THE LYSINE-DEPENDENT STIMULATION OF LYSINE CATABOLISM IN TOBACCO SEEDREQUIRES CALCIUM AND PROTEIN-PHOSPHORYLATION

The accumulation of free lysine in tobacco seed triggers the stimulati on of lysine-ketoglutarate reductase, an enzyme that acts; in lysine c atabolism. The mechanism of lysine-ketoglutarate reductase stimulation was studied in two different systems: (1) developing seeds of wild-ty pe plants in which the low basal lysine-ketoglutarate reductase activi ty can be stimulated by the exogenous addition of lysine; and (2) deve loping seeds of transgenic tobacco plants expressing a bacterial dihyd rodipicolinate synthase in which lysine-ketoglutarate reductase activi ty is stimulated by endogenous lysine overproduction. In both systems, the stimulation of lysine-ketoglutarate reductase activity was signif icantly reduced when treated with the Ca2+ chelator EGTA, Moreover, th e inhibitory effect of EGTA was overcome by the addition of Ca2+ but n ot Mg2+, suggesting that the lysine-dependent activation of lysine-ket oglutarate reductase requires Ca2+. This was further confirmed by a si gnificant stimulation of lysine-ketoglutarate reductase activity follo wing the treatment of wild-type seeds with ionomycin (an ionophore tha t increases Ca2+ flow into the cytoplasm), In addition, treatment of w ild-type seeds with the protein phosphatase inhibitor okadaic acid tri ggered a significant induction in lysine-ketoglutarate reductase activ ity, whereas treatment of the transgenic seeds with the protein kinase inhibitor K-252a caused a significant reduction in its activity. Thus , we conclude that the stimulation of lysine-ketoglutarate reductase a ctivity by lysine in tobacco seed operates through an intracellular si gnaling cascade mediated by Ca2+ and protein phosphorylation.