Myo-inositol 1-phosphate Synthase Research Articles

Primary responses to salt stress in a halophyte, smooth cordgrass (Spartina alterniflora Loisel.)

The response of a grass halophyte Spartina alterniflora at early stages of salt stress was investigated through generation and systematic analysis of expressed sequence tags (ESTs) from both leaf and root tissues. Random EST sequencing produced 1,227 quality ESTs, which were clustered into 127 contigs, and 368 were singletons. Of the 495 unigenes, 27% represented genes for stress response. Comparison of the 368 singletons against the Oryza sativa gene index showed that >85% of these genes had similarity with the rice unigenes. Moreover, the phylogenetic analysis of an EST similar to myo-inositol 1-phosphate synthase of Spartina and some selected grasses and halophytes showed closeness of Spartina with maize and rice. Transcript abundance analysis involving eight known genes of various metabolic pathways and nine transcription factor genes showed temporal and tissue-dependent variation in expression under salinity. Reverse northern analysis of a few selected unknown and ribosomal genes exhibited much higher abundance of transcripts in response to salt stress. The results provide evidence that, in addition to several unknown genes discovered in this study, genes involved in ion transport, osmolyte production, and house-keeping functions may play an important role in the primary responses to salt stress in this grass halophyte.

Protection mechanisms in the resurrection plant Xerophyta viscosa: cloning, expression, characterisation and role of XvINO1, a gene coding for a myo-inositol 1-phosphate synthase.

We have used reverse transcription-PCR coupled with 5'- and 3'-RACE to isolate a full length INO1 cDNA (1692 bp with an ORF of 1530) from the resurrection plant Xerophyta viscosa Baker. XvINO1 encodes 510 amino acids, with a predicted MW of 56.7kD and contains four sequence motifs that are highly conserved in plant myo-inositol-1-phosphate synthases (MIPS, EC5.5.1.4), the enzyme that catalyses the first step in the formation of myo-inositol (Ino). Northern and western analyses show that the transcript and protein are constitutively present in leaves but their expression increases, temporarily, in response to both accumulative salt stress (~300 mM NaCl) and desiccation (to 5% relative water content). Leaf Ino concentration increases 40-fold during the first 6 h of salt stress, and levels of this and other carbohydrates (galactinol, sucrose, raffinose, stachyose and hexoses) remain elevated relative to control leaves for the duration of salt stress treatment. The timing and pattern of accumulation of these carbohydrates differ under desiccation stress and we propose that they perform different functions in the respective stresses. These are elaborated in discussion of our data.

Corrigendum to: Protection mechanisms in the resurrection plant Xerophyta viscosa: cloning, expression, characterisation and role of XvINO1, a gene coding for a myo-inositol 1-phosphate synthase.

We have used reverse transcription-PCR coupled with 52- and 32-RACE to isolate a full length INO1 cDNA (1692bp with an ORF of 1530) from the resurrection plant Xerophyta viscosa Baker. XvINO1 encodes 510 amino acids, with a predicted MW of 56.7kD and contains four sequence motifs that are highly conserved in plant myo-inositol-1-phosphate synthases (MIPS, EC5.5.1.4), the enzyme that catalyses the first step in the formation of myo-inositol (Ino). Northern and western analyses show that the transcript and protein are constitutively present in leaves but their expression increases, temporarily, in response to both accumulative salt stress (~300mM NaCl) and desiccation (to 5% relative water content). Leaf Ino concentration increases 40-fold during the first 6h of salt stress, and levels of this and other carbohydrates (galactinol, sucrose, raffinose, stachyose and hexoses) remain elevated relative to control leaves for the duration of salt stress treatment. The timing and pattern of accumulation of these carbohydrates differ under desiccation stress and we propose that they perform different functions in the respective stresses. These are elaborated in discussion of our data.

