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

Abstract

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

The contribution of plastidial phosphoglucomutase to the control of starch synthesis within the potato tuber

“…Using optimised assays we determined the maximal catalytic activities of alkaline pyrophosphatase, soluble and granule bound starch synthase, branching enzyme, total amylolytic activity, fructokinase and sucrose phosphate synthase (Table 1). Values obtained for wild-type tubers are in close agreement with those published previously (for example Merlo et al 1993;Edwards et al 1999;Lloyd et al 1999;Sweetlove et al 1999). With the exception of the selective velocity (V sel ) assay of sucrose phosphate synthase (SPS; which signi®cantly increased in lines P5 and P9, lines which also contained signi®cant increases in glucose 6-phosphate, Tauberger et al 2000) and, of course, for plastidial PGM, none of the activities were signi®cantly dierent from those observed for the wild type.…”

Cloning and functional analysis of a cDNA encoding a starch synthase from potato ( Solanum tuberosum L.) that is predominantly expressed in leaf tissue

“…This is probably due to the fact that SSI contributes only minor proportions to the total SS activity present in potato tubers, since no appreciable dierences in total SS activity were measured in the transgenic as compared to control tubers. Potato tubers lacking activity of either the SSII or SSIII isoforms individually, or simultaneously, do show major alterations in amylopectin structure when analysed using the methods in this present paper (Lloyd et al 1999). This suggests that it is indeed the small amount of activity that SSI contributes that leads to the lack of alterations in carbohydrate metabolism in SSI antisense tubers, as SSII and SSIII contribute a much greater proportion of the activity to the tuber.…”

“…The phosphate bound to the starch at the C-6 position was determined as glucose-6-phosphate released after acid hydrolysis according to Nielsen et al (1994). Amylopectin was puri®ed according to the method of Tomlinson et al (1997) while analysis of the sidechain distribution of debranched amylopectins by high-performance anion-exchange chromatography using a pulsed amperometric detector (HPAE-PAD) and gel permeation chromatography was performed as described by Lloyd et al (1999).…”

Inhibition of a ubiquitously expressed pectin methyl esterase in Solanum tuberosum L. affects plant growth, leaf growth polarity, and ion partitioning

“…1). Although chimeric antisense constructs have successfully been used (Lloyd et al 1999), other reports have recorded that this technique has failed to inhibit the accumulation of more than one transcript simultaneously.…”

“…Total RNA was extracted from all tissues other than tubers and fruits, while the latter were extracted according to Kay et al (1987). RNA blots were performed as described previously (Lloyd et al 1999) using either a 32 P-labelled 415-bp HindIII/SphI (pest1 specific) or a 471-bp EcoRI/BglII (pest2 specific) cDNA fragment. Equal loading of gels was controlled by hybridization with Rubisco (small subunit, data not shown).…”