Starch is the most widespread and abundant storage carbohydrate in plants. We depend upon starch for our nutrition, exploit its unique properties in industry, and use it as a feedstock for bioethanol production. Here, we review recent advances in research in three key areas. First, we assess progress in identifying the enzymatic machinery required for the synthesis of amylopectin, the glucose polymer responsible for the insoluble nature of starch. Second, we discuss the pathways of starch degradation, focusing on the emerging role of transient glucan phosphorylation in plastids as a mechanism for solubilizing the surface of the starch granule. We contrast this pathway in leaves with the degradation of starch in the endosperm of germinated cereal seeds. Third, we consider the evolution of starch biosynthesis in plants from the ancestral ability to make glycogen. Finally, we discuss how this basic knowledge has been utilized to improve and diversify starch crops.

To determine the enzymatic function of the starch-related R1 protein it was heterologously expressed in Escherichia coli and purified to apparent homogeneity. Incubation of the purified protein with various phosphate donor and acceptor molecules showed that R1 is capable of phosphorylating glucosyl residues of ␣-glucans at both the C-6 and the C-3 positions in a ratio similar to that occurring naturally in starch. Phosphorylation occurs in a dikinase-type reaction in which three substrates, an ␣-polyglucan, ATP, and H2O, are converted into three products, an ␣-polyglucan-P, AMP, and orthophosphate. The use of ATP radioactively labeled at either the ␥ or ␤ positions showed that solely the ␤ phosphate is transferred to the ␣-glucan. The apparent Km of the R1 protein for ATP was calculated to be 0.23 M and for amylopectin 1.7 mg⅐ml ؊1 . The velocity of in vitro phosphorylation strongly depends on the type of the glucan. Glycogen was an extremely poor substrate; however, the efficiency of phosphorylation strongly increased if the glucan chains of glycogen were elongated by phosphorylase. Mg 2؉ ions proved to be essential for activity. Incubation of R1 with radioactively labeled ATP in the absence of an ␣-glucan showed that the protein phosphorylates itself with the ␤, but not with the ␥ phosphate. Autophosphorylation precedes the phosphate transfer to the glucan indicating a ping-pong reaction mechanism.S tarch is the storage carbohydrate most widely distributed in the plant kingdom. In storage organs it serves as a long-term carbon reserve, whereas in photosynthetically competent tissues it is transiently accumulated to provide both reduced carbon and energy during periods unfavorable for photosynthesis. Starch consists of essentially linear (amylose) and highly branched (amylopectin) glucose polymers that are arranged as semicrystalline particles, the starch granules. Amylopectin from many sources contains phosphate-monoesters that are covalently bound at the C6 and C3 positions of the glucosyl residues (1). In potato tuber starch Ϸ0.1-0.5% of the glucose moieties are phosphorylated. The amount of phosphate monoesters in starch strongly influences its physicochemical properties (2) and, therefore, affects the ability of different starches to be used by industry.In potato tubers the starch-bound phosphate comprises a significant proportion of the total tuber phosphate content, but its function in starch metabolism is not clear. The same holds true for the biochemical reaction(s) leading to the formation of the starch phosphate monoesters. A protein (designated as R1) has been recently identified using a proteomic approach, and circumstantial evidence suggests that it is involved in the phosphorylation of starch (2, 3). The C-terminal sequence of the R1 protein shows some homology to bacterial PEP synthases (pyruvate, water dikinase EC 2.7.9.2), which transfer phosphate from ATP in a dikinase reaction to pyruvate and water. Antisense repression of R1 in potato leads to a strong reduction in the amount of starch-boun...

We have cloned a gene involved in starch metabolism that was identified by the ability of its product to bind to potato starch granules. Reduction in the protein level of transgenic potatoes leads to a reduction in the phosphate content of the starch. The complementary result is obtained when the protein is expressed in Escherichia coli, as this leads to an increased phosphate content of the glycogen. It is possible that this protein is responsible for the incorporation of phosphate into starch-like glucans, a process that is not understood at the biochemical level. The reduced phosphate content in potato starch has some secondary effects on its degradability, as the respective plants show a starch excess phenotype in leaves and a reduction in cold-sweetening in tubers.