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Localization of myo-inositol-1-phosphate synthase to the endosperm in developing seeds of Arabidopsis.

ABSTRACT: Expression and localization of myo-inositol-1-phosphate synthase (MIPS) in developing seeds of Arabidopsis thaliana was investigated. MIPS is an essential enzyme for production of inositol and inositol phosphates via its circularization of glucose-6-phosphate as the initial step. myo-inositol-6-phosphate (InsP(6) or phytic acid) is the predominant form of phosphorus found in seeds and accumulates as a consequence of MIPS action. Three MIPS genes have been identified in Arabidopsis, all of which were expressed not only in siliques but in both leaves and roots. Immunoelectron microscopy using a MIPS antibody showed that MIPS localizes to the cytosol primarily in the endosperm during seed development and not in the embryo. This is consistent with results obtained using fluorescent microscopy and western blot analysis that showed a similar pattern of localization. However, InsP(6), which is the final product of inositol phosphate metabolism, was present mainly in the embryo. This suggests that a complex interaction between the endosperm and embryo occurs during the synthesis and subsequent accumulation of InsP(6) in developing seeds of Arabidopsis.

SUBMITTER: Mitsuhashi N

PROVIDER: S-EPMC2504351 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Localization of myo-inositol-1-phosphate synthase to the endosperm in developing seeds of Arabidopsis.

Mitsuhashi Naoto N Kondo Maki M Nakaune Satoru S Ohnishi Miwa M Hayashi Makoto M Hara-Nishimura Ikuko I Richardson Alan A Fukaki Hidehiro H Nishimura Mikio M Mimura Tetsuro T

Journal of experimental botany 20080704 11

Expression and localization of myo-inositol-1-phosphate synthase (MIPS) in developing seeds of Arabidopsis thaliana was investigated. MIPS is an essential enzyme for production of inositol and inositol phosphates via its circularization of glucose-6-phosphate as the initial step. myo-inositol-6-phosphate (InsP(6) or phytic acid) is the predominant form of phosphorus found in seeds and accumulates as a consequence of MIPS action. Three MIPS genes have been identified in Arabidopsis, all of which ...[more]

PMID: 18603618

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Project description:BACKGROUND:Phytic acid (InsP6) is considered as the major source of phosphorus and inositol phosphates in cereal grains. Reduction of phytic acid level in cereal grains is desirable in view of its antinutrient properties to maximize mineral bioavailability and minimize the load of phosphorus waste management. We report here RNAi mediated seed-specific silencing of myo-inositol-3-phosphate synthase (MIPS) gene catalyzing the first step of phytic acid biosynthesis in rice. Moreover, we also studied the possible implications of MIPS silencing on myo-inositol and related metabolism, since, first step of phytic acid biosynthesis is also the rate limiting step of myo-inositol synthesis, catalyzed by MIPS. RESULTS:The resulting transgenic rice plants (T3) showed a 4.59 fold down regulation in MIPS gene expression, which corresponds to a significant decrease in phytate levels and a simultaneous increment in the amount of inorganic phosphate in the seeds. A diminution in the myo-inositol content of transgenic plants was also observed due to disruption of the first step of phytic acid biosynthetic pathway, which further reduced the level of ascorbate and altered abscisic acid (ABA) sensitivity of the transgenic plants. In addition, our results shows that in the transgenic plants, the lower phytate levels has led to an increment of divalent cations, of which a 1.6 fold increase in the iron concentration in milled rice seeds was noteworthy. This increase could be attributed to reduced chelation of divalent metal (iron) cations, which may correlate to higher iron bioavailability in the endosperm of rice grains. CONCLUSION:The present study evidently suggests that seed-specific silencing of MIPS in transgenic rice plants can yield substantial reduction in levels of phytic acid along with an increase in inorganic phosphate content. However, it was also demonstrated that the low phytate seeds had an undesirable diminution in levels of myo-inositol and ascorbate, which probably led to sensitiveness of seeds to abscisic acid during germination. Therefore, it is suggested that though MIPS is the prime target for generation of low phytate transgenic plants, down-regulation of MIPS can have detrimental effect on myo-inositol synthesis and related pathways which are involved in key plant metabolism.

Project description:The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3'-phosphate-1'-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and l-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol and l-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and l-myo-inositol-1-P; the DIPP synthase domain used only l-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using l-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3'-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.

Dataset Information

Localization of myo-inositol-1-phosphate synthase to the endosperm in developing seeds of Arabidopsis.

Publications

Localization of myo-inositol-1-phosphate synthase to the endosperm in developing seeds of Arabidopsis.

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