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.     

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.     

Molecular and Biochemical Characterization of AtPAP15, a Purple Acid Phosphatase with Phytase Activity,in Arabidopsis

Purple acid phosphatase (PAP) catalyzes the hydrolysis of phosphate monoesters and anhydrides to release phosphate within an acidic pH range. Among the 29 PAP-like proteins in Arabidopsis (Arabidopsis thaliana), AtPAP15 (At3g07130) displays a greater degree of amino acid identity with soybean (Glycine max; GmPHY) and tobacco (Nicotiana tabacum) PAP (NtPAP) with phytase activity than the other AtPAPs. In this study, transgenic Arabidopsis that expressed an AtPAP15 promoter∷β-glucuronidase (GUS) fusion protein showed that AtPAP15 expression was developmentally and temporally regulated, with strong GUS staining at the early stages of seedling growth and pollen germination. The expression was also organ/tissue specific, with strongest GUS staining in the vasculature, pollen grains, and roots. The recombinant AtPAP purified from transgenic tobacco exhibited broad substrate specificity with moderate phytase activity. AtPAP15 T-DNA insertion lines exhibited a lower phytase and phosphatase activity in seedling and germinating pollen and lower pollen germination rate compared with the wild type and their complementation lines. Therefore, AtPAP15 likely mobilizes phosphorus reserves in plants, particularly during seed and pollen germination. Since AtPAP15 is not expressed in the root hair or in the epidermal cells, it is unlikely to play any role in external phosphorus assimilation.At pH in the range of 4 to 7, purple acid phosphatases (PAPs) catalyze the hydrolysis of a wide range of activated phosphoric acid monoesters and diesters and anhydrides (Klabunde et al., 1996). They are distinguished from the other phosphatases by their insensitivity to l-(+) tartrate inhibition and therefore are also known as tartrate-resistant acid phosphatases. Their characteristic pink or purple color derives from a charge transfer transition between a Tyr residue and the “chromophoric” ferric ion in the binuclear Fe(III)-Me(II) center, where the metal (Me) is iron, zinc, or manganese (Schenk et al., 1999). PAP proteins are also characterized by seven conserved amino acid residues (shown in boldface) in the five conserved motifs DXG, GDXXY, GNH(D/E), VXXH, and GHXH, which are involved in the coordination of the dimetal nuclear center (Li et al., 2002).PAPs are widespread in mammals, fungi, bacteria, and plants. Interestingly, while only a few copies of PAP-like genes are present in mammalian and fungal genomes (Mullaney and Ullah, 2003; Flanagan et al., 2006), multiple copies are present in plant genomes (Schenk et al., 2000). For example, 29 PAP-like genes have been identified in the Arabidopsis (Arabidopsis thaliana) genome (Li et al., 2002). It is intriguing that so many PAP-like genes are required for plant metabolism; this diverse portfolio of PAP-like genes implies differential functions for them. Plant PAPs are generally considered to mediate phosphorus acquisition and redistribution based on their ability to hydrolyze phosphorus compounds (Cashikar et al., 1997; Bozzo et al., 2004; Lung et al., 2008). However, additional biological roles have been reported for some plant PAPs. For example, the PAPs AtACP5 (AtPAP17), SAP1, and SAP2 (del Pozo et al., 1999; Bozzo et al., 2002) display not only phosphatase but also peroxidase activity, suggesting their involvement in the removal of reactive oxygen compounds in plant organs. GmPAP3, isolated from salted-stressed soybean (Glycine max), reportedly mediates salt tolerance via NaCl and oxidative stress inductions but not by phosphorus starvation (Liao et al., 2003).Some PAP members can hydrolyze phytic acid (myoinositol hexakisphosphate [InsP6]) to inorganic phosphate and free or lower phosphoric esters of myoinositol. Since the major storage form of phosphorus in plant seeds and pollen grains is phytate, PAPs with phytase activity may play a role in seed and pollen germination. However, not all PAPs exhibit phytase activity. The first plant phytase PAP, GmPHY, was isolated from the cotyledon of germinating soybean seedlings (Hegeman and Grabau, 2001). A tobacco (Nicotiana tabacum) root PAP phytase was identified more recently that is likely involved in mobilizing external organic phosphorus in soil (Lung et al., 2008).Relatively little is known about the biochemical properties and physiological roles of the 29 PAP-like Arabidopsis genes (del Pozo et al., 1999; Veljanovski et al., 2006). An enzyme assay involving the glutathione S-transferase (GST)-AtPAP23 fusion protein revealed that the Arabidopsis PAP AtPAP23 exhibits phytase activity (Zhu et al., 2005). A GUS study showed that AtPAP23 is exclusively expressed in the flower of the Arabidopsis plant. In a recent report, a recombinant AtPAP15 expressed in Escherichia coli was also found to exhibit phytase activity; this PAP potentially modulates plant ascorbate synthesis through supply of myoinositol from the phytate hydrolysis reaction (Zhang et al., 2008). However, the possible physiological roles of AtPAP15 in phosphorus mobilization have not been examined.In this study, AtPAP15 expressed in a plant (tobacco) system was biochemically characterized, and its temporal and spatial expression patterns in Arabidopsis were examined. The physiological roles of AtPAP15 in phosphorus mobilization were also delineated.     

