A nonsense mutation in a putative sulphate transporter gene results in low phytic acid in barley

Low phytic acid grains can provide a solution to dietary micronutrient deficiency and environmental pollution. A low phytic acid 1-1 (lpa1-1) barley mutant was identified using forward genetics and the mutant gene was mapped to chromosome 2HL. Comparative genomic analysis revealed that the lpa1-1 gene was located in the syntenic region of the rice Os-lpa-MH86-1 gene on chromosome 4. The gene ortholog of rice Os-lpa-MH86-1 (designated as HvST) was isolated from barley using polymerase chain reaction and mapped to chromosome 2HL in a doubled haploid population of Clipper×Sahara. The results demonstrate the collinearity between the rice Os-lpa-MH86-1 gene and the barley lpa1-1 region. Sequence analysis of HvST revealed a single base pair substitution (C→T transition) in the last exon of the gene in lpa1-1 (M422), which resulted in a nonsense mutation. These results will facilitate our understanding of the molecular mechanisms controlling the low phytic acid phenotype and assist in the development of a diagnostic marker for the selection of the lpa1-1 gene in barley.     

The maize low-phytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene

Reduced phytic acid content in seeds is a desired goal for genetic improvement in several crops. Low-phytic acid mutants have been used in genetic breeding, but it is not known what genes are responsible for the low-phytic acid phenotype. Using a reverse genetics approach, we found that the maize (Zea mays) low-phytic acid lpa2 mutant is caused by mutation in an inositol phosphate kinase gene. The maize inositol phosphate kinase (ZmIpk) gene was identified through sequence comparison with human and Arabidopsis Ins(1,3,4)P(3) 5/6-kinase genes. The purified recombinant ZmIpk protein has kinase activity on several inositol polyphosphates, including Ins(1,3,4)P(3), Ins(3,5,6)P(3), Ins(3,4,5,6)P(4), and Ins(1,2,5,6)P(4). The ZmIpk mRNA is expressed in the embryo, the organ where phytic acid accumulates in maize seeds. The ZmIpk Mutator insertion mutants were identified from a Mutator F(2) family. In the ZmIpk Mu insertion mutants, seed phytic acid content is reduced approximately 30%, and inorganic phosphate is increased about 3-fold. The mutants also accumulate myo-inositol and inositol phosphates as in the lpa2 mutant. Allelic tests showed that the ZmIpk Mu insertion mutants are allelic to the lpa2. Southern-blot analysis, cloning, and sequencing of the ZmIpk gene from lpa2 revealed that the lpa2-1 allele is caused by the genomic sequence rearrangement in the ZmIpk locus and the lpa2-2 allele has a nucleotide mutation that generated a stop codon in the N-terminal region of the ZmIpk open reading frame. These results provide evidence that ZmIpk is one of the kinases responsible for phytic acid biosynthesis in developing maize seeds.     

A paramutation phenomenon is involved in the genetics of maize low phytic acid1-241 (lpa1-241) trait

So far, in maize, three classes of mutants involved in phytic acid biosynthesis have been isolated: lpa1, lpa2 and lpa3. In 2007, a gene tagging experiment performed by Shi et al. found that mutations in ZmMRP4 (multidrug resistance-associated proteins 4) gene cause lpa1 phenotype. In previous studies, we isolated and described a single recessive lpa mutation (originally named lpa241), which was allelic to the lpa1-1 mutant, and was consequently renamed lpa1-241. It showed a decrease in the expression of the myo-inositol (Ins)-3-phosphate synthase gene (mips1S). In this study, we present genetic and molecular analyses of the lpa1-241 mutation that indicate an epigenetic origin of this trait, that is, a paramutagenic interaction that results in meiotically heritable changes in ZmMRP4 gene expression, causing a strong pleiotropic effect on the whole plant. The use of a 5-Azacytidine treatment provided data suggesting an association between gene methylation and the lpa1-241 phenotype. To our knowledge, this is the first report of a paramutagenic activity not involving flavonoid biosynthesis in maize, but regarding a key enzyme of an important metabolic pathway in plants.     

