Two‐step d‐ononitol epimerization pathway in Medicago truncatula

Methylated inositol, d ‐pinitol (3‐O‐methyl‐d ‐chiro‐inositol), is a common constituent in legumes. It is synthesized from myo‐inositol in two reactions: the first reaction, catalyzed by myo‐inositol‐O‐methyltransferase (IMT), consists of a transfer of a methyl group from S‐adenosylmethionine to myo‐inositol with the formation of d ‐ononitol, while the second reaction, catalyzed by d ‐ononitol epimerase (OEP), involves epimerization of d ‐ononitol to d ‐pinitol. To identify the genes involved in d ‐pinitol biosynthesis in a model legume Medicago truncatula, we conducted a BLAST search on its genome using soybean IMT cDNA as a query and found putative IMT (MtIMT) gene. Subsequent co‐expression analysis performed on publicly available microarray data revealed two potential OEP genes: MtOEPA, encoding an aldo‐keto reductase and MtOEPB, encoding a short‐chain dehydrogenase. cDNAs of all three genes were cloned and expressed as recombinant proteins in E. coli. In vitro assays confirmed that putative MtIMT enzyme catalyzes methylation of myo‐inositol to d ‐ononitol and showed that MtOEPA enzyme has NAD⁺‐dependent d ‐ononitol dehydrogenase activity, while MtOEPB enzyme has NADP⁺‐dependent d ‐pinitol dehydrogenase activity. Both enzymes are required for epimerization of d ‐ononitol to d ‐pinitol, which occurs in the presence of NAD⁺ and NADPH. Introduction of MtIMT, MtOEPA, and MtOEPB genes into tobacco plants resulted in production of d ‐ononitol and d ‐pinitol in transformants. As this two‐step pathway of d ‐ononitol epimerization is coupled with a transfer of reducing equivalents from NADPH to NAD⁺, we speculate that one of the functions of this pathway might be regeneration of NADP⁺ during drought stress.     

Plasmodium falciparum is dependent on de novo myo‐inositol biosynthesis for assembly of GPI glycolipids and infectivity

Intra‐erythrocytic stages of the malaria parasite, Plasmodium falciparum, are thought to be dependent on de novo synthesis of phosphatidylinositol, as red blood cells (RBC) lack the capacity to synthesize this phospholipid. The myo‐inositol headgroup of PI can either be synthesized de novo or scavenged from the RBC. An untargeted metabolite profiling of P. falciparum infected RBC showed that trophozoite and schizont stages accumulate high levels of myo‐inositol‐3‐phosphate, indicating increased de novo biosynthesis of myo‐inositol from glucose 6‐phosphate. Metabolic labelling studies with ¹³C‐U‐glucose in the presence and absence of exogenous inositol confirmed that de novo myo‐inositol synthesis occurs in parallel with myo‐inositol salvage pathways. Unexpectedly, while both endogenous and scavenged myo‐inositol was used to synthesize bulk PI, only de novo‐synthesized myo‐inositol was incorporated into GPI glycolipids. Moreover, gene disruption studies suggested that the INO1 gene, encoding myo‐inositol 3‐phosphate synthase, is essential in asexual parasite stages. Together these findings suggest that P. falciparum asexual stages are critically dependent on de novo myo‐inositol biosynthesis for assembly of a sub‐pool of PI species and GPI biosynthesis. These findings highlight unexpected complexity in phospholipid biosynthesis in P. falciparum and a lack of redundancy in some nutrient salvage versus endogenous biosynthesis pathways.     

Receptor‐like kinase OsSIK1 improves drought and salt stress tolerance in rice (Oryza sativa) plants

Receptor‐like kinases (RLKs) play essential roles in plant growth, development and responses to environmental stresses. A putative RLK gene, OsSIK1, with extracellular leucine‐rich repeats was cloned and characterized in rice (Oryza sativa). OsSIK1 exhibits kinase activity in the presence of Mn²⁺, and the OsSIK1 kinase domain has the ability to autophosphorylate and phosphorylate myelin basic protein (MBP). OsSIK1 promoter‐GUS analysis revealed that OsSIK1 is expressed mainly in the stem and spikelet in rice. The expression of OsSIK1 is mainly induced by salt, drought and H₂O₂ treatments. Transgenic rice plants with overexpression of OsSIK1 show higher tolerance to salt and drought stresses than control plants. On the contrary, the knock‐out mutants sik1‐1 and sik1‐2, as well as RNA interference (RNAi) plants, are sensitive to drought and salt stresses. The activities of peroxidase, superoxide dismutase and catalase are enhanced significantly in OsSIK1‐overexpressing plants. Also, the accumulation of H₂O₂ in leaves of OsSIK1‐overexpressing plants is much less than that of the mutants, RNAi plants and control plants, as measured by 3,3′‐diamino benzidine (DAB) staining. We also show that OsSIK1 affects stomatal density in the abaxial and adaxial leaf epidermis of rice. These results indicate that OsSIK1 plays important roles in salt and drought stress tolerance in rice, through the activation of the antioxidative system.     

