Ancient duplication and functional differentiation of phytochelatin synthases is conserved in plant genomes

Despite the paramount importance in metal(loid) detoxification by <i>phytochelatin synthase</i> (<i>PCS</i>) genes, no comprehensive analysis of their evolutionary patterns has been carried out in land plants in general and in crops in particular. A phylogenetic large-scale analysis of gene duplication in angiosperms was carried out followed by <i>in vitro</i> recombinant protein assays as well as complementation analysis (growth, thiol-peptides, elements) of <i>Arabidopsis cad1-3</i> mutant with four representative <i>PCS</i> genes from two model crop species, <i>Malus domestica</i> and <i>Medicago truncatula</i>. We uncovered a so far undetected ancient tandem duplication (D duplication) spanning the whole core eudicotyledon radiation. Complementation with <i>PCS</i> genes from both D-subclades from <i>M. domestica</i> and <i>M. truncatula</i> displayed clear <i>in vivo</i> conservation of the differences between D1 and D2 paralogous proteins in plant growth, phytochelatin, and glutathione pools, as well as element contents under metal(loid) stress. <i>In vitro</i> recombinant PCS analysis identified analogous patterns of differentiation, showing a higher activity of D2 <i>PCS</i> genes, so far largely overlooked, compared to their paralogs from the D1 clade. This suggests that in many other crop species where the duplication is present, the D2 copy might play a significant role in metal(loid) detoxification. The retention of both <i>PCS</i> paralogs and of their functional features for such long divergence time suggests that <i>PCS</i> copy number could be constrained by functional specialization and/or gene dosage sensitivity. These results uncover the patterns of <i>PCS</i> evolution in plant genomes and of functional specialization of their paralogs in the genomes of two important model crops.