Synthesis of Cyclophospho‐Glucoses and Glucitols.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Human MIP synthase splice variants in bipolar disorder

Alternative splicing allows the production of multiple gene products with different functions from a given sequence, affecting cellular function control. Tissue-specific splicing is most prevalent in the brain. We therefore investigate whether splice variants contribute to complex psychiatric disorders. A database search suggested that the myo-inositol-1-phosphate (MIP) synthase gene, possibly involved in pathophysiology of bipolar disorder, has splice variants. Human RNA was purified from lymphocytes and postmortem brain. MIP synthase alternative splice variants were amplified using reverse transcription-polymerase chain reaction. The bioinformatics finding was confirmed in both tissues. No difference in lymphocyte MIP synthase mRNA splice-variant levels was found between bipolar patients and controls. However, patients with family history of a major psychiatric disorder had significantly higher levels of the variant lacking exons 3 and 4 versus patients with no family history and controls. As alternative splicing may be a mechanism by which the approximately 30,000 genes are amplified in mammalian brain, further studies with other candidate genes for psychiatric disorders are needed.

Expression pattern of inositol phosphate-related enzymes in rice ( Oryza sativa L.): Implications for the phytic acid biosynthetic pathway

Phytic acid, myo-inositol-hexa kisphosphate (InsP 6), is a storage form of phosphorus in plants. Despite many physiological investigations of phytic acid accumulation and storage, little is known at the molecular level about its biosynthetic pathway in plants. Recent work has suggested two pathways. One is an inositol lipid-independent pathway that occurs through the sequential phosphorylation of 1 d- myo-inositol 3-phosphate (Ins(3)P). The second is a phospholipase C (PLC)-mediated pathway, in which inositol 1,4,5-tris-phosphate (Ins(1,4,5)P 3) is sequentially phosphorylated to InsP 6. We identified 12 genes from rice ( Oryza sativa L.) that code for the enzymes that may be involved in the metabolism of inositol phosphates. These enzymes include 1 d- myo-inositol 3-phosphate synthase (MIPS), inositol monophosphatase (IMP), inositol 1,4,5-tris-phosphate kinase/inositol polyphosphate kinase (IPK2), inositol 1,3,4,5,6-penta kisphosphate 2-kinase (IPK1), and inositol 1,3,4-tris kisphosphate 5/6-kinase (ITP5/6K). The quantification of absolute amounts of mRNA by real-time RT-PCR revealed the unique expression patterns of these genes. Outstanding up-regulation of the four genes, a MIPS, an IPK1, and two ITP5/6Ks in embryos, suggested that they play a significant role in phytic acid biosynthesis and that the lipid-independent pathway was mainly active in developing seeds. On the other hand, the up-regulation of a MIPS, an IMP, an IPK2, and an ITP5/6K in anthers suggested that a PLC-mediated pathway was active in addition to a lipid-independent pathway in the anthers.

Myo-inositol-1-phosphate (MIP) synthase inhibition: in-vivo study in rats

Lithium and valproate are the prototypic mood stabilizers and have diverse structures and targets. Both drugs influence inositol metabolism. Lithium inhibits IMPase and valproate inhibits MIP synthase. This study shows that MIP synthase inhibition does not replicate or augment the effects of lithium in the inositol sensitive pilocarpine-induced seizures model. This lack of effects may stem from the low contribution of de-novo synthesis to cellular inositol supply or to the inhibition of the de-novo synthesis by lithium itself.

Generation and characterization of two novel low phytate mutations in soybean (Glycine max L. Merr.)

Phytic acid (PA, myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is important to the nutritional quality of soybean meal. Organic phosphorus (P) in PA is indigestible in humans and non-ruminant animals, which affects nutrition and causes P pollution of ground water from animal wastes. Two novel soybean [(Glycine max L. (Merr.)] low phytic acid (lpa) mutations were isolated and characterized. Gm-lpa-TW-1 had a phytic acid P (PA-P) reduction of 66.6% and a sixfold increase in inorganic P (Pi), and Gm-lpa-ZC-2 had a PA-P reduction of 46.3% and a 1.4-fold increase in Pi, compared with their respective non-mutant progenitor lines. The reduction of PA-P and increase of Pi in Gm-lpa-TW-1 were molar equivalent; the decrease of PA-P in Gm-lpa-ZC-2, however, was accompanied by the increase of both Pi and lower inositol phosphates. In both mutant lines, the total P content remained similar to their wild type parents. The two lpa mutations were both inherited in a single recessive gene model but were non-allelic. Sequence data and progeny analysis indicate that Gm-lpa-TW-1 lpa mutation resulted from a 2 bp deletion in the soybean D: -myo-inositol 3-phosphate synthase (MIPS1 EC 5.5.1.4) gene 1 (MIPS1). The lpa mutation in Gm-lpa-ZC-2 was mapped on LG B2, closely linked with microsatellite loci Satt416 and Satt168, at genetic distances of approximately 4.63 and approximately 9.25 cM, respectively. Thus this mutation probably represents a novel soybean lpa locus. The seed emergence rate of Gm-lpa-ZC-2 was similar to its progenitor line and was not affected by seed source and its lpa mutation. However, Gm-lpa-TW-1 had a significantly reduced field emergence when seeds were produced in a subtropic environment. Field tests of the mutants and their progenies further demonstrated that the lpa mutation in Gm-lpa-ZC-2 does not negatively affect plant yield traits. These results will advance understanding of the genetic, biochemical and molecular control of PA synthesis in soybean. The novel lpa mutation in Gm-lpa-ZC-2, together with linked simple sequence repeat (SSR) markers, will be of value for breeding productive lpa soybeans, with meal high in digestible Pi eventually to improve animal nutrition and lessen environmental pollution.

