Phosphonate and α-fluorophosphonate analogs of d-glucose 6-phosphate as active-site probes of 1l-myo-inositol 1-phosphate synthase.

Phosphate ester analogs in which the bridging oxygen is replaced with a methylene or fluoromethylene group are well known non-hydrolyzable mimics of use as inhibitors and substrate analogs for reactions involving phosphate esters. Properties of the replaced oxygen are often best mimicked by a mono-fluoromethylene group, but such groups are challenging to synthesize and can exist as two stereoisomers. Here, we describe the protocol for our method of synthesizing the α-fluoromethylene analogs of d-glucose 6-phosphate (G6P), as well as the methylene and difluoromethylene analogs, and their application in the study of 1l-myo-inositol-1-phosphate synthase (mIPS). mIPS catalyzes the synthesis of 1l-myo-inositol 1-phosphate (mI1P) from G6P, in an NAD-dependent aldol cyclization. Its key role in myo-inositol metabolism makes it a putative target for the treatment of several health disorders. The design of these inhibitors allowed for the possibility of substrate-like behavior, reversible inhibition, or mechanism-based inactivation. In this chapter, the synthesis of these compounds, expression and purification of recombinant hexahistidine-tagged mIPS, the mIPS kinetic assay and methods for determining the behavior of the phosphate analogs in the presence of mIPS, and a docking approach to rationalizing the observed behavior are described.

Myo-inositol phosphate synthase improves heat stress tolerance by ethylene-mediated modulation of chlorophyll content and photosynthetic efficiency.

L-myo-inositol phosphate synthase (MIPS; EC 5.5.1.4) encodes the enzyme synthesizing Myo-inositol for plant growth and development. Myo-inositol and its phosphate derivatives are involved in various physiological functions ranging from cell wall synthesis, chromatin remodeling, signal transduction, and providing stress responses. In the present study, we report that MIPS regulates chlorophyll content and photosynthesis efficiency via the ethylene signaling pathway. We have used Triticum aestivum MIPS-A (TAMIPS-A) for the present study and characterized it by mutant complementation and overexpression studies in Arabidopsis. TaMIPS-A overexpressing Arabidopsis transgenics were analyzed physiologically under thermal stress conditions. Analysis of overexpression TaMIPS-A transgenics under control and thermal stress conditions revealed them to have enhanced photosynthetic potential under heat stress. When TaMIPS-A overexpression (OE) Arabidopsis transgenics are supplemented with either ACC, the ethylene precursor, or AgNO3, the ethylene signaling inhibitor indicated that MIPS regulates the photosynthetic efficiency and chlorophyll content via the ethylene signaling pathway under control and thermal stress. Expression analysis of essential genes involved in the ethylene biosynthetic and signaling pathway corroborated.

Co-culturing experiments reveal the uptake of myo-inositol phosphate synthase (EC 5.5.1.4) in an inositol auxotroph of Saccharomyces cerevisiae.

BACKGROUND: Myo-Inositol Phosphate Synthase (MIP) catalyzes the conversion of glucose 6- phosphate into inositol phosphate, an essential nutrient and cell signaling molecule. Data obtained, first in bovine brain and later in plants, established MIP expression in organelles and in extracellular environments. A physiological role for secreted MIP has remained elusive since its first detection in intercellular space. To provide further insight into the role of MIP in intercellular milieus, we tested the hypothesis that MIP may function as a growth factor, synthesizing inositol phosphate in intercellular locations requiring, but lacking ability to produce or transport adequate quantities of the cell-cell communicator. This idea was experimentally challenged, utilizing a Saccharomyces cerevisiae inositol auxotroph with no MIP enzyme, permeable membranes with a 0.4 µm pore size, and cellular supernatants as external sources of inositol isolated from S. cerevisiae cells containing either wild-type enzyme (Wt-MIP), no MIP enzyme, auxotroph (Aux), or a green fluorescent protein (GFP) tagged reporter enzyme (MIP- GFP) in co- culturing experiments. RESULTS: Resulting cell densities and microscopic studies with corroborating biochemical and molecular analyses, documented sustained growth of Aux cells in cellular supernatant, concomitant with the uptakeof MIP, detected as MIP-GFP reporter enzyme. These findings revealed previously unknown functions, suggesting that the enzyme can: (1) move into and out of intercellular space, (2) traverse cell walls, and (3) act as a growth factor to promote cellular proliferation of an inositol requiring cell. CONCLUSIONS: Co-culturing experiments, designed to test a probable function for MIP secreted in extracellular vesicles, uncovered previously unknown functions for the enzyme and advanced current knowledge concerning spatial control of inositol phosphate biosynthesis. Most importantly, resulting data identified an extracellular vesicle (a non-viral vector) that is capable of synthesizing and transporting inositol phosphate, a biological activity that can be used to enhance specificity of current inositol phosphate therapeutics.

