Similar Protein Phosphatases Control Starch Metabolism in Plants and Glycogen Metabolism in Mammals

Abstract

We report that protein phosphorylation is involved in the control of starch metabolism in Arabidopsis leaves at night. sex4 (starch excess 4) mutants, which have strongly reduced rates of starch metabolism, lack a protein predicted to be a dual specificity protein phosphatase. We have shown that this protein is chloroplastic and can bind to glucans and have presented evidence that it acts to regulate the initial steps of starch degradation at the granule surface. Remarkably, the most closely related protein to SEX4 outside the plant kingdom is laforin, a glucanbinding protein phosphatase required for the metabolism of the mammalian storage carbohydrate glycogen and implicated in a severe form of epilepsy (Lafora disease) in humans.Starch, the main storage carbohydrate of plants, accumulates as a product of photosynthesis in leaves during the day and is converted to sucrose for export from the leaves at night. This conversion of starch to sucrose is one of the largest daily carbon fluxes on the planet, but nothing is known about how the process is initiated and controlled. The amounts of enzymes on the pathway change very little through the diurnal cycle in leaves of the model plant Arabidopsis thaliana, hence flux must be controlled by modulation of their activities (1).Much progress in understanding the pathway has been made through the selection of Arabidopsis mutants impaired in starch degradation at night. All such mutations identified thus far are in genes encoding enzymes of the pathway, rather than proteins likely to be involved in modulation of the activities of these enzymes (2-11). However, a mutation at a locus not yet identified, the starch excess 4 (or SEX4) locus, gives rise to a phenotype indicative of a regulatory defect rather than a defect in a structural enzyme. Mature sex4 leaves contain three to four times more starch than those of wild-type plants, apparently because a reduced capacity for starch degradation at night leads to progressive accumulation of starch over the life of the leaf (12, 13). Starch granules in leaves of the sex4 mutant are much larger and more rounded than those of wild-type plants (14). Measurements of activity and protein of enzymes known to be involved in starch degradation revealed only one significant reduction in the sex4 mutant in the chloroplastic ␣-amylase AMY3 (12, 15). However, although both the activity and amount of protein of AMY3 are strongly reduced, this is not the cause of the deficiency in starch degradation in the sex4 mutant. T-DNA insertion lines lacking AMY3 protein have normal rates of starch degradation (15). The aim of the work described in this paper was to discover the nature of the gene at the SEX4 locus and thus shed light on the regulation of starch degradation. EXPERIMENTAL PROCEDURESPositional Identification of the SEX4 Locus-F2 plants from a cross between sex4-2 (Col-0 background) and Landsberg erecta showing the mutant phenotype were used for mapping. The mapping population (562 plants) was genotyped using SSLP and SNP markers availabl...

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

“…In all of these mutants, rates of starch synthesis and degradation remain high even though the starch content of the leaf is elevated relative to that in wildtype leaves. By contrast, under our growth conditions, starch levels show little change during the diurnal cycle in sex1 mutants, deficient in GWD and hence in starch phosphorylation (Niittylä et al, 2006). The actions of SEX4, the b-amylases, and ISA3 are probably closely linked: we have proposed that dephosphorylation of glucans by SEX4 at the granule surface during starch degradation in the chloroplast at night allows the complete hydrolysis of glucans to maltose and maltotriose by the concerted actions of b-amylases and ISA3 (Kötting et al, 2009).…”

“…This situation contrasts sharply with that in the sex1 mutant, in which the phosphorylation of starch is prevented by the loss of GWD. Under our growth conditions, levels of starch in sex1 rosettes are higher than those in the lsf1/sex4 mutant and change very little over the day/ night cycle (Niittylä et al, 2006).…”

“…LSF1 encodes a 591-amino acid protein containing a domain resembling the catalytic domain of dualspecificity protein phosphatases (DSPc; amino acids 291-441) with 35% identity and 54% similarity to the DSPc domain of SEX4 and a putative carbohydratebinding domain (amino acids 465-545) that has 35% identity and 53% similarity to the carbohydrate-binding domain of SEX4 (Niittylä et al, 2006). The main difference between the two proteins is an N-terminal region of about 200 amino acids present in LSF1 but not SEX4 (Fig.…”

“…To shed light on the function of LSF1, we generated a double mutant (lsf1/sex4) using as parents the SALK lines 102567 (sex4-3; Niittylä et al, 2006) and 053285 (lsf1-1) and compared its phenotype with that of the single sex4 and lsf1 mutants. At the same age, nonflowering rosettes of lsf1 and sex4 weighed about 25% and 65% less, respectively, than those of wild-type plants.…”

“…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).…”