STARCH-EXCESS4 Is a Laforin-Like Phosphoglucan Phosphatase Required for Starch Degradation in Arabidopsis thaliana    

Abstract

Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear a-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases a-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephosphorylation of starch. We propose that glucan phosphorylating enzymes and phosphoglucan phosphatases work in synergy with glucan hydrolases to mediate efficient starch catabolism.

A Putative Phosphatase, LSF1, Is Required for Normal Starch Turnover in Arabidopsis Leaves

“…We discovered recently that substantial amounts of soluble phospho-oligosaccharides accumulate in leaves of sex4 mutant plants but not wildtype plants. This observation provides strong support for the idea that SEX4 dephosphorylates glucans during starch degradation (Kö tting et al, 2009). Soluble phospho-oligosaccharides were present in lsf1/ sex4 plants (Table I) but were not detectable in leaves of either lsf1 or wild-type plants.…”

“…When expressed on a starch basis, the phosphate content of leaf starch was not significantly different in wild-type and lsf1 plants but was lower in both the sex4 mutant (Kö tting et al, 2009) and the double mutant (Table I). The lower levels are accounted for by differences in amylose content between the starches.…”

“…This reduction in AMY3 activity is not responsible for the accumulation of starch in the sex4 mutant: amy3 mutants that lack AMY3 have completely normal rates of starch degradation (Yu et al, 2005). In fact, despite the reduction in its activity, AMY3 plays an important role in determining the sex4 phenotype: together with ISA3 it catalyzes the release from the granule surface of phosphorylated oligosaccharides that accumulate in the stroma of sex4 chloroplasts (Kö tting et al, 2009). To determine whether loss of LSF1 also affects AMY3, we examined amylase activity in leaf extracts by renaturation of SDS-polyacrylamide gels containing amylopectin.…”

“…We discovered that the starch-excess4 (sex4) mutant lacks a phosphatase with a carbohydrate-binding domain (Niittylä et al, 2006). Although it was originally described as a dualspecificity protein phosphatase (PTPKIS1; FordhamSkelton et al, 2002;Kerk et al, 2006), we have shown that the function of the SEX4 phosphatase is to remove phosphate groups added to starch by GWD (and perhaps PWD), thus facilitating the complete degradation of oligosaccharides by b-amylase (Kö tting et al, 2009). Recombinant SEX4 catalyzes the dephosphorylation of glucans, including starch polymers, glycogen, soluble oligosaccharides, and leaf starch granules (Gentry et al, 2007).…”

Regulation of Leaf Starch Degradation by Abscisic Acid Is Important for Osmotic Stress Tolerance in Plants

“…The chains are then simultaneously degraded by a set of glucanhydrolyzing enzymes (including b-amylases [BAM], a-amylases [AMY], and debranching enzymes) and dephosphorylated by phosphoglucan phosphatases (Streb and Zeeman, 2012). These enzymes work in synergy to completely degrade starch (Kötting et al, 2009;Edner et al, 2007). Hydrolysis of starch to maltose by BAM represents the predominant pathway of transitory starch degradation.…”