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Ralf Greiner - One of the best experts on this subject based on the ideXlab platform.
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novel glucose 1 phosphatase with high phytase activity and unusual metal ion activation from soil bacterium pantoea sp strain 3 5 1
Applied and Environmental Microbiology, 2015Co-Authors: Aliya D Suleimanova, Eugene V Shakirov, Ralf Greiner, Inna B. Chastukhina, Astrid Beinhauer, Liia R Valeeva, N P Balaban, M R SharipovaAbstract:Phosphorus is an important macronutrient, but it's availability in soil is limited. Many soil microorganisms improve bioavailability of phosphate by releasing it from various organic compounds, including Phytate. To investigate the diversity of Phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes and urban landscape. Bacterial isolate Pantoea sp. 3.5.1 with the highest level of phytase activity was isolated from forest soil type and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy and biochemically characterized. AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest Km values towards Phytate, glucose-6-phosphate and glucose-1-phosphate. Unexpectedly, a stimulation of enzymatic activity by several divalent metal ions was observed for AgpP enzyme. HPLC and HPIC analyses of Phytate hydrolysis products identify D/L-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel Phytate-hydrolyzing enzymes with unique biochemical features.
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identification of β propeller phytase encoding genes in culturable paenibacillus and bacillus spp from the rhizosphere of pasture plants on volcanic soils
FEMS Microbiology Ecology, 2011Co-Authors: Milko A Jorquera, Maria Teresa Fernandez, Daniela Romero, Daniel Menezesblackburn, Petra Marschner, Ralf Greiner, David E Crowley, Maria MoraAbstract:Phytate is one of the most abundant sources of organic phosphorus (P) in soils, but must be mineralized by phytase-producing bacteria to release P for plant uptake. Microbial inoculants based on Bacillus spp. have been developed commercially, but few studies have evaluated the ecology of these bacteria in the rhizosphere or the types of enzymes that they produce. Here, we studied the diversity of aerobic endospore-forming bacteria (EFB) with the ability to mineralize Phytate in the rhizosphere of pasture plants grown in volcanic soils of southern Chile. PCR methods were used to detect candidate phytase-encoding genes and to identify EFB bacteria that carry these genes. This study revealed that the Phytate-degrading EFB populations of pasture plants included species of Paenibacillus and Bacillus, which carried genes encoding β-propeller phytase (BPP). Assays of enzymatic activity confirmed the ability of these rhizosphere isolates to degrade Phytate. The phytase-encoding genes described here may prove valuable as molecular markers to evaluate the role of EFB in organic P mobilization in the rhizosphere.
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identification of β propeller phytase encoding genes in culturable paenibacillus and bacillus spp from the rhizosphere of pasture plants on volcanic soils
FEMS Microbiology Ecology, 2011Co-Authors: Milko A Jorquera, Maria Teresa Fernandez, Daniela Romero, Daniel Menezesblackburn, Petra Marschner, Ralf Greiner, David E Crowley, Maria MoraAbstract:Phytate is one of the most abundant sources of organic phosphorus (P) in soils, but must be mineralized by phytase-producing bacteria to release P for plant uptake. Microbial inoculants based on Bacillus spp. have been developed commercially, but few studies have evaluated the ecology of these bacteria in the rhizosphere or the types of enzymes that they produce. Here, we studied the diversity of aerobic endospore-forming bacteria (EFB) with the ability to mineralize Phytate in the rhizosphere of pasture plants grown in volcanic soils of southern Chile. PCR methods were used to detect candidate phytase-encoding genes and to identify EFB bacteria that carry these genes. This study revealed that the Phytate-degrading EFB populations of pasture plants included species of Paenibacillus and Bacillus, which carried genes encoding β-propeller phytase (BPP). Assays of enzymatic activity confirmed the ability of these rhizosphere isolates to degrade Phytate. The phytase-encoding genes described here may prove valuable as molecular markers to evaluate the role of EFB in organic P mobilization in the rhizosphere.
