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First published online December 27, 2007; 10.1104/pp.107.113480 Plant Physiology 146:468-477 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Quantitative Conversion of Phytate to Inorganic Phosphorus in Soybean Seeds Expressing a Bacterial Phytase1,[OA]United States Department of Agriculture Agricultural Research Service, Plant Genetics Research Unit, Columbia, Missouri 65211 (K.D.B., H.B.K., A.B., P.R.B.); and Biochemistry Department (P.Z., P.C., J.C.P.), and Plant Transformation Core Facility, Division of Plant Sciences (Z.J.Z.), University of Missouri, Columbia, Missouri 65211
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,  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.
In addition to providing protein and oil, soymeal is a source of phosphorus (P). As in other angiosperm seeds, most soybean seed P is in the form of phytic acid (PA; myoinositol-1,2,3,4,5,6-hexakisphosphate). PA is generally indigestible by monogastric animals such as poultry and swine (Erdman, 1979  ). In addition, in large poultry and swine facilities, PA chelates and lessens the dietary availability of metal ions (iron [Fe], manganese [Mn], magnesium [Mg], zinc [Zn], and copper [Cu]; Vohra et al., 1965). Underutilized PA in animal feeds leads to P pollution of wastewater and runoff from soils to which manure is applied (Sharpley et al., 1994). Feed supplementation with minerals and inorganic P (Pi) is costly and tends to exacerbate the problems of P pollution in the waste stream.
Mutation breeding has lowered seed PA with equivalent increases in seed phosphate in a number of cereal grains (Raboy et al., 2002
Several other loci are mutational targets for lowering seed PA. Wilcox et al. (2000)
Under certain conditions, reduction in seed PA can be deleterious to the plant. Nunes et al. (2006)
A commercially feasible, nonmutational strategy to increase utilization of Pi and bound minerals in PA, as well as to reduce P pollution, is to treat soymeal and other animal feeds with commercial phytase, usually a recombinant form encoded by PhyA of Aspergillus niger. Pen et al. (1993)
An alternative transgenic strategy to seed production of active phytase enzyme is to aim for a direct reduction of PA in feed grains. This approach can target PA synthesis as in the RNAi knockdown of GmMIPS1 or the ABC transporter described above (Nunes et al., 2006
Another popular transgenic strategy is to direct expression of a phytase during embryo development at the site of PA synthesis or storage. Thus, Chiera et al. (2004)
We previously transformed Arabidopsis (Arabidopsis thaliana) with an embryo-expressed phytase. We chose APPA, a periplasmic phytase encoded by the Escherichia coli appA gene (Dassa et al., 1990
Soybean Seeds Expressing a Recombinant Phytase
Soybean (‘Jack’) was transformed (Zhang et al., 1999
Of 14 CAPPA and 17 VAPPA independent lines, T1 seeds of only five CAPPA lines revealed individuals with high available phosphate (Raboy et al., 2002 To confirm the immunodetection of CAPPA, phytase enzyme activity was determined in crude protein extracts from at least three mature seeds of each transgenic line. For samples from most of the lines, phytase activity was nearly undetectable (<0.1 µmol Pi min–1 mg protein–1), while five CAPPA lines had enzymatic activity ranging from 0.5 to 326 µmol Pi min–1 mg protein–1. There was a complex relationship among high-Pi phenotype, cross-reacting APPA protein, and phytase enzyme activity in mature seeds. However, high-phytase-specific activity seeds of line CAPPA 14 consistently had high Pi and cross-reacting APPA protein.
