Additional file 5 of Gene expression profile of the developing endosperm in durum wheat provides insight into starch biosynthesis

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Additional file 1 of Gene expression profile of the developing endosperm in durum wheat provides insight into starch biosynthesis

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Additional file 7 of Gene expression profile of the developing endosperm in durum wheat provides insight into starch biosynthesis

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Additional file 3 of Gene expression profile of the developing endosperm in durum wheat provides insight into starch biosynthesis

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Additional file 6 of Gene expression profile of the developing endosperm in durum wheat provides insight into starch biosynthesis

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Additional file 4 of Gene expression profile of the developing endosperm in durum wheat provides insight into starch biosynthesis

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Additional file 2 of Gene expression profile of the developing endosperm in durum wheat provides insight into starch biosynthesis

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PROTEIN TARGETING TO STARCH Is Required for Localising GRANULE-BOUND STARCH SYNTHASE to Starch Granules and for Normal Amylose Synthesis in Arabidopsis

<div><p>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 <i>ptst</i> 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 <i>ptst</i> mutant starch granules. Pull-down assays with recombinant proteins <i>in vitro</i>, as well as immunoprecipitation assays <i>in planta</i>, 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.</p></div

Proposed model of PTST-mediated GBSS localisation to starch.

<p>(A) GBSS docks onto PTST in the stroma, and the complex binds to starch. (B) PTST dissociates from both GBSS and the starch granule, leaving GBSS on starch to mediate amylose synthesis. (C) PTST returns to the stroma to recruit another GBSS molecule.</p

Localisation of fluorescently tagged PTST and GBSS in tobacco leaves.

<p>(A) PTST-YFP and GBSS-CFP were individually or co-expressed in tobacco epidermal cells and imaged using confocal microscopy. Note that the localisation of GBSS-CFP on starch granules depends on PTST-YFP co-expression. Bar = 10 μm. (B) Same as (A), but additionally using the W217A/W255A non-starch-binding variant of PTST. Bar = 4 μm.</p