Approximately 75% of a mature seed's total P is found as a single compound called phytic acid, formally referred to as myo-inositol 1,2,3,4,5,6-hexakisphosphate or Ins P6. Humans and non- ruminants do not effectively digest grain or legume-derived phytic acid, excreting essentially all phytic acid consumed. This phenomenon has several undesirable outcomes both in terms of human nutrition and animal production. We will focus on the problem of phytic acid in animal production, since it is most relevant in terms of barley end-use.

In the last decade interest in improving P management throughout agricultural production has grown considerably, largely due to a growing concern over the contribution of fertilizer and waste P runoff to water pollution. In addition, grain-based feeds must be supplemented with an "available" form (in nutritional terms) of P to provide for optimal animal growth. An attractive alternative to P supplementation is an industrially produced enzyme called phytase, which when consumed with the grain breaks down phytic acid in the gut, releasing its P for absorption by the animal. More of the animal's requirement for P is therefore provided by grain P, and less is excreted. However, phytase supplementation represents an added cost to production, and is at best only about 50% effective.

The amount of total P in barley grain (typically in the range of 4 mg P per gm seed) roughly approximates the amount of P required by a growing chicken, pig or fish, if it were in an available form. Our approach to this problem was to use genetics to render a greater fraction of seed total P in an available form. As part of this effort we have isolated several non-lethal barley low phytic acid (lpa) mutants. Seeds homozygous for an lpa mutant contain normal levels of seed total P, but greatly reduced levels of phytic acid P. As a result, seed available P (in terms of animal nutrition) should be greatly increased.

We have isolated more than 20 barley lpa mutants and are in the process of conducting allelism tests to determine the number of loci represented. These mutants appear to fall into two phenotypic classes similar to the first two corn mutants we isolated. In seeds homozygous for a "low phytic acid 2"-like mutation, kernel phytic acid P is reduced; this is accompanied by increases both in inorganic P and lower inositol Ps such as myo-inositol penta and tetraphosphates. The first barley mutant observed to have a stable and heritable phenotype was termed barley low phytic acid 1-1 (lpa1-1), indicating that it is the first recessive allele at the lpa1 locus. Homozygosity for this mutation causes about a 50% reduction in kernel phytic acid P, with no obvious change in kernel total P. The reduction in phytic acid P is matched (in terms of P) by an increase in inorganic P. We have also isolated a number of additional lpa1-like mutants that differ in their extent of phytic acid P reduction/inorganic P increase. Two such mutants (referred to by their "M2" numbers pending allelism tests and mapping analyses) merit attention: M2 635 as a homozygote causes about a 75% reduction in kernel phytic acid, while M2 955 causes about a 95% reduction in kernel phytic acid P. Both the original lpa1-1 and M2 635 have little obvious effect on plant growth and productivity (field trials are in progress). However, homozygosity for M2 955 clearly has a large effect on vegetative growth and productivity. Allelism tests of these lpa1-like mutants are in progress, so we do not know if they represent one or more loci.

Our first barley "low phytic acid 2"-like mutation we termed lpa2-1. Homozygosity for this mutation reduces kernel phytic acid P by about 75%. This is accompanied by increases in inorganic P and a series of lower inositol Ps, the predominant member being Ins 1,2,3,4,6-pentakisphosphate. While this mutation is not lethal, like M2 955 it does have a clear effect on plant growth and function. This observation is relevant because it indicates that the genes perturbed in these mutants have a role in vegetative growth and function, in addition to a role in seed phytic acid pathways. We think the lpa1- like mutations are in genes involved in myo-inositol synthesis and metabolism, a critical early step in the pathway to phytic acid. Since myo-inositol is important to many other biochemical pathways and processes in all cells, it is understandable that some of the lpa1-like mutants perturb the whole plant. However, we believe the lpa2-like mutants are in inositol phosphate metabolism, which occurs later in the pathway to phytic acid. The effect of barley lpa2-1 on the whole plant indicates that the pathway to phytic acid is important to whole-plant processes other than P storage in seeds.

Barley lpa1-1 and lpa2-1 were mapped to chromosome 2 and 7, respectively (Larson et al., 1998). We are interested in the comparative genomics of these lpa loci. For example, we hypothesize that lpa1-1 is a mutation in gene encoding the enzyme myo-inositol 1-phosphate synthase (MIPS). A barley MIPS gene maps to chromosome 4, to a site that corresponds with both a corn MIPS and corn lpa1-1 (Larson and Raboy, 1999). However, the chromosome 2 site of barley lpa1-1 may be ancestrally related to this chromosome 4 site. Through an analysis of these genes and mutant loci we therefore hope to determine the kind of genes involved in these pathways, and their number and distribution in these related cereal genomes. Preliminary evidence does indicate that the corn and barley lpa2 loci are ancestrally related (orthologous). Ultimately we hope to clone these genes, and use a biotechnological approach to this problem area.

Breeding efforts are currently focusing on barley lpa-1, and M2 635. This latter mutant has little apparent effect on plant function, like lpa1-1, but as mentioned above causes a larger reduction in grain phytic acid. Drs. Wesenberg and Bregitzer at USDA-ARS, Aberdeen, ID are crossing this trait into elite hulled and hulless barleys, using these and other mutants. We are also cooperating with Dr. Rossnagel and others in similar efforts.

The first animal nutrition study with a barley lpa mutant was in an aquaculture model (Sugiura et al., 1998). This study found that when trout consumed experimental diets largely consisting largely of grain, waste P was reduced by approximately 50% if that grain was lpa1-1, as compared with wild-type grain. A swine nutrition study was designed so that barley represented the sole source of phytic acid in the diet (Trygve Veum and David Ledoux, Univ. Of Missouri, unpublished results). Pigs were fed lpa1-1 barley, or wild-type barley supplemented with P so that the diet contained the level of available P predicted for lpa1-1. Animal performance and bone strength were similar in pigs consuming the two diets, indicating that the predicted level of available P for lpa1-1 is accurate. Pigs consuming the lpa1-1 ration excreted 55% less P than those consuming wild-type barley. Calcium retention was also improved in pigs consuming the lpa1-1 diet.

While a great deal remains to be studied, these initial studies confirm the potential value of a "low phytic acid" approach to end-use improvement of barleys. We believe that at present, progress in "low phytic acid" research with barley, and the genetic resources we and others (Rasmussen and Hatzack, 1998) are developing, may exceed that which have been developed in corn, rice or any other species.

References:

Larson, S. R., K.A. Young, A. Cook, T.K. Blake, V. Raboy. 1998. Linkage mapping of two mutations that reduce phytic acid content of barley grain. Theor. Appl. Genetics: 97: 141-146. 1998.

Larson, S.R., V. Raboy. 1999. Maize and barley myo-Inositol 1-phosphate synthase DNA sequences and linkage mapping: Correspondence with a low phytic acid mutation. Theor. L.) grain mutants Appl. Genetics: In Press. 1998.

Rasmussen, S.K., F. Hatzack. 1998. Identification of two low-phytate barley (Hordeum vulgare L) grain mutants by TLC and genetic analysis. Hereditas 129: 107-112.

Sugiura, S.H., V. Raboy, K.A. Young, F.M. Dong, R.W. Hardy. 1998. Availability of phosphorus and trace elements in low-phytate varieties of barley and corn for rainbow trout (oncorhynchus mykiss). Aquaculture 170: 285- 296.