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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Further Evidence for the Mandatory Nature of Polysaccharide Debranching for the Aggregation of Semicrystalline Starch and for Overlapping Functions of Debranching Enzymes in Arabidopsis Leaves^1,[W]

Unité de Glycobiologie Structurale et Fonctionnelle, UMR8576 CNRS/Université des Sciences et Technologies de Lille, F–59655 Villeneuve d'Ascq, France (F.W., Y.D., N.S., A.D., S.B., C.D.); and UR1268 Biopolymères, Interactions, Assemblages, INRA France, F–44300 Nantes, France (V.P., B.P.)

Four isoforms of debranching enzymes are found in the genome of Arabidopsis (Arabidopsis thaliana): three isoamylases (ISA1, ISA2, and ISA3) and a pullulanase (PU1). Each isoform has a specific function in the starch pathway: synthesis and/or degradation. In this work we have determined the levels of functional redundancy existing between these isoforms by producing and analyzing different combinations of mutations: isa3-1 pu1-1, isa1-1 isa3-1, and isa1-1 isa3-1 pu1-1. While the starch content strongly increased in the isa3-1 pu1-1 double mutant, the latter decreased by over 98% in the isa1-1 isa3-1 genotype and almost vanished in triple mutant combination. In addition, whereas the isa3-1 pu1-1 double mutant synthesizes starch very similar to that of the wild type, the structure of the residual starch present either in isa1-1 isa3-1 or in isa1-1 isa3-1 pu1-1 combination is deeply affected. In the same way, water-soluble polysaccharides that accumulate in the isa1-1 isa3-1 and isa1-1 isa3-1 pu1-1 genotypes display strongly modified structure compared to those found in isa1-1. Taken together, these results show that in addition to its established function in polysaccharide degradation, the activity of ISA3 is partially redundant to that of ISA1 for starch synthesis. Our results also reveal the dual function of pullulanase since it is partially redundant to ISA3 for degradation and to ISA1 for synthesis. Finally, x-ray diffraction analyses suggest that the crystallinity and the presence of the 9- to 10-nm repetition pattern in starch precisely depend on the level of debranching enzyme activity.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Christophe D'Hulst (christophe.dhulst{at}univ-lille1.fr).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.129379

^* Corresponding author; e-mail christophe.dhulst{at}univ-lille1.fr.

Received September 5, 2008; accepted September 22, 2008; published September 24, 2008.

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