Characterization of aspartic proteinases in C. cardunculus L. callus tissue for its prospective transformation

Cardosins A and B are aspartic proteinases from cardoon that share high similarity, although they accumulate in different subcellular compartments in flowers. This fact raises the importance of understanding the mechanisms that account for this specificity. However, it is difficult to study this dynamics in cardoon given its long life cycle and scarcity of studies regarding in vitro cultures, regeneration and transformation procedures. In vitro cultures of cardoon were established and used to characterise cardosins expression and localisation in callus tissue, through RT-PCR, Western blotting and immunofluorescence. We demonstrate that cardosins are expressed and differentially processed in callus tissue, being synthesized de novo. They were found to accumulate in the endoplasmic reticulum, which may be caused by an early removal of the prosegment. This work supports the hypothesis that cardosins might be involved in the basal metabolism of cardoon cells and, accumulated in its precursor or intermediate forms, their activation is probably triggered when the cell metabolism increases in response to developmental signals and specificity of cell functions. This work also allowed to establish cardoon callus tissue as a valuable tool to further study biosynthesis, processing and sorting of cardosins as related to differentiation and regeneration processes.