A novel alpha-amylase gene is transiently upregulated during low temperature exposure in apple fruit

An alpha-amylase gene product was isolated from apple fruit by reverse-tran scriptase PCR using redundant primers, followed by 5' and 3' RACE. The gene is a member of a small gene family. It encodes a putative 46.9 kDa protein that is most similar to an alpha-amylase gene from potato (GenBank accessi on M79328). In apple fruit this new gene was expressed at low levels, as de tected by reverse-transcriptase PCR, in a number of plant tissues and durin g fruit development. Highest levels of mRNA for this transcript were observ ed 3 to 9 days after placing apple fruit at 0.5 degrees C. Phylogenetic ana lysis of amino acid sequence places the potato and apple proteins as a dist inct and separate new subgroup within the plant alpha-amylases, which appea rs to have diverged prior to the split between monocotyledonous and dicotyl edonous plants. These two divergent alpha-amylases lack the standard signal peptide structures found in all other plant alpha-amylases, and have seque nce differences within the B-domain and C-domain. However, comparisons with structures of known starch hydrolases suggest that these differences are u nlikely to affect the enzymatic alpha-1,4-amylase function of the protein. This is the first report of upregulation of a dicotyledonous alpha-amylase in response to low temperature, and confirms the presence of a new family o f alpha-amylases in plants.