The inheritance of Pi in CAPPA seeds was assessed over multiple generations of selfed plants originally derived from two T1 individuals from CAPPA event 14: 14H and 14N (Table I
). Line 14N produced T3 seeds that segregated for the high-Pi phenotype, which, in turn, cosegregated with high-phytase-specific activity; no phytase activity was detected in the seeds with normal Pi levels. In contrast, all seeds tested from selfed line 14H had high Pi and significant phytase enzyme activity. High seed phosphate was stably inherited in each generation between T1 and T5 for line 14H. The inheritance data suggest, therefore, that T2 individuals 14H and 14N were homozygous and hemizygous, respectively, for the functional CAPPA transgene. PCR detection of the CAPPA gene sequence among F2 individuals of a cross of 14H by low-PA line CX1834 (Wilcox et al., 2000
PA Content in CAPPA
PA content was assessed chemically and by electron microscopy. In soybean seeds, PA is complexed with mineral salts in phytin, located within PSVs. Phytin crystals are electron-dense globoid inclusions (Lott and Buttrose, 1978
In the T3 and subsequent generations, PA was also quantified by a modification of a high-performance ion chromatography method (HP-IC; Chen and Li, 2003 ) that distinguishes inositol phosphate metabolism products from intact PA. While PA levels of control seeds typically ranged from 20 to 28 µg PA mg–1, PA in CAPPA samples was consistently at or below the detection limit (2 µg PA mg–1), thus representing 90% reduction in seed PA (Fig. 2
). Neither the control nor CAPPA soybeans contained detectable IP5 (not shown). The chromatographic profiles agreed with those of CX1834, which did not appear to contain inositol phosphate intermediates (Wilcox et al., 2000). The sum of P in Pi and PA was relatively constant for individual seeds containing either high or low levels of phytase enzyme activity.
Subcellular Localization of CAPPA Phytase Cotyledon sections of mature seeds were examined by immunohistochemical analysis employing antibodies against recombinant APPA. Seed phytase accumulated throughout the cotyledon of CAPPA seed (Fig. 3A ), while none was detected in the progenitor seed (Fig. 3B). Because the CAPPA lines expressed phytase without a signal peptide, we expected phytase to have a cytoplasmic location. Higher magnification observation of CAPPA seeds challenged with phytase antibodies revealed prominent labeling in the cytoplasm (Fig. 3C). Preimmune serum did not show this labeling. Slight, but consistent, background labeling of PSVs by preimmune serum (Fig. 3D) was due probably to antibody against seed storage proteins present in rabbit meal. Parallel sections stained with Schiff's reagent confirmed the anatomical features of CAPPA seed (Fig. 3, E and F).
Seed Composition of CAPPA and Progenitor
CAPPA germination and plant development appeared normal in the greenhouse environment. Greenhouse-produced T5 CAPPA and the lines ‘Jack’ (progenitor genotype), ‘Williams 82’, and the low-phytase mutant CX1834 in the ‘Athow’ background (Wilcox et al., 2000
Germination, Field Emergence, and Seed Number of CAPPA Standard laboratory germination tests for field-produced seeds revealed a lower germination percentage for CAPPA compared with the progenitor, ‘Jack’. However, there was no significant difference in field emergence of field- or greenhouse-produced ‘Jack’, CAPPA, ‘Williams 82’, or CX1834 seeds (Table IV ). In addition, when grown under noncompetitive field conditions, CAPPA seeds did not differ significantly from ‘Jack’ in seed weight or in seeds per plant (Table IV). CAPPA seed P-related traits were tested in sequentially propagated field generations (next section).
Production of Sequential Generations of CAPPA in a Field Environment Three generations (T3, T4, and T5) of CAPPA were grown in the same field environment to determine their field phenotypes and phenotypic stability. CAPPA seeds germinated and the plants developed normally in the field environment. Field-produced mature dry seeds, generations T4, T5, and T6, were assayed for PA, Pi, and phytase enzyme activity. Seed phosphate levels were high and PA levels low or undetectable, similar to the seed composition values obtained for greenhouse-grown plants (Table V ). Phytase enzyme activity levels remained high in the field-advanced generations. There was no significant reduction in Pi, PA, or phytase activity in seeds from any of the field-advanced generations. Seeds of ‘Jack’ produced in the field environment had values for PA, Pi, and phytase enzyme activity similar to those of ‘Jack’ seeds produced in the greenhouse environment.
