MODULATING HEAVY METAL ACCUMULATION IN PLANTS: OVEREXPRESSION OF THE PSEUDOMONAS PUTIDA EFFLUX COMPLEX CZCCBA

In the last years, more and more areas were exploited, degraded and polluted by antropic activities. Among the different pollutants released into the environment, heavy metals are one of the most life-threatening, being diffused into the environment through a great variety of activities and industrial processes, such as release of sewage sludge, mining, agricultural practices, and usage of contaminated fertilizers (Hüttermann et al., 1999; Shull, 1998). As, Hg, Ag, Cd e Pb, are examples of heavy metals with no biological functions and toxic to all living organisms even at low concentrations, they can be considered very dangerous to human health (Godbold and Hüttermann, 1985; Breckle, 1991; Nies, 1999). Due to their chemical and physical properties, heavy metals are highly stable and persistent for long periods in the environment. The majority is poorly sensitive to microbial or chemical degradation and usually cannot be biologically destroyed but only modified from one oxidation state or organic complex to another (Garbisu and Alkorta, 2001; Gisbert et al., 2003). Excess concentrations of heavy metals in soil cause strong declines in plant growth and these elements (due to their solubility in water) can easily enter the food-chain, becoming a serious problem to human health. Heavy metal toxicity shows its effect by causing damages at cellular level, leading, for instance, to oxidative stress, perturbation of membrane integrity and permeability, inhibition of enzymatic activities, and interference with protein folding (Schützendübel and Polle, 2002). Plants and microorganisms developed several strategies to survive in dynamic environments and to face different unfavourable conditions, being able to continuously adapt to rapid changes in the environment. During evolution same bacterial and plant species have developed metal homeostasis mechanisms allowing the achievement of tolerance and resistance conditions in order to survive in heavy metals polluted environments (Ramos et al., 2001; Moore and Helmann, 2005; Clemens, 2001). Bacterial resistance mechanisms include the synthesis of metal-chelating molecules (peptides, proteins or polysaccharides), active metal efflux outside the cell and induction of detoxification enzymes that modify a toxic ion into a less toxic or less available form. One of the most important mechanisms of metal resistance is the translocation of toxic ions towards the outside of the cell. Efflux transporters participate in the reduction of heavy metals content into the cell (Nies, 2003). An interesting biotechnological application aimed at improving safe crop production, is the transfer of bacterial mechanisms deputed to extrude heavy metals into plants, that can be evaluated for metal accumulation when grown on slightly polluted soils. In this project, the Pseudomonas putida CzcCBA efflux system (membrane exporter of Cd2+, Zn2+, Co2+), was transferred into plant, in order to modulate heavy metals content. The CzcCBA complex was isolated from P. putida Cd001, a strain of Pseudomonas inhabiting the rhizosphere of an ecotype of A. halleri growing in soils contaminated by high concentrations of Cd, Zn and Pb (Farinati et al., 2009). The CzcCBA complex was chosen among several membrane transporter specifically induced by Cd stress (Manara et al., 2012). Such system is a trans-envelope efflux pump, belonging to the heavy metal efflux family of the Gram-negative bacteria RND-driven tripartite protein complexes (Nies et al., 1989 ). This transporter utilizes the proton motive force, pumping the substrate across the membranes, through a proton/cation antiporter (Goldberg et al., 1999; Nies, 1995). The complex is made up of three proteins defined CzcA, CzcB and CzcC, respectively the RND protein, the Membrane Fusion Protein, and the Outer Membrane Factor. The first is localized at the inner membrane of the cell and allows the movement of metal cations from the cytosol to the periplasmic space, whereas the B component is located in the periplasmic space, directing ions towards the third element, located in the outer membrane, that opens a membrane channel in order to extrude ions from the bacterium (Rensing et al., 1997). The sequences encoding for CzcA, CzcB and CzcC were amplified from the genomic DNA of P. putida Cd001 and cloned into constructs enabling their overexpression in plants under the control of a plant-specific strong promoter. Arabidopsis thaliana, Nicotiana tabacum and Lycopersicon esculentum were used as test species for transformation. Several stable transgenic lines were obtained for each construct. Singly transformed plants were crossed in order to obtain plants expressing partial (CzcA/CzcB) or whole complex (CzcCBA.). The expression levels of transgenes were analyzed by Real Time PCR. Among transgenic lines of the same genotype, different levels of expression were identified. Worth to note that in both Arabidopsis and tobacco plants overexpressing CzcA/CzcB or CzcA/CzcB/CzcC, the expression level of CzcB is always higher in comparison to the other transgenes. The modulation of metal content in transgenic plants was analyzed carrying out accumulation experiments. Transformed plants were cultured hydroponically and treated with 0.45, 0.7, 5 µM (CdSO4) for Arabidopsis, tobacco and tomato plants respectively. Cd accumulated in shoot was analyzed, after 22 days of treatment by comparing transgenic and wild-type plants. Tobacco transgenic lines showed a reduced Cd content in comparison to wild-type plants. Such reduction in Cd level is enhanced progressively in plants overexpressing simultaneously more components of the CzcCBA system. The single expression of CzcA causes (considering all transgenic lines tested) a Cd reduction on average equal to the 30% in comparison to wild-type, while the concomitant presence of CzcA and CzcB led to 39% reduction. Finally, the overexpression of the whole efflux system, result on average in a 55% reduction in shoot Cd content. In Arabidopsis, the presence of CzcA or CzcB alone does not have a clear effect on Cd content into shoot. Differently, the simultaneous presence of both the components, CzcA and CzcB, results on average in a 23% reduction into shoot in comparison to wild-type. The presence of CzcC does not influence Cd accumulation nor in tobacco neither in Arabidopsis transgenic plants. Since in bacteria the CzcCBA transport system is able to export Cd and Zn, the content of the latter was analyzed in tobacco transgenic lines overexpressing the whole complex CzcCBA. Transgenic lines do not show strong differences in Zn content in comparison to wild-type. In order to better characterize the efflux mechanism of CzcCBA in plant, the sub-cellular localization of the three components was analyzed. The following fusion constructs under the control of the 35SCaMV promoter were obtained: CzcA::eGFP, CzcB::dsRED, CzcC::eYFP. These constructs were used both for stable transformation of tobacco and for protoplast transfection. In both cases samples were observed by confocal microscopy. Tobacco protoplast were transiently transformed with the construct of the three proteins fused to fluorescent reporter, and together with sub-cellular markers, in order to identify the precise location. Microscopy analysis indicates a localization on endoplasmic reticulum for CzcA-GFP; however, the protein seems not to be addressed to the lytic vacuole. Preliminary results suggest that CzcB-RFP forms aggregates with cytosolic localization. CzcC-YFP seems to localize diffusely in the cytosol. Further analyses are necessary to determine the possible co-localization of the three components of the CzcCBA system. In conclusion, results of this study showed that overexpression of single components and even more of the whole efflux system CzcCBA, cause a reduction in Cd content into shoot of transformed plants in comparison to wild-type.