The embryo sac of Cynara cardunculus: ultrastructure of the development and localisation of the aspartic proteinase cardosin B

Cynara cardunculus is a native plant with flowers that are used traditionally in the manufacture of ewe’s cheese in the Iberian Peninsula. Milk clotting ability of the plant is attributed to the high concentrations of aspartic proteinases (APs), named cardosins, found in the flowers. Although these enzymes are well characterised on a molecular and biochemical basis, the biological role of the majority of plant APs is yet unassigned. We suspected APs play an important role in ovule function, and we characterised the maturation of the ovules of C. cardunculus and its Polygonum-type embryo sacs. The internal layer of the integument differentiates into an endothelium as described for other Asteraceae, with differentiation of two nucellar layers, a podium and a hypostase coinciding with the onset of pollen receptivity. In flowering plants, programmed cell death (PCD) events are essential for the success of nucellar maturation and consequent differentiation of a fully functional embryo sac. In C. cardunculus, nucellar PCD is integral to the maturation of the embryo sac, which in turn is closely correlated with the accumulation of the AP cardosin B specifically in the hypostase. The onset of cardosin B expression temporally coincides with the degeneration of nucellar cells. In fully mature embryo sacs, cardosin B is localised in both the hypostase and epistase, two regions that differentiate through PCD. Thus, cardosin B localisations closely correlate with events of PCD in the nucellus of C. cardunculus suggesting involvement in ovule and embryo sac development and further suggest the biological significance of APs like cardosin B, in this particular process. This work contributes new data to the plant AP research field and indicates an involvement of cardosin B in the PCD-dependent degeneration of the nucellus.