PHOSPHORYLATION OF NUCLEAR PROTEINS DIRECTS BINDING TO SALICYLIC ACID-RESPONSIVE ELEMENTS

The cis-located DNA sequence as-1 (Activation Sequence-1) from CaMV 35 S promoter has been previously identified as an element that can confe r inducibility by salicylic acid (SA) with immediate early kinetics. T his sequence specifically binds to ASF-1 (Activation Sequence Factor-1 ), previously characterized in tobacco nuclear extracts. To assess whe ther modulation of ASF-1 binding activity can explain the activation o f the as-1 sequence observed in vivo, we performed electrophoretic mob ility shift assays using nuclear extracts from SA-treated and water-tr eated tobacco plants. Our results indicate that treatment of plants wi th SA increases ASF-1 binding to as-1 and to ocs, an as-1-like element from the Agrobacterium octopine synthase gene. In contrast, SA treatm ent has no effect on the binding of GT-1 factor to its target light-in ducible box II element. Furthermore, treatment of nuclear extracts fro m SA-treated plants with alkaline phosphatase decreases ASF-1 binding to the as-1 element. This can be reversed by pretreatment with 10 mM N aF. Accordingly, pretreatment of nuclear extracts from control water-t reated plants with ATP produces an increase in ASF-1 binding activity similar to that observed with SA. This effect of ATP is reversed by tr eatment with alkaline phosphatase and prevented by quercetin, a casein kinase II inhibitor. These results support the hypothesis that a nucl ear protein kinase is involved in the immediate early events of transc riptional activation triggered by SA.