- Top of page
- Materials and Methods
A number of fern species belonging to the order Pteridales, mostly within the genus Pteris, are able to hyperaccumulate arsenic (As) in the fronds to very high concentrations (Ma et al., 2001; Visoottiviseth et al., 2002; Zhao et al., 2002; Meharg, 2003; Srivastava et al., 2006; Wang et al., 2007). Pteris vittata, the first As-hyperaccumulator identified (Ma et al., 2001), can accumulate several thousand mg As kg−1 dry weight in fronds without suffering from As toxicity (Lombi et al., 2002; Tu & Ma, 2002; Caille et al., 2005). Compared with As-nonhyperaccumulating fern species, P. vittata has a higher rate of arsenate uptake and a much enhanced translocation of As from roots to fronds (Huang et al., 2004; Poynton et al., 2004; Caille et al., 2005). For example, within 8 h exposure to arsenate, 76% of the As taken up by P. vittata was transported to the fronds, whereas in the nonhyperaccumulator Pteris tremula the percentage was only 9% (Caille et al., 2005). Similarly, Poynton et al. (2004) showed that 74% of the As taken up during 24 h had been translocated to the shoots in the hyperaccumulator Pteris cretica, compared with only 8.8% in the nonhyperaccumulator Nephrolepis exaltata. It appears that enhanced uptake, translocation and tolerance are three important traits of As hyperaccumulators (McGrath & Zhao, 2003).
Recent studies have shown that arsenate, the predominant form of As in aerobic soils, is taken up by the phosphate transport system in P. vittata and P. cretica (Wang et al., 2002; Poynton et al., 2004). Following the entry of arsenate into the root cells, some arsenate is reduced to arsenite by arsenate reductase. A gene encoding an arsenate reductase, PvACR2, has been cloned from the gametophyte of P. vittata (Ellis et al., 2006). Its expression in the gametophyte is constitutive and unaffected by arsenate exposure. Analysis of As speciation in the roots of both P. vittata and P. cretica shows that arsenate is the dominant species, while arsenite only accounts for 10–40% of the total As (Zhang et al., 2002; Zhao et al., 2002; Poynton et al., 2004; Pickering et al., 2006). By contrast, the fronds contain mainly inorganic arsenite (70–90% of the total As) (Lombi et al., 2002; Wang et al., 2002; Zhang et al., 2002; Zhao et al., 2002; Webb et al., 2003; Poynton et al., 2004; Pickering et al., 2006). The fact that proportionally more arsenite is found in fronds than in roots has led some authors to suggest that arsenate is reduced mainly in fronds (Zhang et al., 2002; Kertulis et al., 2005). However, the differential pattern of As speciation in roots and fronds may also result from a preferential translocation of arsenite from roots to fronds. In fact, Duan et al. (2005) assayed the activity of glutathione-dependent arsenate reductase in roots and fronds of P. vittata and found this activity only in the roots, suggesting that arsenate reduction occurs mainly in roots and that arsenite is subsequently translocated to fronds. By contrast, using an X-ray imaging technique to determine As speciation in different tissues of P. vittata, Pickering et al. (2006) proposed that arsenate is transported through the vascular tissue from the roots to the fronds, where it is reduced to arsenite and stored at high concentrations. Therefore, questions regarding the form of As translocated in xylem and the location of arsenate reduction in P. vittata remain unresolved. Identification of As species in the xylem sap will help to resolve this controversial issue, as well as providing an important clue to the mechanism responsible for the efficient translocation, and thus accumulation, of As in P. vittata.
The main objective of the present study was to determine As speciation in the xylem sap as well as in root and frond tissues of P. vittata supplied with either arsenate or arsenite. An additional objective was to investigate whether arsenite is exuded from roots to the external medium, as has recently been shown in plant species that do not hyperaccumulate As (Xu et al., 2007).
- Top of page
- Materials and Methods
The results of the present study showed that arsenate and arsenite were the main form of As in roots and fronds of P. vittata, respectively, when plants were supplied with arsenate. Arsenate accounted for 60–70% of the total As in roots, but only c. 20% in fronds, with the remainder being present as arsenite. These results are in general agreement with those of previous studies employing different methodologies for As speciation analysis, including HPLC-ICP-MS and X-ray absorption spectrometry (Lombi et al., 2002; Wang et al., 2002; Zhang et al., 2002; Webb et al., 2003; Zhao et al., 2003; Pickering et al., 2006).