Overexpression of MdMIPS1 enhances salt tolerance by improving osmosis, ion balance, and antioxidant activity in transgenic apple.

Myo-inositol and its derivatives play vital roles in plant stress tolerance. Myo-inositol-1-phosphate synthase (MIPS) is the rate-limiting enzyme of myo-inositol biosynthesis. However, the role of apple MIPS-mediated myo-inositol biosynthesis in stress tolerance remains elusive. In this study, we found that ectopic expression of MdMIPS1 from apple increased myo-inositol content and enhanced tolerance to salt and osmotic stresses in transgenic Arabidopsis lines. In transgenic apple lines over-expressing MdMIPS1, the increased myo-inositol levels could promote accumulation of other osmoprotectants such as glucose, sucrose, galactose, and fructose, to alleviate salinity-induced osmotic stress. Also, it was shown that overexpression of MdMIPS1 enhanced salinity tolerance by improving the antioxidant system to scavenge ROS, as well as Na+ and K+ homeostasis. Taken together, our results revealed a protective role of MdMIPS1-mediated myo-inositol biosynthesis in salt tolerance by improving osmotic balance, antioxidant defense system, and ion homeostasis in apple.

Role of myo-inositol during skotomorphogenesis in Arabidopsis.

Myo-inositol is a ubiquitous metabolite of plants. It is synthesized by a highly conserved enzyme L-myo-inositol phosphate synthase (MIPS; EC 5.5.1.4). Myo-inositol is well characterized during abiotic stress tolerance but its role during growth and development is unclear. In this study, we demonstrate that the apical hook maintenance and hypocotyl growth depend on myo-inositol. We discovered the myo-inositol role during hook formation and its maintenance via ethylene pathway in Arabidopsis by supplementation assays and qPCR. Our results suggest an essential requirement of myo-inositol for mediating the ethylene response and its interaction with brassinosteroid to regulate the skotomorphogenesis. A model is proposed outlining how MIPS regulates apical hook formation and hypocotyl growth.

Wheat Myo-inositol phosphate synthase influences plant growth and stress responses via ethylene mediated signaling.

L-myo-inositol phosphate synthase (MIPS; EC 5.5.1.4) is involved in abiotic stress tolerance, however its disruption and overexpression has also been associated with enhanced tolerance to pathogens. The molecular mechanism underlying the role of MIPS in growth, immunity and abiotic stress tolerance remains uncharacterized. We explore the molecular mechanism of MIPS action during growth and heat stress conditions. We raised and characterized the TaMIPS over-expressing rice transgenics which showed a reduced reproductive potential. Transcriptome analysis of overexpression transgenics revealed the activation of ET/JA dependent immune response. Pull-down analysis revealed the interaction of TaMIPS-B with ethylene related proteins. Our results suggest an essential requirement of MIPS for mediating the ethylene response and regulate the growth. A model is proposed outlining how fine tuning of MIPS regulate growth and stress tolerance of the plant.

Accumulation of unacetylatable Snf2p at the INO1 promoter is detrimental to remodeler recycling supply for CUP1 induction.

