(200-24) The pseudoenzyme beta-amylase9 from Arabidopsis binds to and enhances the activity of alpha-amylase3: A possible mechanism to promote stress-induced starch degradation.
Teacher-Scholar Postdoc Fellow University Harrisonburg, Virginia
Body of Abstract: Starch accumulates in plastids to store energy and is degraded at night or under stress by a suite of enzymes including β-amylases (BAM). Presently, a major gap in our understanding of starch degradation is how it is regulated. Of the nine BAM genes in Arabidopsis thaliana, BAM9 encodes a plastid-localized, catalytically-inactive pseudoenzyme suspected to play a regulatory role in starch metabolism. Sequence analysis revealed two conserved surfaces of BAM9 suggesting protein binding sites, so we carried out a yeast 2-hybrid search for BAM9-binding partners. Of the 12 confirmed hits encoding plastidic proteins, three were overlapping partial clones from the α-amylase AMY3, which is known to participate in daytime starch degradation in guard cells and under stress. Structurally, AMY3 contains two N-terminal carbohydrate-binding domains (CBD) and a predicted alpha-alpha hairpin (AAH) region between the second CBD and the catalytic domain. In order to explore the interaction between BAM9 and AMY3, we purified both proteins expressed in E. coli and found that individually, only AMY3 was active, but when combined, AMY3 activity was enhanced about 2.5-fold. To investigate structural features of AMY3 and BAM9, we collected small-angle X-ray scattering (SAXS) data and confirmed that both proteins are monomeric in solution. Moreover, AMY3 is relatively compact supporting a mechanism in which the AAH region of AMY3 blocks access to its active site in the absence of BAM9. Interestingly, each partial clone from the Y2H study contains sequence encoding the AAH region. In order to better understand the patterns of BAM9 expression and suggest a starch degradation regulatory pathway, we constructed transgenic plants expressing a BAM9-promoter:GUS gene. BAM9 appears to be expressed in floral tissues, in guard cells, and in response to wounding and touching trichomes. This suggests a role for calcium in stimulating stress-induced starch degradation by AMY3 through activation by BAM9.