Phytochelatin synthases from cyanobacteria and land plants are less functionally different than previously thought.

The enzyme phytochelatin synthase (PCS) is a ubiquitous enzyme in land plants, and it is involved in the synthesis of some peculiar thiol-peptide compounds, the so-called phytochelatins. Previous studies suggest the PCS can play a role in the response to metal homeostatic needs and toxicity in all tracheopytes. Recently, PCS-like proteins, sharing significant sequence homologies with plant PCSs, were identified also in some prokaryotes, such as the model cyanobacterium Nostoc sp. PCC 7120. Previous evidences suggest that predicted product of PCS gene of Nostoc sp. (alr0975) contains the conserved N-terminal domain but not the variable C-terminal domain found in eukaryotic PC synthases. Proteins encoded by the cyanobacterial genes seem to be progenitor or primitive forms of PCS and to represent an early stage in the evolution of the enzyme in photoautotrophic organisms, since up to now only peptidase and less transpeptidase activity has been observed. However, only a few handful investigations have been carried out to functional characterization of PCS from bryophytes and cyanobacteria, despite their importance as fundamental landmarks in evolution. In the study performed here, we want to investigate functional differences in eukaryotic and prokaryotic PCS, studying the enzyme in the model liverwort Marchantia polymorpha and in three cyanobacterial strains (Nostoc sp. PCC 7120, Geitlerinema sp. PCC 7407 and Gloeobacter violaceous PCC 7421). We isolated and characterized PCS from the liverwort M. polymorpha cloning the only PCS gene (MpPCS) present in its genome. Predicted protein of MpPCS is 530 aminoacids with an overall identity of 49% to Arabidopsis thaliana PCS1 protein. As known for other PCS, also MpPCS presents a highly conserved N-terminal domain and a poorly conserved C-terminus. Results obtained by mass-spectrophotometry showed higher amounts of PC2, PC3 and PC4 in cadmium-exposed M. polymorpha gametophytes compared with metal-untreated gametophytes. We are now going to verify also if significantly higher amounts of PCs are synthesized in the overexpressor yeast strain compared with yeast transformed with an empty vector, thus demonstrating that the encoded enzyme is a functional PCS. Even more so, we investigated cyanobacterial PCS activity and our results, obtained by mass-spectrophotometry, showed PCs synthesis in all cadmium-exposed cyanobacteria studied, thus demostrating that cyanobacterial PCS-like enzymes possess transpeptidase activity and they are functional PCS. These evidence highlight a remarkable evolutionary conservation of PCS activity between cyanobacteria and basal land plants.