Chromatin structure plays a decisive role in gene regulation through the actions of transcriptional activators, coactivators, and epigenetic machinery. These trans-acting factors contribute to gene expression through their interactions with chromatin structure. In yeast INO1 activation, transcriptional activators and coactivators have been defined through intense study but the mechanistic links within these trans-acting factors and their functional implications are not yet fully understood. In this study, we examined the crosstalk within transcriptional coactivators with regard to the implications of Snf2p acetylation during INO1 activation. Through various biochemical analysis, we demonstrated that both Snf2p and Ino80p chromatin remodelers accumulate at the INO1 promoter in the absence of Snf2p acetylation during induction. Furthermore, nucleosome density and histone acetylation patterns remained unaffected by Snf2p acetylation status. We also showed that cells experience increased sensitivity to copper toxicity when remodelers accumulate at the INO1 promoter due to the decreased CUP1 expression. Therefore, our data provide evidence for crosstalk within transcriptional co-activators during INO1 activation. In light of these findings, we propose a model in which acetylation-driven chromatin remodeler recycling allows for efficient regulation of genes that are dependent upon limited co-activators.

A bacterial cell factory converting glucose into scyllo-inositol, a therapeutic agent for Alzheimer's disease.

A rare stereoisomer of inositol, scyllo-inositol, is a therapeutic agent that has shown potential efficacy in preventing Alzheimer's disease. Mycobacterium tuberculosis ino1 encoding myo-inositol-1-phosphate (MI1P) synthase (MI1PS) was introduced into Bacillus subtilis to convert glucose-6-phosphate (G6P) into MI1P. We found that inactivation of pbuE elevated intracellular concentrations of NAD+·NADH as an essential cofactor of MI1PS and was required to activate MI1PS. MI1P thus produced was dephosphorylated into myo-inositol by an intrinsic inositol monophosphatase, YktC, which was subsequently isomerized into scyllo-inositol via a previously established artificial pathway involving two inositol dehydrogenases, IolG and IolW. In addition, both glcP and glcK were overexpressed to feed more G6P and accelerate scyllo-inositol production. Consequently, a B. subtilis cell factory was demonstrated to produce 2 g L-1 scyllo-inositol from 20 g L-1 glucose. This cell factory provides an inexpensive way to produce scyllo-inositol, which will help us to challenge the growing problem of Alzheimer's disease in our aging society.

Mechanism of Long-Range Chromosome Motion Triggered by Gene Activation.

Movement of chromosome sites within interphase cells is critical for numerous pathways including RNA transcription and genome organization. Yet, a mechanism for reorganizing chromatin in response to these events had not been reported. Here, we delineate a molecular chaperone-dependent pathway for relocating activated gene loci in yeast. Our presented data support a model in which a two-authentication system mobilizes a gene promoter through a dynamic network of polymeric nuclear actin. Transcription factor-dependent nucleation of a myosin motor propels the gene locus through the actin matrix, and fidelity of the actin association was ensured by ARP-containing chromatin remodelers. Motor activity of nuclear myosin was dependent on the Hsp90 chaperone. Hsp90 further contributed by biasing the remodeler-actin interaction toward nucleosomes with the non-canonical histone H2A.Z, thereby focusing the pathway on select sites such as transcriptionally active genes. Together, the system provides a rapid and effective means to broadly yet selectively mobilize chromatin sites.

Stereochemistry in the Reaction of the myo-Inositol Phosphate Synthase Ortholog Ari2 during Aristeromycin Biosynthesis.