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the importance of lactic acid bacteria for Phytate degradation during cereal dough fermentation
Journal of Agricultural and Food Chemistry, 2007Co-Authors: A Reale, U. Konietzny, Raffaele Coppola, Elena Sorrentino, Ralf GreinerAbstract:Lactic acid fermentation of cereal flours resulted in a 100 (rye), 95-100 (wheat), and 39-47% (oat) reduction in Phytate content within 24 h. The extent of Phytate degradation was shown to be independent from the lactic acid bacteria strain used for fermentation. However, Phytate degradation during cereal dough fermentation was positively correlated with endogenous plant phytase activity (rye, 6750 mU g(-1); wheat, 2930 mU g(-1); and oat, 23 mU g(-1)), and heat inactivation of the endogenous cereal phytases prior to lactic acid fermentation resulted in a complete loss of Phytate degradation. Phytate degradation was restored after addition of a purified phytase to the liquid dough. Incubation of the cereal flours in buffered solutions resulted in a pH-dependent Phytate degradation. The optimum of Phytate degradation was shown to be around pH 5.5. Studies on phytase production of 50 lactic acid bacteria strains, previously isolated from sourdoughs, did not result in a significant production of intra- as well as extracellular phytase activity. Therefore, lactic acid bacteria do not participate directly in Phytate degradation but provide favorable conditions for the endogenous cereal phytase activity by lowering the pH value.
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distribution of phytase activity total phosphorus and Phytate phosphorus in legume seeds cereals and cereal by products as influenced by harvest year and cultivar
Animal Feed Science and Technology, 2007Co-Authors: Tobias Steiner, Ralf Greiner, R Mosenthin, B Zimmermann, S RothAbstract:Samples of legume seeds, cereals and cereal by-products (n = 113) grown in south-western Germany and originating from different cultivars and harvest years were analyzed for phytase activity, total phosphorus (P) and Phytate P. Phytase activities determined by means of a direct incubation method were lowest in legume seeds and oats (262–496 U/kg dry matter), intermediate in cereals (except oats) (2323–6016 U/kg DM) and highest in cereal by-products (9241–9945 U/kg DM). However, the application of an extraction procedure for the determination of phytase activities in legume seeds resulted in values below the detection limit of 50 U/kg. On average, about 0.67 of total P in legume seeds, cereals and their by-products is bound to Phytate. There was a significant influence (P<0.001) of harvest year (1998–2000) on Phytate P contents in wheat. Furthermore, total P and Phytate P concentrations differed (P<0.05) between different cultivars of wheat. Moreover, phytase activities differed (P=0.023) between different cultivars of barley. Total P and Phytate P concentrations were highly correlated in legume seeds (r = 0.95) and cereal by-products (r = 0.96) and, to a smaller extent, in cereals (r = 0.66). Milling of cereal grains to bran and flour revealed that phytase activity, total P and Phytate P are highly concentrated (P<0.001) in the outer grain layers of cereals. The influence of preservation of intact legume seeds with propionic acid over a period of 4, 8 or 12 weeks resulted only in a marginal decrease in phytase activity. Due to high native phytase activities in cereals (except oats) and their by-products these feedstuffs may contribute substantially to the gastrointestinal hydrolysis of Phytate in non-ruminant animals, whereas the contribution of native phytases originating from legume seeds in terms of improving the availability of plant P seems to be rather limited.
Eugene V Shakirov - One of the best experts on this subject based on the ideXlab platform.
