Victor Raboy

Increasing CO2 threatens human nutrition

Dietary deficiencies of zinc and iron are a substantial global public health problem. An estimated two billion people suffer these deficiencies, causing a loss of 63 million life-years annually. Most of these people depend on C3 grains and legumes as their primary dietary source of zinc and iron. Here we report that C3 grains and legumes have lower concentrations of zinc and iron when grown under field conditions at the elevated atmospheric CO2 concentration predicted for the middle of this century. C3 crops other than legumes also have lower concentrations of protein, whereas C4 crops seem to be less affected. Differences between cultivars of a single crop suggest that breeding for decreased sensitivity to atmospheric CO2 concentration could partly address these new challenges to global health.

Phytic acid and phosphorus in crop seeds and fruits: a global estimate.

A very important mineral storage compound in seeds is phytate, a mixed cation salt of phytic acid (myo-inositol hexakis phosphoric acid). This compound is important for several reasons: (1) It is vital for seed/grain development and successful seedling growth. (2) It is often considered to be an antinutritional substance in human diets, but it may have a positivenutritional role as an anti-oxidant and an anti-cancer agent. (3) It represents a very significant amount of phosphorus being extracted from soilsand subsequently removed with the crop. (4) It plays a role in eutrophication of waterways. A key part of this review is an estimate of the annualtonnage of phosphorus and phytic acid sequestered in up to 4.1 billion metric tonnes of crop seeds and fruits globally each year. We estimate thatnearly 35 million metric tonnes of phytic acid, containing 9.9 million metric tonnes of P, is combined with about 12.5 and 3.9 million metric tonnes of K and Mg respectively, to form each year over 51 million metric tonnes of phytate. The amount of P inthis phytate is equal to nearly 65÷ of the elemental P sold world wide for use in mineral fertilizers. Dry cereal grains account for 69÷ of the total crop seed/fruit production but account for 77÷ of the total phytic acid stored each year. Low phytate mutants, that are now available for some key staple food crops such as maize and barley, offer potential benefits in such areas as the sustainability of lands used to grow crops, the mineral nutrition of humans and animals, and reduction in pollution of waterways.

Genetics and breeding of seed phosphorus and phytic acid

Summary The isolation of cereallow phytic acid (lpa) mutants provides a novel approach to studying the biology of seed phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate or Ins P6), and to dealing with environmental and nutritional problems associated with it. Seed produced bylpa lines contain normal levels of total phosphorus (P), but greatly reduced levels of phytic acid P. Two phenotypically distinct types oflpa mutants have been isolated in maize (Zea mays L.), barley (Hordeum vulgare L.), and rice (Oryza sativa L.). In «lpa1-like» mutants, seed phytic acid P reductions ranging from 50 percnt; to 95 percnt; (in comparison with levels typical of non-mutant seed) are largely matched by corresponding increases in inorganic P. In «lpa2-like» mutants, seed phytic acid P reductions ranging from 50 percnt; to 75 percnt; are matched by increases in both inorganic P and inmyo-inositol (Ins) phosphates containing five or fewer P esters (compared with phytic acids six P esters). In all cases the sum of seed Ins phosphates (including phytic acid) and inorganic P remains constant and similar to that in normal seeds. Somelpa alleles are lethal as homozygotes, others have a negative effect on plant or seed growth and function but are viable, still others have little effect and are being used to breed «low phytate» maize and barley types. Progress inlpa genetics and breeding, and the animal and human nutrition studies conducted with these new crop types, will be reviewed.

