Protein Phosphorylation as a Mechanism for Osmotic-Stress Activation of Sucrose-Phosphate Synthase in Spinach Leaves

Abstract

Experiments were performed to investigate the mechanism of sucrose-phosphate synthase (SPS) activation by osmotic stress i n darkened spinach (Spinacia oleracea L.) leaves. The activation was stable through immunopurification and was not the result of an increased SPS protein level. l h e previously described Ca2+-independent peak III kinase, obtained by ion-exchange chromatography, is confirmed to be the predominant enzyme catalyzing phosphorylation and inactivation of dephosphoserine-158-SPS. A new, Ca'+-dependent SPS-protein kinase activity (peak IV kinase) was also resolved and shown to phosphorylate and activate phosphoserine-158-SPS i n vitro. The peak I V kinase also phosphorylated a synthetic peptide (SP29) based on the amino acid sequence surrounding serine-424, which also contains the motif described for the serine-158 regulatory phosphorylation site; i.e. basic residues at P-3 and P-6 and a hydrophobic residue at P-5. Peak I V kinase had a native molecular weight of approximately 150,000 as shown by gel filtration. The SP29 peptide was not phosphorylated by the inactivating peak III kinase. Osmotically stressed leaves showed increased peak I V kinase activity with the SP29 peptide as a substrate. l r y p t i c 32P-phosphopeptide analysis of SPS from excised spinach leaves fed [32P]inorganic P showed increased phosphorylation of the tryptic peptide containing serine-424. Therefore, at least part of the osmotic stress activation of SPS i n dark leaves results from phosphorylation of serine-424 catalyzed by a Ca'+-dependent, 150-kD protein kinase.

Quantitative Circadian Phosphoproteomic Analysis of Arabidopsis Reveals Extensive Clock Control of Key Components in Physiological, Metabolic, and Signaling Pathways

“…Two are in phase (S121 and S125) and peak at LL25 whereas the third peptide is maximally phosphorylated at subjective dusk (LL37) (S706) (supplemental Table S3). Their anti-phasic nature correlates well with findings from spinach where two sites bring about opposite effects in SPS activity (68,70). Taken together these results suggest a mode of post-translational regulation of SPS in Arabidopsis that is similar to previously studied species, but now implicating additional circadian-mediated phosphoregulation of enzyme activity.…”

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

“…A decline in 3-phosphoglycerate under stress could result from impaired photosynthesis (Kaplan and Guy, 2005;Scarpeci and Valle, 2008) or from the activation of the sucrose biosynthesis in response to stress through sucrose synthase phosphate (Geigenberger et al, 1997), which would compete for assimilates. It was indeed demonstrated that phosphorylation of sucrose synthase phosphate is a mechanism for osmotic stress activation of this enzyme in spinach leaves (Toroser and Huber, 1997).…”

Tansley Review No. 105.

“…In the resurrection plant, Craterostigma plantagineum, which can withstand extreme desiccation, dehydration induces the transcription of one of the two SPS genes, leading to increased levels of SPS protein and activity (Ingram et al, 1997). However, in spinach leaves, the activation of SPS is the main reason for the increase in enzyme activity (Zrenner & Stitt, 1991) and is brought about by the phosphorylation of Ser%#% by a water-stress-induced, Ca# + -dependent protein kinase (Toroser & Huber, 1997). Ser%#% occurs within a sequence motif very similar to that involved in light-dark regulation of SPS ; however, in contrast with the latter, the phosphorylation of Ser%#% leads to activation of the enzyme (Toroser & Huber, 1997).…”

“…They suggested that SPS in the bundle sheath cells might be light-regulated in a different manner to that in the mesophyll cells, to allow sucrose to be synthesized at night from the products of starch breakdown . SPS in spinach leaves is activated by the phosphorylation of Ser%#% in response to water stress (Toroser & Huber, 1997) (see Section III.3 for further discussion), and it is possible that the MgATP# − -dependent activation of SPS in bundle sheath strands is a related phenomenon.…”

“…However, in spinach leaves, the activation of SPS is the main reason for the increase in enzyme activity (Zrenner & Stitt, 1991) and is brought about by the phosphorylation of Ser%#% by a water-stress-induced, Ca# + -dependent protein kinase (Toroser & Huber, 1997). Ser%#% occurs within a sequence motif very similar to that involved in light-dark regulation of SPS ; however, in contrast with the latter, the phosphorylation of Ser%#% leads to activation of the enzyme (Toroser & Huber, 1997). These motifs are conserved in almost all known SPS sequences from C $ plants, suggesting that multi-site regulation of SPS by protein phosphorylation is widespread.…”