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heterologous expression of secreted bacterial bpp and hap phytases in plants stimulates arabidopsis thaliana growth on Phytate
Frontiers in Plant Science, 2018Co-Authors: Lia R. Valeeva, Chuluuntsetseg Nyamsuren, Eugene V Shakirov, Margarita R. SharipovaAbstract:Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo-inositol hexakisphosphate (Phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits towards improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family of phytases) and 168phyA (BPP family) under the control of root-specific inducible promoter Pht1;2. The effects of each phytase expression on growth, morphology and inorganic phosphorus accumulation in plants grown on Phytate hydroponically or in perlite as the only source of phosphorus were investigated. The most enzymatic activity for both phytases was detected in cell wall-bound fractions of roots, indicating that these enzymes were efficiently secreted. Expression of both bacterial phytases in roots improved plant growth on Phytate and resulted in larger rosette leaf area and diameter, higher phosphorus content and increased shoot dry weight, implying that these plants were indeed capable of utilizing Phytate as the source of phosphorus for growth and development. When grown on Phytate the HAP-type phytase outperformed its BPP-type counterpart for plant biomass production, though this effect was only observed in hydroponic conditions and not in perlite. Furthermore, we found no evidence of adverse side effects of microbial phytase expression in A. thaliana on plant physiology and seed germination. Our data highlight important functional differences between these members of bacterial phytase families and indicate that future crop biotechnologies involving such enzymes will require a very careful evaluation of phytase source and activity. Overall, our data suggest feasibility of using bacterial phytases to improve plant growth in conditions of phosphorus deficiency and demonstrate that inducible expression of recombinant enzymes should be investigated further as a viable approach to plant biotechnology.
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microbial phytases and Phytate exploring opportunities for sustainable phosphorus management in agriculture
American Journal of Molecular Biology, 2017Co-Authors: Nelly P. Balaban, Aliya D Suleimanova, N. L. Rudakova, Lia R. Valeeva, Inna B. Chastukhina, Margarita R. Sharipova, Eugene V ShakirovAbstract:Myo-inositol phosphates (Phytates) are important biological molecules produced largely by plants to store phosphorus. Phytate is very abundant in many different soils making up a large portion of all soil phosphorus. This review assesses current phytase science from the perspective of its substrate, Phytate, by examining the intricate relationship between the Phytate-hydrolyzing enzymes and Phytate as their substrate. Specifically, we examine available data on Phytate’s structural features, distribution in nature and functional roles. The role of phytases and their localization in soil and plant tissues are evaluated. We provide a summary of the current biotechnological advances in using industrial or recombinant phytases to improve plant growth and animal nutrition. The prospects of future discovery of novel phytases with improved biochemical properties and bioengineering of existing enzymes are also discussed. Two alternative but complementary directions to increase phosphorus bioavailability through the more efficient utilization of soil Phytate are currently being developed. These approaches take advantage of microbial phytases secreted into rhizosphere either by phytase-producing microbes (biofertilizers) or by genetically engineered plants. More research on Phytate metabolism in soils and plants is needed to promote environmentally friendly, more productive and sustainable agriculture.
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novel glucose 1 phosphatase with high phytase activity and unusual metal ion activation from soil bacterium pantoea sp strain 3 5 1
Applied and Environmental Microbiology, 2015Co-Authors: Aliya D Suleimanova, Eugene V Shakirov, Ralf Greiner, Inna B. Chastukhina, Astrid Beinhauer, Liia R Valeeva, N P Balaban, M R SharipovaAbstract:Phosphorus is an important macronutrient, but it's availability in soil is limited. Many soil microorganisms improve bioavailability of phosphate by releasing it from various organic compounds, including Phytate. To investigate the diversity of Phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes and urban landscape. Bacterial isolate Pantoea sp. 3.5.1 with the highest level of phytase activity was isolated from forest soil type and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy and biochemically characterized. AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest Km values towards Phytate, glucose-6-phosphate and glucose-1-phosphate. Unexpectedly, a stimulation of enzymatic activity by several divalent metal ions was observed for AgpP enzyme. HPLC and HPIC analyses of Phytate hydrolysis products identify D/L-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel Phytate-hydrolyzing enzymes with unique biochemical features.
