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JXB Advance Access originally published online on October 4, 2009
Journal of Experimental Botany 2009 60(15):4423-4440; doi:10.1093/jxb/erp297
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© 2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

The amylose extender mutant of maize conditions novel protein–protein interactions between starch biosynthetic enzymes in amyloplasts

Fushan Liu1, Amina Makhmoudova1, Elizabeth A. Lee2, Robin Wait3, Michael J. Emes1,* and Ian J. Tetlow1

1Department of Molecular and Cellular Biology, College of Biological Sciences, University of Guelph, Guelph, Ontario, N1G 2W1 Canada
2Department of Plant Agriculture, University of Guelph, Guelph, Ontario, N1G 2W1 Canada
3Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College, 1 Aspenlea Road, Hammersmith, London W6 8LH, UK

* To whom correspondence should be addressed: E-mail: memes{at}uoguelph.ca

The amylose extender (ae–) mutant of maize lacks starch branching enzyme IIb (SBEIIb) activity, resulting in amylopectin with reduced branch point frequency, and longer glucan chains. Recent studies indicate isozymes of soluble starch synthases form high molecular weight complexes with SBEII isoforms. This study investigated the effect of the loss of SBEIIb activity on interactions between starch biosynthetic enzymes in maize endosperm amyloplasts. Results show distinct patterns of protein–protein interactions in amyloplasts of ae– mutants compared with the wild type, suggesting functional complementation for loss of SBEIIb by SBEI, SBEIIa, and SP. Coimmunoprecipitation experiments and affinity chromatography using recombinant proteins showed that, in amyloplasts from normal endosperm, protein–protein interactions involving starch synthase I (SSI), SSIIa, and SBEIIb could be detected. By contrast, in ae– amyloplasts, SSI and SSIIa interacted with SBEI, SBEIIa, and SP. All interactions in the wild-type were strongly enhanced by ATP, and broken by alkaline phosphatase, indicating a role for protein phosphorylation in their assembly. Whilst ATP and alkaline phosphatase had no effect on the stability of the protein complexes from ae– endosperm, radiolabelling experiments showed SP and SBEI were both phosphorylated within the mutant protein complex. It is proposed that, during amylopectin biosynthesis, SSI and SSIIa form the core of a phosphorylation-dependent glucan-synthesizing protein complex which, in normal endosperm, recruits SBEIIb, but when SBEIIb is absent (ae–), recruits SBEI, SBEIIa, and SP. Differences in stromal protein complexes are mirrored in the complement of the starch synthesizing enzymes detected in the starch granules of each genotype, reinforcing the hypothesis that the complexes play a functional role in starch biosynthesis.

Key words: Amylopectin, amyloplasts, amylose extender, high-amylose, protein phosphorylation, protein–protein interactions, starch branching enzyme, starch granule associated proteins, starch phosphorylase, starch synthase, starch synthesis

Received 14 August 2009; Revised 7 September 2009 Accepted 7 September 2009


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