Biochemical and molecular characterization of AtPAP26, a vacuolar purple acid phosphatase up-regulated in phosphate-deprived Arabidopsis suspension cells and seedlings

Abstract

A vacuolar acid phosphatase (APase) that accumulates during phosphate (Pi) starvation of Arabidopsis (Arabidopsis thaliana) suspension cells was purified to homogeneity. The final preparation is a purple APase (PAP), as it exhibited a pink color in solution (A(max) = 520 nm). It exists as a 100-kD homodimer composed of 55-kD glycosylated subunits that cross-reacted with an anti-(tomato intracellular PAP)-IgG. BLAST analysis of its 23-amino acid N-terminal sequence revealed that this PAP is encoded by At5g34850 (AtPAP26; one of 29 PAP genes in Arabidopsis) and that a 30-amino acid signal peptide is cleaved from the AtPAP26 preprotein during its translocation into the vacuole. AtPAP26 displays much stronger sequence similarity to orthologs from other plants than to other Arabidopsis PAPs. AtPAP26 exhibited optimal activity at pH 5.6 and broad substrate selectivity. The 5-fold increase in APase activity that occurred in Pi-deprived cells was paralleled by a similar increase in the amount of a 55-kD anti-(tomato PAP or AtPAP26)-IgG immunoreactive polypeptide and a >30-fold reduction in intracellular free Pi concentration. Semiquantitative reverse transcription-PCR indicated that Pi-sufficient, Pi-starved, and Pi-resupplied cells contain similar amounts of AtPAP26 transcripts. Thus, transcriptional controls appear to exert little influence on AtPAP26 levels, relative to translational and/or proteolytic controls. APase activity and AtPAP26 protein levels were also up-regulated in shoots and roots of Pi-deprived Arabidopsis seedlings. We hypothesize that AtPAP26 recycles Pi from intracellular P metabolites in Pi-starved Arabidopsis. As AtPAP26 also exhibited alkaline peroxidase activity, a potential additional role in the metabolism of reactive oxygen species is discussed.

Figure 1.

The 1.25 mm Pi concentration of conventional MS liquid media is insufficient to maintain Arabidopsis suspension cells fully Pi sufficient over 7 d in batch culture. At 0 d, 10-mL aliquots of cells cultured for 7 d in 5 mm Pi were subcultured into 90 mL of fresh MS media containing the indicated Pi concentration. At 7 d, biomass yields and Pi concentration in the CCF were determined. All values represent means ± se of n = 3 separate flasks.

Figure 2.

Up-regulation of intracellular APase in Arabidopsis suspension cells becoming Pi deficient. At 0 d, 100-mL aliquots of cells cultured for 7 d in 5 mm Pi were subcultured into 400 mL of fresh MS media containing 5 mm +Pi or 0 mm −Pi. Time courses for CCF of extracellular Pi (A), intracellular Pi (B), and APase activity of clarified extracts (C) of the +Pi and −Pi cells were determined. All values represent means ± se of n = 3 separate flasks. Where invisible, the error bars are too small to be seen. D, Immunological detection of APase in clarified extracts from the Arabidopsis suspension cells. Purified intracellular PAP (50 ng) from −Pi tomato cells (Bozzo et al., 2004a) and clarified cell-extract proteins (15 μg) from the Arabidopsis cells were resolved by SDS-PAGE and blot transferred to a PVDF membrane. Native intracellular APase of −Pi tomato cells exists as a heterodimer composed of 63- and 57-kD subunits (Bozzo et al., 2004a). The immunoblot was probed with a 1:50 dilution of affinity-purified anti-(tomato intracellular PAP)-IgG, and immunoreactive peptides were detected using an alkaline-phosphatase linked secondary antibody as in Bozzo et al. (2006). “−Pi→+Pi” denotes extracts from 6-d −Pi cells that were resupplied with 2.5 mm Pi and cultured for an additional 2 d. Relative amounts of the 55-kD antigenic polypeptide were quantified by laser densitometry. O, Origin; TD, tracking dye front.

Figure 3.

Visible absorbance spectrum of APase isolated from −Pi Arabidopsis suspension cells. The spectrum was obtained using a solution of 5 mg mL⁻¹ of the purified APase.

Figure 4.

