An Arabidopsis gene encoding a chloroplast-targeted beta-amylase

beta-Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs. Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reas on, it has been assumed that beta-amylases are not involved in the metaboli sm of chloroplast-localized transitory leaf starch. However, we have identi fied a novel beta-amylase gene, designated ct-Bmy, which is located on chro mosome IV of Arabidopsis thaliana. Ct-Bmy encodes a precursor protein which contains a typical N-terminal chloroplast import signal and is highly simi lar at the amino acid level to extrachloroplastic beta-amylases of higher p lants. Expression of the ct-Bmy cDNA in E. coli confirmed that the encoded protein possesses beta-amylase activity. CT-BMY protein, synthesized in vit ro, was efficiently imported by isolated pea chloroplasts and shown to be l ocated in the stroma. In addition, fusions between the predicted CT-BMY tra nsit peptide and jellyfish green fluorescent protein (GFP) or the entire CT -BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabido psis. Expression of the GUS gene fused to ct-Bmy promoter sequences was inv estigated in transgenic tobacco plants. GUS activity was most strongly expr essed in the palisade cell layer in the leaf blade and in chlorenchyma cell s associated with the vascular strands in petioles and stems. Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the fi rst true leaves at approximately 4 weeks.