Periodic Reporting for period 1 - PMTFOS (Plant metallothioneins as potential players in food security)
Reporting period: 2017-08-01 to 2019-07-31
It is estimated that over 2 billion people all over the word suffer from micronutrients deficiency (so called “hidden hunger”). Among several micronutrients, including iron, iodine, and copper, zinc is of particular importance because it is required not for one or a few specific functions, but is involved in almost all conceivable metabolic processes. Therefore, on the one hand zinc deficiency poses a serious threat to humans’ and animals’ life and health and on the other hand it severely impairs plant growth and crop yields. A long term and cost-effective strategy to overcome these issues is biofortification that is based on improved crop cultivars capable to accumulate more micronutrients in their edible parts. The other serious issue emerging in this context are toxic metals like cadmium. The chemical properties of zinc and cadmium are very similar and therefore most proteins involved in zinc uptake from soil and transport and storage within plants also bind toxic cadmium efficiently. Importantly, contamination of the soil with cadmium is not restricted to industrial and mining areas, but recently the amount of this toxic metal has increased also in agricultural land as a result of the usage of cadmium-rich phosphate fertilizers and sewage sludge. Hence, it is crucial to identify proteins not only able to bind zinc but also able to discriminate between essential and non-essential metals. Micronutrient homeostasis in living organisms is governed by complicated networks of countless proteins. Among them, there is a family of proteins called metallothioneins (MTs) that have been shown previously to fold in a metal-dependent fashion. It was proposed that this mechanism is responsible for folding-mediated metal-discrimination.
The overall objective of the PMTFOS project was the comprehensive analysis of the whole family of metallothioneins in Sorghum bicolor. Sorghum is a remarkable crop currently widely cultivated in arid and semi-arid regions of the world due to its unusual drought and heat resistance. More frequent and more widespread drought incidence will likely lead to more global usage of this crop in the future. A cross-disciplinary approach from whole-organism studies to the level of individual proteins was exploited to determine the potential of sorghum metallothioneins for zinc/cadmium discrimination and further for biofortification purposes.
The overall objective of the PMTFOS project was the comprehensive analysis of the whole family of metallothioneins in Sorghum bicolor. Sorghum is a remarkable crop currently widely cultivated in arid and semi-arid regions of the world due to its unusual drought and heat resistance. More frequent and more widespread drought incidence will likely lead to more global usage of this crop in the future. A cross-disciplinary approach from whole-organism studies to the level of individual proteins was exploited to determine the potential of sorghum metallothioneins for zinc/cadmium discrimination and further for biofortification purposes.
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.
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.
Food security is one of the most important global challenges nowadays and the development of stress-resistant nutritious crop plants able to give high yields even in adverse environmental conditions are crucial challenges for European and world agriculture. Therefore, the new knowledge generated during the realization of the PMTFOS project is not only of great importance for scientist working in different areas of food security but also addresses issues that are crucial for the whole society. In the face of the growing world population and decreasing crop yields due to more severe biotic and abiotic stress conditions, the significance of research in the field of food security has indisputably increased. The results obtained have revealed that plant MTs may have great potential for biofortification, but that the ability of zinc/cadmium discrimination differs for individual MTs. Therefore, these results encourage future research in the field of plant science, food security and micronutrients homeostasis.
The PMTFOS project filled the existing knowledge gap about metallothioneins in the economically important staple crop sorghum. A cross-disciplinary approach combining bioinorganic chemistry, molecular biology and plant science was used to comprehensively analyse the whole family of MTs in sorghum. Coherent biophysical data combined with in planta results for the entire family of MTs from a single plant species did not exist before. In addition, CRISPR/Cas9 genome editing was used for the first time in determining the physiological roles of plant metallothioneins.
The PMTFOS project filled the existing knowledge gap about metallothioneins in the economically important staple crop sorghum. A cross-disciplinary approach combining bioinorganic chemistry, molecular biology and plant science was used to comprehensively analyse the whole family of MTs in sorghum. Coherent biophysical data combined with in planta results for the entire family of MTs from a single plant species did not exist before. In addition, CRISPR/Cas9 genome editing was used for the first time in determining the physiological roles of plant metallothioneins.