BINDING-SITE REQUIREMENTS AND DIFFERENTIAL REPRESENTATION OF TGA FACTORS IN NUCLEAR ASF-1 ACTIVITY

Activating sequence factor 1 (ASF-1) is a nuclear DNA-binding activity that is found in monocots and dicots, It interacts with several TGACG -containing elements that have been characterized from viral and T-DNA genes, the prototypes of which are the as-1 element of the CaMV 35S p romoter and the ocs element from the octopine synthase promoter, This class of cis-acting elements can respond to auxin and salicylic acid t reatments, Consistent with these observations, we have shown that ASF- 1 can interact with promoter elements of an auxin-inducible tobacco ge ne GNT35, encoding a glutathione S-transferase. Characterization of th e nuclear factors that make up ASF-1 activity in vivo will be an impor tant step toward understanding this induction phenomenon, The TGA fami ly of basic-leucine-zipper (bZIP) proteins are good candidates for the ASF-1 nuclear factor, However, there may be as many as seven distinct TGA genes in Arabidopsis, five of which have now been reported, In th is study, we expressed the cDNAs that encode four of these five Arabid opsis TGA factors in vitro and compared their DNA-binding behavior usi ng two. types of TGACG-containing elements, With specific antisera pre pared against three of the five known Arabidopsis IGA factors, we also investigated the relative abundance of these three proteins within th e ASF-1 activities of root and leaf nuclear extracts, Our results indi cate that these TGA factors bind to DNA with different degrees of coop erativity and their relative affinity toward as-1 also can differ sign ificantly, The results of a supershift assay suggested that only one o f the three TGA factors represented a significant component of nuclear ASF-1 activity, Arabidopsis TGA2 comprises similar to 33 and 50% of t he ASF-1 activity detected in root and leaf nuclear extracts respectiv ely. These results suggest that each member of the TGA factor family m ay be differentially regulated and that they may play different roles by virtue of their distinct DNA-binding characteristics. Furthermore, since transcripts for each of these factors can be detected in various plant tissues, post-transcriptional regulation may play an important part in determining their contribution to nuclear ASF-1 in a given cel l type.