Characterization of transgenic Trifolium subterraneum L. which expresses phyA and releases extracellular phytase: growth and P nutrition in laboratory media and soil

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.     

Transgenic maize plants expressing a fungal phytase gene

Maize seeds are the major ingredient of commercial pig and poultry feed. Phosphorus in maize seeds exists predominantly in the form of phytate. Phytate phosphorus is not available to monogastric animals and phosphate supplementation is required for optimal animal growth. Undigested phytate in animal manure is considered a major source of phosphorus pollution to the environment from agricultural production. Microbial phytase produced by fermentation as a feed additive is widely used to manage the nutritional and environmental problems caused by phytate, but the approach is associated with production costs for the enzyme and requirement of special cares in feed processing and diet formulation. An alternative approach would be to produce plant seeds that contain high phytase activities. We have over-expressed Aspergillus niger phyA2 gene in maize seeds using a construct driven by the maize embryo-specific globulin-1 promoter. Low-copy-number transgenic lines with simple integration patterns were identified. Western-blot analysis showed that the maize-expressed phytase protein was smaller than that expressed in yeast, apparently due to different glycosylation. Phytase activity in transgenic maize seeds reached approximately 2,200 units per kg seed, about a 50-fold increase compared to non-transgenic maize seeds. The phytase expression was stable across four generations. The transgenic seeds germinated normally. Our results show that the phytase expression lines can be used for development of new maize hybrids to improve phosphorus availability and reduce the impact of animal production on the environment.     

Dual effects of transgenic Brassica napus overexpressing CS gene on tolerances to aluminum toxicity and phosphorus deficiency

Background and aims

Low phosphorus (P) bioavailability and aluminum (Al) toxicity are two major constraints to plant growth in acid soil. To improve the tolerance of Brassica napus to Al toxicity and P deficiency, we generated transgenic canola (Brassica napus cv Westar) lines overexpressing a Pseudomonas aeruginosa citrate synthase (CS) gene and then investigated the effects of CS gene overexpressing in canola on enhancing tolerance to the two constraints.

Methods

The vector construction and plant transformation, molecular identification, estimation of extracellular and cellular citrate and malate concentrations, enzyme activity and gene expression analyse and Al tolerance and P acquisition assays were conducted using both hydroponics and soil culturing in the study.

Results

Both the root citrate and malate concentrations and their exudations in the two transgenic lines significantly increased compared with wild type (WT) following exposure to Al. These increases may be attributed to higher activities of the CS, malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC) enzymes in the TCA cycle and the expression of BnALMT and BnMATE in the transgenic plants following Al exposure. The primary root elongation and prolonged Al treatment (10 days) experiments revealed that the transgenic lines displayed enhanced levels of Al tolerance. In addition, they showed enhanced citrate and malate exudation when grown in P-deficient conditions. Moreover, the enzyme activities of the transgenic lines were significantly higher compared with WT in response to P-deficient stress. The soil culture experiment showed that the transgenic lines possessed improved P uptake from the soil and accumulated more P in their shoots and seeds when FePO₄ was used as the sole P source.

Conclusions

These results indicate that the overexpression of the CS gene in B. napus not only leads to increased citrate synthesis and exudation but also changes malate metabolism, which confers improved tolerances to Al toxicity and P deficiency in the transgenic plants. These findings provide further insight into the dual effects of CS gene overexpression on Al toxicity and P deficiency in plants.     

Enhancing the Thermal Tolerance and Gastric Performance of a Microbial Phytase for Use as a Phosphate-Mobilizing Monogastric-Feed Supplement

The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the appA gene from Escherichia coli, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62°C for 1 h and 27% of its initial activity after 10 min at 85°C, which was a significant improvement over the appA parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.     

Production of two Highly Active Bacterial Phytases with Broad pH Optima in Germinated Transgenic Rice Seeds

Phytate is the main storage form of phosphorus in many plant seeds, but phosphate bound in this form is not available to monogastric animals. Phytase, an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance phosphorus availability in animal diets when engineered in rice seeds as a feed additive. Two genes, derived from a ruminal bacterium Selenomonas ruminantium (SrPf6) and Escherichia coli (appA), encoding highly active phytases were expressed in germinated transgenic rice seeds. Phytase expression was controlled by a germination inducible alpha-amylase gene (alphaAmy8) promoter, and extracellular phytase secretion directed by an betaAmy8 signal peptide sequence. The two phytases were expressed in germinated transgenic rice seeds transiently and in a temporally controlled and tissue-specific manner. No adverse effect on plant development or seed formation was observed. Up to 0.6 and 1.4 U of phytase activity per mg of total extracted cellular proteins were obtained in germinated transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which represent 46-60 times of phytase activities compared to the non-transformant. The appA and SrPf6 phytases produced in germinated transgenic rice seeds had high activity over broad pH ranges of 3.0-5.5 and 2.0-6.0, respectively. Phytase levels and inheritance of transgenes in one highly expressing plant were stable over four generations. Germinated transgenic rice seeds, which produce a highly active recombinant phytase and are rich in hydrolytic enzymes, nutrients and minerals, could potentially be an ideal feed additive for improving the phytate-phosphorus digestibility in monogastric animals.     

