Identification of a Bipartite Nuclear Localization Signal in a Catalytically Inactive β‐Amylase (BAM8) from Arabidopsis

Starch accumulates in leaves during the day and is broken down at night when plants are unable to photosynthesize. Enzymes of the β‐amylase (BAM) family are responsible for degrading starch to maltose, which is then exported to the cytosol where it is converted to hexose‐phosphate. The Arabidopsis thaliana genome contains nine BAM genes ( BAM1‐9 ). Using transgenic plants expressing 35S:BAM‐GFP constructs we determined that BAM7 and ‐8 were localized to the nucleus. The sequences of BAM7 and ‐8 have an N‐terminal domain not found in the other BAMs, that contains a basic‐helix‐loop‐helix motif similar to the known DNA‐binding domains of BZR1 and BES1 . A putative bipartite nuclear localization signal (NLS) was identified in BAM8's N‐terminal sequence. We mutated the putative NLS of BAM8 and transformed the mutant plasmids into Agrobacterium tumefaciens and then inflitrated the cells into Nicotiana benthamiana leaves. Several of the mutant proteins remained in the cytosol confirming the identity of the NLS. In order to see if BAM8 was active in hydrolyzing starch, it was expressed in E. coli . When compared to BAM3, a catalytically active BAM, BAM8 showed no detectable catalytic activity. We hypothesize that BAM7 and ‐8 act as transcription factors to serve as a link between starch availability and control of growth. This work was supported by the Jeffress Memorial Trust.