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

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.

Abstract

The biosynthesis of starch in plant chloroplasts depends on a novel protein that targets starch synthase to the growing starch granules; this represents a potential target for the biotechnological modification of starch. Read the Synopsis.

Author Summary

Starch plays a vital role in our everyday lives. It is not only a major dietary carbohydrate, but is also used to manufacture household products such as pharmaceuticals, paper, and textiles. 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. Being able to control the amount of amylose is therefore a goal for biotechnology. Amylose is made by the enzyme GRANULE BOUND STARCH SYNTHASE (GBSS), which was for decades believed to be the only protein required for amylose production. We now report here that a second protein, PROTEIN TARGETING TO STARCH (PTST), is involved in the process. Mutants lacking the PTST protein in the model plant Arabidopsis thaliana fail to make any amylose in starch. This is because the GBSS protein, which normally binds to starch, cannot bind in the absence of PTST. This discovery sheds new light on a previously unknown protein targeting process by which enzymes are delivered to the starch. Furthermore, our discovery highlights PTST an ideal target gene for biotechnology.