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a USDA-ARS, Forage and Range Research Lab., Utah State Univ., Logan, UT 84322-6300 USA
b USDA-ARS, Dale Bumpers National Rice Research Center, Stuttgart, AR 72610 USA
c USDA-ARS, National Small Grains Germplasm Research Facility, Aberdeen, ID 83210 USA
stlarson{at}cc.usu.edu
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Abbreviations: AFLP, amplified fragment length polymorphism • bp, base pair • HIP, high inorganic phosphorus • HPLC, high performance liquid chromatography • HVPE, high voltage paper electrophoresis • Pi, inorganic P • lpa, low phytic acid • Ins, myo-inositol • MIPS, myo-inositol 1-phosphate synthase • STS-PCR, sequence tagged site-polymerase chain reaction
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Chemically induced, non-lethal recessive mutants that decrease seed phytic acid content have been isolated and genetically mapped in maize (Zea mays L.; Raboy and Gerbasi, 1996; Raboy et al., 2000) and barley (Hordeum vulgare L.; Larson et al., 1998; Rasmussen and Hatzack, 1998). These low phytic acid (lpa) mutations have the potential to alleviate the environmental and nutritional problems associated with phytic acid in animal feeds (Ertl et al., 1998). Low phytic acid crops may also offer improved nutrition for human populations that depend upon grains and legumes as staple foods. Moreover, these lpa mutants will provide a valuable system to study grain phytic acid synthesis.
Unlike the normal genetic and environmental affects that result in quantitative variation in seed total P, lpa mutants show large effects on the partitioning of P into phytic acid P, Pi, and lower Ins polyPs. The lpa mutations of maize and barley are characterized by two chromatographic phenotypes that are circumscribed by two respective genetic complementation groups, designated lpa1 and lpa2, in each species. A wide range of phytic acid reductions are observed among different lpa1 and lpa2 mutants. However, all lpa1 mutants are qualitatively identical in that phytic acid P reductions are counterbalanced by molar-equivalent increases in Pi. The lpa1 loci have been mapped to maize chromosome 1S (centromere-distal; Raboy et al., 2000) and barley chromosome 2H (Larson et al., 1998). In contrast, the phytic acid reductions in lpa2 mutants are matched by increases of Pi and lower Ins polyPs, in approximately equal parts. These lpa2 loci have been mapped to maize chromosome 1S (centromere-proximal; Raboy et al., 2000) and barley chromosome 7H (Larson et al., 1998).
The synthesis of Ins P6 can be summarized in two parts; the early pathway representing Ins and/or Ins P1 synthesis and the late pathway representing Ins polyP metabolism. On the basis of the biochemical phenotypes of barley and maize lpa mutations, it has been hypothesized that lpa1 mutations interrupt Ins or Ins P1 synthesis and/or supply, whereas lpa2 mutations interrupt Ins polyP metabolism (Larson and Raboy, 1999). The first committed step of Ins biosynthesis involves L-myo-inositol 1-phosphate synthase activity (MIPS, also referred to as D-myo-inositol 3-phosphate synthase), the only de novo source of the Ins ring in all organisms (Loewus, 1990). The gene for MIPS was first identified and cloned from yeast (Klig and Henry, 1984; Johnson and Henry, 1989) and subsequently from various higher plant species (Smart and Fleming, 1993; Johnson and Burk, 1995; Wang and Johnson, 1995; Ishitani et al., 1996). Yoshida et al. (1999) recently reported a single copy MIPS gene in rice (Oryza sativa L.) that shows temporal and spatial patterns of transcript accumulation corresponding with seed phytic acid synthesis.
As a first phase in the genetic study of phytic acid synthesis in cereal grains, we have isolated lpa mutants and compared their genetic map positions with those of MIPS loci. Larson and Raboy (1999) found that maize contains several dispersed MIPS loci, one of which maps to a site near and possibly identical to maize lpa1 on chromosome 1S. Barley differs from maize in two ways (Larson and Raboy, 1999). First, the barley genome contains only one MIPS-homologous sequence located on a segment of chromosome 4 that is clearly orthologous to the maize chromosome 1S region containing a MIPS gene and lpa1 mutation. Second, the barley lpa1 mutation maps to a locus on chromosome 2H that is separate from the single copy MIPS gene on chromosome 4H. Here we report the isolation, inheritance and seed P phenotype of the first non-lethal rice lpa1 mutant, and compare its map position to that of a single-copy rice MIPS gene developmentally upregulated at stages corresponding with seed phytic acid synthesis.
