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 layers was developed which utilizes KCl instead of mannitol as the osmoticum. Protoplasts prepared by this method were shown to be hormone-responsive even after polyethyleneglycol-mediated DNA uptake. Both yield and viability were increased by about 50% and 6%, respectively, compared 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 alpha-amylases. The protoplasts were used also to identify the hormone-responsive 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 deletions 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-amylase genes, gRAmy152 and gRAmy56, indicate that the sequences CTTTTG, TAACAAA, and TATCCAC which are conserved in several oc-amylase genes must act in concert to confer hormone-responsiveness to these two promoters 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 expression and GA-responsiveness. Additional sequences present both upstream and downstream of these three conserved elements also enhance hormone-responsiveness of the reporter genes. For the low-pi alpha-amylase gene gKAmy155 promoter, the presence of ACTTGACCAT-CACC (Opaque 2S-like element), a pyrimidine-rich sequence, and TAACAGA alone is not adequate 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 and -197 of the gKAmy 155 promoter to compose an effective GA response complex. ABA- and GA-responsive elements appear to be coincident or close to each other in both high- and low-pi alpha-amylase genes.