Expression of sucrose: fructan 6-fructosyltransferase (6-SFT) and myo -inositol 1-phosphate synthase (MIPS) genes in barley (Hordeum vulgare) leaves

Fructan is an important class of non-structural carbohydrates present in cool-season grasses. Sucrose: fructan 6-fructosyltransferase (6-SFT, EC 2.4.1.10), one of the enzymes thought to be involved in grass fructan biosynthesis, catalyzes the initiation and extension of 2,6-linked fructans.Myo-inositol is a central component in several metabolic pathways in higher plants.Myo-inositol 1-phosphate synthase (MIPS) (EC 5.5.1.4), the first enzyme in inositolde novo biosynthesis, catalyzes the formation ofmyo-inositol 1-phosphate (MIP) from glucose-6-phosphate. The expression of 6-SFT and MIPS genes is compared in barley (Hordeum vulgare L.) leaves under various conditions. In cool temperature treatments, both 6-SFT and MIPS mRNAs accumulate within two days and then decline after four days. Under warm temperatures and continuous illumination, the amount of 6-SFT and MIPS mRNA gradually accumulated in detached leaves and increased significantly by 8 h. In contrast, we observed no significant changes over time in attached (control) leaves. Treating detached leaves with glucose or sucrose in the dark resulted in accumulations of both 6-SFT and MIPS mRNA. Homologous expression patterns for 6-SFT and MIPS genes suggest that they may be similarly regulated in barley leaves. Although sucrose and glucose may play important roles in the expression of 6-SFT and MIPS genes, regulation likely involves multiple factors.     

Co-suppression of AtMIPS demonstrates cooperation of MIPS1, MIPS2 and MIPS3 in maintaining <Emphasis Type="Italic">myo</Emphasis>-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.

Abstract

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.

     

Ectopic expression of the ABA-inducible dehydration-responsive chickpea l-myo-inositol 1-phosphate synthase 2 (CaMIPS2) in Arabidopsis enhances tolerance to salinity and dehydration stress

Myo-inositol participates in many different aspects of plant physiology and myo-inositol 1-phosphate synthase (MIPS; EC 5.5.1.4) catalyzes the rate limiting step of inositol biosynthetic pathway. Chickpea (Cicer arietinum), a drought-tolerant leguminous crop plant, is known to accumulate increased inositol during dehydration stress. Previously, we reported two differentially expressed divergent genes (CaMIPS1 and CaMIPS2) encoding two MIPS isoforms in chickpea. In this communication, we demonstrated that CaMIPS2 is an early dehydration-responsive gene and is also rapidly induced by exogenous ABA application, while CaMIPS1 expression is not much influenced by dehydration or ABA. The regulation of expression of these two genes has been studied by examining their promoter activity through GUS reporter gene and differential promoter activity has been observed. Moreover, unlike CaMIPS1 promoter, CaMIPS2 promoter contains CRT/DRE cis-regulatory element which seems to play a key role in dehydration-induced expression of CaMIPS2. Furthermore, CaMIPS1 and CaMIPS2 have been successfully complemented and shown to repair the defect of seedling growth and altered seed phenotype of Atmips1 mutant. Moreover, Arabidopsis transgenic plants overexpressing CaMIPS1 or CaMIPS2 exhibit improved tolerance to salinity and dehydration stresses and such tolerance of transgenic plants is correlated with their elevated level of inositol. Remarkably, CaMIPS2 transgenic lines perform better in all attributes than CaMIPS1 transformants under such stress conditions, due to comparatively unabated production of inositol by CaMIPS2 enzyme, as this enzyme retains significant activity under stress conditions.     

Inheritance of seed phytate and phosphorus levels in common bean (Phaseolus vulgaris L.) and association with newly-mapped candidate genes

Common bean (Phaseolus vulgaris L.) is an important, high-quality staple food that provides large amounts of protein and mineral micronutrients to the diets of people in many countries. Phytates are a storage form of organic phosphorus which is used by the plant in various stages of growth and development but can have certain anti-nutrient properties due to chelation of minerals such as iron and zinc. At the same time, phytates provide certain health benefits and therefore are the subject of both mutagenesis and breeding programs for functional foods. The objective of this study was to evaluate the quantitative trait loci (QTL) associated with seed phytate and seed phosphorus concentration and content on a per-seed basis and to develop functional molecular markers for genes from the phytic acid synthesis pathway. We used a well-characterized mapping population, DOR364?×?G19833, in three field experiments with three repetitions each and two levels of soil phosphorus fertilization, as well as a large set of previously and newly developed primer pairs for the genes myo-inositol (3)P₁ synthase, myo-inositol kinase and various inositol kinases. We identified an association of phytate concentration QTL with one of two paralogs of the myo-inositol (3)P₁ synthase gene family, located on linkage group b01 and expressed in common bean seed rather than in vegetative tissues. We also identified QTL for phytate concentration on linkage group b06 and phytate content on linkage groups b03, b04 and b10. We provide a synteny analysis based on common bean versus soybean genome comparisons of all the phytic acid pathway genes that were genetically mapped and indicate flanking markers that can be used for marker-assisted selection when the genes themselves are not polymorphic as PCR amplicons. We can conclude that natural variability in phytate levels is controlled by the seed-expressed myo-inositol (3)P₁ synthase gene (MIPS) as well as other loci in the common bean genome. This means that breeding of phytate levels in common bean must take into account allele variability at certain candidate genes, such as this seed MIPS gene, a recently cloned ABC trasnporter and additional QTL for the trait, which underlie the oligogenic inheritance for phytate concentration in common bean.     

