Comparative genetic analysis of,Arabidopsis purple acid phosphatases AtPAP10, AtPAP12, and AtPAP26 provides new insights into their roles in plant adaptation to phosphate deprivation

Induction and secretion of acid phosphatases （APases） is thought to be an adaptive mechanism that helps plants survive and grow under phosphate （Pi） deprivation, in Arabidopsis, there are 29 purple acid phosphatase （AtPAP） genes. To systematically investigate the roles of different AtPAPs, we first identified knockout or knock-down T-DNA lines for all 29 AtPAP genes. Using these atpap mutants combined with in-gel and quantitative APase enzyme assays, we demonstrated that AtPAP12 and AtPAP26 are two major intracellular and secreted APases in Arabidopsis while AtPAPlo is mainly a secreted APase. On Pi-deficient （P-） medium or P- medium supplemented with the organophosphates ADP and fructose-6-phosphate （Fru-6-P）, growth of atpaplo was significantly reduced whereas growth of atpap12 was only moderately reduced, and growth of atpap26 was nearly equal to that of the wild type （WT）. Overexpression of the AtPAP12 or AtPAP26 gene, however, caused plants to grow better on P- or P- medium supplemented with ADP or Fru-6-P. Interest-ingly, Pi levels are essentially the same for the WT and overexpressing lines, although these two types of plants have significantly different growth phenotypes. These results suggest that the APases may have other roles besides enhancing internal Pi recycling or releasing Pi from external organophosphates for plant uptake.     

Improved phosphate metabolism and biomass production by overexpression of <Emphasis Type="Italic">AtPAP18</Emphasis> in tobacco

Limited availability of phosphate ion (Pi) reduces plant growth in natural ecosystems. Here, we report the functional effects of overexpressing an Arabidopsis thaliana purple acid phosphatase encoding gene, AtPAP18, in Nicotiana tabbacum as a crop model plant. Transgenic tobacco plants exhibited significant increases in acid phosphatase activity, total P and Pi contents leading to improved biomass production in both Pi-deficient and Pi-sufficient conditions. Transient expression of AtPAP18::green fluorescent fusion protein in onion epidermal cells indicated that AtPAP18 is a dual-targeted protein, which is detected mainly in the apoplast of the cells after 24 h and in the vacuole after 72 h. Possibly, AtPAP18 protein confers efficient retrieval of Pi from bonded extracellular compounds as well as expendable intracellular Pi-monoesters and anhydrides. These data clearly indicate that overexpression of AtPAP18 gene offers an effective approach for reducing the consumption of chemical Pi fertilizer through increased acquisition of soil Pi and mobilization of internal resources.     

Response to Phosphate Deprivation in Brassica nigra Suspension Cells : Enhancement of Intracellular, Cell Surface, and Secreted Phosphatase Activities Compared to Increases in Pi-Absorption Rate

Suspension cells of Brassica nigra responded to Pi deprivation by increasing their potential for Pi influx and by raising the active levels of intracellular, cell surface, and secreted acid phosphatases. These responses, however, were temporally distinct. Phosphate influx capacity increased 15-fold in parallel to a 10-fold decrease in endogenous Pi during 7 days of culture in basal growth medium. In contrast, intracellular and cell surface phosphatase activities changed only after alterations in cellular phosphorus status had been in place for a number of days. Even in nutrient sufficient cells the secretion of phosphatase remained relatively high as did the activities of the other phosphatases. The cell surface acid phosphatase had a K_m of approximately 10 times that of the influx process and molybdate was a much stronger inhibitor of this phosphatase activity. From these results it appears that Pi absorption and the production or activation of phosphatases are regulated in a distinct manner. In addition, Pi uptake into Brassica nigra cells does not appear to directly involve the cell surface phosphatase under Pi-deficient conditions.     

