Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1

Phytic acid (myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate or Ins P(6)) typically represents approximately 75% to 80% of maize (Zea mays) seed total P. Here we describe the origin, inheritance, and seed phenotype of two non-lethal maize low phytic acid mutants, lpa1-1 and lpa2-1. The loci map to two sites on chromosome 1S. Seed phytic acid P is reduced in these mutants by 50% to 66% but seed total P is unaltered. The decrease in phytic acid P in mature lpa1-1 seeds is accompanied by a corresponding increase in inorganic phosphate (P(i)). In mature lpa2-1 seed it is accompanied by increases in P(i) and at least three other myo-inositol (Ins) phosphates (and/or their respective enantiomers): D-Ins(1,2,4,5,6) P(5); D-Ins (1,4,5,6) P(4); and D-Ins(1,2,6) P(3). In both cases the sum of seed P(i) and Ins phosphates (including phytic acid) is constant and similar to that observed in normal seeds. In both mutants P chemistry appears to be perturbed throughout seed development. Homozygosity for either mutant results in a seed dry weight loss, ranging from 4% to 23%. These results indicate that phytic acid metabolism during seed development is not solely responsible for P homeostasis and indicate that the phytic acid concentration typical of a normal maize seed is not essential to seed function.     

Determination of phytic acid by gas chromatography–mass spectroscopy: application to biological samples

A GC–MS method is reported for the determination of phytic acid based on purification by anion-exchange chromatography, enzymatic hydrolysis of phytic acid to myo-inositol and derivation to trimethylsilyl derivative, with scyllo-inositol as an internal standard. Analytical features of the method are: limit of detection 9 μg l⁻¹ phytic acid, linear working range 18–500 μg l⁻¹ phytic acid, and coefficient of variation 1.9%. The method has been successfully applied to a variety of biological samples: various rat organs (kidney, liver, brain and bone), human plasma and urine and kidney stones. A comparative study of sample treatments, including deproteization, lipid extraction and the presence of a chelator, is also reported. Phytic acid amounts found in rat organs ranged from 1.07 g kg⁻¹ for bone to 32.0 g kg⁻¹ for brain. Phytic acid in human plasma was of the order of 0.14 mg l⁻¹. In kidney stones, phytic acid was found in calcium containing stones.     

Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK

Using a forward genetics approach, we isolated two independent low phytic acid (lpa) rice mutants, N15-186 and N15-375. Both mutants are caused by single gene, recessive non-lethal mutations, which result in approximately 75% (N15-186) and 43% (N15-375) reductions in seed phytic acid (inositol hexakisphosphate). High-performance liquid chromatography and GC–MS analysis of seed extracts from N15-186 indicated that, in addition to phytic acid, inositol monophosphate was significantly reduced whereas inorganic phosphorus and myo-inositol were greatly increased when compared with wild-type. The changes observed in N15-186 resemble those previously described for the maize lpa3 mutant. Analysis of N15-375 revealed changes similar to those observed in previously characterized rice lpa1 mutants (i.e. significant reduction in phytic acid and corresponding increase in inorganic phosphorus with little or no change in inositol phosphate intermediates or myo-inositol). Further genetic analysis of the N15-186 mutant indicated that the mutation, designated lpa N15-186, was located in a region on chromosome 3 between the microsatellite markers RM15875 and RM15907. The rice orthologue of maize lpa3, which encodes a myo-inositol kinase, is in this interval. Sequence analysis of the N15-186 allele of this orthologue (Os03g52760) revealed a single base pair change (C/G to T/A) in the first exon of the gene, which results in a nonsense mutation. Our results indicate that lpa N15-186 is a mutant allele of the rice myo-inositol kinase (OsMIK) gene. Identification and characterization of lpa mutants, such as N15-186, will facilitate studies on the regulation of phytic acid biosynthesis and accumulation and help address questions concerning the contribution of the inositol lipid-dependent and independent biosynthetic pathways to the production of seed phytic acid. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Bone and faecal minerals and scanning electron microscopic assessments of femur in rats fed phytic acid extract from sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis>)

