All sorghum MT proteins were produced in bacteria cells, purified by chromatography and analysed using a range of biophysical techniques including electrospray ionisation mass spectrometry (ESI-MS), UV-Vis spectroscopy and elemental analysis. We found that only one sorghum MT protein (SbMT4) showed strict specificity towards zinc. The other MTs seemed to be involved rather in copper or cadmium binding. In addition, SbMT4 was found to be present only in seeds. Therefore, we concluded that this MT had the highest potential in biofortification of seeds with zinc. Surprisingly, the strict zinc-thionein character of SbMT4 confirmed in these experiments was not clearly reflected in metal-dependent protein folding as assessed by nuclear magnetic resonance (NMR) spectroscopy: It was found that SbMT4 was well-folded in the presence of either zinc or cadmium. In parallel, transgenic Arabidopsis thaliana plants overexpressing SbMT4 were created and grown on cadmium-containing media. Those plants did not accumulate more cadmium, neither in shoots nor in roots, and were not more tolerant towards this toxic metal in comparison to wild type plants. These results suggest that cadmium binding by SbMT4 is not effective in the living plant. Further, a state-of-the-art powerful genome editing approach (clustered regularly interspaced short palindromic repeats; CRIPSR/Cas9) was used to create Arabidopsis plants that lack MT4. Those lines are of crucial importance for establishing the physiological roles of these metallothioneins in plants.
To gain further insight into mechanisms of zinc/cadmium discrimination, site-directed mutagenesis was used to generate proteins with changed amino acid composition. Those mutant proteins were analysed by ESI-MS, elemental analysis and NMR techniques. These mutated proteins had higher specificity towards cadmium and hence no ability to discriminate between zinc and cadmium. Transgenic Arabidopsis plants overexpressing those proteins were also created. Crucially, the plants harbouring mutant proteins accumulated more cadmium and were more tolerant towards cadmium. This confirmed the potential of the original sorghum MT4 protein in discrimination between zinc and cadmium in seeds.
Plant metallothioneins are not only metal-binding proteins but are also considered as stress response proteins. Increased amounts of reactive oxygen species (ROS) are a consequence of most stress conditions including drought. We found that Zn-SbMT4 was able to react with ROS and diminished their reactivity which suggested that MTs may act as antioxidants and hence be linked to fundamental stress responses in plants. Transgenic Arabidopsis plants that overexpress all sorghum MTs separately were created, and their tolerance towards different stress conditions is currently being evaluated.
The PMTFOS project was disseminated in non-scientific publications i.e. “Sorghum during drought: protein protect against withering” on the Science in Poland web page and “Boosting nutrients in crops to beat ‘hidden hunger’ of poor diet” in Horizon: the EU Research & Innovation Magazine. The results obtained during the realization of the PMTFOS project were presented on national and international meetings and conferences i.e. 14th Zinc-Net Meeting in Cambridge (poster presentation) and 7th International Symposium on Metallomics in Warsaw (talk). Currently manuscripts for peer-reviewed scientific journals are being prepared.