Overexpressing AtPAP15 Enhances Phosphorus Efficiency in Soybean

Low phosphorus (P) availability is a major constraint to crop growth and production, including soybean (Glycine max), on a global scale. However, 50% to 80% of the total P in agricultural soils exists as organic phosphate, which is unavailable to plants unless hydrolyzed to release inorganic phosphate. One strategy for improving crop P nutrition is the enhanced activity of acid phosphatases (APases) to obtain or remobilize inorganic phosphate from organic P sources. In this study, we overexpressed an Arabidopsis (Arabidopsis thaliana) purple APase gene (AtPAP15) containing a carrot (Daucus carota) extracellular targeting peptide in soybean hairy roots and found that the APase activity was increased by 1.5-fold in transgenic hairy roots. We subsequently transformed soybean plants with AtPAP15 and studied three homozygous overexpression lines of AtPAP15. The three transgenic lines exhibited significantly improved P efficiency with 117.8%, 56.5%, and 57.8% increases in plant dry weight, and 90.1%, 18.2%, and 62.6% increases in plant P content, respectively, as compared with wild-type plants grown on sand culture containing phytate as the sole P source. The transgenic soybean lines also exhibited a significant level of APase and phytase activity in leaves and root exudates, respectively. Furthermore, the transgenic lines exhibited improved yields when grown on acid soils, with 35.9%, 41.0%, and 59.0% increases in pod number per plant, and 46.0%, 48.3%, and 66.7% increases in seed number per plant. Taken together, to our knowledge, our study is the first report on the improvement of P efficiency in soybean through constitutive expression of a plant APase gene. These findings could have significant implications for improving crop yield on soils low in available P, which is a serious agricultural limitation worldwide.Phosphorus (P) is a critical macronutrient for plant growth and development. Terrestrial plants generally take up soil P in its inorganic form (Pi; Marschner, 1995). However, 50% to 80% of the total P in agricultural soils exists as organic phosphate, in which, up to 60% to 80% is myoinositol hexakisphosphate (phytate; Iyamuremye et al., 1996; Turner et al., 2002; George and Richardson, 2008). Since phytate-P is not directly available to plants, low P availability becomes one of the limiting factors to plant growth.Plants have developed a number of adaptive mechanisms for better growth on low-P soils, including changes in root morphology and architecture, activation of high-affinity Pi transporters, improvement of internal phosphatase activity, and secretion of organic acids and phosphatases (Raghothama, 1999; Vance et al., 2003). Acid phosphatases (APases) are hydrolytic enzymes with acidic pH optima that catalyze the breakdown of P monoesters to release Pi from organic P compounds, and therefore may play an important role in P nutrition (Vincent et al., 1992; Li et al., 2002). APase activity, including extracellular and intracellular APase activity, is generally increased by Pi starvation in higher plants (Duff et al., 1994). Intracellular APases might play a role in internal Pi homeostasis through remobilization of Pi from older leaves and vacuole stores, whereas extracellular APases are believed to be involved in external P acquisition by mobilizing Pi from organic P compounds (Duff et al., 1994). In the last few years, secreted APases have been purified and characterized in some model plants, such as Arabidopsis (Arabidopsis thaliana; Coello, 2002) and tobacco (Nicotiana tabacum; Lung et al., 2008). Furthermore, an Arabidopsis pup3 mutation that underproduced secreted APases in root tissues accumulated 17% less P in shoots when organic P was supplied as the major P source (Tomscha et al., 2004), indicating the possible role of APases during plant growth in response to Pi starvation.Phytase is a special type of APases with the capability to hydrolyze phytate and its derivatives, which are the predominant inositol phosphates present in seeds and soils. It is generally believed that phytase activation in seeds or resynthesis in plants plays important roles in Pi remobilization through hydrolyzing the phytate into Pi during seed germination (Loewus and Murthy, 2000). Furthermore, phytase in roots and/or root exudation has been demonstrated to be important for utilizing Pi from phytate in the growth medium (Asmar, 1997; Li et al., 1997; Hayes et al., 1999; Richardson et al., 2000).AtPAP15, a purple APase with confirmed phytase activity from Arabidopsis, can hydrolyze myoinositol hexakisphosphate, yielding myoinositol and Pi (Zhang et al., 2008). Overexpression of AtPAP15 in Arabidopsis significantly decreased phytate content in leaves (Zhang et al., 2008). Sequence analysis indicates that AtPAP15 exhibits 74% similarity to the soybean (Glycine max) phytase gene, GmPhy (Hegeman and Grabau, 2001). It seems likely that the possible involvement of phytase in plant P nutrition might be conserved among different plant species. But it is still unclear whether AtPAP15 or other phytases can be used to directly help crops, including the major agronomic crop, soybean, to acquire P under low-P conditions.Soybean is one of the most important food crops, accounting for a large segment of the world market in oil crops and also serving as an important protein source for both human consumption and animal feed (Kereszt et al., 2007). Soybean is mainly cultivated in tropic, subtropic, and temperate areas, where the soils are low in P due to intensive erosion, weathering, and strong P fixation by free iron and aluminum oxides (Sample et al., 1980; Stevenson, 1986). Low P availability is especially problematic for soybean, since root nodules responsible for nitrogen fixation have a high P requirement (Robson, 1983; Vance, 2001).In this study, the Arabidopsis PAP15 gene directed by an extracellular targeting sequence from a carrot (Daucus carota) extensin gene was successfully transformed into both soybean hairy roots and whole soybean plants. Overexpression of AtPAP15 not only increased the secretion of APase from transgenic soybean hairy roots and roots of whole transgenic soybean plants, but also significantly improved APase activity in leaves, as well as P efficiency and yield in the transgenic soybean lines. To the best of our knowledge, this is the first report on the improvement of P efficiency in crop plants through constitutive expression of a plant APase gene. This study could have significant implications for improving crop production on low-P soils, which is a serious agronomic limitation worldwide.     