Phytic acid was extracted from sweet potato (Ipomoea batatas) and fed to Wistar rats with or without zinc for 3 weeks. Animals were then sacrificed and bone and faecal minerals were assessed. The ultra-structure of the bones was examined via scanning electron microscopy. Phytic acid extract or commercial phytic acid supplemented diets (D + Zn + PE or D + PE) displayed reduced bone calcium levels (101.27 ± 59.11 and 119.27 ± 45.36 g/kg) compared to the other test groups. Similarly, reduced calcium were observed in the control groups (D + Zn and D) fed formulated diets with or without zinc supplementation (213.14 ± 15.31 and 210 ± 6.88 g/kg) compared to the other test groups. The group fed supplemented commercial phytic acid diet (D + CP) demonstrated the lowest femur magnesium (3.72 ± 0.13 g/kg) while the group fed phytic acid extract supplementation (D + PE) recorded the highest level (4.84 ± 0.26 g/kg) amongst the groups. Femur iron was highest in the group fed commercial phytic acid supplemented diet (D + CP −115.74 ± 2.41 g/kg) compared to the other groups. Faecal magnesium levels were significantly higher in the two test groups fed phytic acid extract with or without zinc (D + Zn + PE or D + PE) compared to all other groups. All the groups which had phytic acid supplemented diets had significantly thinner bone in the trabecular region, compared to the groups fed formulated diet or zinc supplemented formulated diet (D or D + Zn). These observations suggest that the consumption of foods high in phytic acid may contribute to a reduction in the minerals available for essential metabolic processes in rats.     

Effect of phosphorus and zinc nutrition on soybean seed phytic Acid and zinc

The relationships between nutrient P and Zn levels and the phytic acid, P, and Zn concentrations in soybean (Glycine max L. Merr. cv `Williams 79') seed were studied. Phytic acid increased linearly from 4.2 to 19.2 milligrams per gram as nutrient P treatment was varied from 2.0 to 50 milligrams per liter and Zn was held constant at 0.05 milligrams per liter. Leaf P concentration during seed development was found to be closely related to the concentrations of seed P and phytic acid. Leaf and seed Zn concentrations both responded positively to increasing nutrient Zn treatment. The effects of P treatment on plant and seed P and phytic acid were largely independent of the effects of Zn treatment on leaf and seed Zn. Phytic acid to Zn molar ratios ranging from 3.6 to 33.8 were observed.

The effects of nutrient P treatments on the concentrations of phytic acid, seed P, and leaf P were also studied in the P-sensitive (gene np) cultivars `Harosoy' and `Clark' and their respective P-tolerant (gene Np) near-isogenic lines L66-704 and L63-1677. In general, the positive relationships observed among nutrient P, leaf P, seed P, and phytic acid concentrations were similar to those observed in the studies with Williams 79. When fertilized with low or moderate nutrient P (2.5 and 25.0 milligrams P per liter, respectively) no significant differences in any parameter were observed between Harosoy or Clark and their respective P-tolerant isolines. When fertilized with high nutrient P (100 milligrams P per liter), Harosoy seed had a significantly higher concentration of phytic acid (30 milligrams per gram) than did seed of its P-tolerant near-isogenic line L66-704 (24.2 milligrams per gram phytic acid), whereas no significant difference was observed between Clark and its P-tolerant near-isogenic line L63-1677 (22.8 and 21.6 milligrams per gram, respectively). Variation in the phytic acid concentrations in the mature seed of the cultivars and isolines more closely paralleled leaf P concentrations observed during seed development (49 days after flowering), than those observed at the onset of seed development (14 days after flowering). Electrophoresis and ion-exchange chromatography revealed that partially phosphorylated intermediates do not appear when phytic acid accumulation is greatly reduced by limiting the nutrient P or when accumulation is greatly accelerated by excess P.

     

Synchronous fluorescence determination of phytic acid in FOODSTUFFS and urine based on replacement reaction

Introduction – Phytic acid is a ubiquitous and abundant natural component in many plant seeds, fruits and vegetables. Its biological and pharmaceutical functions are still controversial. The examination on the level of phytic acid in foodstuffs and urine can provide valuable information for its dietary intake and metabolism. Objective – To develop a sensitive and reliable synchronous fluorescence protocol for determination of phytic acid in selected foodstuffs and human urine. Methodology – Phytic acid efficiently catches Cu²⁺ ion in previously prepared Cu^II‐2,2′‐bipyridine complex in aqueous solution, releasing the fluorescent 2,2′‐bipyridine molecule and recovering synchronous fluorescence. The recovered fluorescence is proportional to the added phytic acid, by which the levels of phytic acid in the selected foodstuffs and human urine are quantified. Results – A calibration curve with a regression equation of I_f = 37.745 + 39.245c (R² > 0.9988) showed good linearity over the range 0.18–17.50 mg/L phytic acid. The relative standard deviation at 95% confidence degree was less than 2.04% (n = 5), indicating that the procedures are reproducible. The detection and quantification limit of phytic acid were estimated to be 0.12 and 0.18 mg/L, respectively. By the proposed method, phytic acid in the selected foodstuffs and urine was determined to be 3.25–16.76 and 0.43–1.21 mg/L with recoveries of 96.8%–105.6% and 95.1%–104.2%, respectively. The results are in good agreement with those obtained by the reported HPLC technique. Conclusion – The developed method is sensitive, reliable and economical, which permits its practical application in quantitative analyses of trace phytic acid in foodstuffs and urine. Copyright © 2010 John Wiley & Sons, Ltd.     

