Formation and Deposition of Amylose in the Potato Tuber Starch Granule Are Affected by the Reduction of Granule-Bound Starch Synthase Gene Expression.

Abstract

The synthesis of amylose in amyloplasts is catalyzed by granule-bound starch synthase (GBSS). GBSS gene expression was inhibited via antisense RNA in Agrobacterium rhizogenes-transformed potato plants. Analysis of starch production and starch granule composition in transgenic tubers revealed that reduction of GBSS activity always resulted in a reduction of the production of amylose. Field experiments, performed over a 2-year period, showed that stable inhibition of GBSS gene expression can be obtained. Microscopic evaluation of iodine-stained starch granules was shown to be a sensitive system for qualitative and quantitative examination of amylose formation in starch granules of transgenic potato tubers. In plants showing inhibition of GBSS gene expression, the reduced amylose content in tuber starch was not a consequence of a lower amylose content throughout the entire starch granule. Starch granules of transgenic tubers were found to contain amylose at a percentage similar to wild-type starch in a core of varying size at the hilum of each granule. This indicated that reduced GBSS gene expression results in amylose formation in a restricted zone of the granules. The size of this zone is suggested to be dependent on the GBSS protein level. During development of the granules, the available GBSS protein is thought to become limiting, resulting in the formation of starch that lacks amylose. RNA gel blot analysis of tuber tissue showed that inhibition of GBSS gene expression resulted in a reduced GBSS mRNA level but did not affect the expression level of other starch synthesizing enzymes. Antisense RNA could only be detected in leaf tissue of the transgenic plants.

What Controls the Amount and Structure of Starch in Storage Organs?

“…Visser and colleagues have used iodine staining to examine the starch granules of potatoes in which the amount of amylose has been reduced by introduction of an antisense construct for the amf gene (Kuipers et al, 1994). Rather than staining uniformly, these granules stain red with a blue core, indicating that the amylose content of the inner region is far higher than that www.plantphysiol.org on May 12, 2018 -Published by Downloaded from Copyright © 1995 American Society of Plant Biologists.…”

“…Here, too, the avE ilability of plants with altered activities of specific enzymej is important. For example, nove1 information about the synthesis of amylose will be gained from potato tubers wii h reduced levels of the Amf protein (Kuipers et al, 1994).…”

Starch meets biotechnology : in planta modification of starch composition and functionalities

“…Samples were frozen in liquid N2 and stored in -80ºC freezer until RNA isolation was conducted. Total RNA was extracted from potato tuber samples according to Kuipers et al (1994). cDNA was synthesized from 1 μg of total RNA, previously treated with RNase-free DNase I (Invitrogen), by using the iScript TM cDNA Synthesis Kit (Bio-Rad).…”

“…Expression levels of (engineered) laforin from all transformants were determined in triplicate by qRT-PCR using the primers listed in S1 Table. Total RNA was extracted from potato tuber samples according to Kuipers et al (1994) and reverse transcribed using the iScript cDNA synthesis kit from BioRad. qRT-PCR analysis was performed using CFX96 Real-Time PCR machine (BioRad).…”

“…Amylose is an amorphous, essentially linear glucan polymer with α-1,4 linked glucose residues, while amylopectin is a highly branched molecule built of α-1,4 linked glucose residues as backbone and 5% α-1,6 branches (Manners, 1989). In addition to amylose and amylopectin, native starches contain small amounts of phosphate groups monoesterified to the glucose residues (Blennow et al, 2000a;Blennow et al, 2002;Kuipers et al, 1994;Tabata et al, 1975). Most of the phosphate groups are bound to the amylopectin fraction at the C-6 (~70%) and C-3 (~30%) positions of the glucose units (Bay-Smidt et al, 1994;Hizukuri and Takeda, 1970).…”