Conditional identification of phosphate-starvation-response mutants in Arabidopsis thaliana

Plants have evolved elaborate metabolic and developmental adaptations to low phosphorus availability. Biochemical responses to phosphate limitation include increased production and secretion of phosphate-acquisition proteins such as nucleases, acid phosphatases, and high-affinity phosphate transporters. However, the signal transduction pathways that sense phosphate availability and integrate the phosphate-starvation response in plants are unknown. We have devised a screen for conditional mutants in Arabidopsis thaliana (L.) Heynh. to dissect signaling of phosphate limitation. Our genetic screen is based on the facultative ability of wild-type Arabidopsis plants to metabolize exogenous DNA when inorganic phosphate is limiting. After screening 50,000 M2 seedlings, we isolated 22 confirmed mutant lines that showed severely impaired growth on medium containing DNA as the only source of phosphorus, but which recovered on medium containing soluble inorganic phosphate. Characterization of nine such mutant lines demonstrated an inability to utilize either DNA or RNA. One mutant line, psr1 (phosphate starvation response), had significantly reduced activities of phosphate-starvation-inducible isoforms of ribonuclease and acid phosphatase under phosphate-limiting conditions. The data suggest that a subset of the selected mutations impairs the expression of more than one phosphate-starvation-inducible enzyme required for utilization of exogenous nucleic acids, and may thus affect regulatory components of a Pi starvation response pathway in higher plants. Received: 23 September 1999 / Accepted: 10 November 1999     

Differential responses of oat cultivars to phosphate deprivation: plant growth and acid phosphatase activities

The effect of phosphate starvation on growth and acid phosphatases (APases) localization and activity in oat tissues was investigated. Oat cultivars (Avena sativa L.??Arab, Polar, Szakal) were grown for 1?C3?weeks in complete nutrient medium (+P) and without phosphate (?P). Pi concentration in plant tissues decreased strongly after culturing on ?P medium. Pi deficit reduced shoot growth, stimulated root elongation and increased ratio of root/shoot in all oat cultivars. Pi deficit had a greater impact on growth of oat cv. Polar than other varieties. A decrease in the internal Pi status led to an increase of acid phosphatase activities in extracts from shoots and roots, and in root exudates. The highest activity of secreted APases was observed for oat cv. Arab, during the third week of growth under Pi-deficient conditions. The activity of extracellular APase was high in young, growing zones of roots of ?P plants. Histochemical visualization indicated high activity of APases in the epidermis and vascular tissues of ?P plants. Pi deficiency increased intracellular APase activity in shoot mainly in oat cv. Polar, whereas APase activity in roots was the highest in oat cv. Szakal. Protein extracts from roots and shoots were run on native discontinuous PAGE to determine which isoform(s) may be affected by Pi deficiency. Three major APase isoforms were detected in all oat plants; one was strongly induced by Pi deficit. The studied oat cultivars differed in terms of acclimation to deficiency of phosphate??used various pools of APases to acquire Pi from external or internal sources.     

