TRANSCRIPTION EFFICIENCY AND HORMONE-RESPONSIVENESS OF ALPHA-AMYLASE GENE PROMOTERS IN AN IMPROVED TRANSIENT EXPRESSION SYSTEM FROM BARLEY ALEURONE PROTOPLASTS

An improved method for isolation of protoplasts from barley aleurone l ayers was developed which utilizes KCl instead of mannitol as the osmo ticum. Protoplasts prepared by this method were shown to be hormone-re sponsive even after polyethyleneglycol-mediated DNA uptake. Both yield and viability were increased by about 50% and 6%, respectively, compa red to the method we had used previously. These protoplasts were used in a transient expression system to compare the relative transcription rates of promoters from barley genes encoding high-pI and low-pI alph a-amylases. The protoplasts were used also to identify the hormone-res ponsive elements and other enhancer elements in barley cr-amylase gene promoters which have not been studied previously. Promoter fragments from two high-pi and one low-pi alpha-amylase genomic clones and delet ions derived from them were fused to a promoterless vector containing the coding region of bacterial chloramphenicol acetyltransferase (CAT) gene. Deletion studies of promoters from two barley high-pi alpha-amy lase genes, gRAmy152 and gRAmy56, indicate that the sequences CTTTTG, TAACAAA, and TATCCAC which are conserved in several oc-amylase genes m ust act in concert to confer hormone-responsiveness to these two promo ters as has been found for other cc-amylase genes. Removal of any one of these three regions causes a severe reduction in overall level of e xpression and GA-responsiveness. Additional sequences present both ups tream and downstream of these three conserved elements also enhance ho rmone-responsiveness of the reporter genes. For the low-pi alpha-amyla se gene gKAmy155 promoter, the presence of ACTTGACCAT-CACC (Opaque 2S- like element), a pyrimidine-rich sequence, and TAACAGA alone is not ad equate for GA-induced gene expression as has been observed previously for another barley low-pi alpha-amylase gene Amy32b. They have to work cooperatively with other element(s) located between positions -256 an d -197 of the gKAmy 155 promoter to compose an effective GA response c omplex. ABA- and GA-responsive elements appear to be coincident or clo se to each other in both high- and low-pi alpha-amylase genes.