alpha-Amylase is not required for breakdown of transitory starch in Arabidopsis leaves.
The Arabidopsis thaliana genome encodes three alpha-amylase-like proteins (AtAMY1, AtAMY2, and AtAMY3). Only AtAMY3 has a predicted N-terminal transit peptide for plastidial localization. AtAMY3 is an unusually large alpha-amylase (93.5 kDa) with the C-terminal half showing similarity to other known alpha-amylases. When expressed in Escherichia coli, both the whole AtAMY3 protein and the C-terminal half alone show alpha-amylase activity. We show that AtAMY3 is localized in chloroplasts. The starch-excess mutant of Arabidopsis sex4, previously shown to have reduced plastidial alpha-amylase activity, is deficient in AtAMY3 protein. Unexpectedly, T-DNA knock-out mutants of AtAMY3 have the same diurnal pattern of transitory starch metabolism as the wild type. These results show that AtAMY3 is not required for transitory starch breakdown and that the starch-excess phenotype of the sex4 mutant is not caused simply by deficiency of AtAMY3 protein. Knock-out mutants in the predicted non-plastidial alpha-amylases AtAMY1 and AtAMY2 were also isolated, and these displayed normal starch breakdown in the dark as expected for extraplastidial amylases. Furthermore, all three AtAMY double knock-out mutant combinations and the triple knock-out degraded their leaf starch normally. We conclude that alpha-amylase is not necessary for transitory starch breakdown in Arabidopsis leaves.
Yu
Tien-Shin
TS
Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, China.
Zeeman
Samuel C
SC
Thorneycroft
David
D
Fulton
Daniel C
DC
Dunstan
Hannah
H
Lue
Wei-Ling
WL
Hegemann
Björn
B
Tung
Shu-Yun
SY
Umemoto
Takayuki
T
Chapple
Andrew
A
Tsai
Der-Long
DL
Wang
Shue-Mei
SM
Smith
Alison M
AM
Chen
Jychian
J
Smith
Steven M
SM
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D002241:Carbohydrates / Q000737:chemistry; D002736:Chloroplasts / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004269:DNA, Bacterial; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D005821:Genetic Techniques; D018745:Genome, Plant; D015151:Immunoblotting; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D018515:Plant Leaves / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013213:Starch; D013997:Time Factors; D000516:alpha-Amylases / Q000502:physiology*
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D002241:Carbohydrates; D004269:DNA, Bacterial; D018076:DNA, Complementary; C036483:T-DNA; D013213:Starch; D004247:DNA; D000516:alpha-Amylases