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a Univ. of Idaho, Aberdeen Research and Extension Center, P.O. Box 870, Aberdeen, ID 83210
b BASF Corp., Research Triangle Park, NC 27709
c USDA-ARS Small Grains and Potato Research Unit, P.O. Box 607, Aberdeen, ID 83210
* Corresponding author (esouza{at}uidaho.edu).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: AACC, American Association of Cereal Chemistry • EMS, ethyl-methanesulfonate • HIP, high inorganic phosphate • Ins P, inositol phosphate • Ins P6, myo-inositol-1,2,3,4,5,6-hexakisphosphate • lpa, low phytic acid • Pi, inorganic phosphorus
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In humans, diets high in phytate can significantly decrease the absorption of essential micronutrients such as Ca (Kies, 1985), Fe (Brune et al., 1992), and Zn (Sandström et al., 1987). Phytate forms chelates with these divalent minerals, which reduces bioavailability to humans (Jacobsen and Slotfeldt-Ellingsen, 1983). In developing countries, flatbreads may be prepared from whole-wheat flour or high (
95%) extraction flour. The phytate in wheat grain is found predominantly in the aleurone layer, which remains attached to the pericarp during milling and therefore is concentrated in the bran fraction. Although phytate decreases in proportion to fermentation time, in chapati bread, the phytate concentration was only 18 to 24% lower than the concentration in the whole-wheat flour from which it was prepared (Anjum et al., 2002). The phytate concentration in chapati breads was six- to nine-fold higher than in breads prepared from straight-grade flour. However, mineral concentrations generally were significantly higher in whole-grain flour than in straight-grade flour, which might offset the effect of phytate on human nutrition. Human bioavailability studies will be necessary to confirm this hypothesis.
Nonlethal recessive mutations that decrease seed phytic acid concentration have been isolated in maize (Zea mays L.; Raboy and Gerbasi, 1996; Raboy et al., 2000), barley (Hordeum vulgare L.; Larson et al., 1998; Rasmussen and Hatzack, 1998), rice (Oryza sativa L.; Larson et al., 2000), and soybean [Glycine max (L.) Merr.; Hitz et al., 2002; Wilcox et al., 2000]. These low phytic acid (lpa) mutants affect the partitioning of P into phytic acid, inorganic phosphorus (Pi), and Ins P's with five or fewer P esters (Raboy, 2002). The lpa mutations are divided into two groups, designated lpa1 and lpa2. Phytic acid P reductions in lpa1 mutants are counterbalanced by molar-equivalent increases in Pi. In contrast, phytic acid P reductions in lpa2 mutants are counterbalanced by increases in both Pi and non-Ins P6 Ins P's (Ins P's of lower phosphorylation). The isolation and characterization of seed P and Ins P phenotype of a heritable wheat lpa1-like mutant is described here.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.5 mg Pi g–1. Seed with >1.0 mg Pi g–1 were considered HIP phenotypes. One spike from a single M2 plant (A95631S-Js-12-333Mu-4) was identified that appeared heterogeneous for the HIP phenotype. Remnant M3 seed from this spike was planted in the greenhouse. Ten M4 kernels from each M3–derived plant were evaluated for HIP phenotype. Two homozygous M3 plants were identified from the single M2 plant. One M3 plant (A95631S-Js-12-333Mu-4-6) was determined to be homozygous for the HIP phenotype, and another M3 plant (A95631S-Js-12-333Mu-4-8) was determined to be homozygous for the wild-type inorganic P phenotype, on the basis of analyses of 10 M4 kernels from each M3 plant. Remnant M4 kernels from these two plants, hereafter designated as Js-12-LPA and Js-12-WT, were planted in the field at Aberdeen, ID, in 2000. Individual M4 plants were harvested and homozygosity of the HIP phenotype was confirmed by evaluations of single kernels.
Grain Production
Homozygous M5 seed from the 2000 Aberdeen field trial was bulked and planted in El Centro, CA. M6 seed harvested from California was planted in Tetonia, ID, in May 2001. Grain was produced with irrigation by overhead sprinklers using agronomic practices standard for the region. M7 grain was harvested in September 2001.