CAPPA Meal as an Alternative to Fungal Phytase for Digesting PA in Normal Meals Fortification of animal feeds with phytase enzyme is an accepted practice to reduce PA and increase Pi and minerals in the feed. We evaluated the ability of ground CAPPA seed to provide phytase enzyme activity in an in vitro P liberation assay using commercial cornmeal or soybean meal as the PA P source. Cornmeal and soybean meal were supplemented with a commercial source of phytase, ground CAPPA seed, or ground commodity soybean seed (‘Williams 82’; all added as 4 mg/g meal) and compared to samples containing no additive (Fig. 4 ). In both the CAPPA and the commercial phytase treatments, Pi was rapidly liberated, to the same extent, from both cornmeal and soybean meal, reaching a plateau within 4 h. Both treatments with CAPPA and commercial phytase additives released significantly more P than control ‘Williams 82’ additive or the no additive controls (P < 0.001). The gradual increase in P liberated in the ‘Williams 82’ mock treatment and in the no-treatment control may have been due to microbial activity because reactions were at 37°C under nonsterile conditions.
Several mutational and transgenic strategies to reduce PA in soybean have negative aspects. The CX1834 line of soybean (Wilcox et al., 2000 ) exhibits a ratio of Pi P to equivalent P in PA of over 50% compared to 15% in the progenitor (‘Athow’). Though considerable, this increase is still less than the much higher ratio observed in CAPPA, in which PA is often undetectable while total P is not reduced. A drawback to the low-PA trait of CX1834 is that it is due to recessive mutations at two interacting, unlinked loci (Walker et al., 2006). Because the superior Pi P to PA P ratio in CAPPA appears to be due to a single active insert, moving the dominant CAPPA gene into a new variety is more efficient than selecting one of 16 F2 individuals in outcrosses of CX1834.
The soybean mips1 line exhibited a 50% reduction in seed PA as well as reductions in galactinol and raffinosaccharides (Hitz et al., 2002
CAPPA is superior to lowered PA achieved by ectopic expression of a seedling phytase in developing soybean embryos, as reported by Chiera et al. (2004)
The high phytase activity of CAPPA seeds may make its meal a suitable replacement of commercial fungal phytases (product of A. niger PhyA). Ground CAPPA seed as an additive was as effective as commercial phytase in lowering PA in cornmeal and soymeal (Fig. 4). Rodriguez et al. (2000)
The lack of detectable PA in CAPPA seeds had no significant deleterious effect on seed weight, field emergence, or seed number/plant under noncrowded field conditions. Additional germination and field studies, under a variety of conditions, with additional events are needed to test the effect of virtual quantitative conversion of PA to Pi. The high phytase activity of CAPPA seeds was as effective as commercial phytase in lowering PA in cornmeal and soymeal, making CAPPA meal a suitable replacement for commercial fungal phytases (product of A. niger PhyA). The unique combination in CAPPA of nearly quantitative conversion of PA to Pi and high levels of remnant phytase activity in mature seed meal is a distinct improvement over other lines developed via transgenic or mutagenic strategies which, respectively, either lower seed PA or produce seed phytase as single traits.
Transformation of Soybean
DNA fragments containing soybean (Glycine max) seed lectin promoter-phytase constructs, with or without the lectin signal sequence, were excised from the plasmids pBinGLPH7-5 (Coello et al., 2001
Fourteen CAPPA and 17 VAPPA independent T0 plants were recovered, and mature T1 seeds were screened for a high "available" phosphate (Pi), a trait that has been inversely associated with seed PA levels (Raboy et al., 2002
T1 through T5 seeds were produced in either greenhouse with supplemental lighting or in growth chamber environments. Greenhouse settings were 16-h daylength and 30°C/18°C day/night temperatures. Growth chamber settings included 14.5-h daylength and 28°C/22°C day/night temperatures. Plants were grown in 6-inch or 2-gallon pots in PRO-MIX (Premier Horticulture) medium and fertilized with Miracle-Gro or Osmocote Plus (Scotts) according to the manufacturer's recommendations.
