2 10-BP REGIONS ARE CRITICAL FOR PHYTOCHROME REGULATION OF A LEMNA-GIBBA LHCB-GENE PROMOTER

Two small regions of the promoter of an Lhcb gene encoding a light-har vesting chlorophyll alb protein were identified as essential in confer ring phytochrome responsiveness by using a transient expression assay. Initially, 5' deletion analysis of cabAB19, an Lhcb2 gene of Lemna, s howed that sequences within the region from -174 to -104 relative to t he start of transcription were necessary far phytochrome regulation. I nternal deletion and substitution mutants were used to demonstrate tha t no additional phytochrome-responsive regions exist between -1600 and -174 in this promoter. A 171-bp fragment of the promoter extending fr om -239 to -69 was sufficient to impart phytochrome responsiveness to a minimal ubiquitin promoter that was not itself regulated by light. S pecific binding of Lemna proteins to the region necessary for phytochr ome responsiveness was demonstrated using in vitro polyacrylamide gel mobility shift assays and 1,10-phenanthroline copper ion footprinting. Further analysis of the region from -174 to -104 demonstrated that mu tations in two separate 10-bp sequences, from -134 to -125 and from -1 14 to -105, could abolish phytochrome responsiveness; thus, there are two unique regions that are necessary for phytochrome regulation of th is gene. One of these regions contains a CCAAT motif and the other a G ATA motif. These motifs are conserved in the promoters of many Lhcb ge nes and may be important elements in the phytochrome responsiveness of this gene family.