PRODUCTION AND SECRETION OF ALPHA-GALACTOSIDASE AND ENDO-BETA-MANNANASE BY CAROB (CERATONIA-SILIQUA L) ENDOSPERM PROTOPLASTS

Viable protoplasts were isolated for the first time from mature carob (Ceratonia siliqua L.) endosperm tissue. After 5 d of incubation 75% of the protoplasts were viable. During incubation they underwent vacuolation and produced the carob endosperm hydrolases, alpha-galactosidase and endo-beta-mannanase, which were secreted in the incubation medium, The secretion of both enzymes were under Ca2+ control. Many characteristics of alpha-galactosidase and endo-beta-mannanase production by protoplasts were the same as those of whole endosperms: their production did not require any hormonal signal and was inhibited in the presence of ABA or the leachate from the carob endosperm/seed coat. Moderate water stress (-2.0 MPa) neither affected the activity of these hydrolases nor their secretion by endosperm protoplast. However, when the osmoticum of protoplast incubation medium was higher, the production and secretion of both hydrolases were reduced. Comparison of the hydrolases activities in the incubation media of leached carob endosperms, which were incubated under normal and water stress (-1.5 MPa) conditions, with the activities of the protoplast-secreted hydrolases indicated that (i) carob endosperm cell wall acts as a barrier for the secreted enzymes and (ii) that water stress reduces the cell wall porosity of the carob endosperm cells, and thus the release of the secreted alpha-galactosidase and endo-beta-mannanase is inhibited. The isolation of carob endosperm protoplasts offers a potent experimental system for the study of aspects of endosperm cell physiology, such as enzyme secretion.