Studies on avian erythrocyte metabolism: purification and properties of myo-inositol 1-phosphatase form chick erythrocytes

An enzyme capable of hydrolyzing myo-inositol 1-phosphate was identified and partially purified from the erythrocytes of 7-day chicks. It has an apparent molecular weight of approximately 60,000, is heat stable, and has a pH of optimal activity between 6.5 and 7.3. In most regards the kinetic properties are similar to the myo-inositol 1-phosphatases of rat testis, rat mammary gland, bovine brain, and of yeast. The enzyme has an absolute requirement for a divalent cation; Mg²⁺ gave the greatest activity, with an optimal concentration of 2.5 mm in the standard assay employed. Zn²⁺, Co²⁺, and Mn²⁺ supported activity to a lesser degree. Activity was inhibited by NaF, HgCl₂, and p-hydroxymercuribenzoate. myo-Inositol tetrakis (dihydrogen phosphate) and myo-inositol 1,3,4,5,6-pentakis (dihydrogen phosphate) were not substrates for this enzyme and inhibited the hydrolysis of myo-inositol 1-phosphate. Unlike other phosphatases for myo-inositol 1-phosphate, this enzyme cleaved myo-inositol 1-phosphate (K_m = 8.6 × 10^?5 m) and myo-inositol 2-phosphate (K_m = 2.86 × 10^?4 m) at approximately the same rates. It also hydrolyzed 2′-purine and pyrimidine ribonucleotides about as well as myo-inositol 1-phosphate, but was only 20–30% as active against the 3′-ribonucleotides and had scarcely any activity against the 5′-ribonucleotides. The amount of enzyme activity in erythrocytes of embryos, chicks, and mature chickens was the same (~29 μmol/ml rbc/h). The biological function of this enzyme in avian erythrocytes is unclear at this time. Other tissues containing this phosphatase also have an enzyme which synthesizes myo-inositol 1-phosphate from glucose 6-phosphate, but we have been unable to detect the presence of such an enzyme in avian erythrocytes.     

sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as l-myo-inositol 1-phosphate synthase

l-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes the first rate limiting conversion of d-glucose 6-phosphate to l-myo-inositol 1-phosphate in the inositol biosynthetic pathway. In an earlier communication we have reported two forms of MIPS in Synechocystis sp. PCC6803 (Chatterjee et al. in Planta 218:989–998, 2004). One of the forms with a ~50 kDa subunit has been found to be coded by an as yet unassigned ORF, sll1722. In the present study we have purified the second isoform of MIPS as a ~65 kDa protein from the crude extract of Synechocystis sp. PCC6803 to apparent homogeneity and biochemically characterized. MALDI-TOF analysis of the 65 kDa protein led to its identification as acetolactate synthase large subunit (EC 2.2.1.6; ALS), the putatively assigned ORF sll1981 of Synechocystis sp. PCC6803. The PCR amplified ~1.6 kb product of sll1981 was found to functionally complement the yeast inositol auxotroph, FY250 and could be expressed as an immunoreactive ~65 kDa MIPS protein in the natural inositol auxotroph, Schizosaccharomyces pombe. In vitro MIPS activity and cross reactivity against MIPS antibody of purified recombinant sll1981 further consolidated its identity as the second probable MIPS gene in Synechocystis sp. PCC6803. Sequence comparison along with available crystal structure analysis of the yeast MIPS reveals conservation of several amino acids in sll1981 essential for substrate and co-factor binding. Comparison with other prokaryotic and eukaryotic MIPS sequences and phylogenetic analysis, however, revealed that like sll1722, sll1981 is quite divergent from others. It is probable that sll1981 may code for a bifunctional enzyme protein having conserved domains for both MIPS and acetolactate synthase (ALS) activities.Anirban Chatterjee and Krishnarup Ghosh Dastidar contributed equally.     