Accumulation and Storage of Phosphate and Minerals

Developing seeds store reserve levels of phosphorus and minerals, mobilization of which during germination provides the mineral nutrition essential for optimal early seedling growth. This process also functions as a component of the inorganic P and mineral homeostasis mechanisms necessary for nominal cellular function. The storage and resup-ply of phosphorus centers around the synthesis and breakdown of phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate). Phytic acid is often deposited as a mixed, ‘phytin’ salt, primarily of potassium and magnesium. Thus phytin deposition and re-mobilization also represents an important component of mineral cation storage. Exceptions to this may include iron and calcium storage and homeostasis, for which non-phytin mechanisms may play major roles. There has been recent progress in the molecular biology of several components of phosphorus and mineral storage pathways. These include studies of myo-inositol synthesis, some aspects of myo-inositol phosphate metabolism, protein body transport functions, and calcium and iron storage proteins. Recent genetic studies bring into question the paradigm that phytic acid synthesis is essential to phosphorus storage or inorganic P homeostasis. Much additional progress is required, particularly concerning the synthetic pathway to phytic acid, before a thorough or truly detailed understanding of the molecular biology of P and mineral storage processes is obtained.

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

Abstract 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.

Identification and Characterization of a Low Phytic Acid Wheat

crease the absorption of essential micronutrients such as Ca (Kies, 1985), Fe (Brune et al., 1992), and Zn Phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate, or Ins P6) (Sandström et al., 1987). Phytate forms chelates with is the most abundant storage form of P in seeds, yet indigestible by humans and nonruminant livestock. A wheat (Triticum aestivum L.) these divalent minerals, which reduces bioavailability mutant is described herein with greatly reduced seed phytic acid P to humans (Jacobsen and Slotfeldt-Ellingsen, 1983). In but little change in seed total P, similar to lpa1-type mutants described developing countries, flatbreads may be prepared from in other grain species. One nonlethal mutant from 562 ethyl-methanewhole-wheat flour or high (≈95%) extraction flour. The sulfonate (EMS) mutagenized M2 lines was identified with a high phytate in wheat grain is found predominantly in the inorganic phosphate (HIP) phenotype and designated Js-12-LPA. aleurone layer, which remains attached to the pericarp Js-12-LPA homozygotes produced seed in which phytic acid P repreduring milling and therefore is concentrated in the bran sented 48.2% of seed total P, in contrast to 74.7% of seed total P in fraction. Although phytate decreases in proportion to nonmutant or wild-type control, Js-12-WT. The inorganic portion of fermentation time, in chapati bread, the phytate concenseed P was increased from 9.1% in Js-12-WT to 50.1% in Js-12-LPA, tration was only 18 to 24% lower than the concentration with little effect on total seed P. Weight distributions among milling fractions were similar for the Js-12-LPA and Js-12-WT genotypes. in the whole-wheat flour from which it was prepared The low phytic acid trait altered the distribution of total P within (Anjum et al., 2002). The phytate concentration in chathe kernel, increasing the P content of the central endosperm and pati breads was sixto nine-fold higher than in breads decreasing the P content of the bran. The low phytic acid trait deprepared from straight-grade flour. However, mineral creased the phytic acid concentration in the bran by 43% and increased concentrations generally were significantly higher in the inorganic P concentration in the bran nearly four-fold. Inheritance whole-grain flour than in straight-grade flour, which data of F2 and F4:6 families was inconsistent with a single-gene mutation might offset the effect of phytate on human nutrition. and suggests the involvement of two or more genes. This low phytic Human bioavailability studies will be necessary to conacid wheat mutant is a genetic resource for studying the biology of firm this hypothesis. seed phytic acid metabolism and wheat quality improvement. 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), M is ubiquibarley (Hordeum vulgare L.; Larson et al., 1998; Rastous in eukaryotic cells, where it is typically the mussen and Hatzack, 1998), rice (Oryza sativa L.; Larmost abundant inositol phosphate (Ins P) (Sasakawa et son et al., 2000), and soybean [Glycine max (L.) Merr.; al., 1995). First observed as an abundant P-containing Hitz et al., 2002; Wilcox et al., 2000]. These low phytic compound in seeds, Ins P6 in plant species is thus reacid (lpa) mutants affect the partitioning of P into phytic ferred to as “phytic acid” in agronomic literature (Cosacid, inorganic phosphorus (Pi), and Ins P’s with five or grove, 1980). Applied interest in seed phytic acid is fewer P esters (Raboy, 2002). The lpa mutations are the result of its role in human and livestock nutritional divided into two groups, designated lpa1 and lpa2. Phyquality, as well as P management in integrated agricultic acid P reductions in lpa1 mutants are counterbaltural production systems. According to Shears (2001) anced by molar-equivalent increases in Pi. In contrast, and Raboy (1997), Ins P6 is a major pool in both P and phytic acid P reductions in lpa2 mutants are counterbalIns P metabolism. Phytic acid P typically represents anced by increases in both Pi and non-Ins P6 Ins P’s from 65 to 85% of seed total P and 90% of free Ins (Ins P’s of lower phosphorylation). The isolation and polyphosphates (Raboy, 1997). Trace levels ( 10% of characterization of seed P and Ins P phenotype of a total Ins P) of Ins tris-, tetrakis-, and pentakisphosphates heritable wheat lpa1-like mutant is described here. (Ins P’s with three, four, or five phosphomonoesters, respectively), as well as pyrophosphate-containing Ins P’s more highly phosphorylated than Ins P6, also are observed MATERIALS AND METHODS in mature, wild-type seeds (Dorsch et al., 2003). Mutant Isolation In humans, diets high in phytate can significantly deSeed of the breeding line A95631S-Js-12 was mutagenized with 2% EMS. A95631S-Js-12 has the pedigree ‘Kanto 79’/ M. Guttieri, D. Bowen, and E. Souza, Univ. of Idaho, Aberdeen 2*IDO488. IDO488 is a soft white spring wheat breeding line Research and Extension Center, P.O. Box 870, Aberdeen, ID 83210; with the pedigree PI 294994/4*‘Centennial’. M1 plants were J. Dorsch, BASF Corp., Research Triangle Park, NC 27709; V. Raboy, grown in the greenhouse. Approximately 50% of the M1 seeds USDA-ARS Small Grains and Potato Research Unit, P.O. Box 607, either did not germinate or senesced without setting seed. An Aberdeen, ID 83210; J. Dorsch is a former postdoctoral fellow of the USDA-ARS. Research supported by the Idaho Agric. Exp. Stn. M2 row was planted in the field from each of 562 M1 greenProject IDA1222, Manuscript no. 3725. Received 1 May 2003. *Corresponding author (esouza@uidaho.edu). Abbreviations: AACC, American Association of Cereal Chemistry; EMS, ethyl-methanesulfonate; HIP, high inorganic phosphate; Ins P, Published in Crop Sci. 44:418–424 (2004).  Crop Science Society of America inositol phosphate; Ins P6, myo-inositol-1,2,3,4,5,6-hexakisphosphate; lpa, low phytic acid; Pi, inorganic phosphorus. 677 S. Segoe Rd., Madison, WI 53711 USA

