Inhibition of a starch-granule–bound protein leads to modified starch and repression of cold sweetening

Lorberth, Ruth; Ritte, Gerhard; Willmitzer, Lothar; Koßmann, Jens

doi:10.1038/nbt0598-473

Cited by 244 publications

(255 citation statements)

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“…Nielsen et al (1994) have shown that potato tuber starch is phosphorylated during biosynthesis. This result, in combination with the more recent findings (Lorberth et al, 1998;Yu et al, 2001;Ritte et al, 2002), suggests that phosphate incorporation during starch synthesis somehow determines the degradability of the starch particle. However, it is not yet known whether phosphorylation of starch is restricted to the period of biosynthesis.…”

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confidence: 62%

“…However, the analyses of plants with reduced GWD activity indicate that the phosphorylation of starch affects its in vivo degradability. Starch breakdown is strongly impaired in GWD antisense potato plants and in the GWDdeficient starch-excess 1 (sex1) mutants of Arabidopsis (Lorberth et al, 1998;Yu et al, 2001;Ritte et al, 2003). The link between phosphorylation and degradation of starch is presently poorly understood.…”

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confidence: 99%

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Phosphorylation of Transitory Starch Is Increased during Degradation

et al. 2004

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The starch excess phenotype of Arabidopsis mutants defective in the starch phosphorylating enzyme glucan, water dikinase (EC 2.7.9.4) indicates that phosphorylation of starch is required for its degradation. However, the underlying mechanism has not yet been elucidated. In this study, two in vivo systems have been established that allow the analysis of phosphorylation of transitory starch during both biosynthesis in the light and degradation in darkness. First, a photoautotrophic culture of the unicellular green alga Chlamydomonas reinhardtii was used to monitor the incorporation of exogenously supplied 32 P orthophosphate into starch. Illuminated cells incorporated 32 P into starch with a constant rate during 2 h. By contrast, starch phosphorylation in darkened cells exceeded that in illuminated cells within the first 30 min, but subsequently phosphate incorporation declined. Pulse-chase experiments performed with 32 P/ 31 P orthophosphate revealed a high turnover of the starch-bound phosphate esters in darkened cells but no detectable turnover in illuminated cells. Secondly, leaf starch granules were isolated from potato (Solanum tuberosum) plants grown under controlled conditions and glucan chains from the outer granule layer were released by isoamylase. Phosphorylated chains were purified and analyzed using high performance anionexchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry. Glucans released from the surface of starch granules that had been isolated from darkened leaves possessed a considerably higher degree of phosphorylation than those prepared from leaves harvested during the light period. Thus, in the unicellular alga as well as in potato leaves, net starch degradation is accompanied with an increased phosphorylation of starch.

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Phosphorylation of Transitory Starch Is Increased during Degradation

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“…One of the important aims of this work was to produce amylopectins with altered branching structures, and then to try and relate this to altered physical properties of the starches. This may be difficult as the physical properties of the starches are also greatly affected by the phosphate content [27] and, therefore, as both the branching of the amylopectin and the phosphate contents are altered it would be difficult to separate out the effect of altered branching from alterations in phosphate contents.…”

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confidence: 99%

Simultaneous antisense inhibition of two starch-synthase isoforms in potato tubers leads to accumulation of grossly modified amylopectin

Lloyd

LANDSCHÜTZE²,

Kossmann

1999

Biochemical Journal

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A chimaeric antisense construct was used to reduce the activities of the two major starch-synthase isoforms in potato tubers simultaneously. A range of reductions in total starch-synthase activities were found in the resulting transgenic plants, up to a maximum of 90 % inhibition. The reduction in starch-synthase activity had a profound effect on the starch granules, which became extremely distorted in appearance compared with the control lines. Analysis of the starch indicated that the amounts produced in the tubers, and the amylose content of the starch, were not affected by the reduction in activity. In order to understand why the starch granules were distorted, amylopectin was isolated and the constituent chain lengths analysed. This indicated that the amylopectin was very different to that of the

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“…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.…”

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The starch-related R1 protein is an α-glucan, water dikinase

Ritte

Lloyd

Eckermann

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Inhibition of a starch-granule–bound protein leads to modified starch and repression of cold sweetening

Cited by 244 publications

References 16 publications

Phosphorylation of Transitory Starch Is Increased during Degradation

Phosphorylation of Transitory Starch Is Increased during Degradation

Simultaneous antisense inhibition of two starch-synthase isoforms in potato tubers leads to accumulation of grossly modified amylopectin

The starch-related R1 protein is an α-glucan, water dikinase

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