Focus Issue on Plant Cell Walls: Potential Role for Purple Acid Phosphatase in the Dephosphorylation of Wall Proteins in Tobacco Cells

It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as α-xylosidase and β-glucosidase. The dephosphorylation and phosphorylation of recombinant α-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of α-xylosidase and β-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls.Purple acid phosphatase (PAP) belongs to a large family of dinuclear metalloenzymes (LeBansky et al., 1992; Klabunde et al., 1996) and catalyzes the hydrolysis of a wide range of phosphate esters. It is distinguished from other acid phosphatases by its purple color, which is due to a Tyr-to-iron (III) charge transfer transition (Antanaitis et al., 1983). Arabidopsis (Arabidopsis thaliana) contains a large family of PAPs composed of 29 genes, 28 of which have signal peptides that potentially transfer to the wall and/or vacuole. Only a few functions have been suggested for these phosphatases: AtPAP15 seems to modulate ascorbic acid biosynthesis (Zhang et al., 2008), and AtPAP17 may play a role in the metabolism of reactive oxygen species (del Pozo et al., 1999). In other plant species, soybean (Glycine max) GmPAP3 is induced by NaCl stress but not by phosphorus deficiency (Liao et al., 2003), tomato (Solanum lycopersicum) PAP may release phosphate from extracellular phosphate ester under phosphate starvation (Bozzo et al., 2002), and tobacco (Nicotiana tabacum) NtPAP12 could be involved in the deposition of β-glucan (Kaida et al., 2003, 2009; Sano et al., 2003). Mammalian PAPs, which are secretory enzymes, may be involved in iron transport (Nuttleman and Roberts, 1990), generation of reactive oxygen species (Sibille et al., 1987), and bone resorption (Ek-Rylander et al., 1994).We previously demonstrated that the activities of cellulose and callose synthases are enhanced by overexpression of NtPAP12 in tobacco cells (Kaida et al., 2009). The phosphorylation/dephosphorylation process in those synthases may occur directly on the catalytic subunit itself, which has been predicted to be located on the cytoplasmic side of the plasma membrane (Nühse et al., 2004; Taylor, 2007). This is not compatible with the cell wall localization of NtPAP12. The data also indicate that phosphorylation may play a role in regulating the turnover of cellulose synthase by proteolysis through a proteasome-dependent pathway (Taylor, 2007), which again implies a cytoplasmic phosphorylation event. Thus, we suggested that NtPAP12 could be involved in the regulation of cellulose synthase activity, either by acting on an unidentified membrane protein or by enhancing its activity with an effector, which can lead to the promotion of cellulose synthesis. Nevertheless, this phosphatase may be involved in the activation of synthases indirectly by acting on either apoplastic proteins or unidentified membrane proteins, since the level of activation for glucan synthases was only a 2- to 3-fold increase in the transgenic tobacco cells overexpressing NtPAP12 compared with wild-type cells.The extracellular phosphorylation network has been proposed by proteomic analysis of Arabidopsis cells due to the identification of phosphorylated Tyr residues in xyloglucanase, putative lectin receptor-like kinase, and putative chitinase (Ndimba et al., 2003). The change in phosphorylation status was also identified in the extracellular peroxidase in maize (Zea mays) cells (Chivasa et al., 2005b). Another analysis has indicated that some potential phosphorylated proteins might be present in the apoplastic space during wall regeneration (Kwon et al., 2005). We previously showed that tobacco PAP had a higher catalytic efficiency for Tyr phosphopeptides (k_cat/K_m = 1,093–1,335) than for ATP (k_cat/K_m = 333) and p-nitrophenyl-phosphate (k_cat/K_m = 379), suggesting that the enzyme could dephosphorylate the phosphoryl residues of proteins in vivo (Kaida et al., 2008). There is still much to be learned, however, including the role that phosphorylation plays in the functions of these proteins. It is possible, for example, that extracellular PAPs might modify the functions of the phosphoproteins by dephosphorylating those proteins in the apoplasts, but to date no evidence has been reported demonstrating this activity. In this study, we searched for substrates of PAP using phosphoproteomic analyses of apoplastic proteins in tobacco cells.