T Satyanarayana - One of the best experts on this subject based on the ideXlab platform.
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recombinant hap phytase of the thermophilic mold sporotrichum thermophile expression of the codon optimized phytase gene in pichia pastoris and applications
Molecular Biotechnology, 2016Co-Authors: Ibhuti Ranja, T SatyanarayanaAbstract:The codon-optimized phytase gene of the thermophilic mold Sporotrichum thermophile (St-Phy) was expressed in Pichia pastoris. The recombinant P. pastoris harboring the phytase gene (rSt-Phy) yielded a high titer of extracellular phytase (480 ± 23 U/mL) on induction with methanol. The recombinant phytase production was ~40-fold higher than that of the native fungal strain. The purified recombinant phytase (rSt-Phy) has the molecular mass of 70 kDa on SDS-PAGE, with K m and V max (calcium Phytate), k cat and k cat/K m values of 0.147 mM and 183 nmol/mg s, 1.3 × 10(3)/s and 8.84 × 10(6)/M s, respectively. Mg(2+) and Ba(2+) display a slight stimulatory effect, while other cations tested exert inhibitory action on phytase. The enzyme is inhibited by chaotropic agents (guanidinium hydrochloride, potassium iodide, and urea), Woodward's reagent K and 2,3-bunatedione, but resistant to both pepsin and trypsin. The rSt-Phy is useful in the dephytinization of broiler feeds efficiently in simulated gut conditions of chick leading to the liberation of soluble inorganic phosphate with concomitant mitigation in antinutrient effects of Phytates. The addition of vanadate makes it a potential candidate for generating haloperoxidase, which has several applications.
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bioprocess for efficient production of recombinant pichia anomala phytase and its applicability in dephytinizing chick feed and whole wheat flat indian breads
Journal of Industrial Microbiology & Biotechnology, 2015Co-Authors: Swati Joshi, T SatyanarayanaAbstract:The phytase of the yeast Pichia anomala (PPHY) is a suitable biocatalyst as a food and feed additive because of its adequate thermostability, acid stability, protease insensitivity and broad substrate spectrum. The cell-bound nature and low phytase titres are the main bottlenecks for its utility in food and feed industries. In this investigation, we have overcome the problems by constitutive secretory expression of PPHY under glyceraldehyde phosphate dehydrogenase (GAP) promoter. A ~44-fold increase in rPPHY titre has been achieved after optimization of cultural variables by one-variable-at-a-time approach and two factorial statistical design. The use of GAP promoter makes the cultivation of the recombinant P. pastoris straight forward and eliminates the requirement of methanol for induction and hazards associated with its storage. Among metal-Phytate complexes, Ca2+ Phytate is hydrolyzed more efficiently by rPPHY than Co2+, Mn2+, Mg2+, Fe3+ and Zn2+ Phytates. The enzyme is effective in dephytinizing whole wheat unleavened flat Indian breads (naan and tandoori) and different broiler feeds, thus mitigating anti-nutritional effects of Phytates.
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Plant Growth Promotion by an Extracellular HAP-Phytase of a Thermophilic Mold Sporotrichum thermophile
Applied Biochemistry and Biotechnology, 2010Co-Authors: Bijender Singh, T SatyanarayanaAbstract:Phytase of the thermophilic mold Sporotrichum thermophile Apinis hydrolyzed and liberated inorganic phosphate from Ca^+2, Mg^+2, and Co^+2 Phytates more efficiently than those of Al^3+, Fe^2+, Fe^3+, and Zn^2+. The hydrolysis rate was higher at 60 °C as compared to 26 °C. Among all the organic acids tested, citrate was more effective in enhancing solubilization of insoluble Phytate salts by phytase than others. The dry weight and inorganic phosphate contents of the wheat plants were high when supplemented with phytase or fungal spores. The plants provided with 5 mg Phytate per plant exhibited enhanced growth and inorganic phosphate. With increase in the dosage of phytase, there was increase in growth and inorganic phosphate of plants, the highest being at 20 U per plant. The compost made employing the combined native microflora of the wheat straw and S . thermophile promoted growth of the plants. The plant-growth-promoting effect was also higher with the compost made using S. thermophile than that from only the native microflora.