Expression of an Aspergillus niger Phytase Gene (phyA) in Saccharomyces cerevisiae

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase’s activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S. cerevisiae as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60°C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the phyA gene was expressed as an active, extracellular phytase in S. cerevisiae, and its thermostability was affected by glycosylation.     

Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola

Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase. Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication problems.     

Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase

The Aspergillus niger phytase-encoding gene (phyA) has been constitutively expressed in wheat. Transgenic wheat lines were generated by microprojectile bombardment of immature embryos, using the bar-Bialaphos selection system. The bar and the phyA gene expression were controlled by the maize ubiquitin-1 promoter. To ensure secretion and glycosylation of the microbial phytase, an expression cassette was designed (Ubi-SP-Phy) where an -amylase signal peptide sequence was inserted between the promoter and the phytase coding region. A similar cassette was constructed without the signal peptide sequence (Ubi-Phy). Five lines of fertile wheat transformed with the Ubi-SP-Phy were generated and two lines with the Ubi-Phy construct. The inheritance of the phyA gene was monitored through three generations. Western blotting of leaf and seed derived protein revealed the presence of an immunoreacting polypeptide of the size expected for the Aspergillus phytase. Up to 25 days after pollination, the heterologous phytase was exclusively present in the pericarp-seed coat-aleurone fraction. Thereafter, it accumulated in the endosperm in amounts exceeding that found in the seed coat and aleurone. The phyA mRNA and derived protein could at no stage be detected in the embryo. The Ubi-SP-Phy transgenic seeds exhibited up to 4-fold increase of phytase activity while up to 56% increase was found in Ubi-Phy plants. It is concluded that a functional Aspergillus phytase can be produced in significant amounts in wheat grains. This may be of relevance for improving the phytate-phosphorus digestibility when wheat grains are used for non-ruminant animal feed.     

Limitations to the Potential of Transgenic Trifolium subterraneum L. Plants that Exude Phytase when Grown in Soils with a Range of Organic P Content

Growth and P-nutrition of transgenic Trifolium subterraneum L. which express a chimeric fungal phytase gene (ex::phyA) was compared to azygous and wild-type controls in a range of soils that differed in organic P content. Shoot and root growth by plant lines were measured and effects of reducing the influence of soil microorganisms were investigated by pasteurising the soils. Plants that expressed phyA did not have better P-nutrition than control plants after 56 days growth, except in a soil that contained a large concentration of both total organic P and organic P that was amenable to hydrolysis by a plant-derived phytase. Pasteurisation had little effect on the relative P-nutrition of the various plant lines in any of the soils. Roots of transgenic plants that expressed ex::phyA were shorter than controls up to 21 days growth in a number of soils, which resulted in an initial greater P accumulation efficiency. However, greater P accumulation efficiency was only maintained in the soil where significant growth and P nutrition responses were also observed. Availability of inositol phosphates in soil is a major factor that limits the effectiveness of expressing fungal phytase genes in plants as a means to improve P-nutrition. Reducing the influence of rhizosphere microorganisms appeared to have little effect on the P-nutrition of plant lines, but the longer root system produced by control plants may have initially provided them with greater access to soil P resources. This research highlights the inherent difficulty in improving the P-nutrition of plants by the manipulation of single traits in isolation, but does provide some evidence that such approaches can be successful under certain edaphic conditions.     

GmPAP4, a novel purple acid phosphatase gene isolated from soybean (Glycine max), enhanced extracellular phytate utilization in Arabidopsis thaliana

Key message

GmPAP4 , a novel plant PAP gene in soybean, has phytase activity. Over-expressing GmPAP4 can enhance Arabidopsis growth when phytate is the sole P source in culture.