Independent field studies were conducted consisting of a field emergence study and yield component study. Four soybean entries, ‘Williams 82’, ‘Jack’, CAPPA, and CX1834-1-3, were grown at the Bradford Research and Extension Center, near Columbia, MO, in 2006 and 2007. The emergence study plot was disced and cultivated using typical agricultural methods. The experimental design was a randomized complete block with four replications and rows on 0.8-m centers. Seeds were hand planted into rows at a depth of 3 cm every 30 cm to maximize stand count accuracy and minimize seedling competition and spread of disease. Emerged seedlings were counted at 2 weeks after planting (TeKrony and Egli, 1977
The yield component study was cultivated in 2006 and 2007 as described previously. Experimental design was a randomized complete block with rows as previously described. Seeds inoculated with Rhizobium japonicum were hand planted at a depth of 3 cm. Seedlings were later thinned to obtain one plant every 0.8 m. Plants were harvested at the R8 stage (Fehr and Caviness, 1977 In addition, T4 through T6 CAPPA seeds were produced at a research field location in Columbia, MO in the summer of 2006. Seeds were inoculated and hand planted in rows on 11.8-cm centers, and plants were irrigated as needed.
The APPA open reading frame was PCR amplified with Taq polymerase (Promega) so that it lacked 27 N-terminal amino acids, and the 28th codon was converted from CTG (Leu) to ATG (Met), thus creating an NdeI site. Cloning of the amplified product into NdeI, NotI-digested pET28a (Novagen-Merck) created an in-frame N-terminal His(6x) tag with APPA (N-His-APPA). Expression in mid-log cells of the Escherichia coli HMS174 host was induced with 1 mM β-isopropyl thiogalactoside and further growth for 2 h. Cells were disrupted by sonication in 6 M urea, 500 mM NaCl, 5 mM imidazole in 20 mM Tris-HCl, pH 7.9, as recommended by the manufacturer of the Ni2+ affinity column (iminodiacetic acid-substituted His-Bind Resin; Novagen). After removing insolubles, the supernatant was poured over the column that was subsequently washed in the sonication buffer. Bound N-His-APPA was eluted with 1 M imidazole in the same buffer and dialyzed against 10% glycerol, 1 M urea in phosphate-buffered saline. Approximately 0.5 mL (0.16 mg N-His-APPA) was injected subcutaneously into each of two New Zealand white rabbits. Two weeks after a booster injection, rabbits were exsanguinated; the serum fraction, collected and preserved at –20°C in 50% glycerol, was used in immunostaining.
Field-grown soybean seeds at R6 stage (Fehr and Caviness, 1977
Dormant soybean seeds were imbibed in water and incubated at 30°C for 12 h. Seeds were cut into small pieces (1–2 mm) with a double-edged razor blade and fixed immediately in 2.5% glutaraldehyde buffered with 50 mM sodium phosphate, pH 7.2. Fixation was carried out at room temperature for 4 h. The tissue samples were washed four times at 15-min intervals with 50 mM phosphate buffer, pH 7.2, and post-fixed, 1 h at room temperature, with 2% aqueous osmium tetroxide. After extensive rinses in distilled water, the samples were dehydrated in a graded acetone series and infiltrated with Spurr's resin essentially as described (Krishnan et al., 1986
Pi was quantified by slight modifications of the method described by Wilcox et al. (2000)
PA was quantified by modified HP-IC method (Chen and Li, 2003
Protein, oil, and moisture concentration were determined by near infra-red spectroscopy according to standard procedures summarized by Dyer (2004)
For micro- and macronutrient analyses, dried ground seed (0.5 g) was ashed 5 h at 500°C and dissolved in 10 mL of 6 N HCl. Digested samples were diluted with water, filtered, and analyzed for potassium, boron, calcium, Mg, Zn, sodium, Fe, Mn, aluminum, molybedenum, and Cu by ICP-OES (Varian), nitrogen by thermal conductivity of nitrogenous gases with a Leco model FP-428 nitrogen analyzer (Leco), and P by colorimetric analysis (Murphy and Riley, 1962 Oligosaccharides were determined by HP-IC with pulsed amperometric detection employing Agilent 1100 series HPLC and an ESA Coulochem III detector (Agilent Technologies). A 12.5-mg ground seed sample was extracted with 0.5 mL of 50% ETOH at 70°C for 30 min. Samples were then centrifuged 15 min at 16,000g. The supernatant was passed through a 0.2-µm filter. Sugars were separated on a Dionex Carbo Pac PA 10 analytical column (250 mm x 4 mm, 10 µm) connected to a Carbo Pac PA 10 guard column (50 mm x 4 mm). The mobile phase was 90 mM NaOH with a flow rate of 1.5 mL min–1 maintained at 30°C. Detection settings were: time 0, 0.1 v; time 0.41, –2.0 v; time 0.42, 0.6 v; and time 0.44, –0.1 v.
