An Arabidopsis gene encoding a chloroplast-targeted β-amylase
Article first published online: 5 JAN 2002
The Plant Journal
Volume 20, Issue 5, pages 519–527, December 1999
How to Cite
Lao, N. T., Schoneveld, O., Mould, R. M., Hibberd, J. M., Gray, J. C. and Kavanagh, T. A. (1999), An Arabidopsis gene encoding a chloroplast-targeted β-amylase. The Plant Journal, 20: 519–527. doi: 10.1046/j.1365-313X.1999.00625.x
- Issue published online: 5 JAN 2002
- Article first published online: 5 JAN 2002
- Received 25 May 1999; revised 29 September 1999; accepted 12 October 1999.
β-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 reason, it has been assumed that β-amylases are not involved in the metabolism of chloroplast-localized transitory leaf starch. However, we have identified a novel β-amylase gene, designated ct-Bmy, which is located on chromosome IV of Arabidopsis thaliana. Ct-Bmy encodes a precursor protein which contains a typical N-terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic β-amylases of higher plants. Expression of the ct-Bmy cDNA in E. coli confirmed that the encoded protein possesses β-amylase activity. CT-BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma. In addition, fusions between the predicted CT-BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT-BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis. Expression of the GUS gene fused to ct-Bmy promoter sequences was investigated in transgenic tobacco plants. GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells 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 first true leaves at approximately 4 weeks.