Development of breeder-friendly markers for selection of <Emphasis Type="Italic">MIPS1</Emphasis> mutations in soybean

Mutations in the d-myo-inositol 3-phosphate synthase 1 gene (MIPS1) in soybean [Glycine max (L.) Merr.] cause modifications to seed phosphorus and carbohydrate content that improve the nutritional value of food and feed. Molecular markers are an efficient tool for breeding MIPS1 mutant germplasm due to reduced seed germination and field emergence potential. An F₂ population segregating for the MIPS1 mutation found in experimental soybean line V99-5089 was used to develop breeder-friendly markers. Markers were validated in 88 advanced lines from 9 diverse pedigrees. Ten potential simple sequence repeat (SSR) markers, located on Gm11, from the new BARCSOYSSR_1.0 database were tested and four were polymorphic. BARCSOY_11_1495 was 93–97% effective for selecting the mutation. A KBiosciences Competitive Allele Specific PCR (KASPar) assay was developed to select directly for the V99-5089-derived MIPS1 single nucleotide polymorphism (SNP) mutation. The KASPar assay is simple and cost-effective compared to other SNP genotyping assays. The MIPS1 mutation in V99-5089 is likely to have occurred spontaneously. We describe a method of DNA extraction in soybean using a Geno/Grinder for fast and easy tissue maceration.     

Degradation of <Emphasis Type="Italic">myo</Emphasis>-inositol Hexakisphosphate by a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

High-pressure liquid chromatography (HPLC) analysis established myo-inositol pentakisphosphate as the final product of phytate dephosphorylation by the phytate-degrading enzyme from Pantoea agglomerans. Neither product inhibition by phosphate nor inactivation of the Pantoea enzyme during the incubation period were responsible for the limited phytate hydrolysis as shown by addition of phytate-degrading enzyme and phytate, respectively, after the observed stop of enzymatic phytate degradation. In additon, the Pantoea enzyme did not possess activity toward the purified myo-inositol pentakisphosphate. Using a combination of High-Performance Ion Chromatography (HPIC) analysis and kinetic studies, the nature of the generated myo-inositol pentakisphosphate was established. The data demonstrate that the phytate-degrading enzyme from Pantoea agglomerans dephosphorylates myo-inositol hexakisphosphate in a stereospecific way to finally D-myo-inositol(1,2,4,5,6)pentakisphosphate.     

Molecular Cloning,Sequencing, and Expression of a <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine <Emphasis Type="SmallCaps">d</Emphasis>-Fructose 6-Phosphate Amidotransferase Gene from <Emphasis Type="Italic">Volvariella volvacea</Emphasis>

Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K _m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N ³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.     

Functional Characterization of Trehalose Biosynthesis Genes from <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis>: An Osmolyte Involved in Stress Tolerance

Trehalose (1-α-d-glucopyranosyl-1-α-d-glucopyranoside), a non-reducing disaccharide is a major compatible solute, which maintains fluidity of membranes and protects the biological structure of organisms under stress. In this study, trehalose-6-phosphate synthase (otsA) and trehalose-6-phosphate phosphatase (otsB) genes encoding for trehalose biosynthesis from Escherichia coli was cloned as an operon and expressed in E. coli M15(pREP4). The recombinant E. coli strain showed a threefold increase in the activity of otsBA pathway enzymes, compared to the control strain. The transgenic E. coli accumulated up to 0.86 mg/l of trehalose. The sequence of otsA and otsB genes reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in the altered amino acid sequences of the translated proteins.     

An <Emphasis Type="Italic">N</Emphasis>-acyl homolog of mycothiol is produced in marine actinomycetes

Marine actinomycetes have generated much recent interest as a potentially valuable source of novel antibiotics. Like terrestrial actinomycetes the marine actinomycetes are shown here to produce mycothiol as their protective thiol. However, a novel thiol, U25, was produced by MAR2 strain CNQ703 upon progression into stationary phase when secondary metabolite production occurred and became the dominant thiol. MSH and U25 were maintained in a reduced state during early stationary phase, but become significantly oxidized after 10 days in culture. Isolation and structural analysis of the monobromobimane derivative identified U25 as a homolog of mycothiol in which the acetyl group attached to the nitrogen of cysteine is replaced by a propionyl residue. This N-propionyl-desacetyl-mycothiol was present in 13 of the 17 strains of marine actinomycetes examined, including five strains of Salinispora and representatives of the MAR2, MAR3, MAR4 and MAR6 groups. Mycothiol and its precursor, the pseudodisaccharide 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol, were found in all strains. High levels of mycothiol S-conjugate amidase activity, a key enzyme in mycothiol-dependent detoxification, were found in most strains. The results demonstrate that major thiol/disulfide changes accompany secondary metabolite production and suggest that mycothiol-dependent detoxification is important at this developmental stage.     

Generation and characterization of two novel low phytate mutations in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.)

