A Novel Salt-tolerant l-myo-Inositol-1-phosphate Synthase from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice

Abstract

L-myo-Inositol-1-phosphate synthase (EC 5.5.1.4, MIPS), an evolutionarily conserved enzyme protein, catalyzes the synthesis of inositol, which is implicated in a number of metabolic reactions in the biological kingdom. Here we report on the isolation of the gene (PINO1) for a novel salt-tolerant MIPS from the wild halophytic rice, Porteresia coarctata (Roxb.) Tateoka. Identity of the PINO1 gene was confirmed by functional complementation in a yeast inositol auxotrophic strain. Comparison of the nucleotide and deduced amino acid sequences of PINO1 with that of the homologous gene from Oryza sativa L. (RINO1) revealed distinct differences in a stretch of 37 amino acids, between amino acids 174 and 210. Purified bacterially expressed PINO1 protein demonstrated a salt-tolerant character in vitro compared with the salt-sensitive RINO1 protein as with those purified from the native source or an expressed salt-sensitive mutant PINO1 protein wherein amino acids 174 -210 have been deleted. Analysis of the salt effect on oligomerization and tryptophan fluorescence of the RINO1 and PINO1 proteins revealed that the structure of PINO1 protein is stable toward salt environment. Furthermore, introgression of PINO1 rendered transgenic tobacco plants capable of growth in 200 -300 mM NaCl with retention of ϳ40 -80% of the photosynthetic competence with concomitant increased inositol production compared with unstressed control. MIPS protein isolated from PINO1 transgenics showed salt-tolerant property in vitro confirming functional expression in planta of the PINO1 gene. To our knowledge, this is the first report of a salt-tolerant MIPS from any source.Inositols are six-carbon cyclohexane hexitols found ubiquitously in the biological kingdom, and its metabolism plays a vital role in growth regulation, membrane biogenesis, osmotolerance, and in many other processes. As phosphorylated derivatives, its role as a phosphorus store and as a "second messenger" in signal transduction pathways has long been recognized. myo-Inositol, physiologically the most favored stereoisomer among the eight possible geometric isomers of inositol, also enters into an array of biochemical reactions having diverse functions in cellular metabolism both as free and conjugated and phosphorylated or methylated forms (1-3).The primary enzyme for the synthesis of L-myo-inositol 1-phosphate from glucose 6-phosphate is L-myo-inositol-1-phosphate synthase (EC 5.5.1.4; referred to as MIPS), 1 which synthesizes L-myo-inositol 1-phosphate through an internal oxidoreduction reaction involving NAD ϩ . Free inositol is generated by dephosphorylation of the MIPS product by a specific Mg 2ϩ -dependent inositol-1-phosphate phosphatase (EC 3.1.3.25). This mechanism is followed by all myo-inositolproducing organisms throughout the phylogenetic lines, and MIPS has been identified as an evolutionarily conserved protein (4). The structural gene coding for cytosolic MIPS, termed INO1, was first identified in yeast, Saccharomyces cerevisiae (5, 6) and cloned by Klig and Henry (7)....

Life at the extreme: lessons from the genome

“…Differences in individual genes unquestionably have a big role in adaptation to stress. In some cases, they have been inferred from the primary sequences of well-characterized genes, such as the 37-amino-acid stretch in L -myo-inositol-1-phosphate synthase, which distinguishes the salt-tolerant wild rice ( Porteresia coarctata ) from domesticated rice ( Oryza sativa ) [11], or the single-amino-acid variation in AtHKT1;1 (which encodes the high-affinity K + transporter 1) that distinguishes coastal from inland clines of Arabidopsis [12]. In other cases, they have been implicated by the constitutively higher expression - in the absence of stress - of genes that are induced by stress in Arabidopsis , as in the resurrection plant Craterostigma plantagineum [13], the salt-tolerant poplar Populus euphratica [14], or the Arabidopsis relatives T. parvula and Thellungiella salsuginea (formerly T. halophila ) [15-17].…”

L- Myo-Inositol 1-Phosphate Synthase (MIPS) in Chickpea: Gene Duplication and Functional Divergence

“…This differential activity towards high temperature and salt of these two enzymes could be supported by the bioinformatics analysis in respect to the available yeast MIPS crystal structure and salt tolerant PcINO1 (MIPS coding gene from Salt tolerant Porteresia coarctata) protein sequence (Majee et al, 2004). Based on the bioinformatics study, CaMIPS2 appears to be more stable towards destabilizing factors such as high temperature, salt, etc, thereby retains better functionality under such conditions (Kaur et al, 2008).…”

An Insight into the Molecular Basis of Salt Tolerance of l-myo-Inositol 1-P Synthase (PcINO1) from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice

“…Functional viability of each PcINO1 mutant and hybrid genes have been tested by the yeast functional complementation assay as described earlier Majee et al, 2004). Results of such experiments (Fig.…”

“…Funtional Complementation of DPcINO1.1, DPcINO1.2, and OsTPcINO1 in Yeast FY250 (MATae trp1 his3 ura3 leu2 Ino1THIS3) Inositol Auxotrophic Strain A yeast (Saccharomyces cerevisiae)-based functional complementation assay for the confirmation of the MIPS activity of the mutant and hybrid gene products was performed as per procedure described in detail earlier Majee et al, 2004).…”