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Materials and methods |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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20% as compared with wild type) in seed phytic acid P, corresponding to the observed increase in Pi, was detected with HVPE. Remnant M3 seed from these M2 mutants were planted, and the M4 and M5 generation seed was analyzed as above to identify heritable mutations.
Analyses of Seed P and Ins P Fractions
Samples of seeds were dried for 48 h at 60°C, milled to pass through a 2-mm screen, and stored in a desiccator until analysis. Seed total P was determined following wet-ashing of aliquots of tissue (typically 150 mg) and colorimetric assay of digest P (Chen et al., 1956). The ferric-precipitation method was used to determine phytic acid P (Raboy et al., 1990). Aliquots of tissue (typically 0.5–1.0 gm) were extracted in 0.4 M HCl:0.7 M Na2SO4. Phytic acid P was obtained as a ferric precipitate, wet-ashed and assayed for P as in the total P analysis. Seed Pi was determined colorimetrically following extraction of tissue samples (typically 0.5 g in non-mutant seeds and 0.15 g in mutant seeds) in 12.5% (w/v) TCA:25 mM MgCl2. To facilitate comparisons, all seed P fractions are expressed as their P (atomic weight 31) content.
An HVPE assay for acid-extractable Ins Ps was performed as previously described (Raboy et al., 1990). Anion-exchange HPLC analyses of seed Ins Ps were performed by a modification of the methods as described (Phillippy and Bland, 1988; Rounds and Nielsen, 1993). Samples of seeds were dried, milled, and extracted in 0.4 M HCl overnight. Following centrifugation (10000 g, 10 min), supernatants were filtered through Whatman No. 1 filter paper, and passed through Millipore HV 0.45-µm filters. Aliquots were 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 phosphates were then eluted with the following gradient system at a flow rate of 0.5 mL min-1: 0 to1 min 100% Buffer A; 1 to 26 min a concave gradient from 0 to 15% 1 M NaNO3, pH 4.0 (Buffer B); 21 to 41 min a linear gradient from 15 to 100% Buffer B. The column elutent was mixed with colorimetric reagent [0.015% (w/v) FeCl3:0.15% (w/v) sulfosalicylic acid) at a flow rate of 0.5 mL min-1, and passed through a 290-cm reaction coil prior to peak detection via absorbance at 550 nm. Ins P in a sample peak was calculated by means of the following standard curve, obtained via the analysis of four Na Ins P6 standards containing 24.9, 49.7, 74.6 and 99.5 nM Na Ins P6; nM Ins P = 1.66 x 10-5 (Peak Area) - 3.85, R2 = 0.99.
Inheritance and Mapping Studies
A rice lpa1 mapping population was generated from an intersubspecific cross; a true-breeding `Kaybonnet' lpa1 mutant (tropical japonica) x wild-type `ZHE733' (indica). Segregation of the lpa1 trait was determined by progeny testing 137 F2 plants by the HIP assay. Sixteen individual F3 kernels, from each F2 plant were assayed. The F2 plants were classified as homozygous wild type (+/+) if zero F3 kernels were HIP, heterozygous (+/lpa1) if at least one F3 progeny kernel was HIP, and homozygous mutant (lpa1/lpa1) if virtually all F3 progeny kernels were HIP. Allowances were made for up to three phenotypically normal (+/lpa1) F3 kernels identified from several lpa1/lpa1 plants evidently contaminated by genetically dominant wild-type pollen, as determined by the distribution of HIP / wild-type F3 ratios.
DNA samples of all homozygous +/+ or lpa1/lpa1 F2 plants were reconstructed from a bulk of 10 F3 coleoptiles from each F2 family. The AFLP analyses were performed as described by Vos et al. (1995). The AFLP marker loci designations described in this study correspond to the following EcoRI (E) and MseI (M) AFLP primers with two and three selective nucleotides, respectively:
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The E13 primer was end labeled with T4 polynucleotide kinase and ATP[
-33P] before selective amplification. Selective amplification products were fractionated by 6% (w/v) denaturing PAGE (polyacrylamide gel electrophoresis) and visualized by autoradiography. A DNA size standard (10-bp ladder), end labeled with T4 polynucleotide kinase and ATP[-33P], was included in one lane of each gel.
