Type 1 ribosome-inactivating proteins from Phytolacca dioica L. leaves : differential seasonal and age expression, and cellular localization

Phytolacca dioica type 1 ribosome-inactivating proteins (RIPs) were investigated with the following results: i) fully expanded leaves of young P. dioica, up to three year-old, expressed two novel RIPs, dioicin 1 and dioicin 2, being the former also present in adult P. dioica during the first two weeks of leaf development, and the latter continuously synthesized, with no seasonal or ontogenetic constraint; ii) fully expanded leaves of adult P. dioica expressed four RIPs, PD-Ls1-4, exhibiting seasonal variation. RIPs were localized mostly in the extracellular space, but also in the vacuole and in the Golgi apparatus of mesophyll cells. Dioicin 1 and dioicin 2 were rRNA N-b-glycosidases, respectively endowed with the following properties: i) Mr values 30047.00 and 29910.00; ii) pIs 8.74 and 9,37 and, iii) IC50 values 19.74 (@ 0.658 nM) and 6.85 ng/mL (@ 0.229 nM). Furthermore, they showed APG activity and nicked pBR322 dsDNA. The amino acid sequence of dioicin 2 showed the highest identity (81.6%) and similarity (84.6%) with PAP-II from Phytolacca americana while its identity with other RIPs from Phytolaccaceae was around 40%. The differential season and age expression of RIP isoforms suggests that RIPs developed functions, still unknown, but very likely controlled by environmental factor(s).