Nourbakhsh Aida, Collakova Eva, Gillaspy Glenda E
Department of Human and Molecular Genetics, Virginia Commonwealth University Richmond, VA, USA.
Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University Blacksburg, VA, USA.
Front Plant Sci. 2015 Jan 9;5:725. doi: 10.3389/fpls.2014.00725. eCollection 2014.
Synthesis of myo-inositol is crucial in multicellular eukaryotes for production of phosphatidylinositol and inositol phosphate signaling molecules. The myo-inositol monophosphatase (IMP) enzyme is required for the synthesis of myo-inositol, breakdown of inositol (1,4,5)-trisphosphate, a second messenger involved in Ca(2+) signaling, and synthesis of L-galactose, a precursor of ascorbic acid. Two myo-inositol monophosphatase -like (IMPL) genes in Arabidopsis encode chloroplast proteins with homology to the prokaryotic IMPs and one of these, IMPL2, can complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, indicating an important role for IMPL2 in amino acid synthesis. To delineate how this small gene family functions in inositol synthesis and metabolism, we sought to compare recombinant enzyme activities, expression patterns, and impact of genetic loss-of-function mutations for each. Our data show that purified IMPL2 protein is an active histidinol-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme, which has the ability to hydrolyze D-galactose 1-phosphate, and D-myo-inositol 1-phosphate, a breakdown product of D-inositol (1,4,5) trisphosphate. Expression studies indicated that all three genes are expressed in multiple tissues, however, IMPL1 expression is restricted to above-ground tissues only. Identification and characterization of impl1 and impl2 mutants revealed no viable mutants for IMPL1, while two different impl2 mutants were identified and shown to be severely compromised in growth, which can be rescued by histidine. Analyses of metabolite levels in impl2 and complemented mutants reveals impl2 mutant growth is impacted by alterations in the histidine biosynthesis pathway, but does not impact myo-inositol synthesis. Together, these data indicate that IMPL2 functions in the histidine biosynthetic pathway, while IMP and IMPL1 catalyze the hydrolysis of inositol- and galactose-phosphates in the plant cell.
在多细胞真核生物中，肌醇的合成对于磷脂酰肌醇和肌醇磷酸信号分子的产生至关重要。肌醇单磷酸酶（IMP）是肌醇合成、肌醇（1,4,5）-三磷酸（一种参与钙信号传导的第二信使）的分解以及L-半乳糖（抗坏血酸的前体）合成所必需的。拟南芥中的两个类肌醇单磷酸酶（IMPL）基因编码与原核IMP具有同源性的叶绿体蛋白，其中之一IMPL2可以互补在组氨酸合成中存在缺陷的细菌组氨醇1-磷酸磷酸酶突变体，这表明IMPL2在氨基酸合成中具有重要作用。为了阐明这个小基因家族在肌醇合成和代谢中的功能，我们试图比较每种基因的重组酶活性、表达模式以及基因功能丧失突变的影响。我们的数据表明，与IMPL1酶相比，纯化的IMPL2蛋白是一种活性组氨醇-磷酸磷酸酶，IMPL1酶能够水解D-半乳糖1-磷酸和D-肌醇1-磷酸（D-肌醇（1,4,5）三磷酸的分解产物）。表达研究表明，所有三个基因都在多个组织中表达，然而，IMPL1的表达仅限于地上组织。对impl1和impl2突变体的鉴定和表征显示，没有可存活的IMPL1突变体，而鉴定出了两种不同的impl2突变体，并显示它们在生长方面严重受损，组氨酸可以挽救这种情况。对impl2和互补突变体中代谢物水平的分析表明，impl2突变体的生长受到组氨酸生物合成途径改变的影响，但不影响肌醇合成。总之，这些数据表明IMPL2在组氨酸生物合成途径中起作用，而IMP和IMPL1催化植物细胞中肌醇-磷酸和半乳糖-磷酸的水解。