Molecular marker development and linkage analysis in three low phytic acid barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) mutant lines

Phytate is the primary form of phosphorus found in mature cereal grain. This form of phosphorus is not available to monogastric animals due to a lack of the enzyme phytase in their digestive tract. Several barley low phytic acid (lpa) mutants have been identified that contain substantial decreases in seed phytate accompanied by concomitant increases in inorganic phosphorus. Seed homozygous for low phytic acid 1-1 (lpa1-1) or low phytic acid 2-1 (lpa2-1) has a 50% and 70% decrease in seed phytate respectively. These mutations were previously mapped to chromosomes 2HL and 7HL respectively. The RFLP marker ABC153 located in the same region of 2H was converted to a sequence-characterized-amplified-region (SCAR) marker. Segregation analysis of the CDC McGwire × Lp422 doubled haploid population confirmed linkage between the SCAR marker and the lpa1-1 locus with 15% recombination. A third low phytic acid mutant, M635, has a 75% decrease in phytate. This mutation was located to chromosome 1HL by linkage with an inter-simple sequence repeat (ISSR) based marker (LP75) identified through bulked-segregant analysis, and has been designated lpa3-1. Based on analysis of recombination between marker LP75 and low phytic acid in an additional mutant line M955 (95% phytate decrease), lpa3-1 and the mutation in M955 are in the same region on chromosome 1HL, and may be allelic.     

The maize low-phytic acid 3 encodes a myo-inositol kinase that plays a role in phytic acid biosynthesis in developing seeds

Phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate or Ins P6, is the most abundant myo-inositol phosphate in plant cells, but its biosynthesis is poorly understood. Also uncertain is the role of myo-inositol as a precursor of phytic acid biosynthesis. We identified a low-phytic acid mutant, lpa3, in maize. The Mu-insertion mutant has a phenotype of reduced phytic acid, increased myo-inositol and lacks significant amounts of myo-inositol phosphate intermediates in seeds. The gene responsible for the mutation encodes a myo-inositol kinase (MIK). Maize MIK protein contains conserved amino acid residues found in pfkB carbohydrate kinases. The maize lpa3 gene is expressed in developing embryos, where phytic acid is actively synthesized and accumulates to a large amount. Characterization of the lpa3 mutant provides direct evidence for the role of myo-inositol and MIK in phytic acid biosynthesis in developing seeds. Recombinant maize MIK phosphorylates myo-inositol to produce multiple myo-inositol monophosphates, Ins1/3P, Ins4/6P and possibly Ins5P. The characteristics of the lpa3 mutant and MIK suggest that MIK is not a salvage enzyme for myo-inositol recycling and that there are multiple phosphorylation routes to phytic acid in developing seeds. Analysis of the lpa2/lpa3 double mutant implies interactions between the phosphorylation routes.     

Fine mapping of the rice low phytic acid (Lpa1) locus

Phytic acid is the primary storage form of phosphorus (P) in cereal grains. In addition to being essential for normal seedling growth and development, phytic acid plays an important role in human and animal nutrition. The rice low phytic acid mutation lpa1 results in a 45% reduction in seed phytic acid with a molar equivalent increase in inorganic P. The Lpa1 locus was previously mapped to the long arm of chromosome 2. Using microsatellite markers and a recombinant inbred line population, we fine mapped this locus between the markers RM3542 and RM482, which encompass a region of 135 kb. Additional markers were developed from the DNA sequence of this region. Two of these markers further delimited the locus to a 47-kb region containing eight putative open reading frames. Cloning and molecular characterization of the Lpa1 gene will provide insight into phytic acid biosynthesis in plants. The markers reported here should also be useful in introgressing the low phytic acid phenotype into other rice cultivars.The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1

Phytic acid (myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate or Ins P(6)) typically represents approximately 75% to 80% of maize (Zea mays) seed total P. Here we describe the origin, inheritance, and seed phenotype of two non-lethal maize low phytic acid mutants, lpa1-1 and lpa2-1. The loci map to two sites on chromosome 1S. Seed phytic acid P is reduced in these mutants by 50% to 66% but seed total P is unaltered. The decrease in phytic acid P in mature lpa1-1 seeds is accompanied by a corresponding increase in inorganic phosphate (P(i)). In mature lpa2-1 seed it is accompanied by increases in P(i) and at least three other myo-inositol (Ins) phosphates (and/or their respective enantiomers): D-Ins(1,2,4,5,6) P(5); D-Ins (1,4,5,6) P(4); and D-Ins(1,2,6) P(3). In both cases the sum of seed P(i) and Ins phosphates (including phytic acid) is constant and similar to that observed in normal seeds. In both mutants P chemistry appears to be perturbed throughout seed development. Homozygosity for either mutant results in a seed dry weight loss, ranging from 4% to 23%. These results indicate that phytic acid metabolism during seed development is not solely responsible for P homeostasis and indicate that the phytic acid concentration typical of a normal maize seed is not essential to seed function.     