The timing and rate of phytic Acid accumulation in developing soybean seeds

The time-course of phosphorus (P) accumulation in the phytic acid fraction of developing soybean (Glycine max [L.] Merr. cv `Williams 79') seeds as well as the relation of phytic acid P to total P content were determined. Phytic acid was detected early in embryogenesis in field-grown soybeans and accumulated in a linear fashion throughout most of seed development. Although the observed rates of accumulation ranged from 18.7 micrograms phytic acid P per seed per day in pods positioned low on the plant to 33.6 micrograms in pods positioned high on the plant, the final concentrations were the same in all cases. Nearly all of the P translocated to developing seeds was incorporated into phytic acid from the third week after flowering until physiological maturity, with the sum of nonphytic acid P compounds remaining constant. Phytic acid accumulation was also linear throughout development when soybean plants were grown in solutions having nutrient P levels that ranged from severely limiting (2.0 milligrams P per liter) to excess (50 milligrams P per liter). However, there was a pronounced effect on rate of accumulation, which ranged from 7.2 micrograms phytic acid per seed per day with limiting nutrient P to 44.7 micrograms with excess P. The change in level of phytic acid accounted for most of the alteration in total seed P that was caused by altering the P status of the plants. These results support the view that phytic acid synthesis is involved in P homeostasis of the developing soybean seed.     

Fine mapping of the rice low phytic acid (Lpa1) locus

Phytic acid is the primary storage form of phosphorus (P) in cereal grains. In addition to being essential for normal seedling growth and development, phytic acid plays an important role in human and animal nutrition. The rice low phytic acid mutation lpa1 results in a 45% reduction in seed phytic acid with a molar equivalent increase in inorganic P. The Lpa1 locus was previously mapped to the long arm of chromosome 2. Using microsatellite markers and a recombinant inbred line population, we fine mapped this locus between the markers RM3542 and RM482, which encompass a region of 135 kb. Additional markers were developed from the DNA sequence of this region. Two of these markers further delimited the locus to a 47-kb region containing eight putative open reading frames. Cloning and molecular characterization of the Lpa1 gene will provide insight into phytic acid biosynthesis in plants. The markers reported here should also be useful in introgressing the low phytic acid phenotype into other rice cultivars.The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Linkage mapping of two mutations that reduce phytic acid content of barley grain

 This study describes the inheritance and linkage map positions of two low phytic acid barley (Hordeum vulgare) mutations, lpa1-1 and lpa2-1, that dramatically reduce grain phytic acid content and increase inorganic seed phosphorus (P). Wide-cross, F₂ mapping populations were constructed by mating six-rowed varieties, ‘Steptoe’ and/or ‘Morex’, with two-rowed ‘Harrington’lpa donor lines homozygous for either lpa1-1 or lpa2-1. The barley lpa1-1 mutation showed normal inheritance patterns, whereas a deficiency of homozygous lpa2-1/lpa2-1 F₂ plants was observed. We identified a codominant, STS-PCR marker (aMSU21) that cosegregated with lpa1-1 in a population of 41 F₂ plants. The aMSU21 marker was then mapped to a locus on barley chromosome 2H, using a North American Barley Genome Mapping Project (NABGMP) doubled haploid population (‘Harrington’בMorex’). We determined that lpa2-1 is located within a recombination interval of approximately 30 cM between two AFLP markers that were subsequently mapped to barley chromosome 7H by integration with the same NABGMP population. Recent comparative mapping studies indicate conserved genetic map orders of several homologous molecular marker loci in maize and the Triticeae species that also show corresponding linkage to the biochemically similar lpa2 mutations of maize and barley. This observation suggests that barley and maize lpa2 mutations may affect orthologous genes. No such evidence for correspondence of the phenotypically similar lpa1 mutations of barley and maize has been revealed. Received: 22 September 1997 / Accepted: 2 December 1997     

Genetic analysis of phytic acid content in pearl millet

Summary The genetics of phytic acid content in pearl millet (Pennisettum typhoides (Burm) S & H) was studied using a 12 parent diallel. The analysis of variance of diallel progenies exhibited significant genotypic differences. Different analyses, i.e., combining ability analysis, analysis of variance of diallel tables and genetic component analysis, showed that both additive and non additive gene effects were significant. It is suggested that a population improvement is possible by breeding for low phytic acid cultivars of pearl millet.     