Assay for High Inorganic Phosphate Phenotype
Kernels were individually crushed and extracted overnight in 10 µL 0.4 N HCl mg–1 (approximate seed weight). A 10-µL aliquot of each extract was then assayed for Pi in microtiter plates, using a modification of the colorimetric method of Chen et al. (1956). Phenotype was evaluated visually relative to P standards, as described by Larson et al. (2000).
Analyses of Seed Phosphorus Fractions
Analytical protocols for quantitative chemical compositions were as described (Larson et al., 2000; Raboy et al., 2000; Dorsch et al., 2003). Briefly, seed total P was determined colorimetrically (Chen et al., 1956) following wet-ashing of aliquots of tissue. Phytic acid P was determined by the ferric-precipitation method. Inorganic P was determined colorimetrically following extraction of tissue in 12.5% (w/v) 2,4,6-Trichloroanisole:0.25 mM MgCl2. The experiment was conducted as a completely randomized design with three replications. Analysis of variance was used to test the effect of genotype.
Anion-exchange HPLC analyses of seed Ins P's using post-column metal-dye detection were performed as described (Larson et al., 2000; Raboy et al., 2000; Dorsch et al., 2003). Seeds were extracted in 0.4 M HCl (10 v/w) and 200 µL of supernatant was diluted with double-distilled H2O (to 1.0 mL) and passed through a 0.2-µm filter. An aliquot (200 µl) was then fractionated on a Dionex IonPac AS7 anion-exchange column, equipped with a Dionex IonPac AG7 guard column, which had been equilibrated with 10 mM methyl piperazine, pH 4.0 (Buffer A). The Ins P's were eluted with the following gradient system at a flow rate of 1.0 mL min–1: initial condition, 100% Buffer A; 1 to 45 min, a linear gradient from 0 to 80% 0.5 M NaCl pH 4.0, in 10 mM methyl piperazine pH 4.0 (Buffer B); 45 to 60 min, 20 to 100% Buffer A. The column eluent was mixed with metal dye detection colorimetric reagent (0.015% FeCl3:0.15% sulfosalicylic acid in 0.05 M methylpiperazene, pH 4.0) at a flow rate of 0.5 mL min–1, using an Upchurch PEEK high pressure mixing tee (VWR Scientific, Philadelphia, PA) and an Eldex Model B-100-S metering pump (Eldex Laboratories, Inc., Menlo Park, CA), and the mixture passed through a 100-cm reaction coil before peak detection via absorbance at 550 nm. The Ins P in a sample peak was calculated using a standard curve obtained via the analysis of seven Ins P6 standards prepared using commercially-obtained Na Ins P6 (Sigma). These standards contained 25, 50, 75, 100, 125, 150, and 200 nM Na Ins P6, which yielded area units (x106) of 5.9, 14.5, 22.0, 31.1, 39.1, 45.3, and 59.5, respectively.
Milling
Grain (1 kg) from the 2001 Tetonia production was tempered by the standard American Association of Cereal Chemistry (AACC; American Association of Cereal Chemists, 1995) Method 26-10 for soft wheat. Tempered grain was milled using a Brabender Quadrumat Senior Mill (C.W. Brabender Instruments, Inc., South Hackensack, NJ; AACC Method 26-21A). Flour protein concentration was determined with a near-infrared analyzer (Instalab 600, Dickey-John Corp., Auburn, IL, AACC method 39-10A), calibrated by automated combustion analysis of total N content (LECO Model NFP-428, LECO Corp., St. Joseph, MO), and corrected to 120 g kg–1 moisture.
Elemental Analysis
Mineral element composition of milling fractions was determined by the University of Idaho Analytical Services Laboratory using a Perkin-Elmer Optima 3200 ICP-OES (Inductively Coupled Plasma–Optical Emission Spectrometer) to quantify aqueous constituents following nitric acid digestion of the milling fractions. Mineral concentrations tested included Al, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Pb, S, V, Zn, and Y. The experimental design was a randomized complete block with two replications. Analysis of variance was used to test the effect of genotype, milling fraction, and the interaction between genotype and milling fraction on concentration of each element.
