Site-Directed Mutations at Phosphorylation Sites in Zea mays PHO1 Reveal Modulation of Enzymatic Activity by Phosphorylation at S566 in the L80 Region

Abstract

Starch phosphorylase (PHO) is a pivotal enzyme within the GT35-glycogen-phosphorylase (GT; glycosyltransferases) superfamily. Despite the ongoing debate surrounding the precise role of PHO1, evidence points to its substantial influence on starch biosynthesis, supported by its gene expression profile and subcellular localization. Key to PHO1 function is the enzymatic regulation via phosphorylation; a myriad of such modification sites has been unveiled in model crops. However, the functional implications of these sites remain to be elucidated. In this study, we utilized site-directed mutagenesis on the phosphorylation sites of Zea mays PHO1, replacing serine residues with alanine, glutamic acid, and aspartic acid, to discern the effects of phosphorylation. Our findings indicate that phosphorylation exerts no impact on the stability or localization of PHO1. Nonetheless, our enzymatic assays unveiled a crucial role for phosphorylation at the S566 residue within the L80 region of the PHO1 structure, suggesting a potential modulation or enhancement of PHO1 activity. These data advance our understanding of starch biosynthesis regulation and present potential targets for crop yield optimization.

Keywords: activity; phosphorylation; plastidial starch phosphorylase; subcellular localization.

Figure 1

Induced expression of the recombinant plasmid and PHO1 rabbit antiserum-specific detection and verification of ZmPHO1 antibodies in maize tissue. (A) Symbolic representation of domains on the primary structure of Zea mays PHO1 (ZmPHO1); (B) recombinant plasmid vector construction flow chart. (C) PHO1-C-3′ end PCR amplification. (D) PGEX-6T-1-PHO1-C recombinant plasmid digestion identification. (E) IPTG induced expression of PHO1-C protein; (F) the targeted protein purified using GST-tag purification resin, with BSA as a loading control; (G) detection of specificity of antibody against ZmPHO1 of maize seed; the dilution ratio of the PHO1-C antibody was 1:1000.

Figure 2

Western blot detection phosphorylation of Zea mays PHO1 (ZmPHO1). Negative and positive signs indicate the addition and absence of alkaline phosphatase. The dilution ratio of ZmPHO1 antibody was 1:1000 and the dilution ratio of β-Actin was 1:10,000. The amount of protein loaded was 30 µg.

Figure 3

Mass spectrometry identification of ZmPHO1 phosphopeptide. (A) Mass spectrometry identification of ZmPHO1 phosphorylated peptide “DRDVQGPVSPAEGLPSVLNS”, the red letter (S) is the phosphorylation site at 69; (B) mass spectrometry identification of ZmPHO1 phosphorylated peptide “LESEEVEAEEESSEDELDPFVK”, the red letter (S) is the phosphorylation site at 566.

Figure 4

Expression and subcellular localization of mutated and non-mutated recombinant PHO1 in Zea mays protoplast of the leaves. (A) pCAMBIA2300-eGFP. (B) pCAMBIA2300-ZmPHO1-eGFP. (C) pCAMBIA2300-ZmPHO1-S566A-eGFP. (D) pCAMBIA2300-ZmPHO1-S566E-eGFP. (E) pCAMBIA2300-ZmPHO1-S69A-eGFP. (F) pCAMBIA2300-ZmPHO1-S69E-eGFP. All of the expressed GFP-tagged PHO1 is found in the chloroplast. There are no significant differences in the subcellular localization found for both phosphorylation site mutations.

Figure 5

The activity of recombinant PHO1. (A) The activity of recombinant PHO1 with maltodextrin substrate with no pre-treatment with ATP. (B) The activity of phosphorylated PHO1 with maltodextrin substrate. Non-mutated recombinant PHO1 was used as a negative control without pretreatment with ATP. Activities were calculated as nmol/10 min/mg. Significantly different means (at p < 0.05) from the one-way ANOVA followed by LSD. a–c represents a significant difference.