myo-Inositol-1-phosphate Synthase Is Required for Polar Auxin Transport and Organ Development

myo-Inositol-1-phosphate synthase is a conserved enzyme that catalyzes the first committed and rate-limiting step in inositol biosynthesis. Despite its wide occurrence in all eukaryotes, the role of myo-inositol-1-phosphate synthase and de novo inositol biosynthesis in cell signaling and organism development has been unclear. In this study, we isolated loss-of-function mutants in the Arabidopsis MIPS1 gene from different ecotypes. It was found that all null mips1 mutants are defective in embryogenesis, cotyledon venation patterning, root growth, and root cap development. The mutant roots are also agravitropic and have reduced basipetal auxin transport. mips1 mutants have significantly reduced levels of major phosphatidylinositols and exhibit much slower rates of endocytosis. Treatment with brefeldin A induces slower PIN2 protein aggregation in mips1, indicating altered PIN2 trafficking. Our results demonstrate that MIPS1 is critical for maintaining phosphatidylinositol levels and affects pattern formation in plants likely through regulation of auxin distribution.     

A limitation of the continuous spectrophotometric assay for the measurement of myo-inositol-1-phosphate synthase activity

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay—the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg²⁺ and is not enhanced by other divalent metal ions (Zn²⁺ and Mn²⁺), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors.     

D-myo-inositol-3-phosphate affects phosphatidylinositol-mediated endomembrane function in Arabidopsis and is essential for auxin-regulated embryogenesis

In animal cells, myo-inositol is an important regulatory molecule in several physiological and biochemical processes, including signal transduction and membrane biogenesis. However, the fundamental biological functions of myo-inositol are still far from clear in plants. Here, we report the genetic characterization of three Arabidopsis thaliana genes encoding d-myo-inositol-3-phosphate synthase (MIPS), which catalyzes the rate-limiting step in de novo synthesis of myo-inositol. Each of the three MIPS genes rescued the yeast ino1 mutant, which is defective in yeast MIPS gene INO1, and they had different dynamic expression patterns during Arabidopsis embryo development. Although single mips mutants showed no obvious phenotypes, the mips1 mips2 double mutant and the mips1 mips2 mips3 triple mutant were embryo lethal, whereas the mips1 mips3 and mips1 mips2^+/− double mutants had abnormal embryos. The mips phenotypes resembled those of auxin mutants. Indeed, the double and triple mips mutants displayed abnormal expression patterns of DR5:green fluorescent protein, an auxin-responsive fusion protein, and they had altered PIN1 subcellular localization. Also, membrane trafficking was affected in mips1 mips3. Interestingly, overexpression of PHOSPHATIDYLINOSITOL SYNTHASE2, which converts myo-inositol to membrane phosphatidylinositol (PtdIns), largely rescued the cotyledon and endomembrane defects in mips1 mips3. We conclude that myo-inositol serves as the main substrate for synthesizing PtdIns and phosphatidylinositides, which are essential for endomembrane structure and trafficking and thus for auxin-regulated embryogenesis.     

Partial Purification and Characterization of Indol-3-Ylacetylglucosemyo-Inositol Indol-3-Ylacetyltransferase (Indoleacetic Acid-Inositol Synthase)

A procedure is described for the purification of the enzyme indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAA-myo-inositol synthase). This enzyme catalyzes the transfer of indol-3-ylacetate from 1-0-indol-3-ylacetyl-β-d-glucose to myo-inositol to form indol-3-ylacetyl-myo-inositol and glucose. A hexokinase or glucose oxidase based assay system is described. The enzyme has been purified approximately 16,000-fold, has an isoelectric point of pH 6.1 and yields three catalytically inactive bands upon acrylamide gel electrophoresis of the native protein. The enzyme shows maximum transferase activity with myo-inositol but shows some transferase activity with scyllo-inositol and myo-inosose-2. No transfer of IAA occurs with myo-inositol-d-galactopyranose, cyclohexanol, mannitol, or glycerol as acyl acceptor. The affinity of the enzyme for 1-0-indol-3-ylacetyl-β-d-glucose is, K_m = 30 micromolar, and for myo-inositol is, K_m = 4 millimolar. The enzyme does not catalyze the exchange incorporation of glucose into IAA-glucose indicating the reaction mechanism involves binding of IAA glucose to the enzyme with subsequent hydrolytic cleavage of the acyl moiety by the hydroxyl of myo-inositol to form IAA myo-inositol ester.     

