In vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

Jose A. Cuesta-Seijo, Morten M. Nielsen, Christian Ruzanski, Katarzyna Krucewicz, Sophie Beeren, Maja G. Rydhal, Yayoi Yoshimura, Alexander Striebeck, Mohammed S. Motawia, William G. T. Willats, Monica M. Palcic

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Abstract

Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.
Original languageEnglish
Article number1265
JournalFrontiers in Plant Science
Volume6
Number of pages17
ISSN1664-462X
DOIs
Publication statusPublished - 2016
Externally publishedYes

Bibliographical note

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Keywords

  • Substrate Specificity
  • Kinetics
  • Affinity
  • Stability
  • Barley
  • Biochemical characterization
  • Starch synthases
  • Expression levels

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