Light Induces Phosphorylation of Glucan Water Dikinase, Which Precedes Starch Degradation in Turions of the Duckweed Spirodela polyrhiza    

Abstract

Degradation of storage starch in turions, survival organs of Spirodela polyrhiza, is induced by light. Starch granules isolated from irradiated (24 h red light) or dark-stored turions were used as an in vitro test system to study initial events of starch degradation. The starch-associated pool of glucan water dikinase (GWD) was investigated by two-dimensional gel electrophoresis and by western blotting using antibodies raised against GWD. Application of this technique allowed us to detect spots of GWD, which are light induced and absent on immunoblots prepared from dark-adapted plants. These spots, showing increased signal intensity following incubation of the starch granules with ATP, became labeled by randomized [bg-33 P]ATP but not by [g-33 P]ATP and were removed by acid phosphatase treatment. This strongly suggests that they represent a phosphorylated form(s) of GWD. The same light signal that induces starch degradation was thus demonstrated for the first time to induce autophosphorylation of starch-associated GWD. The in vitro assay system has been used to study further effects of the light signal that induces autophosphorylation of GWD and starch degradation. In comparison with starch granules from dark-adapted plants, those from irradiated plants showed increase in (1) binding capacity of GWD by ATP treatment decreased after phosphatase treatment; (2) incorporation of the b-phosphate group of ATP into starch granules; and (3) rate of degradation of isolated granules by starch-associated proteins, further enhanced by phosphorylation of starch. The presented results provide evidence that autophosphorylation of GWD precedes the initiation of starch degradation under physiological conditions. Degradation of starch in cereal endosperm has been thoroughly investigated over a long period (see Ritchie et al., 2000). However, the mechanisms discovered in these systems are not applicable to the metabolic mechanisms of starch in other plant cells, e.g. transitory starch in chloroplasts of leaves or storage starch in amyloplasts of tubers, roots, or turions (see Smith et al., 2003). The main reason for this difference is the a-cellular character of endosperm at the time of starch degradation. The network of regulatory processes is certainly different in living cells. In the last few years, a hitherto unexpected aspect of starch degradation has been revealed (see Blennow et al., 2002). A protein was discovered associated with starch granule surfaces (Lorberth et al., 1998;Ritte et al., 2000b), which showed reversible binding depending on the metabolic status of the cell (Ritte et al., 2000b;Reimann et al., 2002). By analysis of antisense plants and mutants, it became clear that this protein is necessary for phosphorylation of starch and that starch degradation is impaired in its absence (Lorberth et al., 1998;Yu et al., 2001). Later, the biochemical function of this protein, now named a-glucan water dikinase (GWD; EC 2.7.9.4), was revealed Mikkelsen et al., 2004). This protein becomes autophosphorylated by the b-phosp...

Identification of a Novel Enzyme Required for Starch Metabolism in Arabidopsis Leaves. The Phosphoglucan, Water Dikinase

“…It has recently been reported that the in vitro degradation of granules isolated from turions of the duckweed by starch associated proteins (including a putative GWD ortholog) could be increased by addition of ATP, thereby enabling starch phosphorylation (Reimann et al, 2004). We have obtained similar results using potato leaf starch.…”

Dynamics of starch granule biogenesis – the role of redox-regulated enzymes and low-affinity carbohydrate-binding modules

“…For some proteins, partitioning between the starch granule and the stroma has been reported to respond dynamically to the diurnal cycle of photosynthesis and carbon fl ow. Important examples include the diurnal change in partitioning of GWD (Ritte et al 2002;Mikkelsen et al 2005), α-amylase (Reimann et al 2004) and the dual specifi city phosphatatse SEX4 (Sokolov et al 2006). As several of these enzymes contain SBDs the question arises whether these modules contribute to the dynamic partitioning and what would be the functional requirement of this type of SBD.…”

Starch

“…B-type crystalline polymorphs are typically more resistant towards amylolytic hydrolysis in vitro (Wickramasinghea et al 2009;Planchot et al 1997) forming a rational explanation for the phosphorylationdependent amorphisation of only this type of starch. In turions GWD is potentially autophosphorylated, or at least subject to major conformational changes, prior to starch phosphorylation (Reimann et al 2004). In potato (Solanum tuberosum) and pea (Pisum sativum) leaf cells, GWD is found bound to starch granule at night where starch degradation takes place (Ritte et al 2000a, b).…”