SDS-PAGE and immunoblot analysis of purified PAP from −Pi Arabidopsis suspension cells. A, SDS-PAGE (10% separating gel) of purified Arabidopsis APase. Lane 1 contains 6.5 μg of various protein molecular mass standards, whereas lane 2 contains 2 μg of the purified PAP. Protein staining was performed using Coomassie Blue R-250. B, SDS-PAGE of 5 μg of the purified PAP was followed by glycoprotein staining using a periodic acid-Schiff procedure (Gradilone et al., 1998). C and D, Immunoblotting was performed using a 1:50 dilution of affinity-purified anti-(tomato intracellular PAP)-IgG (C; Bozzo et al., 2006) or a 1:1,000 dilution of anti-(AtPAP26)-immune serum (D), and antigenic polypeptides were visualized using an alkaline-phosphatase-linked secondary antibody. Lanes 1 and 2 of each segment contain 15 ng of purified PAP from −Pi Arabidopsis and tomato suspension cells, respectively. Lane 3 and 4 of segment D contain clarified cell-extract proteins (15 μg) from the +Pi and −Pi 7-d Arabidopsis cells, respectively. O, Origin; TD, tracking dye front.

Figure 5.

Deduced amino acid sequence alignment of Arabidopsis AtPAP26 with orthologs from various plants. The PAP sequences included are from Arabidopsis (AAW29950.1), tomato (BT014303), soybean (AAN85417), rice (BAD37373), and onion (BAB60719). Identical and similar amino acids are indicated by shaded black and gray, respectively. Conserved sequence motifs containing potential metal-ligating residues (Li et al., 2002) are marked by asterisks. The arrow indicates the predicted cleavage site of the AtPAP26 signal peptide. The 23-amino acid N-terminal sequence obtained by automated Edman degradation of the 55-kD subunit of the PAP purified from the −Pi Arabidopsis suspension cells appears in bold font above the deduced sequences. The N-terminal sequence and a tryptic peptide sequence obtained for the 57-kD subunit of the PAP purified from −Pi tomato suspension cells (Bozzo et al., 2004a) are also indicated.

Figure 6.

Semiquantitative RT-PCR analysis of AtPAP26 gene expression in Pi-sufficient (+), Pi-starved (−), and Pi-resupplied Arabidopsis suspension cells. The cells were cultured as described in the legend for Figure 2. Levels of mRNA were analyzed by RT-PCR using primers specific for the genes AtPAP26, AtPAP17, and Actin 2. AtPAP17 was used as a positive control (del Pozo et al., 1999; Li et al., 2002; Müller et al., 2004), whereas Arabidopsis Actin 2 was used as a reference control to ensure equal template loading. All PCR products were taken at cycle numbers determined to be nonsaturating. Control RT-PCR reactions lacking RT did not show any bands. The AtPAP17 and AtPAP26 primers amplified 469-bp and 610-bp fragments of the 5′ regions of their respective cDNAs, as expected. “Pi-refed” denotes extracts from 6-d −Pi cells that were resupplied with 2.5 mm Pi and cultured for an additional 24 and 48 h.

Figure 7.

Immunological PAP detection and corresponding APase activity of clarified extracts from roots and shoots of +Pi versus −Pi Arabidopsis seedlings. The seedlings were germinated and cultivated on MS media containing 1.25 mm Pi for 14 d, transferred to fresh MS media containing 1.25 or 0 mm Pi (+Pi and −Pi, respectively), and cultivated for an additional 10 d prior to extraction and analysis of root and shoot tissues. Purified AtPAP26 (20 ng) from −Pi Arabidopsis suspension cells and clarified extract proteins (15 μg) from shoots and roots of the +Pi and −Pi Arabidopsis seedlings were resolved by SDS-PAGE and blot transferred onto a PVDF membrane. The immunoblot was probed with a 1:1,000 dilution of anti-(AtPAP26)-immune serum and immunoreactive peptides detected using an alkaline-phosphatase linked secondary antibody. Relative amounts of the antigenic 55-kD polypeptide were quantified by laser densitometry. O, Origin; TD, tracking dye front. Corresponding specific APase activities are indicated below each lane. APase activities were determined using assay A, represent the means of n = 4 separate extractions, and are reproducible to within ±20% of the mean value.

Figure 8.

Phosphatase versus peroxidase activity of purified AtPAP26 as a function of assay pH. Assays were performed as described in the “Materials and Methods” except that they were buffered by a mixture of 25 mm sodium acetate, 25 mm MES, and 25 mm Bis-Tris propane. All values represent the means of n = 3 separate determinations and are reproducible to within ±10% of the mean value.