Segregation Studies
Segregation of the HIP phenotype was evaluated in the cross Js-12-LPA/IDO563. IDO563 is an elite soft white spring breeding line of the pedigree ‘Kanto 79’/2*IDO488 and is a sister selection to the line A95631S-Js-12 used for mutagenesis. The F1 generation was grown in the greenhouse to produce F2 seed. Eighty-six F2 plants then were grown in the greenhouse. Six plants produced <20 kernels or produced kernels with an average weight of <10 mg and were excluded from the segregation analysis. These small kernels were highly shriveled and had not undergone normal seed development. The F2 population of 80 plants was the largest F2 population available for the trait and, assuming normal segregation, the size was sufficiently large to differentiate between the expected segregation of a single recessive mutation (expected number of homozygous recessive individuals: 20 ± 4) and the most likely alternative to a single recessive mutation, recessive mutations at two loci (expected number of double recessive individuals in a population of 80: 5 ± 2) with a probability exceeding 99.9%. At the initiation of the F2 study, the likelihood of the trait being conditioned by three recessive mutations was not considered.
Twenty F3 kernels harvested from each F2 plant were evaluated for the HIP phenotype. A total of 80 F2 plants were evaluated for the HIP phenotype of progeny (F3) kernels using the HIP assay described above coupled with measurement of absorbance at 820 nm in a Dynamax MRX microplate reader (Dynex Technologies, Chantilly, VA). F3 kernels were classified relative to the means and standard deviations of the Pi content of 80 kernels of each of the parent genotypes, IDO563 or Js-12-LPA. Individual F3 kernels were classified as wild-type if the Pi concentration in the kernel was within the interval of two standard deviations above and two standard deviations below the IDO563 mean (0.24 ± 0.14 mg g–1). Individual F3 kernels were classified as HIP if the Pi concentration in the kernel was within the interval of two standard deviations above and two standard deviations below the Js-12-LPA mean (1.19 ± 0.44 mg g–1). The wild-type and HIP classifications did not overlap. F3 kernels having Pi concentrations between these two classifications were categorized as intermediate.
F2 genotype was inferred from F2:3 phenotypes by classification into five categories as follows: (i) F2 plants were classified as homozygous wild-type if no progeny (F2:3) kernels were HIP and all F2:3 kernels with intermediate Pi concentrations had Pi concentrations below the mid-parent mean. (ii) F2 plants were classified as heterozygous if at least one progeny (F2:3) kernel was HIP and at least one progeny (F2:3) kernel was wild-type. (iii) F2 plants were also classified as heterozygous when HIP kernels were observed, no wild-type F2:3 kernels were observed, and kernels of intermediate Pi concentration had concentrations below the mid-parent mean. (iv) F2 plants were classified as homozygous for the low phytic acid trait if no progeny (F2:3) kernels were wild-type and all F2:3 kernels with intermediate Pi concentrations had Pi concentrations above the mid-parent mean. (v) F2 plants were classified as homozygous for the low phytic acid trait if all progeny (F2:3) kernels were HIP.
A single F3 seed of each F2 plant was planted and advanced in the greenhouse. This process was repeated to produce F4 plants through single-seed descent. F4:5 families were planted in the field at Aberdeen, ID, in 2002. F4:6 seed was harvested from those families. Ten individual kernels from each family were evaluated for the HIP phenotype. Total P and the distribution of P as Pi and phytic acid P were determined on whole grain meal of each family as described above. On the basis of our observations of the F2 families, we assumed that the progeny rows through genetic segregation should fit into three categories: the two parental phenotypes and intermediate phenotypes. Because the distribution of phosphorus as inorganic P, phytic acid P, and other forms is a multivariate distribution we grouped F4:6 families into three categories by Euclidean distances using nearest centroid sorting (Anderberg, 1973; SAS Institute, 1997). The clustered procedure used PROC FASTCLUS (SAS Institute, 1997) in SAS using MAXCLUSTERS = 3 with the variables for clustering as Pi, phytic acid P, and other P, all standardized to a percentage of total P.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Milling Analysis
Grain of Js-12-LPA and Js-12-WT produced at Tetonia milled similarly on the Brabender Quadrumat Senior mill (Table 1). Total flour yields were 580 g kg–1 for both genotypes. The bran yield was approximately 380 g kg–1 for both genotypes. Weight distributions among the flour fractions were essentially identical for Js-12-LPA and Js-12-WT; 27% of flour of both genotypes was recovered in first pass through mill rolls (break flour), and 73% of the flour was recovered in the second pass through the mill rolls (reduction flour).
