The main aim of the proposed project is to identify the molecular and chemical mechanisms underlying tolerance to heavy metals in Viola spp. The project will focus on an example of heavy metal excluder – Viola tricolor represented by two genotypes: metallicolous (MET) – growing at polluted sites and non-metallicolous (NMET) – growing at non-polluted sites, both differing morphologically, genetically and in capability of accumulating heavy metals to indicate genotype-dependent basis of tolerance to heavy metals. Violaceae family, including genus Viola (violets) is rich in metallophytes – species occurring in habitats with soils extremely polluted with heavy metals (e.g., serpentine, calamine, cupriferous or arsenic). These species must have developed the mechanisms which make them tolerant to the excess of heavy metals. Many of violets are metal excluders, which means that, in contrary to hyperacumulators, they prevent the translocation of heavy metals to the aerial parts of plant. While the molecular and chemical basis of tolerance mechanisms of hyperaccumulators are widely investigated, especially in species from the Brassicaceae family, little is known about non-model heavy metal excluders. Therefore, the following questions are raised: 1) what molecular mechanisms are engaged in exclusion of heavy metals (Zn and Pb) in violets, making them heavy metal tolerant? 2) what are the differences in gene expression level and profile between these two genotypes (MET vs NMET)? 3) do some chemical compounds (metabolites) produced by violets trap and detoxify heavy metals? 4) which tissues (especially of roots) are specifically involved in heavy metal binding and detoxification? We hypothesize that V. tricolor develops either universal (production of non-enzymatic antioxidants and protein heavy metal transporters), or specific to violets (e.g., cyclotides) mechanisms of tolerance to Zn and Pb, both unexplored so far. The following techniques and experimental systems will be used: (Task 1) treatment of plants with heavy metals (Zn and Pb in concentration ≤ 1000 ppm) under experimental conditions; (Task 2) measurement of heavy metal content in roots and leaves with atomic absorption spectrometry (AAS); (Task 3) RNA-seq transcriptome profiling for the determination of plant response to heavy metals at the gene expression level; (Task 4 - 6) application of different mass spectrometry and chromatography techniques (MALDI-MSI – matrix assisted laser desorption ionization mass spectrometry imaging; LC-MS – liquid chromatography mass spectrometry; LC-MS/MS – tandem MS) for metabolomic profiling and mining in response to heavy metals on the plant organ and tissue level, as well as for indication of compounds that bind and detoxify heavy metals.