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.     

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.     

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.     

Differential responses of rice acid phosphatase activities and isoforms to phosphorus deprivation

Acid phosphatases (APases) play a role in the release of phosphate in organic complexes in soil. We investigated tissue- and isoform-specific responses of APases to phosphorus (P) deficiency in three rice genotypes; Dasan-byeo, Sobi-byeo, and Palawan. The levels of shoot APase activity per protein were similar in the three genotypes. They significantly decreased with P deprivation that was longer than seven days. Root APase activity per protein was two- to three-fold higher in Dasan than in Sobi and Palawan. In all genotypes the APase activity increased in P-deficient plants, but the increase was higher in Sobi and Palawan. After 21 days of P deprivation, secreted APase activity increased more than eight-fold in Dasan and two-fold in Sobi and Palawan. Isoform profiles of shoot and root APases were most diverse in Dasan. The activities of the major isoforms in P-deficient shoots decreased in all three genotypes. Depending on the genotypes, further increases in constitutive isoforms and new induction of one to four isoforms occurred in P-deficient roots. The results indicate that tissue and genotype differences in the response of APase to P deficiency are primarily facilitated by the different responses of the isoforms.     

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.     

The Arabidopsis gene hypersensitive to phosphate starvation 3 encodes ethylene overproduction 1

When plants are subjected to a deficiency in inorganic phosphate (Pi), they exhibit an array of responses to cope with this nutritional stress. In this work, we have characterized two Arabidopsis mutants, hps3-1 and hps3-2 (hypersensitive to Pi starvation 3), that have altered expression of Pi starvation-induced (PSI) genes and enhanced production of acid phosphatase (APase) when grown under either Pi sufficiency or deficiency conditions. hps3-1 and hps3-2, however, accumulate less anthocyanin than the wild type when grown on a Pi-deficient medium. Molecular cloning indicated that the phenotypes of hps3 mutants were caused by mutations within the ETO1 (ETHYLENE OVERPRODUCTION 1) gene. In Arabidopsis, ETO1 encodes a negative regulator of ethylene biosynthesis, and mutation of ETO1 causes Arabidopsis seedlings to overproduce ethylene. The ethylene biosynthesis inhibitor aminoethoxyvinyl glycine or the ethylene perception inhibitor Ag(+) suppressed all the mutant phenotypes of hps3. Taken together, these results provide further genetic evidence that ethylene is an important regulator of multiple plant responses to Pi starvation. Furthermore, we found that a change in ethylene level has differential effects on the expression of PSI genes, maintenance of Pi homeostasis, production of APase and accumulation of anthocyanin. We also demonstrated that ethylene signaling mainly regulates the activity of root surface-associated APases rather than total APase activity.     

The role of acid phosphatases in plant phosphorus metabolism

Hydrolysis of phosphate esters is a critical process in the energy metabolism and metabolic regulation of plant cells. This review summarizes the characteristics and putative roles of plant acid phosphatase (APase). Although immunologically closely related, plant APases display remarkable heterogeneity with regards to their kinetic and molecular properties, and subcellular location. The secreted APases of roots and cell cultures are relatively non-specific enzymes that appear to be important in the hydrolysis and mobilization of P_i from extracellular phosphomonoesters for plant nutrition. Intracellular APases are undoubtedly involved in the routine utilization of P_i reserves or other P_i-containing compounds. A special class of intracellular APase exists that demonstrate a clear-cut (but generally nonabsolute) substrate selectivity. These APases are hypothesized to have distinct metabolic functions and include: phytase, phosphoglycolate phosphatase, 3-phosphoglycerate phosphatase, phosphoenolpyruvate phosphatase, and phosphotyrosyl-protein phosphatase. APase expression is regulated by a variety of developmental and environmental factors. P_i starvation induces de novo synthesis of extra- and intracellular APases in cell cultures as well as in whole plants. Recommendations are made to achieve uniformity in the analyses of the different APase isoforms normally encountered within and between different plant tissues.     

Molecular control of acid phosphatase secretion into the rhizosphere of proteoid roots from phosphorus-stressed white lupin

White lupin (Lupinus albus) grown under P deficiency displays a suite of highly coordinated adaptive responses. Included among these is secretion of copious amounts of acid phosphatase (APase). Although numerous reports document that plants secrete APases in response to P deficiency, little is known of the biochemical and molecular events involved in this process. Here we characterize the secreted APase protein, cDNA, and gene from white lupin. The secreted APase enzyme is a glycoprotein with broad substrate specificity. It is synthesized as a preprotein with a deduced M(r) of 52,000 containing a 31-amino acid presequence. Analysis of the presequence predicts that the protein is targeted to outside the cell. The processed protein has a predicted M(r) of 49,000 but migrates as a protein with M(r) of 70,000 on sodium dodecyl sulfate gels. This is likely due to glycosylation. Enhanced expression is fairly specific to proteoid roots of P-stressed plants and involves enhanced synthesis of both enzyme protein and mRNA. Secreted APase appears to be encoded by a single gene containing seven exons interrupted by six introns. The 5'-upstream putative promoter of the white lupin-secreted APase contains a 50-base pair region having 72% identity to an Arabidopsis APase promoter that is responsive to P deficiency. The white lupin-secreted APase promoter and targeting sequence may be useful tools for genetically engineering important proteins from plant roots.     