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improved phytase production by a thermophilic mould sporotrichum thermophile in submerged fermentation due to statistical optimization
Bioresource Technology, 2008Co-Authors: Ijende Singh, T SatyanarayanaAbstract:Abstract Culture variables affecting phytase production by a thermophilic mould Sporotrichum thermophile in submerged fermentation were optimized. Soluble starch, peptone, Tween-80 and sodium Phytate were identified by Plackett–Burman design as the most significant factors to affect phytase production. The 24 full factorial central composite design of response surface methodology was applied for optimizing the concentrations of the significant variables and to delineate their interactions. Starch, Tween-80, peptone and sodium Phytate at 0.4%, 1.0%, 0.3% and 0.3% supported maximum enzyme titres, respectively. An overall 3.73-fold improvement in phytase production was achieved due to optimization. When sodium Phytate was substituted with wheat bran (3%), the phytase titre in the former was comparable with that in the latter.
Maria Mora - One of the best experts on this subject based on the ideXlab platform.
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identification of β propeller phytase encoding genes in culturable paenibacillus and bacillus spp from the rhizosphere of pasture plants on volcanic soils
FEMS Microbiology Ecology, 2011Co-Authors: Milko A Jorquera, Maria Teresa Fernandez, Daniela Romero, Daniel Menezesblackburn, Petra Marschner, Ralf Greiner, David E Crowley, Maria MoraAbstract:Phytate is one of the most abundant sources of organic phosphorus (P) in soils, but must be mineralized by phytase-producing bacteria to release P for plant uptake. Microbial inoculants based on Bacillus spp. have been developed commercially, but few studies have evaluated the ecology of these bacteria in the rhizosphere or the types of enzymes that they produce. Here, we studied the diversity of aerobic endospore-forming bacteria (EFB) with the ability to mineralize Phytate in the rhizosphere of pasture plants grown in volcanic soils of southern Chile. PCR methods were used to detect candidate phytase-encoding genes and to identify EFB bacteria that carry these genes. This study revealed that the Phytate-degrading EFB populations of pasture plants included species of Paenibacillus and Bacillus, which carried genes encoding β-propeller phytase (BPP). Assays of enzymatic activity confirmed the ability of these rhizosphere isolates to degrade Phytate. The phytase-encoding genes described here may prove valuable as molecular markers to evaluate the role of EFB in organic P mobilization in the rhizosphere.
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identification of β propeller phytase encoding genes in culturable paenibacillus and bacillus spp from the rhizosphere of pasture plants on volcanic soils
FEMS Microbiology Ecology, 2011Co-Authors: Milko A Jorquera, Maria Teresa Fernandez, Daniela Romero, Daniel Menezesblackburn, Petra Marschner, Ralf Greiner, David E Crowley, Maria MoraAbstract:Phytate is one of the most abundant sources of organic phosphorus (P) in soils, but must be mineralized by phytase-producing bacteria to release P for plant uptake. Microbial inoculants based on Bacillus spp. have been developed commercially, but few studies have evaluated the ecology of these bacteria in the rhizosphere or the types of enzymes that they produce. Here, we studied the diversity of aerobic endospore-forming bacteria (EFB) with the ability to mineralize Phytate in the rhizosphere of pasture plants grown in volcanic soils of southern Chile. PCR methods were used to detect candidate phytase-encoding genes and to identify EFB bacteria that carry these genes. This study revealed that the Phytate-degrading EFB populations of pasture plants included species of Paenibacillus and Bacillus, which carried genes encoding β-propeller phytase (BPP). Assays of enzymatic activity confirmed the ability of these rhizosphere isolates to degrade Phytate. The phytase-encoding genes described here may prove valuable as molecular markers to evaluate the role of EFB in organic P mobilization in the rhizosphere.
