Mutational analysis of a plant heat shock element

A total of 32 mutations were generated within the TATA-proximal site 1 (-72 to -47) of soybean heat shock gene Gmhsp17.5E in order to functionally define the optimal configuration of sequences within the heat shock element (HSE). Mutants were tested in vivo utilizing sunflower tumors transformed by a T-DNA based vector. Promoter activity was determined by S1 nuclease hybrid protection analysis of tumor transcripts. A total of five repeats (5'-nGAAn-3' or 5'-nTTCn-3') which comprise the HSE at site 1 were required for full transcription induction by heat stress. Analysis of non-conserved bases flanking the central trinucleotide block indicated that 5'-aGAAg'-3' is the optimum sequence for the 5 bp repeat.