The ABCs of low-phytate crops

The nutritional quality of maize and soybean seeds is improved by embryo-specific silencing of an ABC transporter.

Linkage mapping of maize and barley myo-inositol 1-phosphate synthase DNA sequences : correspondence with a low phytic acid mutation

Abstract We sequenced and genetically mapped the myo-inositol 1-phosphate synthase (MIPS) genes of maize (Zea mays L.) and barley (Hordeum vulgare L). Our objective was to determine whether the genetic map positions of these MIPS loci correspond with the location of the low phtyic acid 1 (lpa1) mutations that were previously identified in maize and barley. Seven MIPS-homologous sequences were mapped to positions on maize chromosomes 1S, 4L, 5S, 6S, 8L, 9S and 9L, and a similar number of divergent MIPS sequences were amplified from maize. To the extent that we can compare across different genetic mapping populations, the position of the MIPS gene on maize chromosome 1S is identical to the location of the maize lpa1 mutation. However, only one MIPS sequence was identified in barley and this gene was mapped to a locus on chromosome 4H that is separate from the barley lpa1 mutation on chromosome 2H. Although several RFLP markers linked to the barley MIPS gene on chromosome 4H also detect loci near barley lpa1 on chromosome 2H, our experiments failed to reveal a second MIPS gene that could be associated with the barley lpa1 mutation. Therefore, genetic mapping results from this study support the MIPS candidate-gene hypothesis for maize lpa1, but do not support the MIPS candidate-gene-hypothesis for barley lpa1. These opposing results contradict the hypothesis that maize lpa1 and barley lpa1 are mutations of orthologous genes, which is suggested by the similar biochemical phenotypes of these mutants. Yet, comparisons of RFLP mapping studies show loci that are homologous between maize chromosome 1S, barley chromosome 4H and barley chromosome 2H, including regions flanking the respective MIPS and/or lpa1 loci. This putative relationship, between the regions flanking the lpa1 mutations on maize 1S and barley 2H, also supports the assertion that these mutations are orthologous despite contradictory results between our maize and barley candidate-gene experiments.