AtPAP2 is a tail-anchored protein in the outer membrane of chloroplasts and mitochondria

To date, Arabidopsis purple acid phosphatase 2 (AtPAP2) is the only known plant protein that is dual-targeted to chloroplasts and mitochondria by a C-terminal targeting signal. Using in vitro organelle import and green fluorescence protein (GFP) localization assays, we showed that AtPAP2 is located on, but not imported across the outer membrane (OM) of chloroplasts and mitochondria and exposed its N-terminal enzymatic domain to the cytosol. It was also found that a short stretch of 30 amino acids (a.a.) at the C-terminal region (a.a. 615-644) that contains a stretch of 18 hydrophobic residues, a WYAK motif and 8 hydrophilic residues is sufficient for dual-targeting. Mutation of WYAK to WYAE had no effect on dual-targeting ability suggesting that the charge within this flanking region alone is not an important determinant for dual-targeting.        

Functional characterization of an Arabidopsis prolyl aminopeptidase AtPAP1 in response to salt and drought stresses

It has been over 50 years since the first prolyl aminopeptidase gene was identified in Escherichia coli (EC 3.4.11.5). However, up to now, few prolyl aminopeptidases have been reported to regulate osmotic stress tolerance, especially in plant. In this study, we focused on characterization of the biological functions of the Arabidopsis prolyl aminopeptidase AtPAP1 (At2g14260), which positively regulated plant tolerance to salt and drought stresses. Protein sequence alignment revealed that AtPAP1 was evolutionarily conserved among different plant species, and the smaller molecular weight and phylogenetic tree indicated that AtPAP1 belonged to the S33.001 subfamily. By using quantitative real-time PCR assays, we demonstrated that expression of the AtPAP1 gene was rapidly induced by salt and drought stresses. We also found that knockout of the AtPAP1 gene decreased, while AtPAP1 overexpression enhanced plant tolerance to salt and drought stresses. Measurements of the proline contents and the prolyl aminopeptidase activity suggested that the transgenic plants accumulated more free proline and exhibited higher prolyl aminopeptidase activity than the wild type or knockout plants under control conditions, as well as salt and drought stresses. Furthermore, through the GUS activity analysis, we also demonstrated that the AtPAP1 promoter is stress inducible and tissue specific. The AtPAP1-GFP fusion protein was found to localize in the cytoplasm of the onion epidermal cells. In conclusion, we showed that the Arabidopsis AtPAP1 gene could positively regulate plant tolerance to salt and drought stress, maybe by acting as a prolyl aminopeptidase and thereby increasing the concentration of free proline in plant cells.     

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.     

Distribution of N,P, K,Ca, Mg,S, Zn,Fe, Mn and Cu in groundnut

Summary Concentration of N, P, K, Ca, Mg and S in summer groundnut crop was higher than in kharif while Zn, Fe, Mn and Cu contents were higher in summer crop. Kernel's N, P and Zn; Leaflet's Ca and Mn; Stem's K and Fe; Root's S and Cu and Petiole's Mg contents were highest. Shell's N, P, K, Mg, S, Zn and Cu; Kernel's Ca, Fe and Mn contents were the least. N, P, K, S, Zn and Cu concentrations decreased linearly as the crop grew. Ca, Mg, Fe and Mn concentrations did not display any distinct pattern. Ca concentration was positively correlated with pod yield in both the seasons.     

Simultaneous measurement of the trace elements Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum and their reference ranges by ICP-MS

The goal of this article was to establish reference ranges of the concentration of trace elements in human serum and to compare these results with those reported by other authors. We describe the sample preparation and measurement conditions that allow the rapid, precise, and accurate determination of Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum samples (n=110) by inductively coupled plasma-mass spectrometry (ICP-MS). Accuracy and precision were determined by analyzing three reconstituted reference serum samples by comparison with other methods and by the standard addition procedure. The advantages of the ICP-MS method include short time of analysis of the elements mentioned, low detection limit, high precision, and high accuracy. Disadventages include a high risk of contamination due to the presence of some of the elements of interest in the environment, the relatively delicate sample handling, and the high cost of the equipment.