A Mutant of Arabidopsis Lacking a Chloroplastic Isoamylase Accumulates Both Starch and Phytoglycogen

Abstract

In this study, our goal was to evaluate the role of starch debranching enzymes in the determination of the structure of amylopectin. We screened mutant populations of Arabidopsis for plants with alterations in the structure of leaf starch by using iodine staining. The leaves of two mutant lines stained reddish brown, whereas wild-type leaves stained brownish black, indicating that a more highly branched polyglucan than amylopectin was present. The mutants were allelic, and the mutation mapped to position 18.8 on chromosome 1. One mutant line lacked the transcript for a gene with sequence similarity to higher plant debranching enzymes, and both mutants lacked a chloroplastic starch-hydrolyzing enzyme. This enzyme was identified as a debranching enzyme of the isoamylase type. The loss of this isoamylase resulted in a 90% reduction in the accumulation of starch in this mutant line when compared with the wild type and in the accumulation of the highly branched water-soluble polysaccharide phytoglycogen. Both normal starch and phytoglycogen accumulated simultaneously in the same chloroplasts in the mutant lines, suggesting that isoamylase has an indirect rather than a direct role in determining amylopectin structure.

Starch Synthesis in Arabidopsis. Granule Synthesis, Composition, and Structure

“…We reasoned that a soluble ␣-1,4-, ␣-1,6-linked glucan might be more sensitive to the actions of starch-degrading enzymes during its synthesis than starch. Therefore, we performed similar pulse-chase experiments on the Arabidopsis mutant dbe1, which lacks an isoform of the debranching enzyme isoamylase (Zeeman et al, 1998b). This mutant accumulates the soluble, highly branched glucan phytoglycogen, which does not form semicrystalline granules but remains soluble in the stroma of the chloroplast.…”

“…It is accumulated together with small amounts of starch during photosynthesis and degraded during the subsequent dark period. During the pulse, starch and phytoglycogen were labeled in the ratio 5:1, reflecting the relative rates of synthesis of the two glucans in dbe1 leaves (Zeeman et al, 1998b). No 14 C was lost from either starch or phytoglycogen during the chase (Table I).…”

“…The SEs were added together. Rees, 1999;Yu et al, 2001), sex4-1 (Zeeman et al, 1998a;Zeeman and ap Rees, 1999) and dbe1-1 (Zeeman et al, 1998b) were grown in peat-based compost in a growth chamber with a 12-h-light/12-h-dark cycle. The irradiance was 170 mol photons m Ϫ2 s Ϫ1 , the temperature 20°C, and the humidity 75%, unless otherwise specified.…”

“…Typically, values of approximately 15% or less have been found, whereas most storage starches contain between 20% and 30% amylose. In wild-type Arabidopsis leaves, the amylose content of the starch is low (Zeeman et al, 1998b), but in starch-excess mutants, increased amylose contents have been reported (Critchley et al, 2001;Yu et al, 2001). We have established a robust method for the measurement of amylose content of Arabidopsis starch and used this method to investigate the conditions of amylose synthesis in wild-type leaves and in starch-excess mutant lines.…”

Comparative proteome analysis of A- and B-type starch granule-associated proteins in bread wheat (Triticum aestivum L.) and Aegilops crassa

“…6 these two steps, some improper branches are removed by starch debranching enzymes such as isozymes and pullulan that efficiently hydrolyze alpha-(1-6)-linkages in amylopectin [36][37][38][39]. The biosynthesis of starch granules is the result of the coordinated actions of all enzymes described above that begins with the immediate soluble precursor of ADPGlc and end with the formation of a starch granule [16].…”