Functional identification of sll1383 from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp PCC 6803 as L-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning,expression and characterization

The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is ∼870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K _m) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 ± 0.0003 mM) compared to IMPase(s) from other sources but in comparison V _max (∼0.033 μmol Pi/min/mg protein) was low. Li⁺ was found to be an inhibitor (IC₅₀ 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na⁺, K⁺ NH₄⁺) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg²⁺ dependent, which can be substituted partially by Mn²⁺. However, unlike other IMPase(s), the enzyme is optimally active at ∼42°C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.     

Permeabilized Escherichia coli Whole Cells Containing Co‐Expressed Two Thermophilic Enzymes Facilitate the Synthesis of scyllo‐Inositol from myo‐Inositol

Scyllo‐inositol (SI), a stereoisomer of inositol, is regarded as a promising therapeutic agent for Alzheimer's disease. Here, an in vitro cofactor‐balance biotransformation for the production of SI from myo‐inositol (MI) by thermophilic myo‐inositol 2‐dehydrogenase (IDH) and scyllo‐inositol 2‐dehydrogenase (SIDH) is presented. These two enzymes (i.e., IDH and SIDH from Geobacillus kaustophilus) are co‐expressed in Escherichia coli BL21(DE3), and E. coli cells containing the two enzymes are permeabilized by heat treatment as whole‐cell catalysts to convert MI to SI. After condition optimizations about permeabilized temperature, reaction temperature, and initial MI concentration, about 82 g L^?1 of SI is produced from 250 g L^?1 of MI within 24 h without any cofactor supplementation. This final titer of SI produced is the highest to the authors’ limited knowledge. This study provides a promising method for the large‐scale industrial production of SI.     

Enhanced salt tolerance of transgenic tobacco plants by co-expression of PcINO1 and McIMT1 is accompanied by increased level of myo-inositol and methylated inositol

Introgression and functional expression of either the PcINO1 (l-myo-inositol 1-phosphate synthase or MIPS coding gene from the wild halophytic rice, Porteresia coarctata) or McIMTI (inositol methyl transferase, IMTI coding gene from common ice plant Mesembryanthemum crystallinum) has earlier been shown to confer salt tolerance to transgenic tobacco plants (Sheveleva et al., Plant Physiol 115:1211–1219, 1997; Majee et al., J Biol Chem 279:28539–28552, 2004). In this communication, we show that transgenic tobacco plants co-expressing PcINO1 and McIMT1 gene either in cytosol or in chloroplasts accumulate higher amount of total inositol (free and methyl inositol) compared to non-transgenic plants. These transgenic plants were more competent in terms of growth potential and photosynthetic activity and were less prone to oxidative stress under salt stress. A positive correlation between the elevated level of total inositol and methylated inositol and the capability of the double transgenic plants to withstand a higher degree of salt stress compared to the plants expressing either PcINO1 or McIMT1 alone is inferred.     

Phosphatidylinositol‐hydrolyzing phospholipase C4 modulates rice response to salt and drought

Phosphatidylinositol‐specific phospholipase C (PI‐PLC) is involved in stress signalling but its signalling function remains largely unknown in crop plants. Here, we report that the PI‐PLC4 from rice (Oryza sativa cv), OsPLC4, plays a positive role in osmotic stress response. Two independent knockout mutants, plc4‐1 and plc4‐2, exhibited decreased seedling growth and survival rate whereas overexpression of OsPLC4 improved survival rate under high salinity and water deficiency, compared with wild type (WT). OsPLC4 hydrolyses PI, phosphatidylinositol 4‐phosphate (PI4P), and phosphatidylinositol‐4,5‐bisphosphate (PIP₂) to generate diacylglycerol (DAG) in vitro. Knockout of OsPLC4 attenuated salt‐induced increase of phosphatidic acid (PA) whereas overexpression of OsPLC4 decreased the level of PI4P and PIP₂ under salt treatment. Applications of DAG or PA restored the growth defect of plc4‐1 to WT but DAG kinase inhibitor 1 blocked the complementary effect of DAG in plc4‐1 under salt stress. In addition, the loss of OsPLC4 compromised the increase of inositol triphosphate and free cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) and inhibited the induction of genes involved in Ca²⁺ sensor and osmotic stress response to salt stress. The results indicate that OsPLC4 modulates the activity of two signalling pathways, PA and Ca²⁺, to affect rice seedling response to osmotic stress.