Synthesis of cyclophospho-glucoses and glucitols

The syntheses of cyclophosphodiesters of 5-C-(hydroxymethyl)-hexoses and hexitols and of 6-C-(hydroxymethyl)-hexoses are reported, along with that of 6-deoxy- gluco-heptose 7-phosphate. These compounds proved to be reasonable substrate mimics and show inhibitory activity against human d- myo-inositol 3-phosphate synthase.

The development of genetically modified maize for abiotic stress tolerance

Drought is one of the most serious abiotic stresses affecting crops in Africa. Our group has been addressing the problem using genes obtained from the resurrection plant, Xerophyta viscosa. This South African indigenous monocot, which grows in cracks in rocks in the Drakensberg range of mountains in KwaZulu-Natal province, can tolerate losses of up to 95% relative water content. Upon rehydration, it can “resurrect” within 72 h. It can also tolerate high and low temperatures ranging from 40 °C to below freezing. We have used genes from this plant to transform maize and the model plant, Arabidopsis thaliana, to determine which could be used to confer drought resistance. XvSAP1 codes for a signalling membrane hydrophobic protein with little similarity to any protein in gene banks. It was transformed into A. thaliana and constitutive expression led to higher growth rates during osmotic, high salinity and high temperature stress compared with control plants. Non-transgenic plants had shorter roots, leaf expansion was inhibited and leaves were more chlorotic than those of the transgenic plants. This gene has been transformed into maize and results of exposure to abiotic stresses will be presented. XvAld1 codes for an aldose reductase that converts glucose to sorbitol. Transgenic A. thaliana were more tolerant to osmotic stress, high temperatures and salinity than non-transgenic controls. XvPrx2 encodes a type II peroxiredoxin which is induced in X. viscosa by dehydration, low temperature and ABA. Transgenic maize plants have been made carrying this gene and results of exposure to abiotic stresses will be presented. XvPer1 encodes a 1-Cys peroxiredoxin which is induced in X. viscosa by dehydration, heat, high light intensity and salinity. In initial experiments, transgenic maize expressing this gene showed some tolerance to high light intensity but this was not maintained. Other X. viscosa genes under study include XvERD15 (early-responsive to dehydration), XvINO1 coding for Myo-inositol 1-phosphate synthase, XvGolS coding for galactinol synthase, XvVHA-c″1 encoding a vacuolar H+ATPase subunit c″. The status of their studies will be presented. Future work will include the transformation of maize with XvALD, the generation of more maize plants expressing XvSAP1 and XvPrx2. These will be driven by a stress inducible promoter.

Localization of myo-inositol phosphate synthase (GmMIPS-1) during the early stages of soybean seed development

As a precursor to a large variety of compounds, myo-inositol is a central molecule required for cell metabolism and plant growth. The de novo synthesis of myo-inositol requires the activity of the enzyme D-myo-inositol-3-phosphate synthase (MIPS). MIPS cDNAs encoding one or more isoforms have been cloned from a number of species, nevertheless, little is known about the regulation of MIPS expression in developing seed. Seed-specific expression of a soybean isoform (GmMIPS-1) has been demonstrated, but tissue-specific localization during embryo development has not been reported. Using immunolocalization techniques, a specialized area of GmMIPS-1 expression was identified in the outer integumentary layer during early soybean seed development. In addition, localization data provided evidence that MIPS was associated with oxalate crystal idioblasts.