The effect of phosphate concentration on phytase production and the reduction of phytic acid content in canola meal by Aspergillus carbonarius during a solid-state fermentation process

The use of canola meal, an abundant side-product of canola oil processing in Canada, as animal feed is hampered by high phytic acid levels that reduce metal cation availability. Aspergillus carbonarius grows well in a solid canola meal medium, produces phytase and reduces the phytic acid content to zero. Inorganic phosphate addition at a concentration of 1 mg and 5 mg/110 g solid-state culture system results in better growth of the microorganism, higher rates and levels of phytase production, and faster reduction of phytic acid content. Phosphate concentrations of 50mg and 100 mg/110 g inoculated system had a negative effect affecting primarily the initial rates of biomass and phytase production and phytic acid content reduction. Models that predict biomass production (expressed as glucosamine content) and phytase, as well as the reduction of phytic acid content in the solid-state cultures supplemented with phosphate are reported. They fit the experimental results reasonably well (with a maximum deviation of 7%).     

蒲公英植酸对沙门氏菌抑制作用及其抑菌机理研究

为探究蒲公英植酸对沙门氏菌的抑制作用及其抑菌机理。本文利用沉淀法和离子交换法提取蒲公英植酸,滤纸片法分析蒲公英植酸对沙门氏菌(Salmonella)的抑菌作用,倍比稀释法研究蒲公英植酸的最低抑菌浓度。通过分析沙门氏菌的细胞通透性和生长动力学,结合扫描电镜和荧光显微镜研究了蒲公英植酸对沙门氏菌的抑菌机理,表明蒲公英植酸对沙门氏菌具有很好的抑菌能力,其最小抑菌浓度为0.2 mg/mL。而且植酸对沙门氏菌的抑制作用是通过破坏细胞膜达到抑菌的效果,并且植酸浓度越高,抑菌效果越显著。这表明蒲公英植酸可以有效地抑制沙门氏菌生长,其主要是通过破坏菌体细胞膜完整性,增加细胞薄膜的通透性,使细胞内容物外溢达到抑制细菌生长的目的。     

Effect of phytic acid,tannic acid and pectin on fasting iron bioavailability both in the presence and absence of calcium

ObjectiveTo determine the effect of phytic acid, tannic acid and pectin on fasting non-heme iron bioavailability in both the presence and absence of calcium.Research methodsTwenty-eight apparently healthy adult females participated in two iron absorption studies using radioactive iron isotopes (⁵⁹Fe and ⁵⁵Fe). One group received 5 mg of iron (as FeSO₄) alone (control), together with 10 mg of phytic acid, 100 mg of tannic acid and 250 mg of pectin (study A), on different days. The second group received the same iron doses and compounds as the other group, plus 800 mg of calcium (CaCl₂) (study B). The compounds were administered after an overnight fast, and no food or beverages were consumed for the following 3 h. Iron status and circulating radioactivity were measured in venous blood samples.ResultsThe geometric means of iron bioavailability (range ± 1SD) for iron alone, iron with phytic acid, iron with tannic acid, and iron with citrus pectin were 25.0% (11.9–52.0); 18.9% (9.9–35.8); 16.8% (8.7–32.3); and 21.1% (10.2–43.9), respectively (repeated-measures ANOVA, p < 0.02 (Dunnett's post hoc: control vs tannic acid p < 0.05). When 800 mg of calcium was added (study B), iron bioavailability was 16.7% (10.1–27.5); 13.2% (7.1–24.6); 14.8% (8.8–25.1); and 12.6% (5.5–28.8), respectively (repeated-measures ANOVA, NS).ConclusionsTannic acid decreases the fasting bioavailability of non-heme iron, however this effect did not exist in the presence of calcium. No effect was observed by phytic acid or citrus pectin on fasting non-heme iron bioavailability in both the presence and absence of calcium.     

Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds

Phytic acid in cereal grains and oilseeds is poorly digested by monogastric animals and negatively affects animal nutrition and the environment. However, breeding programs involving mutants with less phytic acid and more inorganic phosphate (P(i)) have been frustrated by undesirable agronomic characteristics associated with the phytic acid-reducing mutations. We show that maize lpa1 mutants are defective in a multidrug resistance-associated protein (MRP) ATP-binding cassette (ABC) transporter that is expressed most highly in embryos, but also in immature endosperm, germinating seed and vegetative tissues. Silencing expression of this transporter in an embryo-specific manner produced low-phytic-acid, high-Pi transgenic maize seeds that germinate normally and do not show any significant reduction in seed dry weight. This dominant transgenic approach obviates the need for incorporating recessive lpa1 mutations to create maize hybrids with reduced phytic acid. Suppressing the homologous soybean MRP gene also generated low-phytic-acid seed, suggesting that the strategy might be feasible for many crops.     