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

 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. Received: 24 August 1998 / Accepted: 19 December 1998     

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

 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. Received: 22 September 1997 / Accepted: 2 December 1997     

Gene identification and allele-specific marker development for two allelic low phytic acid mutations in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Phytic acid (PA, myo-inositol 1,2,3,4,5,6-hexakisphosphate) is an important anti-nutritional component in cereal and legume grains. PA forms of phosphorus (P) and its salts with micronutrient cations, such as iron and zinc, are indigestible in humans and non-ruminant animals, and hence could affect food/feed nutritional value and cause P pollution of ground water from animal waste. We previously developed a set of low phytic acid (LPA) rice mutants with the aim to increase their nutritional quality. Among them, one line, i.e., Os-lpa-XQZ-1 (hereafter lpa 1-2), was identified to have a mutation allelic to the KBNT lpa 1-1 mutation (hereafter lpa 1-1), which was already delimited to a 47-kb region on chromosome 2. In this study, we searched the candidate gene for these two allelic LPA mutations using T-DNA insertion mutants, mutation detection by CEL I facilitated mismatch cleavage, and gene sequencing. The TIGR locus LOC_Os02g57400 was revealed as the candidate gene hosting these two mutations. Sequence analysis showed that the lpa 1-1 is a single base pair substitution mutation, while lpa 1-2 involves a 1,475-bp fragment deletion. A CAPS marker (LPA1_CAPS) was developed for distinguishing the lpa 1-1 allele from lpa 1-2 and WT alleles, and InDel marker (LPA1_InDel) was developed for differentiating the lpa 1-2 allele from lpa 1-1 and WT ones. Analysis of two populations derived from the two mutants with wild-type varieties confirmed the complete co-segregation of these two markers and LPA phenotype. The LOC_Os02g57400 is predicted to encode, through alternative splicing, four possible proteins that are homologous to the 2-phosphoglycerate kinase reported in hyperthermophilic and thermophilic bacteria. The identification of the LPA gene and development of allele-specific markers are of importance not only for breeding LPA varieties, but also for advancing genetics and genomics of phytic acid biosynthesis in rice and other plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Seed phosphorus and inositol phosphate phenotype of barley low phytic acid genotypes

myo-Inositol-1,2,3,4,5,6-hexakisphosphate (Ins P(6) or "phytic acid") typically represents approximately 75% of the total phosphorus and >80% of soluble myo-inositol (Ins) phosphates in seeds. The seed phosphorus and Ins phosphate phenotypes of four non-lethal barley (Hordeum vulgare L.) low phytic acid mutations are described. In seeds homozygous for M 635 and M 955 reductions in Ins P(6), approximately 75 and >90% respectively, are accompanied by reductions in other Ins phosphates and molar-equivalent increases in Pi. This phenotype suggests a block in supply of substrate Ins. In seeds homozygous for barley low phytic acid 1-1 (lpa1-1), a 45% decrease in Ins P(6) is mostly matched by an increase in Pi but also accompanied by small increases in Ins(1,2,3,4,6)P(5). In seeds homozygous for barley lpa2-1, reductions in seed Ins P(6) are accompanied by increases in both Pi and in several Ins phosphates, a phenotype that suggests a lesion in Ins phosphate metabolism, rather than Ins supply. The increased Ins phosphates in barley lpa2-1 seed are: Ins(1,2,3,4,6)P(5); Ins(1,2,4,6)P(4) and/or its enantiomer Ins(2,3,4,6)P(4); Ins(1,2,3,4)P(4) and/or its enantiomer Ins(1,2,3,6)P(4); Ins(1,2,6)P(3) and/or its enantiomer Ins(2,3,4)P(3); Ins(1,5,6)P(3) and/or its enantiomer Ins(3,4,5)P(3) (the methods used here cannot distinguish between enantiomers). This primarily "5-OH" series of Ins phosphates differs from the "1-/3-OH" series observed at elevated levels in seed of the maize lpa2 genotype, but previous chromosomal mapping data indicated that the maize and barley lpa2 loci might be orthologs of a single ancestral gene. Therefore one hypothesis that might explain the differing lpa2 phenotypes is that their common ancestral gene encodes a multi-functional, Ins phosphate kinase with both "1-/-3-" and "5-kinase" activities. A putative pyrophosphate-containing Ins phosphate, possibly an Ins P(7), was also observed in the mature seed of all barley genotypes except lpa2-1. Barley M 955 indicates that at least for this species, the ability to accumulate Ins P(6) can be nearly abolished while retaining at least short-term ( approximately 1.0 years) viability.     