The myo-inositol-1-phosphate synthase (MIPS) ortholog Ari2, which is encoded in the aristeromycin biosynthetic gene cluster, catalyzes the formation of five-membered cyclitol phosphate using d-fructose 6-phosphate (F6P) as a substrate. To understand the stereochemistry during the Ari2 reaction in vivo, we carried out feeding experiments with (6S)-d-[6-2H1]- and (6R)-d-[6-2H1]glucose in the aristeromycin-producing strain Streptomyces citricolor. We observed retention of the 2H atom of (6S)-d-[6-2H1]glucose and no incorporation of the 2H atom from (6R)-d-[6-2H1]glucose in aristeromycin. This indicates that Ari2 abstracts the pro-R proton at C6 of F6P after oxidation of C5-OH by nicotinamide adenine dinucleotide (NAD+) to generate the enolate intermediate, which then attacks the C2 ketone to form the C-C bond via aldol-type condensation. The reaction of Ari2 with (6S)-d-[6-2H1]- and (6R)-d-[6-2H1]F6P in vitro exhibited identical stereochemistry compared with that observed during the feeding experiments. Furthermore, analysis of the crystal structure of Ari2, including NAD+ as a ligand, revealed the active site of Ari2 to be similar to that of MIPS of Mycobacterium tuberculosis, supporting the similarity of the reaction mechanisms of Ari2 and MIPS.

Seed targeted RNAi-mediated silencing of GmMIPS1 limits phytate accumulation and improves mineral bioavailability in soybean.

Phytic acid (PA), the major phosphorus reserve in soybean seeds (60-80%), is a potent ion chelator, causing deficiencies that leads to malnutrition. Several forward and reverse genetics approaches have ever since been explored to reduce its phytate levels to improve the micronutrient and phosphorous availability. Transgenic technology has met with success by suppressing the expression of the PA biosynthesis-related genes in several crops for manipulating their phytate content. In our study, we targeted the disruption of the expression of myo-inositol-3-phosphate synthase (MIPS1), the first and the rate limiting enzyme in PA biosynthesis in soybean seeds, by both antisense (AS) and RNAi approaches, using a seed specific promoter, vicilin. PCR and Southern analysis revealed stable integration of transgene in the advanced progenies. The transgenic seeds (T4) of AS (MS14-28-12-29-3-5) and RNAi (MI51-32-22-1-13-6) soybean lines showed 38.75% and 41.34% reduction in phytate levels respectively, compared to non-transgenic (NT) controls without compromised growth and seed development. The electron microscopic examination also revealed reduced globoid crystals in the Protein storage vacoules (PSVs) of mature T4 seeds compared to NT seed controls. A significant increase in the contents of Fe2+ (15.4%, 21.7%), Zn2+ (7.45%, 11.15%) and Ca2+ (10.4%, 15.35%) were observed in MS14-28-12-29-3-5 and MI51-32-22-1-13-6 transgenic lines, respectively, compared to NT implicating improved mineral bioavailability. This study signifies proof-of-concept demonstration of seed-specific PA reduction and paves the path towards low phytate soybean through pathway engineering using the new and precise editing tools.

Water Deficit Elicits a Transcriptional Response of Genes Governing d-pinitol Biosynthesis in Soybean (Glycine max).

d-pinitol is the most commonly accumulated sugar alcohol in the Leguminosae family and has been observed to increase significantly in response to abiotic stress. While previous studies have identified genes involved in d-pinitol synthesis, no study has investigated transcript expression in planta. The present study quantified the expression of several genes involved in d-pinitol synthesis in different plant tissues and investigated the accumulation of d-pinitol, myo-inositol and other metabolites in response to a progressive soil drought in soybean (Glycine max). Expression of myo-inositol 1-phosphate synthase (INPS), the gene responsible for the conversion of glucose-6-phosphate to myo-inositol-1-phosphate, was significantly up regulated in response to a water deficit for the first two sampling weeks. Expression of myo-inositol O-methyl transferase (IMT1), the gene responsible for the conversion of myo-inositol into d-ononitol was only up regulated in stems at sampling week 3. Assessment of metabolites showed significant changes in their concentration in leaves and stems. d-Pinitol concentration was significantly higher in all organs sampled from water deficit plants for all three sampling weeks. In contrast, myo-inositol, had significantly lower concentrations in leaf samples despite up regulation of INPS suggesting the transcriptionally regulated flux of carbon through the myo-inositol pool is important during water deficit.

