Planta DOI 10.1007/s00425-010-1276-9 123 ORIGINAL ARTICLE Dissecting cardosin B traYcking pathways in heterologous systems Diana Soares da Costa · Susana Pereira · Ian Moore · José Pissarra Received: 28 June 2010 / Accepted: 7 September 2010  Springer-Verlag 2010 Abstract In cardoon pistils, while cardosin A is detected in the vacuoles of stigmatic papillae, cardosin B accumu- lates in the extracellular matrix of the transmitting tissue. Given cardosins’ high homology and yet diVerent cellular localisation, cardosins represent a potentially useful model to understand and study the structural and functional plas- ticity of plant secretory pathways. The vacuolar targeting of cardosin A was replicated in heterologous species so the targeting of cardosin B was examined in these systems. Inducible expression in transgenic Arabidopsis and tran- sient expression in tobacco epidermal cells were used in parallel to study cardosin B intracellular traYcking and localisation. Cardosin B was successfully expressed in both systems where it accumulated mainly in the vacuole but it was also detected in the cell wall. The glycosylation pattern of cardosin B in these systems was in accordance with that observed in cardoon high-mannose-type glycans, suggest- ing that either the glycans are inaccessible to the Golgi pro- cessing enzymes due to cardosin B conformation or the protein leaves the Golgi in an early step before Golgi-mod- ifying enzymes are able to modify the glycans. Concerning cardosin B traYcking pathway, it is transported through the Golgi in a RAB-D2a-dependent route, and is delivered to the vacuole via the prevacuolar compartment in a RAB- F2b-dependent pathway. Since cardosin B is secreted in cardoon pistils, its localisation in the vacuoles in cardoon ovary and in heterologous systems, suggests that the diVer- ential targeting of cardosins A and B in cardoon pistils results principally from diVerences in the cells in which these two proteins are expressed. Keywords Aspartic proteinases · Cardosins · Dexamethasone-inducible expression · RabGTPases · Transient expression Abbreviations AP Aspartic proteinases ER Endoplasmic reticulum PCD Programmed cell death PSI Plant-speciWc insert Introduction Cardosins are aspartic proteinases from the Xowers of Cynara cardunculus L. (cardoon). Cardosin A is the most studied enzyme of cardoon Xowers, due to its abundance in these tissues which facilitated its isolation and therefore its characterisation (Ramalho-Santos et al. 1997). Although less abundant, cardosin B has also been isolated and char- acterized at the molecular and biochemical levels, though to a less extent (Vieira et al. 2001). Cardosins diverge in terms of tissue and intracellular localisation: cardosin A was mainly detected in the protein storage vacuoles of the stig- matic papillae (Ramalho-Santos et al. 1997), whereas card- osin B accumulates in the extracellular matrix of the stylar transmitting tissue (Vieira et al. 2001). Considering that both enzymes share a high similarity of the primary struc- ture (73%), their distinct biochemical behaviours could be accounted, at least in part, by the diVerences in the amino D. S. da Costa (&) · S. Pereira · J. Pissarra Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre, s/nº, 4169-007 Porto, Portugal e-mail: diana.costa@fc.up.pt I. Moore Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX13RB, UK