Variation in the enzyme activity and gene expression of myo-inositol-3-phosphate synthase and phytate accumulation during seed development in common bean (Phaseolus vulgaris L.)

The most abundant form in which phosphorus occurs in seeds is phytate (myo-inositol hexakisphosphate), mostly known as an antinutrient for animals, given its ability to complex proteins and minerals, despite its antioxidant and anticarcinogenic properties. However, phytate synthesis is still poorly understood, both in terms of its regulation and metabolic route, and relatively few works have addressed the control mechanism of phytate accumulation during seed development. Aiming at understanding the control mechanism of phytate synthesis, we examined myo-inositol-3-phosphate synthase (MIPS) (EC 5.5.1.4) activity and gene expression during seed development of common bean. Phytate concentration was low at the initial stage of seed development, coinciding with a period of the most intense seed metabolism, but followed by a period of high enzymatic activity and gene expression of MIPS when a decrease in its specific activity and transcription was detected throughout seed development until 20 days after flowering; however, the specific activity of MIPS dropped more expressively than the gene expression, matching with higher phytate concentration. Hence, we show that there is evidence of one control point regulating phytate synthesis with MIPS enzyme.

Diurnal expression of five protein phosphatase type 2C genes in the common ice plant, Mesembryanthemum crystallinum.

The common ice plant, Mesembryanthemum crystallinum L., is a eu-halophytic model species with an environmental stress-initiated switch from C3 photosynthesis to crassulacean acid metabolism (CAM). Phosphoenolpyruvate carboxylase activity in 6-week-old plants exposed to salt stress for 5 days was ~15-fold higher than before stress, indicating the salinity-dependent induction of the C3 to CAM transition. Five plant protein phosphatase type 2C (PP2C) genes were cloned, representative of five of the 10 plant PP2C sub-families. We measured mRNA levels of these PP2Cs and of myo-inositol 1-phosphate synthase (Inps1) in 6-week-old plants before (C3) and after (CAM) salt stress. Remarkably, four PP2C genes and Inps1 were expressed with a diurnal fluctuation in plants in C3 mode. After salt-induced CAM induction, the six genes were expressed with more prominent fluctuations than before stress, suggesting that these PP2C genes may be involved in the diurnal regulation of protein phosphorylation in CAM. Under continuous light treatment the expression of two PP2C genes continued to fluctuate, indicating that their expression is controlled by circadian rhythm.

Molecular breeding for transgenic rice with low-phytic-acid phenotype through manipulating myo-inositol 3-phosphate synthase gene

In most plant seeds, phosphorus is stored primarily as myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP6; phytic acid). Reducing the phytic acid content of seeds is a major breeding target, both to increase the availability of mineral nutrients and to decrease the environmental load of phosphorus. The first step in phytic acid biosynthesis and inositol metabolism is catalyzed by 1d-myo-inositol 3-phosphate (Ins(3)P1) synthase. In this study, we aimed to reduce phytic acid levels in rice seeds by manipulating the expression of the rice Ins(3)P1 synthase gene RINO1 using transgenic methods. RINO1 cDNA was transformed into rice plants in the antisense orientation under the control of the rice major storage protein glutelin GluB-1 promoter. The T4 generation of a stable transgenic line that contained four copies of the transgene showed little morphological differences compared to non-transgenic rice. In the T5 seeds of this line, severe reductions in RINO1 protein levels were observed during the late maturing stages of ripening. Most of the T5 seeds contained higher amounts of inorganic phosphates (Pi), without a reduction in total phosphorus levels, compared to non-transgenic seeds. Ion chromatography analysis suggested that the increase in available Pi is accompanied by a molar-equivalent decrease in phytic acid P. The expression patterns of RINO1 and GluB-1 were examined by quantitative real-time reverse transcriptase-polymerase chain-reaction (RT-PCR). Potential strategies for further molecular breeding to reduce phytic acid levels in seeds are discussed.

