Rice SPK, a calmodulin-like domain protein kinase, is required for storage product accumulation during seed development: phosphorylation of sucrose synthase is a possible factor

Abstract

Suc, an end product of photosynthesis, is metabolized by Suc synthase in sink organs as an initial step in the biosynthesis of storage products. Suc synthase activity is known to be regulated by reversible phosphorylation, but the details of this process are unclear at present. Rice SPK, a calcium-dependent protein kinase, is expressed uniquely in the endosperm of immature seed, and its involvement in the biosynthetic pathways of storage products was suggested. Antisense SPK transformants lacked the ability to accumulate storage products such as starch, but produced watery seed with a large amount of Suc instead, as the result of an inhibition of Suc degradation. Analysis of in vitro phosphorylation indicated that SPK phosphorylated specifically a Ser residue in Suc synthase that has been shown to be important for its activity in the degradation of Suc. This finding suggests that SPK is involved in the activation of Suc synthase. It appears that SPK is a Suc synthase kinase that may be important for supplying substrates for the biosynthesis of storage products.

Figure 1.

Detection of SPK mRNA in an Immature Rice Seed by in Situ Hybridization. (A) The region expressing Spk is shown in purple. The specific RNA of the antisense strand was used as a probe. (B) and (C) Magnified images corresponding to the boxes in (A). (D) and (E) Results of the control experiment using sense strand RNA as a probe. AL, aleurone layer; EM, embryo; EN, endosperm.

Figure 2.

Effect of the Antisense Spk Gene on Its Transformants. Phenotypic features of immature seed 2 weeks after pollination of the wild type (cv Nipponbare) (A) and of a representative antisense transformant (ASPK19) (B) are shown. HU, hull; EN, endosperm; EN′, watery endosperm.

Figure 3.

Protein Accumulation in Immature Seed of the Transformants and Wild-Type Plants. (A) Protein accumulated in immature seed of the transformant ASPK19 and the wild type detected by SDS-PAGE. Lane 1, crude extract; lane 2, soluble fraction; lane 3, insoluble fraction. Each lane contained one-fifth of a total crude extract or of each fraction that was prepared independently from a single grain. (B) Protein composition of immature seed of the transformant ASPK19 or the wild type. Each lane contained 25 μg of total protein from the crude extract. (C) Detection of RBE1 (85 kD) and SPK (68 kD) in immature seed of the transformant ASPK19 or the wild type by protein gel blot analysis. Each lane contained one-fifth of a total crude extract from a single grain. The proteins corresponding to RBE1 and SPK are indicated by arrows. TF, transformant ASPK19; WT, wild type.

Figure 4.

SPK May Have CDPK Activity. (A) Calcium dependence of SPK autophosphorylation. The effects of the addition of 100 μM Ca²⁺ (as CaCl₂) or 100 μM EGTA to the reaction (+) were examined. (B) Effects of inhibitors. Results in the presence of 1 μM (lane 1), 5 μM (lane 2), or 10 μM (lane 3) staurosporine (STA), ML-9, W-7, or with no inhibitor (CON) are shown. Each lane contained 3.0 μg of GST–SPK in the reaction mixture.

Figure 5.

In Vitro Phosphorylation of GST–Suc Synthase by SPK. In vitro phosphorylation of GST–Suc synthase (Susy) and RBE1 (RBE) in the presence or absence of GST–SPK (SPK). The presence of these proteins in the reaction (2.5 μg of GST–SPK, 3.5 μg of GST–Suc synthase, and 3.0 μg of RBE1) is indicated (+). Top, Coomassie blue–stained proteins (CBB); bottom, corresponding autoradiograms (AR).

Figure 6.

Detection of in Vitro Phosphorylation of Proteins by SPK in Immature Seed. Proteins on a 10% SDS–polyacrylamide gel are shown as an autoradiogram. Lanes 1 and 2 contained crude extracts from the wild-type plant (cv Nipponbare). Lane 2 shows the results of kinase reactions in the presence of 100 μM Ca²⁺.

Figure 7.

Immunoprecipitation of Suc Synthase in Immature Seed and Phosphorylation by SPK. Phosphorylation of the constituents of immunoprecipitates obtained with anti-Suc synthase antibody is shown. Lanes show the results of reactions in the presence (+) or absence of a crude extract of immature seed (C.E.), anti-Suc synthase antibody (anti-Susy), and GST–SPK. CBB indicates Coomassie blue–stained proteins, and AR shows the corresponding autoradiograms.

Figure 8.

In Vitro Phosphorylation of GST–Suc Synthase by the Crude Extract from the Immature Seed of the Wild-Type Plant and the Watery Seed of the Antisense Transformants. Each lane contained 3.0 μg of GST–Suc synthase as the substrate. Lanes 1 and 2 contained 8.0 μg of total proteins in the crude extract from the immature seed of cv Nipponbare. Lane 2 also included 1 mM EGTA in the reaction mixture. Lane 3 contained 8.0 μg of total proteins in the crude extract from the watery seed of the antisense transformant ASPK19. Proteins resolved on a 10% SDS–polyacrylamide gel are shown in the autoradiogram.

Figure 9.

Detection of Suc Synthase in the Watery Seed of the Antisense Transformant by Protein Gel Blot Analysis. Each lane contained one-fifth of a total crude extract from a single grain of the antisense SPK transformant line ASPK19 (TF) and the wild-type plant (WT). The 85-kD protein corresponding to Suc synthase (Susy) is indicated by the arrow.

Figure 10.

Determination of the Phosphorylation Site in Suc Synthase. (A) Alignment of the amino acid sequences of Suc synthases in the region around the potential target site. The consensus amino acid residues, Arg and Ser, are boxed. Zm1, Zm2, Rs1, Rs2, and Rs3 denote Suc synthases encoded by maize Msus1, maize Msus2, rice Rsus1, rice Rsus2, and rice Rsus3, respectively. RtoG, mutant with substitution at the Arg residue; StoV, mutant with substitution at the Ser residue. Numbers indicate the positions of amino acid residues around the potential phosphorylation site. For accession numbers, see Methods. (B) In vitro phosphorylation of the site-specifically mutated and wild-type Suc synthases. Lanes show the reaction with GST–Suc synthase (wild type [WT]), with GST–RtoG (RtoG), and with GST–StoV (StoV). CBB and AR indicate the Coomassie blue–stained proteins and the corresponding autoradiograms, respectively.