Arabidopsis purple acid phosphatase 10 is a component of plant adaptive mechanism to phosphate limitation

Abstract

When grown with inadequate quantities of inorganic phosphate (Pi), plants synthesize and secret acid phosphatases into the rhizosphere. These secreted acid phosphatases are thought to release the Pi group from organophosphates present in the surrounding environment and to thereby increase Pi availability to plants. So far, however, the genetic evidence to support this hypothesis is still lacking. Previously, we showed that overexpression of Arabidopsis purple acid phosphatase 10 (AtPAP10) improved the growth of plants on Pi-deficient medium (P^- medium) supplemented with the organophosphate compound ADP; in contrast, the growth of atpap10 mutant lines was reduced on the same medium. In the current research, we determined the growth performance of these lines on P^- medium supplemented with four other organophosphates. The results showed that AtPAP10 could utilize rhizosphere organophosphates other than ADP for plant growth but with different utilization efficiencies. This work provides further genetic evidence that AtPAP10 phosphatase is a component of plant adaptive mechanism to Pi limitation.

Although total phosphorus (P) is abundant in most soils, the concentration of inorganic phosphate (Pi) in most natural and arable lands is currently below the level required for optimal plant growth.^1,2 This is because most P exists in the form of organophosphate, which cannot be taken up via phosphate transporters on the root surface.³ Under low Pi conditions, plants synthesize acid phosphatases and secrete them into the rhizosphere.^4,5 The secreted acid phosphatases are believed to scavenge Pi from organophosphate compounds in the rhizosphere and to thus increase Pi availability to plants.⁶ Though this hypothesis has been proposed for several decades, definitive genetic data in its support are still lacking. In 2010, Hurley et al. showed that an Arabidopsis T-DNA knockout line of a purple acid phosphatase, AtPAP26, exhibits impaired growth under Pi deficiency.⁷ Because AtPAP26 is targeted to both vacuoles and rhizosphere, it is unclear whether impaired growth was due to the loss-of-function of an intracellular phosphatase involved in Pi recycling and remobilization or loss-of-function of a secreted enzyme participating in the release of the Pi group from external organophosphates. Some studies had previously shown that overexpression of a plant phytase or a purple acid phosphatase can increase plant growth on a medium with the organophosphate phytate as the sole source of P.^8-10 So far, however, there were no reports about the growth performance of their corresponding knockout lines on P^- medium with supplemented phytate or other organophosphates.

Using genetic and molecular approaches, we recently identified Arabidopsis purple acid phosphatase 10 (AtPAP10) as a Pi starvation-induced acid phosphatase that is predominantly associated with the root surface after its secretion.¹¹ Recombinant AtPAP10 protein has phosphatase activity against a variety of organophosphates, including a moderate activity against phytate. Furthermore, we showed that on a P^- medium supplemented with ADP, an organophosphate compound, AtPAP10 overexpressing lines grew better than the WT; in contrast, multiple atpap10 mutant lines showed reduced growth on these media. These results demonstrated that AtPAP10 plays an important role in the utilization of an external organophosphate compound under Pi deficiency and thus contributes to plant tolerance to Pi limitation. However, it is not known whether AtPAP10 can also acts on other organophosphates to increase plant tolerance to Pi limitation.

