N-Linked Glycosylation Modulates Golgi-Independent Vacuolar Sorting Mediated by the Plant Specific Insert

In plant cells, the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant-Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSI domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlike its native glycosylated form, is able to bypass ER-to-Golgi blockage and accumulate in the vacuole. This is also true when the PSI domain is analyzed in the context of the full-length cardosin. Regardless of opening exciting research gaps, the results obtained so far need a more comprehensive study of the mechanisms behind this unconventional direct sorting to the vacuole.

The Plant Specific Insert as an Unconventional Signal in the Route to Plant Vacuoles

In plant cells the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSIs domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlike its native glycosylated form, is able to bypass ER-to-Golgi blockage and accumulate in the vacuole. This is also true when the PSI domain is analyzed in the context of the full-length cardosin. Regardless of opening exciting research gaps, the results obtained so far need a more comprehensive study of the mechanisms behind this unconventional direct sorting to the vacuole.

Structural characterization of the stigma-style complex of Cynara cardunculus (Asteraceae) and immunolocalization of cardosins A and B during floral development

Studies were carried out on the structure of the stigma and style of Cynara cardunculus L. (cardoon) during flower development. The stigma is of the dry type with a papillate cuticularized epidermis. During development, the unicellular papillae become match-stick shaped, cuticularize, and show an increase in vacuolar volume. In mature papillae, two morphologically different vacuoles were observed, one electron-dense and the other electron-transparent, putatively corresponding to distinct vacuolar populations. These vacuoles label differently for cardosin A, specifically detected in the electron-dense compartments. The style is solid with a cuticularized epidermis and a central core of transmitting tissue (TT) several cell layers thick. The TT cells show abundant rough endoplasmic reticulum and Golgi bodies, associated with active secretion. During maturation, TT cells become increasingly separated by a polysaccharide-rich extracellular matrix. Communication between TT cells is maintained via plasmodesmata in longitudinal walls. Distribution of cardosins A and B in developing C. cardunculus flowers was also characterized. The presence of aspartic proteinases (APs) in flowers is unusual, generally occurring at low levels. Cardosins A and B are always present in cardoon florets and localize at distinct pistil levels: stigma (papillae) and style (TT), respectively. This differential localization suggests distinct biological functions for cardosins, most likely essential for reproduction in this species.