Pteris vittata takes up arsenate via the phosphate transport pathway (Wang et al., 2002; Poynton et al., 2004). This is further confirmed by a large inhibitory effect of phosphate on the depletion of arsenate from the nutrient solution and on As accumulation by the plants observed in the present study (Figs 3, 5). The time-course experiment showed that a portion of arsenate was rapidly reduced to arsenite in roots, although the percentage of arsenite in roots remained relatively constant (30–40%) from 1 to 24 h (Fig. 2). However, until now it has not been resolved whether roots or fronds are the main location of arsenate reduction in P. vittata, and what is the main form of As transported from roots to fronds. Our results showed that As was transported in the xylem sap of P. vittata predominantly (93–98%) as arsenite, regardless of whether arsenate or arsenite was supplied to the plants (Figs 1, 4). This also means that roots are the main site of arsenate reduction in P. vittata, and that, following arsenate reduction, arsenite is preferentially loaded into the xylem. This conclusion is supported by the study of Duan et al. (2005), who reported measurable activity of glutathione-dependent arsenate reductase only in the roots of P. vittata, and not in the fronds. In Expt 2 of the present study, as well as in the study of Wang et al. (2002), proportionally more As was distributed from roots to fronds in the short term (8–24 h) when As was supplied as arsenite than as arsenate. Furthermore, addition of L-buthionine-sulphoximine (BSO) to the nutrient solution was found to inhibit glutathione biosynthesis in the roots of P. vittata markedly, leading to decreased arsenate reduction in roots (a smaller proportion of arsenite) and markedly decreased translocation of As to the fronds (by 40–50%) (Zhao et al., 2003). These results are consistent with a model in which arsenate is reduced to arsenite in the roots of P. vittata first, before being transported to the fronds as arsenite. In both the present study on P. vittata and that of Poynton et al. (2004) on P. cretica, phosphate had no significant effect on As translocation from roots and fronds. Inhibition of As translocation by phosphate might be expected if As were translocated as arsenate. However, phosphate would have no effect if As is translocated as arsenite, as shown in the present study. The fact that the percentage of arsenite in the fronds (c. 80%) was smaller than that in the xylem sap (93–98%) suggests that some arsenite may be oxidized to arsenate in the fronds. It is also possible that redox cycling of As may take place in the frond to some extent, although direct evidence for this has yet to be obtained.
The results of the present study do not support the conclusion of Kertulis et al. (2005) and Pickering et al. (2006) that P. vittata transports mainly arsenate from roots to fronds, and that the latter are the main location of arsenate reduction. In the study of Kertulis et al. (2005), ‘xylem sap’ was collected from intact fronds using a Scholander pressure chamber. Whether the sap obtained in this way really represents the xylem sap originating from the root system remains to be tested. One surprising result from the study of Kertulis et al. (2005) is that the As concentration in the sap was lower than that in the external solution, which is at variance with the fact that P. vittata is extremely efficient at As translocation from roots to shoots (Tu & Ma, 2002; Caille et al., 2005). In our study, xylem sap was obtained from the natural root pressure within a short period after stem excision. This is the method that is used most often to obtain xylem sap. Furthermore, the sap samples obtained by Kertulis et al. (2005) were stored at −80°C for a unspecified time period before As speciation analysis. Arsenite is thermodynamically unstable in the oxygenated environment, and it is possible that arsenite is partly oxidized to arsenate during storage. In our study, xylem sap samples were diluted with an EDTA solution, which helps to preserve As speciation (Bednar et al., 2002), followed by immediate analysis of As speciation using HPLC-ICP-MS. The fact that most of the sap As was arsenite in our study indicates that little oxidation occurred, thus giving us confidence in the As speciation data. In the study of Pickering et al. (2006), intact fronds were subjected to X-ray absorption spectrometry (XAS) imaging for different As species. They found that arsenate was the dominant species in the central region of the mid-vein of rachis, although the rachis as a whole contained more arsenite (76%) than arsenate (24%). It should be pointed out that the XAS imaging information was for the rachis tissue, not the xylem sap per se. Arsenic speciation in the xylem sap may be different from that retained by the vascular bundles.