The Effects of Inorganic Nitrogen form and CO2 Concentration on Wheat Yield and Nutrient Accumulation and Distribution

Inorganic N is available to plants from the soil as ammonium (NH4+) and nitrate (NO3-). We studied how wheat grown hydroponically to senescence in controlled environmental chambers is affected by N form (NH4+ vs. NO3−) and CO2 concentration (“subambient,” “ambient,” and “elevated”) in terms of biomass, yield, and nutrient accumulation and partitioning. Wheat supplied with NH4+ as a sole N source had the strongest response to CO2 concentration. Plants exposed to subambient and ambient CO2 concentrations typically had the greatest biomass and nutrient accumulation under both N forms. In general NH4+-supplied plants had higher concentrations of total N, P, K, S, Ca, Zn, Fe, and Cu, while NO3--supplied plants had higher concentrations of Mg, B, Mn, and NO3- - N. NH4+-supplied plants contained amounts of phytate similar to NO3−-supplied plants but had higher bioavailable Zn, which could have consequences for human health. NH4+-supplied plants allocated more nutrients and biomass to aboveground tissues whereas NO3+-supplied plants allocated more nutrients to the roots. The two inorganic nitrogen forms influenced plant growth and nutrient status so distinctly that they should be treated as separate nutrients. Moreover, plant growth and nutrient status varied in a non-linear manner with atmospheric CO2 concentration.

Neither a Zinc Supplement nor Phytate-Reduced Maize nor Their Combination Enhance Growth of 6- to 12-Month-Old Guatemalan Infants

After age 6 mo, the combination of breast-feeding and unfortified plant-based complementary feeding provides inadequate zinc (Zn). Additionally, high phytate intakes compromise the bioavailability of zinc. Our principal objective in this randomized controlled, doubly masked trial was to determine the effect of substituting low-phytate maize, a daily 5-mg zinc supplement, or both, in infants between ages 6-12 mo on impaired linear growth velocity, a common feature of zinc deficiency. In the Western Highlands of Guatemala, 412 infants were randomized to receive low-phytate or control maize. Within each maize group, infants were further randomized to receive a zinc supplement or placebo. Length, weight, and head circumference were measured at 6, 9, and 12 mo of age. There were no significant differences between the 2 maize groups or between the Zn supplement and placebo groups and no treatment interaction was observed for length-for-age (LAZ), weight-for-length (WLZ) or head circumference Z-scores. Overall mean (+/- SD) Z-scores at 6 mo for combined treatment groups were: LAZ, -2.1 +/- 1.1; WLZ, 0.7 +/- 1.0; and head circumference Z-score, -0.7.0 +/- 1.0. At 12 mo, these had declined further to: LAZ, -2.5 +/- 1.1; WLZ, -0.0 +/- 0.9; and head circumference Z-score, -0.9 +/- 1.1; 83.3% were stunted and 2% were wasted. Low linear growth in older Guatemalan infants was not improved with either low-phytate maize or a daily 5-mg zinc supplement. Low contribution of maize to the complementary food of the infants negated any potential advantage of feeding low-phytate maize.