Soybeans were powdered in liquid nitrogen, and 25 mg of either fresh or lyophilized sample was extracted in 1 mL of enzyme buffer (0.1 M sodium acetate, pH 5.5, 1 mM CaCl2, and 0.1 g L–1 Tween 20) by thorough vortex mixing, followed by incubation on ice 10 min. Samples were centrifuged 5 min at 15,000g, 4°C. The supernatants were then either diluted up to 50-fold in enzyme buffer or sampled without further dilution. With solutions cooled on ice, a 40-µL aliquot of sample was combined with 680 µL of reaction buffer (0.1 M sodium acetate, pH 4.5) and 80 µL of 12.5 mM PA. For the zero time point, 125 µL of the mixture was removed and mixed with 125 µL of cold 20% TCA, vortexed, and placed on ice. The remaining reactions were incubated 15 min in a 55°C or 37°C water bath. Reactions (125 µL) were stopped as for the zero time point. All samples were centrifuged 5 min at 15,000g, 4°C. Pi content was quantified as above with 100 µL of sample combined with 100 µL of colorimetric reagent, except that standards were assayed in 10% TCA rather than water. Extracted protein was quantified against bovine serum albumin standard according to the recommendations of the manufacturer of the Bio-Rad protein assay. Results were converted to phytase specific activity: µmol Pi released min–1 mg protein–1.
Coarse-ground whole seed of CAPPA (T4 generation) and ‘Williams-82’ were ground to fine powders in a small grinder (SmartGrind; Black & Decker). Commercial phytase (Natuphos 5,000 units g–1; BASF) was ground to a fine powder in a mortar. Fine-ground CAPPA, ‘Williams 82’ seed, or commercial phytase (40 mg) was mixed with 10 g commercially available soybean meal and cornmeal (MFA). One-gram samples of mixed and control meals were suspended in 10 mL of reaction buffer (0.1 M sodium acetate, pH 4.5) in 15-mL screw-top plastic tubes and incubated at 37°C on a rotary shaker. One tube in each treatment was randomly sampled at 0, 1, 2, 4, 8, 12, 16, 20, or 24 h and 400 µL was centrifuged, 200 µL of the supernatant was transferred to a fresh tube, mixed with 200 µL 20% TCA, and stored on ice prior to storage at –20°C. Samples were thawed and centrifuged 5 min at 15,000g, 4°C and 10 µL of supernatant was combined with 90 µL of 10% TCA in 0.1 M sodium acetate, pH 4.5, and 100 µL of colorimetric reagent (see above). Pi was quantified as above, except that standards were assayed in 0.05 M sodium acetate, pH 4.5, containing 10% TCA rather than water. Results were converted to µg P g meal–1. Treatments and controls were done in triplicate.
Data were analyzed by PROC Mixed routine (SAS, 1990
We thank Sean Pfaff, Paul Little, and Justin Kleffner for excellent technical assistance. Received November 19, 2007; accepted December 13, 2007; published December 27, 2007.
1 This work was supported by the National Center of Soybean Biotechnology (University of Missouri). 
2 Present address: Department of Nature Sciences, College of Agriculture and Technology, SUNY, Cobleskill, NY 12043.
3 Present address: Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, 04510 México DF, México. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Joe C. Polacco (polaccoj{at}missouri.edu).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.113480 * Corresponding author; e-mail polaccoj{at}missouri.edu.
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ABSTRACT
RESULTS
0.5 µg P mg–1) typical of the progenitor. In contrast, analysis of samples of the remaining five CAPPA lines revealed individual seeds with high Pi containing up to 6 µg P mg–1.