First published online December 23, 2004; 10.1104/pp.104.055954
Plant Physiology 137:242-252 (2005)
© 2005 American Society of Plant Biologists
BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES
Identification of a Novel Enzyme Required for Starch Metabolism in Arabidopsis Leaves. The Phosphoglucan, Water Dikinase1,[w]
Oliver Kötting,
Kerstin Pusch,
Axel Tiessen,
Peter Geigenberger,
Martin Steup and
Gerhard Ritte*
Plant Physiology, Institute of Biochemistry and Biology, University of Potsdam, 14476 Golm, Germany (O.K., K.P., M.S., G.R.); and Max Planck Institute of Molecular Plant Physiology, 14476 Golm, Germany (A.T., P.G.)
The phosphorylation of amylopectin by the glucan, water dikinase (GWD; EC 2.7.9.4) is an essential step within starch metabolism. This is indicated by the starch excess phenotype of GWD-deficient plants, such as the sex1-3 mutant of Arabidopsis (Arabidopsis thaliana). To identify starch-related enzymes that rely on glucan-bound phosphate, we studied the binding of proteins extracted from Arabidopsis wild-type leaves to either phosphorylated or nonphosphorylated starch granules. Granules prepared from the sex1-3 mutant were prephosphorylated in vitro using recombinant potato (Solanum tuberosum) GWD. As a control, the unmodified, phosphate free granules were used. An as-yet uncharacterized protein was identified that preferentially binds to the phosphorylated starch. The C-terminal part of this protein exhibits similarity to that of GWD. The novel protein phosphorylates starch granules, but only following prephosphorylation with GWD. The enzyme transfers the  -P of ATP to the phosphoglucan, whereas the  -P is released as orthophosphate. Therefore, the novel protein is designated as phosphoglucan, water dikinase (PWD). Unlike GWD that phosphorylates preferentially the C6 position of the glucose units, PWD phosphorylates predominantly (or exclusively) the C3 position. Western-blot analysis of protoplast and chloroplast fractions from Arabidopsis leaves reveals a plastidic location of PWD. Binding of PWD to starch granules strongly increases during net starch breakdown. Transgenic Arabidopsis plants in which the expression of PWD was reduced by either RNAi or a T-DNA insertion exhibit a starch excess phenotype. Thus, in Arabidopsis leaves starch turnover requires a close collaboration of PWD and GWD.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant nos. SFB 429 TP–B2 to M.S. and TP–B7 to G.R. and P.G.).
[w] The online version of this article contains Web-only data.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.055954.
* Corresponding author; e-mail ritte{at}rz.uni-potsdam.de; fax 49–331–977–2512.
Received November 3, 2004;
returned for revision November 16, 2004;
accepted November 16, 2004.
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