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

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.     

pho3: a phosphorus-deficient mutant of Arabidopsis thaliana (L.) Heynh

A novel P-deficient mutant of Arabidopsis thaliana, pho3, was isolated by screening for root acid phosphatase (APase) activity in plants grown under low-P conditions. pho3 had 30% less APase activity in roots than the wild type and, in contrast to wild-type plants, root APase activity did not increase in response to growth in low P. However, shoot APase activity was higher in pho3 than in the wild-type plants. In addition, the pho3 mutant had a P-deficient phenotype, even when grown in P-sufficient conditions. The total P content of 11-d-old pho3 plants, grown in agar media with a plentiful supply of P, was about 25% lower than the wild-type level in the shoot, and about 65% lower in the roots. In the rosette leaves of mature soil-grown pho3 plants the total P content was again reduced, to about 50% of wild-type levels. pho3 exhibited a number of characteristics normally associated with low-P stress, including severely reduced growth, increased anthocyanin content (at least 100-fold greater than the wild type in soil-grown plants) and starch accumulation. The results suggest that the mutant is unable to respond to low internal P levels, and may lack a transporter or a signalling component involved in regulating P nutrition. Received: 21 March 2000 / Accepted: 15 August 2000     

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.     

Induction of maize acid phosphatase activities under phosphorus starvation

Large variation in phosphorus-(P) acquisition efficiency exists among maize inbred and hybrid genotypes. Acid phosphatases are a type of enzyme that affects P acquisition and P-use efficiency in plants. The objectives of this research were (1) to characterize acid phosphatase activity in maize grown hydroponically under P starvation, and (2) to determine if there is differential induction of acid phosphatases in two maize genotypes previously characterized as P efficient (Mo17) and P inefficient (B73). B73 and Mo17 seedlings were grown hydroponically and both intracellular and secreted acid phosphatase activities were characterized. Fresh seedling weight of both genotypes decreased under P starvation, but percent fresh weight allocated to roots increased 14 days after P starvation in B73. Soluble protein concentration in shoots and roots was affected little, but secreted protein decreased by 40 and 20% in B73 and Mo17 seedlings grown without P for 14 days. Intracellular and secreted acid phosphate activity increased substantially in leaves and roots in B73 and Mo17 in response to P starvation. Secreted APase activity per unit protein increased 310 and 300% in B73 and Mo17, respectively, 7 days after P withdrawal. One of the minor isozymes identified on non-denaturing PAGE, was increased specifically in response to P starvation in both maize genotypes. The patterns and levels of change in APase activities in B73 and Mo17 were not sufficiently different to account for the diverse growth response of these genotypes in low-P conditions. The results suggest that APases may not be a major mechanism for scavenging or acquiring P and changes in APases may reflect a state of P stress in both varieties. Other factors such as root architecture, secretion of low-molecular weight carboxylates and microbial interactions might explain the difference between these two genotypes.     

磷胁迫诱导大豆叶片酸性磷酸酶同工酶的表达

通过田间试验对两种磷处理的274个大豆基因型叶片酸性磷酸酶活性进行筛选,行将其中8个进行营养液栽培试验以研究磷胁迫对其叶片酸性磷酸酶同工酶表达的影响。结果表明,大豆叶片酸性磷酸酶活性存在着明显的基因型差异,不施磷处理提高了大部分(约60％)供试基因型叶片酸性磷酸酶的活性。营养液栽培试验表明,低磷处理普遍提高了所有8个供试大豆基因型叶片酸性磷酸酶的活性。等电聚焦电泳结果表明,供试大豆基因型的老叶和新叶中均有6条酸性磷酸酶的同工酶带。低磷处理显著增加了叶片酸性磷酸酶酶带的活性,但是没有诱导新的酸性磷酸酶酶带产生。研究发现叶片酸性磷酸酶活性可作为反映大豆磷肋迫的酶学指标;磷胁迫诱导大豆叶片酸性磷酸酶活性的增加是由于已有同工酶活性的提高而不是由于特异性酶带的产生。