Abstract

Phosphorus (P) is an important macronutrient for plant growth and development. However, most of the total P in soils is fixed into organic phosphate (Po). Purple acid phosphatase (PAP) can hydrolyze Po in the soil to liberate inorganic phosphate and enhance plant P utilization. We isolated a novel PAP gene, GmPAP4, from soybean (Glycine max). It had an open reading frame of 1,329 bp, encoding 442 amino acid residues. Sequence alignment and phylogenetics analysis indicated that GmPAP4 was similar to other plant PAPs with large molecular masses. Quantitative real-time PCR analysis showed that the induced expression of GmPAP4 was greater in P-efficient genotype Zhonghuang15 (ZH15) than in P-inefficient genotype Niumaohuang (NMH) during the periods of flowering (28–35 days post phytate stress; DPP) and pod formation (49–63 DPP). Moreover, peak expression, at 63 DPP, was about 3-fold higher in ‘ZH15’ than in ‘NMH’. Sub-cellular localization showed that GmPAP4 might be on plasma membrane or in cytoplasm. Over-expressing GmPAP4 in Arabidopsis resulted in significant rises in P acquisition and utilization compared with the wild-type (WT). Under phytate condition, transgenic Arabidopsis plants showed increases of approximately 132.7 % in dry weight and 162.6 % in shoot P content compared with the WT. Furthermore, when phytate was added as the sole P source in cultures, the activity of acid phosphatase was significantly higher in transgenic plants. Therefore, GmPAP4 is a novel PAP gene that functions in plant’s utilization of organic phosphate especially under phytate condition.     

Does the combination of citrate and phytase exudation in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> promote the acquisition of endogenous soil organic phosphorus?

Background and Aims

Plant acquisition of endogenous forms of soil phosphorus (P) could reduce external P requirements in agricultural systems. This study investigated the interaction of citrate and phytase exudation in controlling the accumulation of P and depletion of soil organic P by transgenic Nicotiana tabacum plants.

Methods

N. tabacum plant lines including wild-type, vector controls, transgenic plants with single-trait expression of a citrate transporter (A. thaliana frd3) or fungal phytases (phyA: A. niger, P. lycii) and crossed plant lines expressing both traits, were characterized for citrate efflux and phytase exudation. Monocultures and intercropped combinations of single-trait plants were grown in a low available P soil (12 weeks). Plant biomass, shoot P accumulation, rhizosphere soil pH and citrate-extractable-P fractions were determined. Land Equivalent Ratio and complementarity effect was determined in intercropped treatments and multiple-linear-regression was used to predict shoot P accumulation based on plant exudation and soil P depletion.

Results

Crossed plant lines with co-expression of citrate and phytase accumulated more shoot P than single-trait and intercropped plant treatments. Shoot P accumulation was predicted based on phytase-labile soil P, citrate efflux, and phytase activity (Rsq=0.58, P < .0001). Positive complementarity occurred between intercropped citrate- and phytase-exuding plants, with the greatest gains in shoot P occurring in plant treatments with A. niger phyA expression.

Conclusions

We show for the first time that trait synergism associated with the exudation of citrate and phytase by tobacco can be linked to the improved acquisition of P and the depletion of soil organic P.

     

Biolistic transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) with the <Emphasis Type="Italic">phyA</Emphasis> gene from <Emphasis Type="Italic">Aspergillus ficuum</Emphasis>

Transgenic cotton with an increased level of phytase activity was generated from cotton (Gossypium hirsutum L.) cv. ND94-7 by subjecting shoot-apex explants to particle bombardment. These tissues were transformed with plasmid pC-KSA2300 carrying a selectable marker (for kanamycin) and a target gene (phytase, or phyA, from Aspergillus ficuum). Primary plants were regenerated in a medium containing 75 mg l⁻¹ kanamycin. Of 1,534 shoot apices, 52 (3.4%) survived on this selection medium. Southern and Northern blot analyses confirmed that phyA was stably integrated and expressed in those primary transgenics. The progenies of the primary transgenic plants were found to have a 3.1- to 3.2-fold increase in root extracellular phytase activity, resulting in improved phosphorus (P) nutrition. Growth also was enhanced when they were supplied with phytate, and their P content was equivalent to that of wildtype plants supplied with inorganic phosphate. These results demonstrate that the expression of phyA in cotton plants improves their ability to utilize organic P in response to a deficiency.     

Gene Silencing of BnTT10 Family Genes Causes Retarded Pigmentation and Lignin Reduction in the Seed Coat of Brassica napus

Yellow-seed (i.e., yellow seed coat) is one of the most important agronomic traits of Brassica plants, which is correlated with seed oil and meal qualities. Previous studies on the Brassicaceae, including Arabidopsis and Brassica species, proposed that the seed-color trait is correlative to flavonoid and lignin biosynthesis, at the molecular level. In Arabidopsis thaliana, the oxidative polymerization of flavonoid and biosynthesis of lignin has been demonstrated to be catalyzed by laccase 15, a functional enzyme encoded by the AtTT10 gene. In this study, eight Brassica TT10 genes (three from B. napus, three from B. rapa and two from B. oleracea) were isolated and their roles in flavonoid oxidation/polymerization and lignin biosynthesis were investigated. Based on our phylogenetic analysis, these genes could be divided into two groups with obvious structural and functional differentiation. Expression studies showed that Brassica TT10 genes are active in developing seeds, but with differential expression patterns in yellow- and black-seeded near-isogenic lines. For functional analyses, three black-seeded B. napus cultivars were chosen for transgenic studies. Transgenic B. napus plants expressing antisense TT10 constructs exhibited retarded pigmentation in the seed coat. Chemical composition analysis revealed increased levels of soluble proanthocyanidins, and decreased extractable lignin in the seed coats of these transgenic plants compared with that of the controls. These findings indicate a role for the Brassica TT10 genes in proanthocyanidin polymerization and lignin biosynthesis, as well as seed coat pigmentation in B. napus.     

