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Further Evidence for the Mandatory Nature of Polysaccharide Debranching for the Aggregation of Semi-Crystalline Starch and for Overlapping Functions of Debranching Enzymes in Arabidopsis Leaves.
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Edité par CCSD ; Oxford University Press ; American Society of Plant Biologists -
International audience. Four isoforms of debranching enzymes (DBEs) are found in the genome of A. 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 isa3-1 pu1-1 double mutants, the latter decreased by over 98% in the isa1-1 isa3-1 genotype and almost vanished in triple mutant combination. In addition, whereas isa3-1 pu1-1 double mutants synthesizes starch very similar to that of the WT, the structure of the residual starch present either in isa1-1 isa3-1 or in isa1-1 isa3-1 pu1-1 combinations 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 9-10 nm repetition pattern in starch precisely depend on the level of DBEs activity.
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