Regulation of the activity of Korean radish cationic peroxidase promoter during dedifferentiation and differentiation

Studies of the regulation of the activity of the Korean radish cationic peroxidase ( KRCP ) promoter during dedifferentiation and redifferentiation are reported here. Histochemical staining with 5-bromo-4-chloro-indolyl glucuronide (X-gluc) showed that only dedifferentiated marginal cells of leaf discs of the transgenic plants, but not of the interior region, were stained blue, as leaf discs were incubated on dedifferentiation-inducing medium from 5 days after callus induction (DACI). The levels of cationic peroxidase activity and of KRCP transcripts in Korean radish seedlings ( Raphanus sativus L. F1 Handsome Fall) were also upregulated by a low ratio of cytokinin to auxin, but not by high concentrations of cytokinin. To identify important cis -regulatory regions controlling callus-specific expression, a series of 5′ promoter deletions was carried out with KRCP::GUS gene fusion systems. The data suggest that at least two positively regulatory regions are involved in the KRCP::GUS expression during dedifferentiation induced by a low ratio of cytokinin to auxin: one from –471 to –242 and another from –241 to +196. GUS expression, however, was quickly decreased to a basal level during regeneration of root and shoot. Thus, the downstream region between +197 and +698 seems to be enough to suppress GUS expression of all constructs during regeneration. We further show that the 142-bp fragment (–471 to –328) has at least one cis -element to bind to the nuclear proteins from Korean radish seedlings induced by dedifferentiation.