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The concentration of P as phytic acid in the reduction flour of Js-12-LPA was lower than the concentration of P as phytic acid in reduction flour of Js-12-WT (Table 1). Moreover, concentrations of inorganic P in Js-12-LPA break and reduction flour were nearly four-fold greater than in Js-12-WT break and reduction flour. The concentration of P as phytic acid was 43% lower in bran of Js-12-LPA relative to bran of Js-12-WT.
A relatively low percentage of total P in break and reduction flour was represented by phytic acid P and inorganic P (25 to 43%). While in the bran fractions, 86 to 89% of total P was represented by phytic acid P and inorganic P (Table 1). Lysophospholipids represent 86 to 94% of total starch lipids, and lysophospholipid content can be quantified by multiplying starch P concentration by 16.5 (Morrison, 1964; Morrison et al., 1975; Raeker et al., 1998). Phosphorus content of 12 highly purified soft wheat starches ranged from 0.48 to 0.60 mg g–1 dry starch (Raeker et al., 1998). As starch accounts for about 65% of soft wheat flour weight, we can extrapolate that starch-bound P represents between 0.31 and 0.39 mg g–1 flour. Therefore, in this study, much of the break and reduction flour P not represented by inorganic P or phytic acid P may be starch-bound lysophospholipid P.
Concentrations of Cd, Co, Cr, Na, Y, and V in all milling fractions were below the analytical limits of detection. Nickel and Pb concentrations in all milling fractions were below the limits of detection, but were above the minimum detection limits in the whole grain sample (Table 2). Concentrations of all minerals were greatest in the bran. Therefore, differences in mineral concentration between the genotypes were most apparent in the bran fraction. The concentrations of only two minerals, Cu and Zn, were significantly affected by the mutant phenotype (Table 2). Js-12-LPA had 21% lower Cu concentration than Js-12-WT in whole grain and 16.5% lower Cu concentration in the bran. Zinc concentration in bran of Js-12-LPA was 33% lower than in bran of Js-12-WT. However, concentrations of Zn in the flour and shorts of the Js-12-LPA and Js-12-WT were similar. The depression in Cu and Zn concentration in bran may be a nutritionally significant detrimental effect of the mutant and will require further characterization in nutrition studies.
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0.001), 8.0 (P 0.018), and 102.5 (P 0.001), respectively. The observed segregation best fits the expected 1:14:1 ratio of a two-gene trait. However, the observed segregation is not good fit to a two-gene model, perhaps because of imperfect classification of progeny or distorted segregation.
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2 = 0.60, 2 df, P 0.74) and does not fit the one gene model of 35:11:35 for the 81 families (2 = 208, 2 df, P 0.001). The mean kernel weight of families from the three clusters did not differ significantly. The mean kernel weight of families from the high inorganic P/low phytic acid P cluster was 37.1 mg kernel–1, while the mean kernel weight of families from the low inorganic P group/high phytic acid P cluster was 35.1 mg kernel–1, and the mean kernel weight of families from the large intermediate cluster was 34.7 mg kernel–1. This suggests that the mutations conferring the low phytic acid trait do not affect general grain development but are specific to P storage.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50 to 95%). This may reflect a greater buffering capacity conferred by the hexaploid wheat genome relative to the diploid barley and maize genomes. Accumulations of lower inositol polyphosphates were not observed in Js-12-LPA, indicating that this mutant is not of the lpa2-type. Flour extractions of both Js-12-LPA and Js-12-WT wheat produced at Tetonia were relatively low. But in our experience, these extractions are typical of late-planted grain produced at Tetonia. The 2001 Tetonia crop was planted unusually late because of the late timing of the harvest of the Southern California increase. Therefore, our initial conclusion is that there are not obvious adverse effects of the HIP phenotype on milling.