An Arabidopsis purple acid phosphatase with phytase activity increases foliar ascorbate

Ascorbate (AsA) is the most abundant antioxidant in plant cells and a cofactor for a large number of key enzymes. However, the mechanism of how AsA levels are regulated in plant cells remains unknown. The Arabidopsis (Arabidopsis thaliana) activation-tagged mutant AT23040 showed a pleiotropic phenotype, including ozone resistance, rapid growth, and leaves containing higher AsA than wild-type plants. The phenotype was caused by activation of a purple acid phosphatase (PAP) gene, AtPAP15, which contains a dinuclear metal center in the active site. AtPAP15 was universally expressed in all tested organs in wild-type plants. Overexpression of AtPAP15 with the 35S cauliflower mosaic virus promoter produced mutants with up to 2-fold increased foliar AsA, 20% to 30% decrease in foliar phytate, enhanced salt tolerance, and decreased abscisic acid sensitivity. Two independent SALK T-DNA insertion mutants in AtPAP15 had 30% less foliar AsA and 15% to 20% more phytate than wild-type plants and decreased tolerance to abiotic stresses. Enzyme activity of partially purified AtPAP15 from plant crude extract and recombinant AtPAP15 expressed in bacteria and yeast was highest when phytate was used as substrate, indicating that AtPAP15 is a phytase. Recombinant AtPAP15 also showed enzyme activity on the substrate myoinositol-1-phosphate, indicating that the AtPAP15 is a phytase that hydrolyzes myoinositol hexakisphosphate to yield myoinositol and free phosphate. Myoinositol is a known precursor for AsA biosynthesis in plants. Thus, AtPAP15 may modulate AsA levels by controlling the input of myoinositol into this branch of AsA biosynthesis in Arabidopsis.     

Acid phosphatases and growth of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) cultivars under diverse phosphorus nutrition

The morphological and physiological responses of barley to moderate Pi deficiency and the ability of barley to grow on phytate were investigated. Barley cultivars (Hordeum vulgare L., Promyk, Skald and Stratus) were grown for 1–3 weeks on different nutrient media with contrasting phosphorus source: KH₂PO₄ (control), phytic acid (PA) and without phosphate (−P). The growth on −P medium strongly decreased Pi concentration in the tissues; culture on PA medium generally had no effect on Pi level. Decreased content of Pi reduced shoot and root mass but root elongation was not affected; Pi deficit had slightly greater impact on growth of barley cv. Promyk than other varieties. Barley varieties cultured on PA medium showed similar growth to control. Extracellular acid phosphatase activities (APases) in −P roots were similar to control, but in PA plants were lower. Histochemical visualization indicated for high APases activity mainly in the vascular tissues of roots and in rhizodermis. Pi deficiency increased internal APase activities mainly in shoot of barley cv. Stratus and roots of cv Promyk; growth on PA medium had no effect or decreased APase activity. Protein extracts from roots and shoots were run on native discontinuous PAGE to determine which isoforms may be affected by Pi deficiency or growth on PA medium; two of four isoforms in roots were strongly induced by conditions of Pi deficit, especially in barley cv. Promyk. In conclusion, barley cultivars grew equally well both on medium with Pi and where the Pi was replaced with phytate and only slightly differed in terms of acclimation to moderate deficiency of phosphate; they generally used similar pools of acid phosphatases to acquire Pi from external or internal sources.