The role of phytic Acid in the wheat grain

The concentrations of adenosine triphosphate and phytic acid in testa, embryo plus scutellum, aleurone, and endosperm fractions from grain of Triticum vulgare cv. Insignia have been determined during development under both normal conditions and those of water stress. Phytic acid was not detected in the endosperm. In the embryo plus scutellum and aleurone fractions there was a rapid build-up of phytic acid, but the adenosine triphosphate level did not change markedly at this time. These results are not consistent with physiological roles previously suggested for phytic acid other than the role of phytin as a phosphorus and cation store for the germinating seed.     

Seed phosphorus and inositol phosphate phenotype of barley low phytic acid genotypes

myo-Inositol-1,2,3,4,5,6-hexakisphosphate (Ins P(6) or "phytic acid") typically represents approximately 75% of the total phosphorus and >80% of soluble myo-inositol (Ins) phosphates in seeds. The seed phosphorus and Ins phosphate phenotypes of four non-lethal barley (Hordeum vulgare L.) low phytic acid mutations are described. In seeds homozygous for M 635 and M 955 reductions in Ins P(6), approximately 75 and >90% respectively, are accompanied by reductions in other Ins phosphates and molar-equivalent increases in Pi. This phenotype suggests a block in supply of substrate Ins. In seeds homozygous for barley low phytic acid 1-1 (lpa1-1), a 45% decrease in Ins P(6) is mostly matched by an increase in Pi but also accompanied by small increases in Ins(1,2,3,4,6)P(5). In seeds homozygous for barley lpa2-1, reductions in seed Ins P(6) are accompanied by increases in both Pi and in several Ins phosphates, a phenotype that suggests a lesion in Ins phosphate metabolism, rather than Ins supply. The increased Ins phosphates in barley lpa2-1 seed are: Ins(1,2,3,4,6)P(5); Ins(1,2,4,6)P(4) and/or its enantiomer Ins(2,3,4,6)P(4); Ins(1,2,3,4)P(4) and/or its enantiomer Ins(1,2,3,6)P(4); Ins(1,2,6)P(3) and/or its enantiomer Ins(2,3,4)P(3); Ins(1,5,6)P(3) and/or its enantiomer Ins(3,4,5)P(3) (the methods used here cannot distinguish between enantiomers). This primarily "5-OH" series of Ins phosphates differs from the "1-/3-OH" series observed at elevated levels in seed of the maize lpa2 genotype, but previous chromosomal mapping data indicated that the maize and barley lpa2 loci might be orthologs of a single ancestral gene. Therefore one hypothesis that might explain the differing lpa2 phenotypes is that their common ancestral gene encodes a multi-functional, Ins phosphate kinase with both "1-/-3-" and "5-kinase" activities. A putative pyrophosphate-containing Ins phosphate, possibly an Ins P(7), was also observed in the mature seed of all barley genotypes except lpa2-1. Barley M 955 indicates that at least for this species, the ability to accumulate Ins P(6) can be nearly abolished while retaining at least short-term ( approximately 1.0 years) viability.     

Isolation and characterisation of an lpa (low phytic acid) mutant in common bean (Phaseolus vulgaris L.)

Phytic acid is considered as one of the major antinutritional compounds in cereal and legume seeds. The development of lpa (low phytic acid) grains, resulting in increased mineral cation availability, is considered a major goal in the improvement of the nutritional quality of seed crops, especially those largely consumed in developing countries. From a mutagenised population of common bean we isolated a homozygous lpa mutant line (lpa-280-10) showing, compared to wild type, a 90% reduction of phytic acid, a 25% reduction of raffinosaccharides and a much higher amount of free or weakly bound iron cations in the seed. Genetic analysis showed that the lpa character is due to a recessive mutation that segregates in a monogenic, Mendelian fashion. Germination tests performed using varying ageing or stress conditions, clearly showed that the bean line lpa-280-10 has a better germination response than the wild type. These data, together with those obtained from 2 years of agronomic trials showing that the mutant seed yield is close to that of its parents and other evidence, indicate that the new lpa-280-10 mutation might be the first devoid of visible macroscopic negative effects in plants, pods and seeds. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.