Mutants of Arabidopsis Lacking a Chloroplastic Isoamylase Accumulate Phytoglycogen and an Abnormal Form of Amylopectin  

Abstract

Mutant lines defective for each of the four starch debranching enzyme (DBE) genes (AtISA1, AtISA2, AtISA3, and AtPU1) detected in the nuclear genome of Arabidopsis (Arabidopsis thaliana) were produced and analyzed. Our results indicate that both AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of DBE named Iso1, the major isoamylase activity found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content in both lines and to the accumulation of water-soluble polysaccharides whose structure is similar to glycogen. In addition, the residual amylopectin structure in the corresponding mutant lines displays a strong modification when compared to the wild type, suggesting a direct, rather than an indirect, function of Iso1 during the synthesis of amylopectin. Mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure. This result suggests that this isoamylase-type of DBE plays a major role during starch mobilization. The analysis of the Atpu1 single-mutant lines did not lead to a distinctive phenotype. However, Atisa2/Atpu1 double-mutant lines display a 92% decrease in starch content. This suggests that the function of pullulanase partly overlaps that of Iso1, although its implication remains negligible when Iso1 is present within the cell.

Recent progress toward understanding the role of starch biosynthetic enzymes in the cereal endosperm

“…This proposes that phytoglycogen is a separate soluble product rather than an intermediate for amylopectin synthesis [6,97]. This model is consistent with that in DBE mutants, the accumulation of phytoglycogen is always at the expense of amylopectin [89,99,113].…”

“…However, this enzyme is believed to be associated with starch biosynthesis in leaves and potato tuber and is required, along with ISA1, for the activity of the ISA heteromeric enzyme [98,99,101,102]. This hetero-oligomer is less important compared to the homo-oligomers of ISA1 in rice and maize endosperm for the trimming of improper branches [103][104][105].…”

“…ISA3 is mainly expressed in rice leaves and to a lesser extent in endosperm [100], and appears to be essential for starch degradation in Arabidopsis leaves [99]. The function of PUL is less understood than that of ISA.…”

“…In Arabidopsis and potato, accumulation of phytoglycogen is due to the loss of either ISA1 or ISA2. In addition to a reduction in starch content, a loss of ISA1 has been linked to a reduction in granule size and an increase in granule number [93,99], indicating ISA1 may be involved in granule initiation. In maize, a loss of ISA1 had a pleiotropic effects on other enzymes, particularly SBEIIa [91], while the presence of a catalytically inactive ISA1 eliminates some of these effects, suggesting that the ISA1 has both enzymatic and non-enzymatic functions and may form enzymatic complex with other enzymes.…”

Convergent Evolution of Polysaccharide Debranching Defines a Common Mechanism for Starch Accumulation in Cyanobacteria and Plants

“…From these observations, several groups proposed that DBE was selectively responsible for removal of misplaced branches, preventing polysaccharide aggregation in an otherwise hydrosoluble precursor . This model, known as the preamylopectin trimming model, is well sustained by the evidence gathered through mutant analysis in all plant systems examined to date, including Arabidopsis thaliana (Wattebled et al, 2005). However, direct proof of this model will only be obtained by achieving in vitro synthesis of starch granules.…”