In a number of As-nonhyperaccumulator species, arsenite was found to be the main species of As in the xylem sap, accounting for c. 60% in Brassica juncea (Pickering et al., 2000) and sunflower (Helianthus annuus; Raab et al., 2005), 87% in cucumber (Cucumis sativus; Mihucz et al., 2005) and 90–96% in tomato (Lycopersicon esculentum; Xu et al., 2007). Furthermore, Raab et al. (2005) showed that arsenite in the xylem sap of sunflower was present in inorganic form, not complexed by thiol compounds. The results suggest that transport of arsenite in xylem is a common feature in both As hyperaccumulators and nonhyperaccumulators.
The translocation of arsenite from roots to fronds was extremely efficient in P. vittata, with the As concentration in the xylem sap being 18–73 times greater than that in the external solution. For a comparison, the As concentration in the xylem sap of tomato was only 1.5–10% of the external solution (Xu et al., 2007), c. 4% in B. juncea (Pickering et al., 2000) and 17.5% in cucumber (Mihucz et al., 2005). Our unpublished data showed a range of 2–50% for several cereal species including barley (Hordeum vulgare), maize (Zea mays), wheat (Triticum aestivum), and rice (Oryza sativa). Using the ratio of xylem sap As to external As concentration as an indicator of xylem mobility, P. vittata is two to three orders of magnitude higher than the As nonhyperaccumulators already described. Therefore, efficient loading of arsenite to the xylem is likely to be a key step of As hyperaccumulation in P. vittata. The second contributing factor to the efficient xylem transport of arsenite may be that very little arsenite in the roots of P. vittata is complexed by thiol-containing compounds such as phytochelatins (Zhao et al., 2003; Raab et al., 2004). Complexation of arsenite by thiols is an important mechanism of As detoxification in nonhyperaccumulating plants (Sneller et al., 1999; Schmöger et al., 2000; Raab et al., 2005); complexation may decrease translocation of As from roots to shoots, especially if the arsenite–thiol complexes are subsequently sequestered in the root vacuoles. This interpretation reconciles two pieces of seemingly conflicting evidence regarding the xylem mobility of As: the extremely high mobility of arsenite in P. vittata as demonstrated in the present study and the recent report by Dhankher et al. (2006) that silencing arsenate reductase in the roots of Arabidopsis thaliana markedly increased As accumulation in the shoots. Dhankher et al. (2006) proposed that arsenate is the most mobile form of arsenic in the majority of plant species and that arsenite stays sequestered in roots. This model clearly does not apply to P. vittata. In A. thaliana, and perhaps also other As-nonhyperaccumulating plant species, arsenite may be less mobile than arsenate because of arsenite complexation and sequestration in roots, and possibly also because of the lack of an efficient xylem loading system for arsenite.
The third possible contributing factor to the highly efficient translocation of arsenite in P. vittata is the lack of a strong efflux of arsenite from roots cells to the external solution (Fig. 3). Recently, it has been reported that the efflux of arsenite to the external medium, following arsenate reduction in roots, is a prominent process in tomato and rice (Xu et al., 2007). We have found that this is also the case in a range of other plant species including Arabidopsis thaliana, Holcus lanatus, wheat, barley and maize. Interestingly, P. vittata appeared to release very little arsenite to the nutrient solution during the 24-h exposure period. It thus appears that, although P. vittata possesses a highly efficient efflux system for arsenite for xylem loading, there is little efflux of arsenite to the external medium.
In conclusion, our study has identified arsenite as the predominant form of As transported in the xylem from roots to fronds of P. vittata. This transport is extremely efficient, possibly owing to one or more of the following reasons: efficient reduction of arsenate to arsenite in roots; hyper-expression of an arsenite efflux system toward xylem loading; lack of arsenite complexation by thiol compounds and sequestration in the root vacuoles; and weak efflux of arsenite to the external medium. Unravelling the mechanism responsible for the efficient xylem transport of arsenite in P. vittata is imperative to understanding of the As-hyperaccumulation phenotype.