Introgression of a novel salt-tolerant L- myo-inositol 1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka ( PcINO1) confers salt tolerance to evolutionary diverse organisms

We have previously demonstrated that introgression of PcINO1 gene from Porteresia coarctata (Roxb.) Tateoka, coding for a novel salt-tolerant L- myo-inositol 1-phosphate synthase (MIPS) protein, confers salt tolerance to transgenic tobacco plants (Majee, M., Maitra, S., Dastidar, K.G., Pattnaik, S., Chatterjee, A., Hait, N.C., Das, K.P. and Majumder, A.L. (2004) A novel salt-tolerant L- myo-inositol-1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice: molecular cloning, bacterial overexpression, characterization, and functional introgression into tobacco-conferring salt-tolerance phenotype. J. Biol. Chem. 279, 28539–28552). In this communication we have shown that functional introgression of the PcINO1 gene confers salt-tolerance to evolutionary diverse organisms from prokaryotes to eukaryotes including crop plants albeit to a variable extent. A direct correlation between unabated increased synthesis of inositol under salinity stress by the PcINO1 gene product and salt tolerance has been demonstrated for all the systems pointing towards the universality of the application across evolutionary divergent taxa.

Characterization of an Arabidopsis inositol 1,3,4,5,6-pentakisphosphate 2-kinase (AtIPK1)

The metabolic pathway(s) by which plants synthesize InsP6 (inositol 1,2,3,4,5,6-hexakisphosphate) remains largely undefined [Shears (1998) Biochim. Biophys. Acta 1436, 49-67], while the identities of the genes that encode enzymes catalysing individual steps in these pathways are, with the notable exception of myo-inositol phosphate synthase and ZmIpk [Shi, Wang, Wu, Hazebroek, Meeley and Ertl (2003) Plant Physiol. 131, 507-515], unidentified. A yeast enzyme, ScIPK1, catalyses the synthesis of InsP6 by 2-phosphorylation of Ins(1,3,4,5,6)P5 (inositol 1,3,4,5,6-pentakisphosphate). A human orthologue, HsIPK1, is able to substitute for yeast ScIPK1, restoring InsP6 production in a Saccharomyces cerevisiae mutant strain lacking the ScIPK1 open reading frame (ScIpk1Delta). We have identified an Arabidopsis genomic sequence, AtIPK1, encoding an Ins(1,3,4,5,6)P5 2-kinase. Inclusion of the AtIPK1 protein in alignments of amino acid sequences reveals that human and Arabidopis kinases are more similar to each other than to the S. cerevisiae enzyme, and further identifies an additional motif. Recombinant AtIPK1 protein expressed in Escherichia coli catalysed the synthesis of InsP6 from Ins(1,3,4,5,6)P5. The enzyme obeyed Michaelis-Menten kinetics with an apparent V(max) of 35 nmol x min(-1) x (mg of protein)(-1) and a K(m) for Ins(1,3,4,5,6)P5 of 22 microM at 0.4 mM ATP. RT (reverse transcriptase)-PCR analysis of AtIPK1 transcripts revealed that AtIPK1 is expressed in siliques, leaves and cauline leaves. In situ hybridization experiments further revealed strong expression of AtIPK1 in male and female organs of flower buds. Expression of AtIPK1 protein in an ScIpk1Delta mutant strain restored InsP6 production and rescued the temperature-sensitive growth phenotype of the yeast.

The Maize lpa241 Mutation Causes a Remarkable Variability of Expression and Some Pleiotropic Effects

Phytic acid is a nearly ubiquitous component of plant seeds, supplying both phosphate (P) and cations during germination. However, during digestion, the phytic acid form of P is not bioavailable for monogastric animals. A possible solution to this problem is the isolation of cereal mutants accumulating less phytic P and more free P and cations in the seed (low phytic acid, lpa). In a previously published study, we described a single, recessive lpa mutation (named lpa241) in maize (Zea mays L) mapping on chromosome 1S in the same location of the lpa1‐1 mutant, showing a decrease in the expression of the first enzyme of phytic acid pathway, myo‐inositol‐3‐phosphate synthase (MIPS). The goal of this study was to gain further information about the nature of lpa241 mutation as well as its effects on seed physiology and plant growth. We present new results of genetic, molecular, and histological analyses demonstrating that lpa241 is indeed allelic to lpa1‐1, its MIPS1S coding region does not show a relevant molecular lesion, is accompanied by a strong reduction in globoid number and size, and shows highly variable expression causing some negative pleiotropic effects related to embryo development and size, germination rate, seedling growth rate, and ear size. The origin and the consequences of the lpa241 mutation led us to advance the hypothesis of an epigenetic nature of the mutation and to emphasize the role of myo‐inositol and its derivatives during seed maturation, germination, and seedling growth.

