Abstract
Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The aim of this work was to further investigate the function of one of these phosphatases in tobacco (Nicotiana tabacum), NtPAP12, using transgenic cells overexpressing the enzyme. The transgenic cells exhibited a higher level of phosphatase activity in their walls. The corresponding protoplasts regenerating a cell wall exhibited a higher rate of beta-glucan synthesis and cellulose deposition was increased in the walls of the transgenic cells. A higher level of plasma membrane glucan synthase activities was also measured in detergent extracts of membrane fractions from the transgenic line, while no activation of Golgi-bound glycan synthases was detected. Enzymatic hydrolysis and methylation analysis were performed on the products synthesized in vitro by the plasma membrane enzymes from the wild-type and transgenic lines extracted with digitonin and incubated with radioactive UDP-glucose. The data showed that the glucans consisted of callose and cellulose and that the amount of each glucan synthesized by the enzyme preparation from the transgenic cells was significantly higher than in the case of the wild-type cells. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis.
Highlights
Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth
Fluorescence microscopy showed that the purple acid phosphatase could be visualized between the plasma membrane sheets and the polylysine-coated coverslip immediately after the beginning of the incubation, and its deposition increased with incubation time (Fig. 1). b-Glucan microstructures had been formed on the plasma membrane sheets between the sheets and the polylysine-coated coverslip (Fig. 1)
This is supported by several lines of evidence: (1) the transgenic protoplasts regenerating a cell wall exhibit a higher rate of b-glucan synthesis; (2)
Summary
Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis. We suggested that the wall-bound purple acid phosphatase NtPAP12 from tobacco (Nicotiana tabacum) functions as a protein phosphatase (Kaida et al, 2008) and promotes cellulose synthesis (Sano et al, 2003). To shed light on the possible function of NtPAP12 in the regulation of b-glucan and cellulose biosynthesis, expression of the phosphatase was induced at the early stage of wall regeneration in tobacco protoplasts (Kaida et al, 2003). Our work reveals that NtPAP12 is involved in the regulation of callose and cellulose synthases and, indirectly, in cell growth and morphogenesis
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