Characterization of Arabidopsis thaliana transgenic lines expressing synthetic heavy metal binding oligopeptides
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- Institutt for biologi 
Heavy metal pollution is an increasing environmental problem, and their bio-toxic effects are of great concern. Some organisms have developed strategies to withstand metal excess. A particular case is the so-called metal hyperaccumulator plants which can grow and reproduce on metalliferous soil. The behaviour and capabilities of metal hyperaccumulators could be highly beneficial from an environmental point of view by their potential use in phytoremediation - the using of plants to remove contaminants from soil. To provide clues for optimizing biotechnical approaches aiming at engineering transgenic plants suited for phytoremediation, the non- hyperaccumulator plant Arabidopsis thaliana (A. thaliana) is widely used. One novel approach currently being tested, is the ectopic expression of synthetic heavy metal binding oligopeptides (MeBPep) targeted to the inner face of the plasma membrane. In the present project, transgenic lines of A. thaliana expressing a MeBPep consisting of six histidine residues (His6), in the accession Colombia-0 background and the AtHMA4 overexpressing background, were grown on heavy metal containing medium for 21 days. The purpose was to characterize their tolerance to nickel (Ni), zinc (Zn) and cadmium (Cd) by monitoring the seedlings growth, morphology, physiology and the expression of genes involved in metal homeostasis. An evident trend was observed, in which the lines overexpressing a His6 oligopeptide seemed to grow longer primary roots and have increased biomass production compared to the control lines on Ni-supplemented medium. Although differences were not statistically significant, these results imply that these transgenic lines have enhanced tolerance to Ni. In addition, our results corroborate with previously finding as the overexpression of the HMA4 transporter was found to elevate Zn-tolerance, visualised through longer primary roots of A. thaliana seedlings. Expression analysis of selected genes involved in metal homeostasis showed that ALMT1 were affected by Ni exposure, but no difference was observed between His6-expressing and control lines. As a follow-up on membrane bound MeBPeps consisting of six amino acid residues, longer MeBPeps (36-mers) were cloned into a plant transformation vector for further use in research on the subject. Moreover, as it has been hypothesized that metal hyperaccumulation in plants has evolved as a defence strategy against natural enemies, the effect of heavy metal exposure on the accumulation of glucosinolates (GSL), secondary metabolites known to be involved in plant defences, was monitored to assess whether the two defence strategies act exclusionary on each other or in a complementary way. The results revealed that Zn-exposure significantly reduced the amount of accumulated GSL in shoot tissue of A. thaliana seedlings, which points towards a trade-off between Zn-mediated defence and the secondary metabolite defence system.