ABC transporters, especially from the MRP subfamily, are frequently involved in the detoxification of various xenobiotics, among which, heavy metals are found. Here, we tried to decipher the function of a previously uncharacterized A. thaliana gene, AtMRP6, which is flanked by two other MRPs gene on chromosome III, AtMRP3 and AtMRP7.
Analysis of AtMRP6 gene expression by RT-Q-PCR as well as by promoter GUS analysis, demonstrated that this gene is weakly expressed and has a restricted pattern of expression, mainly in germinating seeds and seedlings. Subcellular localization of AtMRP6 in planta was attempted through two different approaches. First, CaMV35s transgenic plants expressing AtMRP6-GFP were generated. Strikingly, whereas empty vector and AtMRP6 antisens plants were easily obtained, it was never the case for the sense construction, probably indicating a toxicity of this gene product under over-expressing conditions. As an alternative way to address the localization of the transporter, mesophyll cell protoplasts were transfected with AtMRP6-GFP by the classical polyethylene glycol method. No fluorescence could be observed in these conditions whereas, in control cells expressing the GFP alone, fluorescence was detected in the cytoplasm and in the nucleus. The subcellular localization of AtMRP6 could not be determined however, our experiments highlighted the difficulties when working with this gene. In addition, heterologous expression of transporters in yeast constitutes an elegant approach to screening for complementation of various mutants and also to perform flux experiments with radiolabelled compounds. In the case of AtMRP6, no complementation of the Δycf1 mutant could be obtained in this study: AtMRP6 being truncated (figure 2A). We assume that this truncation of the protein was probably due to a toxicity of the transporter for the host. The development of such host toxicity is also consistent with an almost systematic mutation of the corresponding plasmid that occurred in bacteria at 37°C. When looking for an alternative expression system for AtMRP6, HEK-293 cells were transfected. As shown in figure 2B–C, AtMRP6 expression was successfully obtained. However, despite many efforts (assays with various plasmids such as pCi, pcDNA6 or pEGFP, optimization of the Kozak sequence, use of different cationic lipid transfection reagents), the yield of expression was too weak to initiate any flux experiment.
Results obtained in this study by RT-Q-PCR (figure 5D) and within a previous transcriptomic analysis , demonstrate that AtMRP6 expression is up-regulated in roots within 30-hr by 5 μM Cd. Interestingly, not only AtMRP6, but the three members of the gene cluster were also up-regulated by after Cd exposition. These results are in accordance with an enhanced level of both AtMRP3 and AtMRP6 transcripts, reported previously in cDNA microarray experiments . It has already been reported that AtMRP3 can be important in Cd detoxification since its heterologous expression in the yeast strain deprived of ycf1 restores Cd tolerance . However, in Arabidopsis, despite the fact that Cd-related induction of AtMRP3 is correlated with Cd uptake after a short metal exposure , whether AtMRP3 is involved in Cd transport or in the detoxification of toxic compounds produced after the metal stress awaits future studies. In the case of AtMRP7, very little data is available about its tissue expression  and its function is still unknown. A fourth gene, located upstream of the MRP cluster, is also up-regulated in roots by Cd treatment: it encodes a mitochondrial-localized serine acetyl-transferase, SAT3 or serat2.2 (At3g13110; ). This enzyme catalyzes the formation of O-acetyl-Ser from L-Ser and acetyl-CoA, which is used in cysteine synthesis, an important component of glutathione. Expression of the bacterial enzyme in tobacco led to an increase in cysteine and glutathione contents . Moreover, the high activity of SAT is associated with nickel tolerance in Thlaspi nickel hyper-accumulators  suggesting a major role of SAT in heavy metal resistance. Recently, expression of SAT3 has been achieved in tobacco; however no experiments have been performed in relation to Cd . All these results suggest that these four genes (AtMRP3, AtMRP6, AtMRP7 and SAT3), oriented in the same transcription direction on chromosome III, are members of a Cd-responding cluster. This hypothesis is also supported by the fact that all these genes are up-regulated by a Cd treatment into the same organ (roots) and in the same time scale (24-hr for SAT3, ; 30-hr for the three MRP genes). Identification of such Cd-responsive elements would be useful in the context of phytoremediation strategies either to drive the expression of cadmium-transporter or reporter genes that might be used as biosensors of contaminated soils.
At the sight of the expression pattern of this gene (figure 3), a phenotype was expected at root level in T-DNA KO lines. One cannot exclude that the neighboring MRP genes might complement the deletion of AtMRP6. For this reason, the expression levels of AtMRP3 and AtMRP7 were compared in wild type plants and in Atmrp6 genetic backgrounds. No significant difference in their expression levels was detected in the presence or in the absence of cadmium (data not shown). Thus, it is possible that if a mechanism of gene compensation is taking place in Atmrp6 KO plants, it involves (an)other gene(s) than AtMRP3 and AtMRP7 or that the basal levels of expression of AtMRP3/7 are sufficient to compensate for the absence of AtMRP6. Alternatively, these two genes could be up-regulated in the few cells expressing AtMRP6 in roots without significantly affecting their global root-expression level. The screening of several dozen conditions to observe a phenotype for Atmrp6 KO plants allowed us to show that, in the presence of Cd, the deletion of AtMRP6 has a small but significant impact on the development of primary leaves whereas roots elongation and ramification were unaffected. This phenotype was lost in 3- to 5-week-old plant, probably because at this developmental stage, Cd translocation from root to shoot is much lower, as already reported for AtMRP3 .