The dual-targeted purple acid phosphatase isozyme AtPAP26 is essential for efficient acclimation of Arabidopsis to nutritional phosphate deprivation

Abstract

Induction of intracellular and secreted acid phosphatases (APases) is a widespread response of orthophosphate (Pi)-starved (-Pi) plants. APases catalyze Pi hydrolysis from a broad range of phosphomonoesters at an acidic pH. The largest class of nonspecific plant APases is comprised of the purple APases (PAPs). Although the biochemical properties, subcellular location, and expression of several plant PAPs have been described, their physiological functions have not been fully resolved. Recent biochemical studies indicated that AtPAP26, one of 29 PAPs encoded by the Arabidopsis (Arabidopsis thaliana) genome, is the predominant intracellular APase, as well as a major secreted APase isozyme up-regulated by -Pi Arabidopsis. An atpap26 T-DNA insertion mutant lacking AtPAP26 transcripts and 55-kD immunoreactive AtPAP26 polypeptides exhibited: (1) 9- and 5-fold lower shoot and root APase activity, respectively, which did not change in response to Pi starvation, (2) a 40% decrease in secreted APase activity during Pi deprivation, (3) 35% and 50% reductions in free and total Pi concentration, respectively, as well as 5-fold higher anthocyanin levels in shoots of soil-grown -Pi plants, and (4) impaired shoot and root development when subjected to Pi deficiency. By contrast, no deleterious influence of AtPAP26 loss of function occurred under Pi-replete conditions, or during nitrogen or potassium-limited growth, or oxidative stress. Transient expression of AtPAP26-mCherry in Arabidopsis suspension cells verified that AtPAP26 is targeted to the cell vacuole. Our results confirm that AtPAP26 is a principal contributor to Pi stress-inducible APase activity, and that it plays an important role in the Pi metabolism of -Pi Arabidopsis.

Figure 1.

Confirmation of T-DNA insert location and copy number in an atpap26 T-DNA insertional mutant. A, Schematic representation of AtPAP26 gene (At5g34850); white boxes and solid lines represent exons and introns, respectively. T-DNA insertion location is indicated by atpap26 T-DNA, while arrows represent primers used for RT-PCR and genotyping. B, Assessment of T-DNA location and homozygosity of mutants via PCR-based screening of gDNA template isolated from +Pi seedlings. PCR products were amplified from Col-0 and atpap26 gDNA in a 30-cycle PCR reaction containing the indicated primers. The letter M denotes a 100-bp ladder for confirmation of product size. C, Analysis of T-DNA insert number by Southern-blot analysis of atpap26 gDNA probed with NPTII. Arrows indicate the base pair length of EcoRI and HindIII digested phage λ-DNA markers labeled with dig (Roche).

Figure 2.

AtPAP26 is the predominant intracellular APase isozyme up-regulated by −Pi Arabidopsis and whose expression is nullified in atpap26 mutant seedlings. RNA and soluble proteins were isolated from seedlings cultivated in liquid media containing 0.2 mm Pi for 7 d prior to transfer into media containing 0 (−Pi) or 1.5 mm Pi (+Pi) for an additional 7 d. A, Levels of mRNA were analyzed by semiquantitative RT-PCR using gene-specific primers for AtPAP12, AtPAP17, AtPAP26, AtPPCK1, and AtACT2. AtACT2 was used as a reference to ensure equal template loading. Control RT-PCR reactions lacking reverse transcriptase did not show any PCR product. B, Purified native AtPAP26 from −Pi Arabidopsis suspension cells (50 ng/lane; Veljanovski et al., 2006) and clarified extract proteins from shoots (2 μg/lane) and roots (4 μg/lane) of the +Pi and −Pi seedlings were resolved by SDS-PAGE and electroblotted onto a poly(vinylidene difluoride) membrane. Following oxidation of antigenic glycosyl groups with sodium-m-periodate (Laine, 1988), blots were probed with a 1,000-fold dilution of anti-(native AtPAP26)-immune serum and immunoreactive polypeptides detected using an alkaline-phosphatase-linked secondary antibody and chromogenic detection (Veljanovski et al., 2006). C, APase activity of clarified extracts represent means (±SE) of duplicate assays on n = 3 biological replicates; asterisks denote values that are significantly different from Col-0 (P < 0.01). D, Clarified extracts from −Pi shoots of Col-0 and atpap26 were resolved by nondenaturing PAGE and subjected to in-gel APase activity staining (50 μg protein/lane) or immunoblotting with anti-(native AtPAP26)-immune serum (7 μg protein/lane). O, Origin; TD, tracking dye front.