Stability of the Metabolite Signature Resulting from the MIPS1 Mutation in Low Phytic Acid Soybean ( Glycine max L. Merr.) Mutants upon Cross-Breeding.

The low phytic acid ( lpa) soybean ( Glycine max L. Merr.) mutant Gm-lpa-TW-1-M, resulting from a 2 bp deletion in GmMIPS1, was crossed with a commercial cultivar. F3 and F5 progenies were subjected to nontargeted GC-based metabolite profiling, allowing analysis of a broad array of low molecular weight constituents. In the homozygous lpa mutant progenies the intended phytic acid reduction was accompanied by remarkable metabolic changes of nutritionally relevant constituents such as reduced contents of raffinose oligosaccharides and galactosyl cyclitols as well as increased concentrations in sucrose and various free amino acids. The mutation-induced metabolite signature was nearly unaffected by the cross-breeding and consistently expressed over generations and in different growing seasons. Therefore, not only the primary MIPS1 lpa mutant but also its progenies might be valuable genetic resources for commercial breeding programs to produce soybean seeds stably exhibiting improved phytate-related and nutritional properties.

A Cotton (Gossypium hirsutum) Myo-Inositol-1-Phosphate Synthase (GhMIPS1D) Gene Promotes Root Cell Elongation in Arabidopsis.

Myo-inositol-1-phosphate synthase (MIPS, EC 5.5.1.4) plays important roles in plant growth and development, stress responses, and cellular signal transduction. MIPS genes were found preferably expressed during fiber cell initiation and early fast elongation in upland cotton (Gossypium hirsutum), however, current understanding of the function and regulatory mechanism of MIPS genes to involve in cotton fiber cell growth is limited. Here, by genome-wide analysis, we identified four GhMIPS genes anchoring onto four chromosomes in G. hirsutum and analyzed their phylogenetic relationship, evolutionary dynamics, gene structure and motif distribution, which indicates that MIPS genes are highly conserved from prokaryotes to green plants, with further exon-intron structure analysis showing more diverse in Brassicales plants. Of the four GhMIPS members, based on the significant accumulated expression of GhMIPS1D at the early stage of fiber fast elongating development, thereby, the GhMIPS1D was selected to investigate the function of participating in plant development and cell growth, with ectopic expression in the loss-of-function Arabidopsis mips1 mutants. The results showed that GhMIPS1D is a functional gene to fully compensate the abnormal phenotypes of the deformed cotyledon, dwarfed plants, increased inflorescence branches, and reduced primary root lengths in Arabidopsis mips1 mutants. Furthermore, shortened root cells were recovered and normal root cells were significantly promoted by ectopic expression of GhMIPS1D in Arabidopsis mips1 mutant and wild-type plants respectively. These results serve as a foundation for understanding the MIPS family genes in cotton, and suggest that GhMIPS1D may function as a positive regulator for plant cell elongation.

MIPS: Functional dynamics in evolutionary pathways of plant kingdom.

The myo-inositol biosynthesis pathway triggering protein MIPS is best known for its necessity, ubiquitous nature and occurrence throughout all living kingdom. However, the functional disparity of MIPS genes in green plant is still viable. The present work considered a comprehensive genome-wide analysis from sequenced plants to identify MIPS homologs in respective organisms and their genomic architecture. Variation of MIPS gene expression in twelve different species in diverse conditions has also been analysed. All MIPS genes share a conserved sequence property in most of its coding region, but its regulatory elements, gene structure and expression network vary significantly. Phylogenetic inference confirms the evolution of MIPS from a single common algal ancestor to seed plants and acquiring functional variation through genomic control. This paper represents MIPS as a model for studying gene duplication, functional divergence and diversification events in plant lineages.

Myo-inositol-1-phosphate synthase (Ino-1) functions as a protection mechanism in Corynebacterium glutamicum under oxidative stress.

