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% o f the protoplasts were viable. During incubation they underwent vacuol ation 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 characte ristics of alpha-galactosidase and endo-beta-mannanase production by p rotoplasts were the same as those of whole endosperms: their productio n did not require any hormonal signal and was inhibited in the presenc e of ABA or the leachate from the carob endosperm/seed coat. Moderate water stress (-2.0 MPa) neither affected the activity of these hydrola ses nor their secretion by endosperm protoplast. However, when the osm oticum of protoplast incubation medium was higher, the production and secretion of both hydrolases were reduced. Comparison of the hydrolase s activities in the incubation media of leached carob endosperms, whic h were incubated under normal and water stress (-1.5 MPa) conditions, with the activities of the protoplast-secreted hydrolases indicated th at (i) carob endosperm cell wall acts as a barrier for the secreted en zymes and (ii) that water stress reduces the cell wall porosity of the carob endosperm cells, and thus the release of the secreted alpha-gal actosidase and endo-beta-mannanase is inhibited. The isolation of caro b endosperm protoplasts offers a potent experimental system for the st udy of aspects of endosperm cell physiology, such as enzyme secretion.