The synthesis of inositol hexakisphosphate. Characterization of human inositol 1,3,4,5,6-pentakisphosphate 2-kinase

The enzyme(s) responsible for the production of inositol hexakisphosphate (InsP(6)) in vertebrate cells are unknown. In fungal cells, a 2-kinase designated Ipk1 is responsible for synthesis of InsP(6) by phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (InsP(5)). Based on limited conserved sequence motifs among five Ipk1 proteins from different fungal species, we have identified a human genomic DNA sequence on chromosome 9 that encodes human inositol 1,3,4,5,6-pentakisphosphate 2-kinase (InsP(5) 2-kinase). Recombinant human enzyme was produced in Sf21 cells, purified, and shown to catalyze the synthesis of InsP(6) or phytic acid in vitro. The recombinant protein converted 31 nmol of InsP(5) to InsP(6)/min/mg of protein (V(max)). The Michaelis-Menten constant for InsP(5) was 0.4 microM and for ATP was 21 microM. Saccharomyces cerevisiae lacking IPK1 do not produce InsP(6) and show lethality in combination with a gle1 mutant allele. Here we show that expression of the human InsP(5) 2-kinase in a yeast ipk1 null strain restored the synthesis of InsP(6) and rescued the gle1-2 ipk1-4 lethal phenotype. Northern analysis on human tissues showed expression of the human InsP(5) 2-kinase mRNA predominantly in brain, heart, placenta, and testis. The isolation of the gene responsible for InsP(6) synthesis in mammalian cells will allow for further studies of the InsP(6) signaling functions.     

Phenotypic,genetic and molecular characterization of a maize low phytic acid mutant (lpa241)

Phytic acid, myo-inositol 1,2,3,4,5,6-hexakisphosphate, is the major storage compound of phosphorous (P) in plants, predominantly accumulating in seeds (up to 4–5% of dry weight) and pollen. In cereals, phytic acid is deposited in embryo and aleurone grain tissues as a mixed "phytate" salt of potassium and magnesium, although phytates contain other mineral cations such as iron and zinc. During germination, phytates are broken down by the action of phytases, releasing their P, minerals and myo-inositol which become available to the growing seedling. Phytic acid represents an anti-nutritional factor for animals, and isolation of maize low phytic acid (lpa) mutants provides a novel approach to study its biochemical pathway and to tackle the nutritional problems associated with it. Following chemical mutagenesis of pollen, we have isolated a viable recessive mutant named lpa 241 showing about 90% reduction of phytic acid and about a tenfold increase in seed-free phosphate content. Although germination rate was decreased by about 30% compared to wild-type, developement of mutant plants was apparentely unaffected. The results of the genetic, biochemical and molecular characterization experiments carried out by SSR mapping, MDD-HPLC and RT-PCR are consistent with a mutation affecting the MIPS1S gene, coding for the first enzyme of the phytic acid biosynthetic pathway.Communicated by F. Salamini     

Marker-assisted selection for low phytic acid (lpa1-1) with single nucleotide polymorphism marker and amplified fragment length polymorphisms for background selection in a maize backcross breeding programme

