A critical role for disproportionating enzyme in starch breakdown is revealed by a knock‐out mutation in Arabidopsis

Abstract

SummaryDisproportionating enzyme (D-enzyme) is a plastidial a-1,4-glucanotransferase but its role in starch metabolism is unclear. Using a reverse genetics approach we have isolated a mutant of Arabidopsis thaliana in which the gene encoding this enzyme (DPE1) is disrupted by a T-DNA insertion. While Denzyme activity is eliminated in the homozygous dpe1±1 mutant, changes in activities of other enzymes of starch metabolism are relatively small. During the diurnal cycle, the amount of leaf starch is higher in dpe1±1 than in wild type and the amylose to amylopectin ratio is increased, but amylopectin structure is unaltered. The amounts of starch synthesised and degraded are lower in dpe1±1 than in wild type. However, the lower amount of starch synthesised and the higher proportion of amylose are both eliminated when plants are completely de-starched by a period of prolonged darkness prior to the light period. During starch degradation, a large accumulation of malto-oligosaccharides occurs in dpe1±1 but not in wild type. These data show that D-enzyme is required for malto-oligosaccharide metabolism during starch degradation. The slower rate of starch degradation in dpe1±1 suggests that maltooligosaccharides affect an enzyme that attacks the starch granule, or that D-enzyme itself can act directly on starch. The effects on starch synthesis and composition in dpe1±1 under normal diurnal conditions are probably a consequence of metabolism at the start of the light period, of the high levels of maltooligosaccharides generated during the dark period. We conclude that the primary function of D-enzyme is in starch degradation.

Gene Regulation and Survival under Hypoxia Requires Starch Availability and Metabolism

“…Frozen samples were homogenized and then extracted in 80% ethanol. Glc, Fru, and Suc were measured enzymatically (soluble sugars) (Guglielminetti et al, 1995a), and starch was assayed in the insoluble pellet (starch) (Critchley et al, 2001). …”

Repression of a Novel Isoform of Disproportionating Enzyme (stDPE2) in Potato Leads to Inhibition of Starch Degradation in Leaves But Not Tubers Stored at Low Temperature

“…This is because analysis of an Arabidopsis dpe1 mutant demonstrated that it accumulated malto-oligosaccharides, especially malto-triose, which is its preferred substrate (Critchley et al, 2001). The amounts (Table I) and types (Fig.…”

“…Mutant and transgenic plants, which lack its activity almost entirely, have been isolated (Takaha et al, 1998;Colleoni et al, 1999aColleoni et al, , 1999bCritchley et al, 2001;Wattebled et al, 2003), and based on studies on these, two contradictory proposals have been made as to its role. Following analysis of the Chlamydomonas reinhardtii sta11 mutant, it was suggested that D-enzyme is involved directly in the synthesis of the amylopectin fraction of starch (Colleoni et al, 1999a(Colleoni et al, , 1999bWattebled et al, 2003); however, no evidence for this could be found in an Arabidopsis D-enzyme mutant (dpe1; Critchley et al, 2001). It was demonstrated in this mutant that starch degradation was impaired, and it was proposed that the main role of D-enzyme is in malto-oligosaccharide metabolism during mobilization of starch in the dark period.…”

“…Analysis of the Arabidopsis genome, however, has revealed that it contains two genes that appear to code for different D-enzyme isoforms. The mutation studied by Critchley et al (2001) was in a gene lying on chromosome 5 (At5g64860), with the other putative isoform being on chromosome 2 (At2g40840). Recently two studies have identified Arabidopsis plants mutated in the gene coding for this second isoform (Lu and Sharkey, 2004;Chia et al, 2004).…”

Spatiotemporal Profiling of Starch Biosynthesis and Degradation in the Developing Barley Grain

“…Four BAM isoforms of Arabidopsis are plastid localized, and three of them are involved in degradation of transient starch to maltose (Fulton et al, 2008). ISA3 (Wattebled et al, 2005;Delatte et al, 2006) is also capable of degrading transient starch to maltooligosaccharides, which are further converted either to Glc by plastidial disproportionating enzyme 1 (DPE1; Critchley et al, 2001) or by BAM to maltose. Produced maltose is metabolized to Glc in cytosol by DPE2 (Lu and Sharkey, 2004).…”