Valproate decreases inositol biosynthesis

Lithium and valproate (VPA) are used for treating bipolar disorder. The mechanism of mood stabilization has not been elucidated, but the role of inositol has gained substantial support. Lithium inhibition of inositol monophosphatase, an enzyme required for inositol recycling and de novo synthesis, suggested the hypothesis that lithium depletes brain inositol and attenuates phosphoinositide signaling. Valproate also depletes inositol in yeast, Dictyostelium, and rat neurons. This raised the possibility that the effect is the result of myo-inositol-1-phosphate (MIP) synthase inhibition. Inositol was measured by gas chromatography. Human prefrontal cortex MIP synthase activity was assayed in crude homogenate. INO1 was assessed by Northern blotting. Growth cones morphology was evaluated in cultured rat neurons. We found a 20% in vivo reduction of inositol in mouse frontal cortex after acute VPA administration. As hypothesized, inositol reduction resulted from decreased MIP synthase activity: .21-.28 mmol/LVPA reduced the activity by 50%. Among psychotropic drugs, the effect is specific to VPA. Accordingly, only VPA upregulates the yeast INO1 gene coding for MIP synthase. The VPA derivative N-methyl-2,2,3,3,-tetramethyl-cyclopropane carboxamide reduces MIP synthase activity and has an affect similar to that of VPA on rat neurons, whereas another VPA derivative, valpromide, poorly affects the activity and has no affect on neurons. The rate-limiting step of inositol biosynthesis, catalyzed by MIP synthase, is inhibited by VPA; inositol depletion is a first event shown to be common to lithium and VPA.

E2F1 regulation of the human myo-inositol 1-phosphate synthase ( ISYNA1) gene promoter

Human myo-inositol 1-phosphate synthase (IP synthase; E.C. 5.5.1.4), encoded by ISYNA1, catalyzes the de novo synthesis of inositol 1-phosphate from glucose 6-phosphate. It is a potential target for mood-stabilizing drugs such as lithium and valproate. But, very little is known about the regulation of human IP synthase. Here, we have characterized the minimal promoter of ISYNA1 and show that it is upregulated by E2F1. Upregulation occurs in a dose-dependent fashion and can be suppressed by ectopic expression of Rb. EMSA and antibody supershift analysis identified a functional E2F binding motif at −117. Complex formation at this site was competed by an excess of unlabeled Sp1 oligo consistent with the −117 E2F site overlapping an Sp1 motif. Because the −117 E2F motif is not a high-affinity binding site, we propose that the upregulation of ISYNA1 occurs through the cooperative interaction of several low-affinity E2F binding motifs present in the minimal promoter.

Expression and nucleotide sequence of an INS (3) P1 synthase gene associated with low-phytate kernels in maize (Zea mays L.).

Most of the phosphorus (P) in maize (Zea mays L.) kernels is in the form of phytic acid. A low phytic acid (lpa) maize mutant, lpa1-1, displays levels reduced by 66%. A goal of genetic breeding is development of low phytic acid feedstocks to improve P nutrition of nonruminant animals and reduce the adverse environmental impacts of excess P in manure. The genetic basis of the lpa1-1 mutation is not known, but previous genetic mapping has shown both the mutant phenotype and the Ins (3) P(1) synthase (MIPS) gene, which encodes the first enzyme, myo-inositol phosphate synthase, in the phytic acid biosynthetic pathway, map to the same chromosomal region in maize. Research was conducted to determine the expression of the MIPS gene in lpa1-1 and wild-type kernels with similar genetic backgrounds and to ascertain if variation in the MIPS coding sequence could be inferred to be the basis of the mutation. MIPS enzyme activity determined by TLC was reduced 2-3-fold in mutant kernels. RT-PCR-based experiments using primers specific for the 1S-MIPS sequence indicated gene expression was reduced 50-60% in the mutant. Sequence analysis of the MIPS genomic sequence revealed 10 exons and 9 introns that are identical in both mutant and wild-type developing kernels. These findings support an association between reduced MIPS gene activity and low phytic acid content, but additional research should examine the promoter, the 5'UTR, or transcriptional controlling elements of the MIPS gene to ascertain the possible presence of a genetic lesion in those regions.