds are dependent on the stage of development, the mode of culture and other environmental factors.
Low Mg status may enhance Cd detoxification processes
We observed the induction of four DETOXIFICATION family members (DTX1, 2, 3 and 4) (Table S1). In Arabidopsis, the DTX family consists of 56 members and belongs to the multidrug and toxic efflux (MATE) superfamily. To date, these transporters have been poorly characterized; nevertheless, some members have been shown to transport ethidium bromide and secondary metabolites (Li et al., 2002). Interestingly, one DTX member could be involved in Cd efflux out of the cell (Li et al., 2002). On the basis of these observations, it is possible that DTXs mediate at least in part the beneficial effect of low Mg status against Cd exposure by lowering the cytosolic Cd concentration (Fig. 4). In order to evaluate the impact of DTX activity on the Cd protection phenotype, we challenged dtx2 and dtx3 knockout mutants with Mg starvation followed by Cd treatment. However, no change in the phenotype compared with the wild type was observed (data not shown). In addition, we also observed in Mg-deficient leaves a strong induction of MULTIDRUG RESISTANCE PROTEIN 3 (MRP3) (Table S1, Fig. 4), which encodes a vacuolar ABC transporter that transports glutathione conjugates and chlorophyll catabolites (Tommasini et al., 1998) and has a possible role in toxic metal detoxification (Zientara et al., 2009).
Several knockout mutants of Mg deficiency-responsive genes (dtx and rrtf1) were challenged with a Cd excess, but the protective effect of Mg deficiency persisted in these mutant backgrounds. At this stage we cannot exclude the possibility that the protective effect of low Mg status is polygenic. To dissect the Cd–Mg interaction, further studies are underway. In conclusion, the low Mg status protective effect against Cd is pleiotropic and could be caused by modification of Fe, Mn, Zn and S leaf tissue concentrations, enhancement of antioxidative capacity, detoxification and/or protection of the photosynthetic apparatus.
This work was supported by a grant from the Interuniversity Attraction Poles Programme (IUAP VI/33), initiated by the Belgian State, Science Policy Office, FRFC (no. 2.4.583.08F), Crédit aux Chercheurs (no. 1.5.019.08) from the Fonds National de la Recherche Scientifique (FNRS-FRS) and Fondation Jaumotte-Demoulin at ULB. C.H. is a postdoctoral fellow of the FNRS. We thank C.L. Meyer for statistical advice and P. Salis for technical assistance.
While our paper was in press, Chou et al. (2011) published that Mg deficiency also protected rice seedlings from Cd stress. However these authors reported higher Cd concentrations in Mg-deficient plants compared with Mg-sufficient plants upon Cd exposure.