Reactive oxygen species (ROS) generated in aerobic metabolism and oxidative stress lead to macromolecules damage, such as to proteins, lipids, and DNA, which can be eliminated by the redox buffer mycothiol (AcCys-GlcN-Ins, MSH). Myo-inositol-phosphate synthase (Ino-1) catalyzes the first committed step in the synthesis of MSH, thus playing a critical role in the growth of the organism. Although Ino-1s have been systematically studied in eukaryotes, their physiological and biochemical functions remain largely unknown in bacteria. In this study, we report that Ino-1 plays an important role in oxidative stress resistance in the gram-positive Actinobacteria Corynebacterium glutamicum. Deletion of the ino-1 gene resulted in a decrease in cell viability, an increase in ROS production, and the aggravation of protein carbonylation levels under various stress conditions. The physiological roles of Ino-1 in the resistance to oxidative stresses were corroborated by the absence of MSH in the Δino-1 mutant. In addition, we found that the homologous expression of Ino-1 in C. glutamicum yielded a functionally active protein, while when expressed in Escherichia coliBL21(DE3), it lacked measurable activity. An examination of the molecular mass (Mr) suggested that Ino-1 expressed in E. coliBL21(DE3) was not folded in a catalytically competent conformation. Together, the results unequivocally showed that Ino-1 was important for the mediation of oxidative resistance by C. glutamicum.

Preparation of 1L-myo-Inositol 1-Phosphate as a Substrate of Phosphatidylinositol Phosphate Synthase.

Novel drugs possessing a mechanism of action specific to pathogenic mycobacteria, including Mycobacterium tuberculosis, are needed. In 2010, we discovered that the biosynthetic pathway of phosphatidylinositol, which is a membrane phospholipid, differs between humans and mycobacteria. The key enzyme responsible for this difference is phosphatidylinositol phosphate (PIP) synthase, which is present only in a few bacteria belonging to the phylum Actinobacteria. Discovering compounds that inhibit the activity of this enzyme will lead to the development of new drugs specific to pathogenic mycobacteria. Measuring PIP synthase activity requires the isotope-labeled substrate 1l-myo-inositol 1-phosphate (1l-Ino-1P). Because this substrate is not commercially available, we synthesized it from [14C] glucose 6-phosphate ([14C] Glc-6P), using a crude enzyme solution isolated from the methanoarchaeon 1l-Ino-1P synthase. The activity of 1l-Ino-1P synthase in the crude enzyme mixture was low, and quantitative analysis of the synthesized 1l-Ino-1P was inaccurate due to impurities present in the crude enzyme mixture. In the present study, we describe a method for synthesizing 1l-Ino-1P using a solution containing recombinant 1l-Ino-1P synthase derived from the hyperthermophilic archaeon Aeropyrum pernix. In addition, we elucidate the conditions leading to the almost complete conversion of Glc-6P into 1l-Ino-1P using this enzyme. Quantitation of the synthesized 1l -Ino-1P was performed by colorimetry and gas liquid chromatography. Further, we confirmed that isotope-labeled 1l-Ino-1P, which is difficult to quantitate by gas liquid chromatography, can be accurately quantified by colorimetry. We also confirmed that 1d-inositol 1-phosphate cannot be a substrate for PIP synthase.

Disruption of INOS, a Gene Encoding myo-Inositol Phosphate Synthase, Causes Male Sterility in Drosophila melanogaster.

