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

Abstract

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.

Figure 1.

Electron microscopic analysis of mature seeds. Cotyledon sections of ‘Williams 82’ (A) and CAPPA (B) represent genotypes with normal and low PA levels, respectively. Phytin globoid cavities are the small white circular areas present in the ‘Williams 82’ PSVs. cw, Cell wall; N, nucleus; LB, lipid bodies.

Figure 2.

Reduction in PA P correlates with increased Pi and phytase enzyme activity in mature CAPPA seed samples. A, Individual mature seed samples were subjected to three analyses: PA P, Pi P, and phytase enzyme activity. The sum of Pi P and PA P is represented for each T₃ seed sample. Samples 1 through 10 were derived from 14H (CAPPA/CAPPA), while samples 11 to 15 were derived from 14N (CAPPA/−) and were segregating for the transgene event. B, Phytase enzyme activity was determined for individual seed samples carried out at 55°C.

Figure 3.

Distribution of CAPPA antigen in immature (R6) CAPPA line cotyledons. A and B, Total cotyledon sections of CAPPA (A) and progenitor cultivar ‘Jack’ (B) immunostained with anti-APPA serum and counterstained with haematoxylin. C and D, Photomicrographs of a similarly treated longitudinal cotyledon section of CAPPA (C) at higher magnification and a control section treated with preimmune serum (D). E and F, Parallel sections of CAPPA and ‘Jack’ were stained with Schiff's reagent to observe the anatomical features.

Figure 4.

Release of phosphate from soybean meal by CAPPA meal and fungal phytase. Commercial cornmeal (CM) or soybean meal (SM) was incubated without additions or with additions (4 mg/g meal) of ground ‘Williams 82’ soybean (CM+W82; SM+W82), of ground CAPPA soybean (CM+CAPPA; SM+CAPPA), or of commercial phytase (CM+PHYT; SM+PHYT). Lines are plotted means of phosphate determinations for each treatment and time.