The sweet potato sporamin promoter confers high-level phytase expression and improves organic phosphorus acquisition and tuber yield of transgenic potato

The sweet potato sporamin promoter was used to control the expression in transgenic potato of the E. coli appA gene, which encodes a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The sporamin promoter was highly active in leaves, stems and different size tubers of transgenic potato, with levels of phytase expression ranging from 3.8 to 7.4% of total soluble proteins. Phytase expression levels in transgenic potato tubers were stable over several cycles of propagation. Field tests showed that tuber size, number and yield increased in transgenic potato. Improved phosphorus (P) acquisition when phytate was provided as a sole P source and enhanced microtuber formation in cultured transgenic potato seedlings when phytate was provided as an additional P source were observed, which may account for the increase in leaf chloroplast accumulation (important for photosynthesis) and tuber yield of field-grown transgenic potato supplemented with organic fertilizers. Animal feeding tests indicated that the potato-produced phytase supplement was as effective as a commercially available microbial phytase in increasing the availability of phytate-P to weanling pigs. This study demonstrates that the sporamin promoter can effectively direct high-level recombinant protein expression in potato tubers. Moreover, overexpression of phytase in transgenic potato not only offers an ideal feed additive for improving phytate-P digestibility in monogastric animals but also improves tuber yield, enhances P acquisition from organic fertilizers, and has a potential for phytoremediation.     

Quantitative conversion of phytate to inorganic phosphorus in soybean seeds expressing a bacterial phytase

Phytic acid (PA) contains the major portion of the phosphorus in the soybean (Glycine max) seed and chelates divalent cations. During germination, both minerals and phosphate are released upon phytase-catalyzed degradation of PA. We generated a soybean line (CAPPA) in which an Escherichia coli periplasmic phytase, the product of the appA gene, was expressed in the cytoplasm of developing cotyledons. CAPPA exhibited high levels of phytase expression, >or=90% reduction in seed PA, and concomitant increases in total free phosphate. These traits were stable, and, although resulted in a trend for reduced emergence and a statistically significant reduction in germination rates, had no effect on the number of seeds per plant or seed weight. Because phytate is not digested by monogastric animals, untreated soymeal does not provide monogastrics with sufficient phosphorus and minerals, and PA in the waste stream leads to phosphorus runoff. The expression of a cytoplasmic phytase in the CAPPA line therefore improves phosphorus availability and surpasses gains achieved by other reported transgenic and mutational strategies by combining in seeds both high phytase expression and significant increases in available phosphorus. Thus, in addition to its value as a high-phosphate meal source, soymeal from CAPPA could be used to convert PA of admixed meals, such as cornmeal, directly to utilizable inorganic phosphorus.     

Seed-specific silencing of OsMRP5 reduces seed phytic acid and weight in rice

Phytic acid (PA) is poorly digested by humans and monogastric animals and negatively affects human/animal nutrition and the environment. Rice mutants with reduced PA content have been developed but are often associated with reduced seed weight and viability, lacking breeding value. In the present study, a new approach was explored to reduce seed PA while attaining competitive yield. The OsMRP5 gene, of which mutations are known to reduce seed PA as well as seed yield and viability, was down-regulated specifically in rice seeds by using an artificial microRNA driven by the rice seed specific promoter Ole18. Seed PA contents were reduced by 35.8–71.9 % in brown rice grains of transgenic plants compared to their respective null plants (non-transgenic plants derived from the same event). No consistent significant differences of plant height or number of tillers per plant were observed, but significantly lower seed weights (up to 17.8 % reduction) were detected in all transgenic lines compared to null plants, accompanied by reductions of seed germination and seedling emergence. It was observed that the silencing of the OsMRP5 gene increased the inorganic P (Pi) levels (up to 7.5 times) in amounts more than the reduction of PA-P in brown rice. This indicates a reduction in P content in other cellular compounds, such as lipids and nucleic acids, which may affect overall seed development. Put together, the present study demonstrated that seed specific silencing of OsMRP5 could significantly reduce the PA content and increase Pi levels in seeds; however, it also significantly lowers seed weight in rice. Discussions were made regarding future directions towards producing agronomically competitive and nutritionally valuable low PA rice.