The F2 intercross model of MAPMAKER version 3.0b (Lander et al., 1987) was used to identify AFLP markers genetically linked to the rice lpa1 mutation. The F2 backcross model of MAPMAKER was used to map genetically AFLP markers linked to rice lpa1 in a population comprised of 135 IR64 (indica) and Azucena (tropical japonica) F1 doubled haploids, characterized with 175 skeletal RFLP markers (Guiderdoni et al. 1992; Huang et al. 1994; and Huang et al. 1997; and kindly provided by B. Courtois, IRRI, Manila, The Philippines). This population was also used to map the rice MIPS gene as described below.
Microsatellite primers pairs were custom made for loci corresponding with the chromosomal map location of rice lpa1, based on sequences described by Panaud et al. (1996) and Chen et al. (1997), with 6-FAM 5' conjugates on the forward primer sequences. Microsatellite amplification products and ROX-labeled internal lane size standards, were fractionated on 6% (w/v) denaturing PAGE gels and analyzed by mens of an automated, fluorescent DNA sequencer.
PCR primers (Saiki et al., 1985) for the rice MIPS gene were designed from the rice MIPS sequence reported by Yoshida et al. (1999) and selected from sites intended to amplify the same 3' regions corresponding to the MIPS STS-PCR restriction site polymorphisms mapped to barley chromosome 4 and maize chromosome 1S (Larson and Raboy, 1999). The sequence of these primers were as follows (F = plus strand, R = minus strand):
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These primers are referenced using numbers relative to the translation initiation codon of the barley, maize, and rice MIPS genes. STS-PCR analysis was performed as described by Larson and Raboy (1999), except that the final magnesium concentration was adjusted to 1 mM and the annealing temperature was 60°C.
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Results |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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95% of acid-extractable Ins P in these wild-type rice seeds. M3 seed representing a total of 3632 M2 rows were screened for HIP, and putative mutants were observed to segregate in two M2 rows; Orion M2-70 and Kaybonnet M2-2045. Initial assays of M3 seed produced by 10 Orion M2-70 M2 plants indicated that 8 of 10 plants produced one or more seed displaying a HIP phenotype, with an elevated Pi concentration representing about 50 to 75% of the seed total P concentration typical of non-mutant seed (Fig. 1A)
. However, the Orion M2-70 mutant was not obtained as a viable homozygote in the M3 generation (Fig. 1A) nor in the M4 (Fig. 1B) generation. Frequent sterility was observed in M3 plants, and the HIP phenotype was observed in tests of only three of 14 M3 plants. No further studies of this apparently lethal mutant have been conducted to date.
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Map Position of Rice MIPS
The STS-PCR primers used in this study (OsMIPS1083F + OsMIPS1520R) specifically amplified a 
800-bp product (see Uncut portion of Fig. 5)
from five genotypes of rice genomic DNA. This product is similar to STS-PCR sequences obtained from barley and maize (Larson and Raboy, 1999), but varies by length of three introns, at identical locations. The 800-bp MIPS STS-PCR product of five rice genotypes was digested with nine, four-base cutter restriction enzymes that yielded a total of 25 fragments (results not shown). The 310-bp ScrFI fragments observed in IR64 and ZHE733 (indica genotypes) were evidently cleaved at another ScrFI site to produce 270-bp fragments in Azucena and Kaybonnet (japonica genotypes) (Fig. 5). Segregation analysis of this 40-bp length polymorphism in the DH mapping population indicated that this MIPS marker maps to a locus on rice chromosome 3 that is evidently orthologous to the MIPS loci on maize chromosome 1S and Triticeae 4H (Fig. 4). By two-point map analysis, this MIPS gene was 7 cM (LOD = 18.1) from the RG100 locus. Multi-locus map analysis of marker in this region indicated scoring errors in more than one of these loci and that MIPS may actually be within 3 cM of RG100. Nevertheless, the general order of markers in our analysis (Fig. 4) is congruent with previously reported maps for this DH mapping population (Guiderdoni et al., 1992; Huang et al., 1994, 1997) and other rice mapping studies.
