Simultaneous Repression of GLUCAN WATER DIKINASE 1 and STARCH BRANCHING ENZYME 1 in Potato Tubers Leads to Starch With Increased Amylose and Novel Industrial Properties

Abstract

This study examines how post-transcriptional gene silencing of STARCH BRANCHING ENZYME 1 (SBE1) and GLUCAN WATER DIKINASE 1 (GWD1) affects the structure and properties of potato tuber starch. Silencing of either gene individually or simultaneously altered starch chemistry physical properties. Repression of StGWD1 reduced phosphate content, while repression of StSBE1 increased it. The phosphate content of starch isolated from plants where both genes were repressed was increased compared to StGWD1 repressed lines, but lower than both the SBE1 repressed lines and the untransformed control. Constituent chain lengths of starches from all lines were altered, and amylose content was increased in the gwd1 and sbe1/gwd1 double repressed lines, which also accumulated small numbers of lobed starch granules. Pasting properties were also affected, with starch from StSBE1-repressed lines demonstrating increased peak and trough viscosities and gwd1 lines showing decreased peak and trough viscosities, compared with the control. Peak and trough viscosities were lowest in the sbe1/gwd1 repressed lines. We believe that these data demonstrate that alterations in starch phosphate influence the degree of branching within starch and offer a novel in planta strategy for optimizing the industrial properties of potato storage starch.

Keywords: Glucan water dikinase; Starch branching enzyme; Starch structure.

FIGURE 1

Screening of transgenic plants. (A) Semi‐quantitative RT‐PCR to examine the accumulation of SBE1, SBE2, GWD1, or EF1α (housekeeping control) transcripts in transgenic lines where either SBE1, GWD1, or both genes were simultaneously (SBE1/GWD1) repressed. The negative control is a sample containing no cDNA but undergoing the same PCR reaction. (B) Immunoblot examining GWD1 protein in 25 µg crude protein extracts from tubers. Full electrophoresis gels and immunoblots, as well as Coomassie stained polyacrylamide gels, are shown in Figures S1 and S2.

FIGURE 2

Pot trials of transgenic plants. (A) Plants after 26 weeks of growth, (B) tuber morphology, (C) tuber yield, and (D) average mass per tuber. Data represents the mean ± SEM of measurements from at least five plants. A 5% level of significance was tested using the Bonferroni–Holm post hoc test after one‐way analysis of variance.

FIGURE 3

Difference plots of debranched tuber starch. Isolated starches from the lines were debranched, dephosphorylated, and separated by anion exchange chromatography (HPAEC‐PAD). Data indicate the differences between starches from the indicated transgenic lines and the wild‐type. Error bars represent SEM of three replicates. If not visible, they sit within the symbol.

FIGURE 4

Scanning electron micrographs of purified tuber starch granules. Inset pictures show compound granules identified in the sbe1/gwd1 lines. White scale bars represent 20 µm, and black scale bars represent 10 µm.

FIGURE 5

Viscosity profiles of various tuber starches measured as a starch/water paste in a rapid visco‐analyzer. Graph profiles represent the mean of three measurements, and table data represent the means ± SEM of at least three measurements for pooled starch samples isolated from tubers of at least five plants per line. Data were analyzed by one‐way analysis of variance followed by a Bonferroni post hoc test. Letters represent groups with similar means at the 5% significance level. Viscosity is measured as centipoise (cP).