Figure 3.

Immunological AtPAP detection, in-gel APase activity staining, and corresponding APase-specific activities of proteins secreted into the media by +Pi and −Pi Col-0 and atpap26 seedlings. Growth medium containing secreted proteins was passed through a 0.45 μm filter and concentrated >250-fold with Amicon Ultra-15 ultrafiltration devices (30,000 M_r cutoff; Millipore) at room temperature. A and B, Concentrated proteins (15 μg/lane) as well as homogeneous AtPAP12 and AtPAP26 from −Pi Arabidopsis suspension cells (Tran et al., 2010b; 20 ng each) were subjected to SDS-PAGE and immunoblotting as described in the legend for Figure 2, except that the immunoreactive polypeptides were visualized using a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescent detection (ECL Plus, GE Healthcare). C, Concentrated secreted proteins (30 μg/lane) and homogenous AtPAP26 (1 μg; Tran et al., 2010b) were subjected to nondenaturing PAGE and in-gel APase activity staining as described in the legend for Figure 2D. APase activity was also assayed from the concentrated liquid media collected from seedlings cultivated as described in the legend for Figure 2. All APase activities represent the means of duplicate determinations on n = 3 biological replicates and are reproducible to within ±15% of the mean value.

Figure 4.

Influence of nutrient deprivation or oxidative stress on growth of atpap26 and Col-0 seedlings. A, Shoot fresh weight (FW) of seedlings cultivated for 14 d under continuous illumination (100 μmol m⁻² s⁻¹ photosynthetically active radiation) on agar-solidified 0.5× Murashige and Skoog media containing 1% Suc and 1.5 mm or 50 μm Pi. B, Shoot fresh weight of seedlings cultivated on agar media containing 1.5 mm Pi for 7 d, then grown for an additional 14 d on media containing 1.5 mm or 50 μm Pi, or on +Pi media lacking nitrogen (−N) or potassium (−K), or containing 1 μm paraquat (PQ). C, Primary root length of seedlings cultivated on vertically oriented plates for 6, 9, or 12 d as described in A. All values in A to C represent means ± SE of n = 16 seedlings from four different plates; asterisks denote values that are significantly different from Col-0 (P < 0.01).

Figure 5.

Effect of Pi deprivation on soil-grown atpap26 and Col-0 seedlings. A, Seedlings were cultivated for 7 d on Pi-fertilized soil, then transplanted into a Pi-deficient soil mixture and grown for an additional 14 d; fertilization occurred biweekly with 0.25× Hoagland media containing 2 mm Pi (+Pi) or 0 mm Pi (−Pi). Pots shown are representative of 10 replicates. B, Rosette dry weight of seedlings cultivated as in A. C to E, Anthocyanin (C), and free and total Pi concentrations (D and E) of rosette leaves of seedlings cultivated as in A. All values are means ± se (n = 20 for B, 5 for C, and 10 for D and E); asterisks denote values that are significantly different from Col-0 (P < 0.01).

Figure 6.

AtPAP26-mCherry localizes to lytic vacuoles of transiently transformed Arabidopsis suspension cells. Heterotrophic suspension cells were transiently cotransformed via biolistic bombardment with AtPAP26-mCherry and sporamin NTPP-GFP. Following bombardment, cells were incubated for 8 h to allow for gene expression and protein sorting, then fixed in formaldehyde and viewed using epifluorescence microscopy. Note that the fluorescence patterns attributable to coexpressed AtPAP26-mCherry (A) and sporamin NTPP-GFP (B) colocalize in the same cell, as evidenced by yellow color in the merged image (C). Also shown (D), is the differential interference contrast (DIC) image of the cell depicted in A to C. This result is representative of ≥25 cells from at least two independent biolistic bombardments. Bar = 10 μm.