title: Beta-AMYLASE4, a noncatalytic protein required for starch breakdown, acts upstream of three active beta-amylases in Arabidopsis chloroplasts.
abstract: This work investigated the roles of beta-amylases in the breakdown of leaf starch. Of the nine beta-amylase (BAM)-like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable beta-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized beta-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active beta-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total beta-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that beta-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.
authors.affiliation: Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3JH, United Kingdom.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Fulton
authors.forename: Michaela
authors.initials: M
authors.lastname: Stettler
authors.forename: Tabea
authors.initials: T
authors.lastname: Mettler
authors.forename: Cara K
authors.initials: CK
authors.lastname: Vaughan
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Perigio
authors.initials: P
authors.lastname: Francisco
authors.forename: Manuel
authors.initials: M
authors.lastname: Gil
authors.forename: Heike
authors.initials: H
authors.lastname: Reinhold
authors.forename: Simona
authors.initials: S
authors.lastname: Eicke
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Messerli
authors.forename: Gary
authors.initials: G
authors.lastname: Dorken
authors.forename: Karen
authors.initials: K
authors.lastname: Halliday
authors.forename: Alison M
authors.initials: AM
authors.lastname: Smith
authors.forename: Steven M
authors.initials: SM
authors.lastname: Smith
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Zeeman
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000378:metabolism*; D001614:beta-Amylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D013213:Starch; D001614:beta-Amylase