sll1722, an unassigned open reading frame of<Emphasis Type="Italic"> Synechocystis</Emphasis> PCC 6803, codes for <Emphasis Type="SmallCaps">l</Emphasis>-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate synthase

l-myo-Inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes conversion of glucose 6-phosphate to l-myo-inositol 1-phosphate, the first and the rate-limiting step in the production of inositol, and has been reported from evolutionarily diverse organisms. Two forms of the enzyme have been characterized from higher plants, viz. cytosolic and chloroplastic, and the presence of MIPS has been earlier reported from the cyanobacteria (e.g. Spirulina sp.), the presumed chloroplast progenitors. The present study demonstrates possible multiple forms of MIPS and identifies the gene for one of them in the cyanobacterium Synechocystis sp. PCC 6803. Following detection of at least two immunologically cross-reactive MIPS forms, we have been able to identify from the fully sequenced Synechocystis genome an as yet unassigned open reading frame (ORF), sll1722, coding for the approx. 50-kDa MIPS protein, by using biochemical, molecular and bioinformatics tools. The DNA fragment corresponding to sll1722 was PCR-amplified and functional identity of the gene was confirmed by a complementation assay in Saccharomyces cerevisiae mutants containing a disrupted INO1 gene for the yeast MIPS. The sll1722 PCR product was cloned in Escherichia coli expression vector pET20b and the isopropyl -d-thiogalactopyranoside (IPTG)-induced overexpressed protein product was characterized following complete purification. Comparison of the sll1722 sequences with other MIPS sequences and its phylogenetic analysis revealed that the Synechocystis MIPS gene is quite divergent from the others.Abbreviations CBB Coomassie Brilliant Blue - EST Expressed sequence tag - G6P d-Glucose 6-phosphate - IPTG Isopropyl -d-thiogalactopyranoside - MIPS l–myo-Inositol 1-phosphate synthase - ORF Open reading frame     

Partial purification and characterization of L-myo-inositol-1-phosphate synthase of pteridophytic origin

Myo-Inositol is an important metabolite for normal growth and development of all living organisms. The cellular level of myo-inositol is controlled by the enzyme L-myo- inositol-1-phosphate synthase (MIPS) [EC 5.5.1.4]. Appreciable level of MIPS activity was detected from the common pteridophytes like Dicranopteris, Diplazium, Diplopterygium, Equisetum, Lycopodium, Polypodium, Pteridium, Selaginella etc. available in Darjeeling Himalayas. The enzyme was partially purified from the reproductive pinnules of Diplopterygium glaucum (Thunb.) Nakai. The purification obtained was about 81 fold and the recovery was about 13.5 %. The final enzyme preparation specifically utilized D-Glucose-6-phosphate and NAD+ as its substrate and co-factor respectively. It shows pH optima between 7.0 and 7.5 while the temperature maximum was at 35 °C. The enzyme activity was slightly inhibited by Na⁺ and Cd²⁺ and highly inhibited by Li⁺ and Hg²⁺. The K _rn values for D-glucose-6-phosphate and NAD+ was found to be as 0.83 mM and 0.44 mM respectively while the V _max values were 1.42 mM and 1.8 mM for D-glucose-6-phosphate and NAD+ respectively. The present study indicates the universal occurrence of this enzyme in all plant groups.     

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.     

Functional dissection of a napin gene promoter: identification of promoter elements required for embryo and endosperm-specific transcription

The promoter region (?309 to +44) of the Brassica napus storage protein gene napA was studied in transgenic tobacco by successive 5′ as well as internal deletions fused to the reporter gene GUS (β-glucuronidase). The expression in the two main tissues of the seed, the endosperm and the embryo, was shown to be differentially regulated. This tissue-specific regulation within the seed was found to affect the developmental expression during seed development. The region between ?309 to ?152, which has a large effect on quantitative expression, was shown to harbour four elements regulating embryo and one regulating endosperm expression. This region also displayed enhancer activity. Deletion of eight bp from position ?152 to position ?144 totally abolished the activity of the napA promoter. This deletion disrupted a cis element with similarity to an ABA-responsive element (ABRE) overlapping with an E-box, demonstrating its crucial importance for quantitative expression. An internal deletion of the region ?133 to ?120, resulted in increased activity in both leaves and endosperm and a decreased activity in the embryo. Within this region, a cis element similar to the (CA)_n element, found in other storage protein promoters, was identified. This suggest that the (CA)_n element is important for conferring seed specificity by serving both as an activator and a repressor element.     

Evidence for existence and a tentative identification of coenzyme in yeast thiamine pyrophosphokinase.

Thiamine pyrophosphokinase (E.C. 2.7.6.2.) from $\text{Saccharomyces cerevisiae}$ was found to require the presence of a non-protein, non-metal compound for its activity. $\text{myo}$-Inositol was found capable of stimulating the kinase activity in the presumably resolved but otherwise crude sample of the enzyme. The hexytol was also found capable of inducing the enzyme in growing yeast cells. The cultured yeast cells, in which the kinase had been induced, were used as source of the enzyme for its purification. The compound that had been left adsorbed to the final column of DEAE-Sephadex was proved to have a coenzyme activity towards the enzyme and tentatively identified with $\text{myo}$-inositol 1-pyrophosphate. A sample of synthetic $\text{myo}$-inositol 1-pyrophosphate was made and its coenzyme activity was observed.