Richard J Simpson - One of the best experts on this subject based on the ideXlab platform.
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expression of a fungal phytase gene in nicotiana tabacum improves phosphorus nutrition of plants grown in amended soils
Plant Biotechnology Journal, 2005Co-Authors: Timothy S George, Richard J Simpson, P A Hadobas, Alan RichardsonAbstract:Transgenic Nicotiana tabacum plants expressing a chimeric phytase gene (ex::phyA) from the soil fungus Aspergillus niger were generated. Three independently transformed lines showed increased extracellular phytase activity compared with a vector control and wild-type plants, both of which had no detectable extracellular phytase. Transgenic N. tabacum plants grown in sterile agar supplied with phosphorus (P) as Phytate accumulated 3.7-fold more P than vector control plants. Despite this, the expression of ex::phyA in plants did not lead to an improved accumulation of P from two unamended P-deficient soils. However, when soils were amended with either Phytate or phosphate and lime, transgenic plants accumulated up to 52% more P than controls. Positive responses by transgenic plants were, in some instances, coincident with a putative increase in soil Phytate. We conclude that the development of plants that exude phytase to the soil may not ensure improved plant P nutrition, as the availability of Phytate in the soil also appears to be critical. Nevertheless, if plants that express ex::phyA are combined with soil amendments that promote the availability of Phytate, there is the potential to enhance the P nutrition of crop plants and to improve the efficiency of P fertilizer use in agricultural systems.
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characterization of transgenic trifolium subterraneum l which expresses phya and releases extracellular phytase growth and p nutrition in laboratory media and soil
Plant Cell and Environment, 2004Co-Authors: Timothy S George, P A Hadobas, Alan Richardson, Richard J SimpsonAbstract:Transgenic Trifolium subterraneum expressing a phytase gene (phyA) from Aspergillus niger were generated. Five independently transformed lines showed an average 77-fold increase in exuded phytase activity in comparison with null segregant and wild-type controls. Unlike other phosphatases, exuded phytase activity was unaffected by P supply, verifying the constitutive expression of phyA. Transgenic T. subterraneum grown in agar with P supplied as Phytate, took up 1.3- to 3.6-fold more P than controls and had equivalent P uptake to plants supplied with orthophosphate. This unique phenotype was compromised when the plants were grown in soil. None of the five lines showed increased shoot biomass or total P uptake in an unfertilized, low-P soil taken from under permanent pasture. With addition of P, one of the five transgenic lines had consistently greater P nutrition compared with control plants. Despite variable growth and P nutrition responses, P uptake per root length was on average greater for transgenic lines. Exudation of phytase by transgenic T. subterraneum allowed utilization of P from Phytate in non-sorbing, sterile laboratory media, but was less effective when plants were grown in soil. Release of extracellular phytase is therefore not the only requirement for the acquisition of P from endogenous soil Phytate by plants.
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Phytate as a source of phosphorus for the growth of transgenic trifolium subterraneum
Plant nutrition: food security and sustainability of agro-ecosystems through basic and applied research. Fourteenth International Plant Nutrition Coll, 2001Co-Authors: Alan Richardson, P A Hadobas, Richard J SimpsonAbstract:Although Phytate is an abundant form of soil organic P, plants generally have only a limited capacity to obtain phosphorus (P) directly from this substrate. Inability of plants to effectively use Phytate-P is associated with poor substrate availability in soil environments, and insufficient extracellular root phytase activity. We therefore generated transgenic lines of Trifolium subterraneum L. that expressed the phytase (phyA) gene from Aspergillus niger. Analysis of segregating T1 populations of these plants showed that the presence of an extracellular phytase was effective in improving the P nutrition of the plants when supplied with Phytate and grown in agar under sterile conditions.