Inositol is a precursor for the phospholipid membrane component phosphatidylinositol (PI), involved in signal transduction pathways, endoplasmic reticulum stress, and osmoregulation. Alterations of inositol metabolism have been implicated in human reproductive issues, the therapeutic effects of drugs used to treat epilepsy and bipolar disorder, spinal cord defects, and diseases including diabetes and Alzheimer's. The sole known inositol synthetic enzyme is myo-inositol synthase (MIPS), and the homolog in Drosophilia melanogaster is encoded by the Inos gene. Three identical deletion strains (inosΔDF /CyO) were constructed, confirmed by PCR and sequencing, and homozygotes (inosΔDF /inosΔDF ) were shown to lack the transcript encoding the MIPS enzyme. Without inositol, homozygous inosΔDF deletion fertilized eggs develop only to the first-instar larval stage. When transferred as pupae to food without inositol, however, inosΔDF homozygotes die significantly sooner than wild-type flies. Even with dietary inositol the homozygous inosΔDF males are sterile. An inos allele, with a P-element inserted into the first intron, fails to complement this male sterile phenotype. An additional copy of the Inos gene inserted into another chromosome rescues all the phenotypes. These genetic and phenotypic analyses establish D. melanogaster as an excellent model organism in which to examine the role of inositol synthesis in development and reproduction.

Co-suppression of AtMIPS demonstrates cooperation of MIPS1, MIPS2 and MIPS3 in maintaining myo-inositol synthesis.

KEY MESSAGE: Co-suppressed MIPS2 transgenic lines allow bypass of the embryo lethal phenotype of the previously published triple knock-out and demonstrate the effects of MIPS on later stages of development. Regulation of inositol production is of interest broadly for its effects on plant growth and development. The enzyme L-myo-inositol 1-phosphate synthase (MIPS, also known as IPS) isomerizes D-glucose-6-P to D-inositol 3-P, and this is the rate-limiting step in inositol production. In Arabidopsis thaliana, the MIPS enzyme is encoded by three different genes, (AtMIPS1, AtMIPS2 and AtMIPS3), each of which has been shown to produce proteins with biochemically similar properties but differential expression patterns. Here, we report phenotypic and biochemical effects of MIPS co-suppression. We show that some plants engineered to overexpress MIPS2 in fact show reduced expression of AtMIPS1, AtMIPS2 and AtMIPS3, and show altered vegetative phenotype, reduced size and root length, and delayed flowering. Additionally, these plants show reduced inositol, increased glucose levels, and alteration of other metabolites. Our results suggest that the three AtMIPS genes work together to impact the overall synthesis of myo-inositol and overall inositol homeostasis.

Overexpression of PeMIPS1 confers tolerance to salt and copper stresses by scavenging reactive oxygen species in transgenic poplar.

Myo-inositol is a vital compound in plants. As the key rate-limiting enzyme in myo-inositol biosynthesis, l-myo-inositol-1-phosphate synthase (MIPS) is regarded as a determinant of the myo-inositol content in plants. The up-regulation of MIPS genes can increase the myo-inositol content, thereby enhancing the plant's resistance to a variety of stresses. However, there are few reports on the roles of myo-inositol and the identification of MIPS in woody trees. In this study, a MIPS gene, named as PeMIPS1, was characterized from Populus euphratica Oliv. The heterologous expression of PeMIPS1 compensated for inositol production in the yeast inositol auxotrophic mutant ino1 and the phenotypic lesions of the atmips1-2 mutant, an Arabidopsis MIPS1 knock-out mutant. A subcellular location analysis showed that the PeMIPS1-GFP fusion was localized in the nucleus and cytoplasm, but not in the chloroplasts, indicating that PeMIPS1 represented the cytosolic form of MIPS in P. euphratica. Interestingly, PeMIPS1 was not only inducible by drought and high salinity, but also by CuSO4 treatment. The transgenic poplar lines overexpressing PeMIPS1 had greater plant heights, shoot biomasses and survival rates than the wild type during the salt- or copper-stress treatment, and this was accompanied by an increase in the myo-inositol content. The overexpression of PeMIPS1 resulted in the increased activities of antioxidant enzymes and the accumulation of ascorbate, a key nonenzymatic antioxidant in plant, which partly accounted for the enhanced reactive oxygen species-scavenging capacity and the lowered hydrogen peroxide and malondialdehyde levels in the transgenic poplar. To the best of our knowledge, this study is the first to report the roles of MIPS genes in the tolerance to copper stress.