Abstract
The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis.
Highlights
Starch is a vitally important plant-derived material that is widely used as food and in manufacturing of non-food products
Plants produce starch as a means of storing energy; it is composed of two glucose polymers —amylopectin and amylose
While amylose is present in a smaller quantity than amylopectin, it has a major impact on starch processing
Summary
Starch is a vitally important plant-derived material that is widely used as food and in manufacturing of non-food products. In its native form, starch exists as semi-crystalline, insoluble granules that are typically between 1 to 100 μm in diameter, depending on botanical source [2]. It is composed of polymers of glucose, in which α-1,4 bonds connect the glucosyl residues into linear chains and α-1,6 bonds form branch points. The branches in amylopectin are arranged in a clustered structure that allows adjacent chains to form double helices [3]. The packing of these helices result in the crystalline regions of starch. Starch from Arabidopsis leaves contains about 6% amylose [4], while starch in most cereals and storage organs contain about 20%–30% [5]
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