The level of phytic acid is difficult to assess in a maize breeding programme, therefore a co-dominant single nucleotide polymorphism (SNP) marker was used to detect the single recessive low phytic acid (lpa1-1) gene in a BC2F1 population developed from a locally adapted tropical normal inbred line (P 16) and CM 32 (lpa1-1 donor). High-resolution melt analysis of the lpa1-1 SNP marker was able to identify 11 homozygous recessive and 17 heterozygote genotypes for the lpa1-1 mutation. The SNP R ² values for the heterozygotes were higher (90.95?C99.59%) than the lpa1-1 recessives (82.81?C99.58%). The selected BC2F1 lines were fingerprinted with six amplified fragment length polymorphism (AFLP) EcoRI/MseI primer combinations to determine the amount of recurrent parent genome present. The 277 AFLP markers were clearly able to differentiate all the BC2F1 lines from each other and the parental controls with a similarity range from 62.12 to 92.15%. It is expected in the BC2 generation to find 87.5% similarity to the recurrent parent, however in this study higher levels of similarity in 13 BC2F1 lines (six heterozygotes and seven homozygous recessive) with 92.15?C83.33% similarity were observed. The use of marker-assisted selection for foreground and background selection greatly increased the efficiency of detection of the homozygous recessive (99.58%) and heterozygous (99.59%) genotypes as well as improving the recovery of the recurrent parent (92.15%) in the BC2F1 generation of the maize backcross breeding programme.     

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

Phytic acid, myo-inositol-hexakisphosphate (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 1D-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 1D-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-pentakisphosphate 2-kinase (IPK1), and inositol 1,3,4-triskisphosphate 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.     

The low phytic acid1-241 (lpa1-241) maize mutation alters the accumulation of anthocyanin pigment in the kernel

The lpa1 mutations in maize are caused by lesions in the ZmMRP4 (multidrug resistance-associated proteins 4) gene. In previous studies (Raboy et al. in Plant Physiol 124:355–368, 2000; Pilu et al. in Theor Appl Genet 107:980–987, 2003a; Shi et al. Nat Biotechnol 25:930–937, 2007), several mutations have been isolated in this locus causing a reduction of phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate, or InsP₆) content and an equivalent increasing of free phosphate. In particular, the lpa1-241 mutation causes a reduction of up to 90% of phytic acid, associated with strong pleiotropic effects on the whole plant. In this work, we show, for the first time to our knowledge, an interaction between the accumulation of anthocyanin pigments in the kernel and the lpa mutations. In fact the lpa1-241 mutant accumulates a higher level of anthocyanins as compared to wild type either in the embryo (about 3.8-fold) or in the aleurone layer (about 0.3-fold) in a genotype able to accumulate anthocyanin. Furthermore, we demonstrate that these pigments are mislocalised in the cytoplasm, conferring a blue pigmentation of the scutellum, because of the neutral/basic pH of this cellular compartment. As a matter of fact, the propionate treatment, causing a specific acidification of the cytoplasm, restored the red pigmentation of the scutellum in the mutant and expression analysis showed a reduction of ZmMRP3 anthocyanins’ transporter gene expression. On the whole, these data strongly suggest a possible interaction between the lpa mutation and anthocyanin accumulation and compartmentalisation in the kernel.     

Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds

Phytic acid in cereal grains and oilseeds is poorly digested by monogastric animals and negatively affects animal nutrition and the environment. However, breeding programs involving mutants with less phytic acid and more inorganic phosphate (P(i)) have been frustrated by undesirable agronomic characteristics associated with the phytic acid-reducing mutations. We show that maize lpa1 mutants are defective in a multidrug resistance-associated protein (MRP) ATP-binding cassette (ABC) transporter that is expressed most highly in embryos, but also in immature endosperm, germinating seed and vegetative tissues. Silencing expression of this transporter in an embryo-specific manner produced low-phytic-acid, high-Pi transgenic maize seeds that germinate normally and do not show any significant reduction in seed dry weight. This dominant transgenic approach obviates the need for incorporating recessive lpa1 mutations to create maize hybrids with reduced phytic acid. Suppressing the homologous soybean MRP gene also generated low-phytic-acid seed, suggesting that the strategy might be feasible for many crops.     

Effect of myo-inositol(1,4,5)trisphosphate on the hydrolysis of phytic acid by phytase

Phytase is a monomeric enzyme of molecular mass 160 kDa which catalyzes the hydrolysis of phytic acid (D-myo inositol hexakisphosphate, InsP6) in a stepwise manner to myo-inositol. The enzyme-InsPn (n = 1-6) interaction at the catalytic site has a dissociation constant in the micro molar range. There also exists in the enzyme, a non-catalytic site specific for InsP3 with dissociation constant in the nano molar range. We have probed the effect of the high affinity InsP3 binding on the dissociation constant (Kd) of the phytase-InsP6 interaction and the kinetics of hydrolysis. These studies demonstrate the effect exerted by the high affinity InsP3 binding on the catalytic site of the enzyme.