Phytic acid was primarily sequestered in the bran of both Js-12-LPA and Js-12-WT wheat. However, the HIP phenotype altered the distribution and compositional profile of P within the kernel. Phytic acid P concentration was reduced 43% in bran of Js-12-LPA, while inorganic P concentration was increased three- to four-fold in all milling fractions of Js-12-LPA relative to Js-12-WT. Phytic acid becomes a component of animal feed through bran. Nonruminant animals do not utilize phytic acid P, which is primarily excreted and becomes an important environmental concern. Therefore, the Js-12-LPA trait may have a positive impact on the nutritional quality of the wheat bran fed to animals and may reduce the environmental impact of animal waste P.
Phytic acid chelates divalent cations, which are principally sequestered in the bran. It is possible that the decreased concentration of Cu and Zn in bran of Js-12-LPA was a consequence of the decrease in phytic acid concentration. To date, no other lpa mutation has been found to reproducibly impact seed or seed fraction mineral content. These minerals did not appear to be present in higher concentrations in other milling fractions of Js-12-LPA. Further studies are underway in our laboratory to evaluate the relationship between the lpa trait and mineral content in wheat.
The mutagenesis of Js-12 produced a heritable low phytic acid phenotype. The distribution of phosphorus as inorganic P relative to phytic acid P is a quantitative trait affected by seed characteristics. This limits the resolution of our segregation data. The simplest explanation of mutagenesis for a new phenotype is the assumption of perturbation of a single gene. Mapping of low phytic acid mutants of rice, barley, and maize has indicated that these are single-gene perturbations (Larson et al., 1998, 2000; Raboy et al., 2001). However, our inheritance data of F2 and F4:6 families is inconsistent with the assumption of a single locus model and suggests the involvement of multiple loci in Js-12-LPA. The observed frequencies of parental phenotypes are most consistent with a two-gene model. Mapping studies with Js-12-LPA are needed to confirm this hypothesis. Other possible explanations for the observed data in the F2 population include a naturally occurring mutation in the IDO563 parent and a mutation with partial or ‘leaky’ expression. The first alternative seems unlikely because IDO563 is a first backcross sib of the donor germplasm used for mutagenesis and should be similar to the donor line, given that very limited variation has been reported in wheat or other agronomic crops in the absence of induced mutations (Raboy, 2002). The second alternative also seemed unlikely given the phenotypes observed in the RI lines. A leaky mutation should produce many lines with variable expression for the HIP phenotype. Instead, we observed that most of the genotypes were internally consistent. The number of RI lines with variation in HIP similar to the total range observed for the cross was small enough in number to be consistent with the expected number of heterozygotes in a two-gene model.
The low phytic acid trait is heritable and was recovered with sufficient frequency to be used in breeding. Backcross breeding using the HIP phenotype has successfully moved the Js-12-LPA phenotype into advanced spring wheat breeding materials (Souza and Guttieri, 2003, unpublished data).
The full HIP phenotype requires further investigation. Ideally, the suppression of phytic acid should have minimal pleiotropic effects on the normal functionality of the wheat kernel within the milling and baking industry. Moreover, the HIP phenotype ideally should be agronomically neutral. The Js-12-LPA wheat characterized in this study is agronomically unacceptable because of reduced stature, markedly weak straw, and dramatically reduced grain yield. Genotypes derived from mutagenesis often have undesirable traits that will segregate independently of the desired trait. Studies to address the agronomic and end-use quality effects of the HIP phenotype currently are underway in our laboratory.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication May 1, 2003.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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., C.S. Gaines, P.L. Finney, and T. Donelson. 1998. Granule size distribution and chemical composition of starches from 12 soft wheat cultivars. Cereal Chem. 75:721–728.This article has been cited by other articles:
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P. Bregitzer and V. Raboy Effects of Four Independent Low-Phytate Mutations on Barley Agronomic Performance Crop Sci., April 25, 2006; 46(3): 1318 - 1322. [Abstract] [Full Text] [PDF]
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