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Discussion |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Inheritance studies of lpa1 mutations indicate that they are recessive and that translocation of gene products or metabolites from the maternal plant to the developing seed does not appear to complement the loss of function in homozygous, lpa1-1/lpa1-1 seeds produced by heterozygous, +/lpa1-1 maternal rice, barley (Larson et al., 1998), or maize (Raboy et al., 2000) plants. Therefore the phytic acid reductions in lpa1 seeds are a result of metabolic changes that affect the developing seed per se. Sakri and Shannon (1975) demonstrated that [2-3H]myo-inositol, injected into developing wheat spikes, was translocated to the kernels where it appeared in the phytic acid-rich bran fraction. This observation suggests that seed-specific MIPS activity may not be the only source of Ins available for phytic acid synthesis. However, Yoshida et al. (1999) recently reported a single copy MIPS gene in rice that shows temporal and spatial patterns of elevated transcript accumulation corresponding with phytic acid synthesis during seed development. The mapping experiments described in this study demonstrated that this MIPS gene maps to a locus on rice chromosome 3, which is orthologous to the MIPS gene near maize lpa1 on chromosome 1S and the barley MIPS gene on Triticeae chromosome 4. Homozygosity for several maize lpa1 mutations, which map near the canonical MIPS gene on maize chromosome 1S, result in a near absence of seed phytic acid (unpublished data, 1995). Therefore, expression studies of this canonical MIPS gene in rice (Yoshida et al., 1999) and genetic map location near maize lpa1 suggest that this MIPS gene may be an appropriate target for seed-specific control of Ins polyphosphate metabolism.
Since rice lpa1 and barley lpa1 loci are not coincident with single-copy, canonical MIPS genes identified in these species, they may represent additional, noncanonical MIPS targets of potential value in manipulating seed phytic acid content. It is possible that barley lpa1 and/or rice lpa1-1 are mutations of cryptic MIPS genes; sequences encoding a protein with MIPS activity but having little or no homology to the canonical MIPS sequence . Another possibility is that these barley and rice lpa1 mutations affect trans-acting regulatory factors that control expression or activity of MIPS.
MIPS regulatory loci have not yet been identified in higher plant species; however, at least 10 different Ins auxotrophy genes have been identified in yeast (Culbertson and Henry, 1975), albeit several of these have pleiotropic effects on carbon source utilization. The majority of yeast auxotroph mutants map to the ino1 locus, the MIPS structural gene (Donahue and Henry, 1981); however, two other loci, ino2 and ino4, also show more than one allelic representative. Studies suggest that gene products of ino2 and ino4 interact as transcription factors of MIPS (Ambroziak and Henry, 1994; Hoshizaki et al., 1990). A search of Genbank failed to reveal any rice sequences homologous to the yeast ino2 or ino4 nucleotide sequences (results not shown). Another possibility is that barley lpa1 and/or rice lpa1 may be structural loci encoding one of at least two other functions believed to play a role in Ins synthesis and supply: Ins 1-phosphatase or Ins 1-kinase. On the basis of the low level of Ins polyphosphates other than phytic acid observed in lpa1 mutants, we still believe the effect of these mutations precedes Ins phosphorylation pathways.
The issue of dietary phytic acid in human nutrition and health is complex. Dietary phytic acid may have positive roles as an anti-oxidant or anti-cancer agent, or negative roles as a contributor to mineral deficiency (Harland and Morris, 1995). The impact or role of phytic acid in human nutrition may vary as a result of dietary differences within and among populations. For example, phytic acid's negative roles may outweigh any positive roles for growing children and child-bearing women in developing countries that depend on whole grains foods for the bulk of nutritional calories. The potential value of a low phytate rice is further complicated by the fact most grain phytic acid is localized in the aleurone and scutellum (O'Dell et al., 1972; Yoshida et al., 1999). Since rice is primarily consumed as a milled product, most grain phytic acid is removed in the bran fraction. Thus, in terms of nutritional value, milled rice produced from an lpa1-1 line may not differ greatly from milled rice from a wild-type line. Since phytic acid represents the major sink for P and mineral deposition in the aleurone layer, studies are underway to determine if in addition to altering the chemistry of seed P, mutations like rice lpa1-1 alter the distribution of P and nutritionally important minerals like iron and zinc in the seed. Any increases in the level of these nutrients in the endosperm fraction (i.e., milled rice) would represent an improvement in nutritional value. In any case, the bran fraction of rice lpa genotypes would be of greater nutritional value when used in animal feeds, and reduce the environmental problems associated with manure phosphates from non-ruminant livestock.
Mutants like rice lpa1-1 are currently being used to breed first generation low phytate crops for use in foods and feeds, although the agronomic acceptability of lpa genotypes has yet to be determined. Regardless of whether or not lpa genotypes are widely adopted, isogenic lines with and without these lpa mutations will enable new experimental approaches to examine the nutritional merits of phytic acid in human health and livestock production.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Received for publication November 20, 1999.
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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