Here we examined the growth of AtPAP10-overexpressing lines and the atpap10 mutant lines on P^- medium supplemented with four other organophosphates. The P^- medium was full-strength MS medium¹² without inorganic phosphate. The four organophosphates tested in this study were fructose-6-phosphate (Fru-6-P), phosphoenolpyruvate (PEP), phytate, and phosphoserine (P-Ser). The reason that these four compounds were chosen in this study was because they might have different nutritional values. Among these four compounds, phytate and P-Ser have been shown to be hydrolyzed by recombinant AtPAP10 enzymes.¹¹ Our previous results have indicated that the growth of AtPAP10-overexpressing lines, multiple atpap10 mutant lines, and the WT did not significantly differ on P⁺ or P^- medium, so the comparative study in this work was performed only on the P^- medium supplemented with various types of organophosphates. We first compared the growth performance between the WT and four atpap10 mutant lines. These four atpap10 mutant lines included two T-DNA insertion lines and two lines with point mutations within the AtPAP10 genes.¹¹ The seeds of the WT and four atpap10 mutant lines were sown directly on P^- medium supplemented with various amount of the four organophosphate compounds. Fourteen days after germination (DAG), the shoot and root biomass (fresh weight, FW) were determined for each line. On the P^- medium supplemented with 10, 50 or 150 µM Fru-6-P, shoot and root fresh weights were significantly lower for the four atpap10 mutant lines than for the WT (Fig. 1A); the reductions in fresh weights ranged from 40–50% for shoots and 15–30% for roots. The similar results were obtained when these lines were grown on the P^- medium supplemented with PEP, although the overall difference of growth between the WT and four mutant lines was smaller than that on P^- medium with Fru-6-P (Fig. 1B). When the P^- medium was supplemented with phytate, shoot and root growth increased for all the lines as the concentration of phytate increased (Fig. 1C), however, growth did not significantly differ among the genotypes. This later finding indicated that Arabidopsis plants can utilize phytate as a P source but that AtPAP10 does not play a prominent role in its utilization (although the recombinant AtPAP10 protein displays moderate activity against phytate in enzyme assays).¹² Addition of a fourth organophosphate, phosphoserine (P-Ser), to the P^- medium did not increase growth of any of the plants (Fig. 1D). This is surprising because the recombinant AtPAP10 protein exhibited a high activity against P-Ser in enzyme assays.¹² The cause for the inconsistency between in vitro biochemical data and in vivo physiological effects requires further investigation.

Figure 1. Shoot and root fresh weights of the four atpap10 mutant lines and the WT when grown for 14 d on the P^- medium supplemented with different quantities of Fru-6-P (A), PEP (B), phytate (C) and P-Ser (D). The quantities of organophosphates added to the P^- medium are indicated at the bottom. The experiments were repeated three times with similar results. Values represent means with SE of three replicates (40 seedlings per replicate). Asterisks indicate significant differences compared with the WT (p < 0.05, t-test).

Next, we compared the growth between the three AtPAP10 overexpressing lines and the WT on various media. The AtPAP10-overexpressing lines were generated by transforming the AtPAP10 gene into WT plants under the control of the 35S Cauliflower Mosaic Virus gene promoter.¹¹ In contrast to the atpap10 mutant lines, three overexpressing lines produced more shoot and root biomasses than the WT on the P^- medium supplemented with Fru-6-P or PEP (Fig. 2A and B). Similarly, no growth advantage was observed for the overexpressing lines when grown on P^- medium supplemented with phytate or Ser-P (Fig. 2C and D). Taken together, our results provided further genetic evidence that AtPAP10 is involved in plant adaptation to Pi limitation by enabling the use of a variety of organophosphates in the rhizosphere.

Figure 2. Shoot and root fresh weights of the three AtPAP10 overexpressing lines (lines 5, 10 and 17) and the WT when grown for 14 d on the P^- medium supplemented with different quantities of Fru-6-P (A), PEP (B), phytate (C) and P-Ser (D). The quantities of organophosphates added to the P^- medium are indicated at the bottom. The experiment were repeated three times with similar results. Values represent means with SE of three replicates (40 seedlings per replicate). Asterisks indicate significant differences compared with the WT (p < 0.05, t-test).

Arabidopsis has 29 purple acid phosphatase genes.¹³ Among these, AtPAP10, AtPAP12 and AtPAP26 form a closely related subgroup and are all secreted enzymes.^11,14 These three enzymes have both overlapping and distinct substrate spectra against different organophosphates. Thus, it will be interesting to use both their overexpressing and mutant lines to compare the roles of each enzyme in the utilization of external organophosphate under Pi deficiency. If these three enzymes differ in their efficiency for utilizing different external organophosphates, plants with high P-nutrition efficiency might be generated by stacking the three overexpressing traits into one line.

Conclusions

Our previous work demonstrated that Arabidopsis purple acid phosphatase AtPAP10 can release Pi from external organophosphate ADP and thereby increase Pi availability and increase plant tolerance to low Pi stress. By testing four additional organophosphates, the current study showed that AtPAP10 could also act on two other organophosphates in the rhizosphere and thereby make plant more adapted to Pi limitation. These work provides unequivocal evidence for the generally held but experimentally unconfirmed belief that secreted acid phosphatase plays an important role in plant adaptation to Pi limitation by enabling plants to utilize organophosphates in the rhizosphere.