In vitro biosynthesis of phosphorylated starch in intact potato amyloplasts

Abstract

Intact amyloplasts from potato (Solanum tuberosum L.) were used to study starch biosynthesis and phosphorylation. Assessed by the degree of intactness and by the level of cytosolic and vacuolar contamination, the best preparations were selected by searching for amyloplasts containing small starch grains. The isolated, small amyloplasts were 80% intact and were free from cytosolic and vacuolar contamination. Biosynthetic studies of the amyloplasts showed that [1-14C]glucose-6-phosphate (Glc-6-P) was an efficient precursor for starch synthesis in a manner highly dependent on amyloplast integrity. Starch biosynthesis from [1-14C]Glc-1-P in small, intact amyloplasts was 5-fold lower and largely independent of amyloplast intactness. When [33P]Glc-6-P was administered to the amyloplasts, radiophosphorylated starch was produced. Isoamylase treatment of the starch followed by high-performance anion-exchange chromatography with pulsed amperometric detection revealed the separated phosphorylated alpha-glucans. Acid hydrolysis of the phosphorylated alpha-glucans and high-performance anion-exchange chromatography analyses showed that the incorporated phosphate was preferentially positioned at C-6 of the Glc moiety. The incorporation of radiolabel from Glc-1-P into starch in preparations of amyloplasts containing large grains was independent of intactness and most likely catalyzed by starch phosphorylase bound to naked starch grains.

Figure 1

Starch biosynthesis from [1-¹⁴C]Glc-6-P and [1-¹⁴C]Glc-1-P in experiments with ruptured (□) and intact (▪) potato tuber amyloplasts. Small amyloplasts were selected for the experiment. The amyloplasts were ruptured by treatment with 0.1% Triton X-100. The amyloplasts were incubated for 1 h with 4 mm exogenously added ATP and 3PGA and 2 mm metabolite (Glc-6-P or Glc-1-P) with 6.3 kBq [1-¹⁴C]Glc-6-P or [1-¹⁴C]Glc-1-P. The amount incorporated was normalized on the basis of the activity of AGPase in the plastid preparations. Each column represents a mean of three replicates. Each separate amyloplast preparation is numbered using roman numerals. U, Units.

Figure 2

Starch biosynthesis from [1-¹⁴C]Glc-6-P and [1-¹⁴C]Glc-1-P in experiments with ruptured (□) and intact (▪) plastids prepared from large amyloplasts. The amyloplasts were incubated as described in the legend to Figure 1. Each set of experiments is numbered. The amount of hexose residues incorporated is calculated as described in the legend to Figure 1. U, Units.

Figure 3

Verification of incorporated phosphate into starch in experiments with small amyloplasts using [³³P]Glc-6-P and effect of β-amylase. A, HPAEC elution profile of linear phosphorylated oligosaccharides together with the corresponding radioactivity. B, Detector response and corresponding radioactivity after β-amylase treatment of the linear phosphorylated oligosaccharides. In both panels peaks of radioactivity were integrated and normalized to 100%. nC, Nanocoulombs.

Figure 4

Verification of phosphate incorporated into starch at C-6 and C-3 in the Glc residues in experiments with small amyloplasts using [³³P]Glc-6-P. A, HPAEC elution profile of phosphorylated dextrins treated with α-amylase and corresponding radioactivity. B, Detector response and radioactivity after acid hydrolysis: 2 h in 0.7 n HCl at 100°C. nC, Nanocoulombs.

Figure 5

HPAEC elution profile of phosphorylated acid-hydrolyzed dextrins, as in Figure 4B, showing the elution time of internal standards of Glc-3-P and Glc-6-P. A, No internal standard added; B and C, Glc-3-P and Glc-6-P added as internal standards, respectively. nC, Nanocoulombs.