Arsenic response of AtPCS1- and CePCS-expressing plants – Effects of external As(V) concentration on As-accumulation pattern and NPT metabolism
Introduction
Arsenic is a highly toxic element, present in the environment in two main inorganic forms: arsenite(III) and arsenate(V) (Norman, 1998). Arsenate, being an analogue of phosphate, is transported into plant cells via phosphate transporters (Meharg and MacNair, 1992) and interferes with essential metabolic processes like ATP synthesis. As(III) crosses plant plasma membrane through aquaglyceroporins (Meharg and Jardine, 2003) and binds to sulfhydryl groups of proteins, leading to disruption of cell metabolism.
Plants detoxify As by reducing As(V) to As(III), which is subsequently complexed by thiol peptides (Bleeker et al., 2006). Many reports describe that, under As(III) and As(V) exposure, plants accumulate small cysteine-containing thiol peptides – phytochelatins (PCs) (Sneller et al., 1999; Schmöger et al., 2000). They are synthesized from glutathione by the enzyme PC synthase (PCS) in the presence of heavy metal ions or metalloids (Clemens et al., 1999). It was reported that PCs form various complexes with arsenite, such as As(III)—(PC2)2, GS–As(III)–PC2, As(III)–PC3 and monomethylarsonic(III)–PC2 (Raab et al., 2005); however, the subsequent metabolism of these complexes is poorly understood. It has been postulated that the As(III)–thiol complexes are sequestered into the vacuole by ABC-type transporters, although so far the protein with this transport activity remains unidentified.
Due to the important role of PCs in As-detoxification, several attempts were made to enhance their synthesis in plants. The enzymes from the glutathione biosynthesis pathway or PCS were overexpressed, leading to an increase in PC levels and As-tolerance – an important result from the phytoremediation point of view (Li et al. 2004, Li et al. 2005; Gasic and Korban, 2007; Guo et al., 2008). However, in most of those studies, As-accumulation in above-ground tissues was not substantially increased (Li et al. 2004, Li et al. 2005; Gasic and Korban, 2007), except for dual-gene transformations with GSH1 and AsPCS1 (Guo et al., 2008). Surprisingly, in some of those reports on PCS overexpression, the enhanced As-tolerance was paradoxically accompanied with cadmium hypersensitivity (Li et al. 2004, Li et al. 2005), whereas other studies demonstrated an increase in tolerance to both As and Cd (Gasic and Korban, 2007; Guo et al., 2008). The reason for those contradictory results on As- and Cd-tolerance levels of PCS-expressing plants remains unknown. It is possible that different PCS genes used for the transformation and/or various plant species tested might contribute to the generation of these phenotypes.
In previous work (Wojas et al., 2008), we demonstrated that the PCS genes used for plant transformation strongly influence the cadmium tolerance of plants. We used a novel approach – two different PCS genes were introduced in one model species – tobacco. It appeared that plants overexpressing the AtPCS1 gene from Arabidopsis were hypersensitive to Cd2+, whereas the tobacco expressing CePCS from Caenorhabditis elegans was more Cd-tolerant. Those differences in Cd-tolerance were accompanied by substantial changes in thiol metabolism. Thus, it was demonstrated for the first time that the overexpression of two different PCS enzymes in the same plant species could result in distinctive metabolic changes accompanied by differences in cadmium tolerance between the transformants.
In view of the above information, it appears that our novel approach – overexpression of two different PCS genes in one model plant species can be useful also as an attempt to understand the mechanisms of As-detoxification, including the metabolic pathways associated with As complexation by PCs.
Therefore, in the present study, we compare As-tolerance, accumulation and non-protein thiol (NPT) levels of AtPCS1 and CePCS transformants. This approach allows us to determine the potential differences in plant response to As(V), depending on the PCS gene used for plant transformation. Thus, the results presented here have important implications for better understanding PC function in As-tolerance as a prerequisite for plant genetic engineering for phytoremediation.
Section snippets
Plants and growth conditions
Experiments were performed on tobacco (Nicotiana tabacum var. Xanthi) wild-type and transgenic plants overexpressing AtPCS1 (two independent lines PaII4, PaII12) and CePCS (two independent lines PcII3, PcII4) generated by Wojas et al. (2008). Seeds were sterilized and germinated on sterile agar plates, positioned vertically, on quarter-strength Knop's medium (containing 1% agar, 2% sucrose and, for the selection of transgenic plants, 10 μg/mL of Basta). Seedlings were grown for 3 weeks on plates
As-tolerance level of PCS overexpressing plants
To evaluate changes in As-tolerance due to AtPCS1 and CePCS overexpression, tolerance tests on the agar plates with the range of As(V) concentrations were performed. They revealed that tobacco seedlings expressing AtPCS1 and CePCS were more tolerant to As(V) than wild-type ones when tested at 20, 50 and 75 μM As(V) (Fig. 1A and B). However, the increase in As(V) tolerance was the most pronounced in CePCS1 expressing plants; they had longer roots relative to both AtPCS1 and WT plants even at the
Discussion
The role of PCs, synthesized from GSH by PCS (Clemens et al., 1999), in Cd and As-detoxification, hence tolerance, has been demonstrated for numerous plant species (Sneller et al., 1999; Schmöger et al., 2000; Hartley-Whitaker et al., 2001). However, the overexpression of PCS genes in different plants resulted in contrasting phenotypes in the presence of Cd2+, ranging from Cd hypersensitivity (Li et al., 2004) to higher Cd-tolerance (Gasic and Korban, 2007). In contrast, upon As exposure, PCS
Acknowledgements
This work was financially supported by the FP5 EU grant METALLOPHYTES (QLRT-385 200000479), FP6 EU grant PHIME (FOOD-CT-2006-016253), MNiSW Grant no. K118/T09/2005, COST Action 859, and EU Mazowieckie Stypendium Doktoranckie.
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