Phytic acid (PA, myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is important to the nutritional quality of soybean meal. Organic phosphorus (P) in PA is indigestible in humans and non-ruminant animals, which affects nutrition and causes P pollution of ground water from animal wastes. Two novel soybean [(Glycine max L. (Merr.)] low phytic acid (lpa) mutations were isolated and characterized. Gm-lpa-TW-1 had a phytic acid P (PA-P) reduction of 66.6% and a sixfold increase in inorganic P (Pi), and Gm-lpa-ZC-2 had a PA-P reduction of 46.3% and a 1.4-fold increase in Pi, compared with their respective non-mutant progenitor lines. The reduction of PA-P and increase of Pi in Gm-lpa-TW-1 were molar equivalent; the decrease of PA-P in Gm-lpa-ZC-2, however, was accompanied by the increase of both Pi and lower inositol phosphates. In both mutant lines, the total P content remained similar to their wild type parents. The two lpa mutations were both inherited in a single recessive gene model but were non-allelic. Sequence data and progeny analysis indicate that Gm-lpa-TW-1 lpa mutation resulted from a 2 bp deletion in the soybean d-myo-inositol 3-phosphate synthase (MIPS1 EC 5.5.1.4) gene 1 (MIPS1). The lpa mutation in Gm-lpa-ZC-2 was mapped on LG B2, closely linked with microsatellite loci Satt416 and Satt168, at genetic distances of ∼4.63 and ∼9.25 cM, respectively. Thus this mutation probably represents a novel soybean lpa locus. The seed emergence rate of Gm-lpa-ZC-2 was similar to its progenitor line and was not affected by seed source and its lpa mutation. However, Gm-lpa-TW-1 had a significantly reduced field emergence when seeds were produced in a subtropic environment. Field tests of the mutants and their progenies further demonstrated that the lpa mutation in Gm-lpa-ZC-2 does not negatively affect plant yield traits. These results will advance understanding of the genetic, biochemical and molecular control of PA synthesis in soybean. The novel lpa mutation in Gm-lpa-ZC-2, together with linked simple sequence repeat (SSR) markers, will be of value for breeding productive lpa soybeans, with meal high in digestible Pi eventually to improve animal nutrition and lessen environmental pollution.     

Raffinose in seedlings of winter vetch (<Emphasis Type="Italic">Vicia villosa</Emphasis> Roth.) under osmotic stress and followed by recovery

During germination of winter vetch (Vicia villosa Roth.) seeds, the degradation of raffinose family oligosaccharides and galactosyl pinitols occurred faster in axis than in cotyledons. After 7 days of germination, all α-d-galactosides disappeared and the soluble carbohydrates in seedling tissues consisted of d-pinitol, sucrose, fructose, glucose and myo-inositol. Osmotic stress caused by incubation of seedlings in PEG 8000 solution (−0.5, −1.0, and −1.5 MPa) for 48 h induced the activity of crucial enzymes of the RFOs pathway, i.e. galactinol synthase and raffinose synthase, in both the root and epicotyl but not in cotyledons. The root and epicotyl accumulated elevated amounts of galactinol and raffinose as the osmotic potential was lowered. This process was transient because when PEG solution was replaced with water, galactinol and raffinose were degraded, thus confirming their direct involvement in the response of tissues to osmotic stress. Among other soluble carbohydrates, only sucrose accumulated in response to stress. The results did not show potential role of d-pinitol in the adjustment of winter vetch seedlings to osmotic stress.     

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.     

HPLC and GC analyses of in vitro-grown leaves of the cancer bush <Emphasis Type="Italic">Lessertia</Emphasis> (<Emphasis Type="Italic">Sutherlandia</Emphasis>) <Emphasis Type="Italic">frutescens</Emphasis> L. reveal higher yields of bioactive compounds

Lessertia frutescens L., commonly known as cancer-bush, is a medicinally reputed plant species indigenous to southern Africa. Field leaf extracts of this species are known to exhibit many curative properties. However, little is known about the bioactive compounds that are present in in vitro leaf extracts and seed extracts. The objective of this study was to verify the presence of and quantify l-canavanine, gamma amino butyric acid (GABA), arginine and d-pinitol in the seeds, field leaves and in vitro leaves of L. frutescens using gas and liquid chromatography. Methanolic extracts of in vitro leaves, field leaves and seeds were used. MRM chromatograms were recorded for l-canavanine and arginine using tandem mass spectrometry. GC chromatograms were recorded for GABA and d-pinitol using gas chromatography. d-Pinitol was found to be most abundant and was 14.75 and 18.17 mg/g in in vitro and field leaf extracts respectively, followed by GABA (7.29 and 3.48 mg/g), arginine (7.08 and 0.35 mg/g) and l-canavanine (0.55 and 0.08 mg/g). In the seed extracts, GABA content was found to be the highest (1.69 mg/g) followed by l-canavanine (0.37 mg/g), then d-pinitol (0.25 mg/g), and arginine (0.02 mg/g). In vitro leaves had higher quantities of all compounds, except for d-pinitol. This study therefore highlights the potential of bulking in vitro leaves for the extraction of the medicinal compounds, l-canavanine and GABA.     

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.

     

Inositol monophosphate phosphatase genes of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome.     

In <Emphasis Type="Italic">silico</Emphasis> study on the substrate binding manner in human <Emphasis Type="Italic">myo</Emphasis>-inositol monophosphatase 2

The human IMPA2 gene encoding myo-inositol monophosphatase 2 is highly implicated with bipolar disorder but the substrates and the reaction mechanism of myo-inositol monophosphatase 2 have not been well elucidated.9 In the present study, we constructed 3D models of three- and two-Mg²⁺-ion bound myo-inositol monophosphatase 2, and studied substrate-binding manners using the docking program AutoDock3. The subsequent study showed that the three-metal-ion model could interact with myo-inositol monophosphates, as follows: The phosphate moiety coordinated three Mg²⁺ ions, and the inositol ring formed hydrogen bonds with the amino acids conserved in the family. Furthermore, the OH group vicinal to the phosphate group formed a hydrogen bond with a non-bridging oxygen atom of the phosphate. These interactions have been proposed as crucial for forming the transitional state, bipyramidal structure in the bovine myo-inositol monophosphatase. We therefore propose that the human myo-inositol monophosphatase 2 interacts with myo-inositol monophosphates in the three-metal-ion bound form, and proceeds the dephosphorylation through the three-metal-ion theory.     

Identification and characterization of class 1 <Emphasis Type="Italic">DXS</Emphasis> gene encoding 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose-5-phosphate synthase,the first committed enzyme of the MEP pathway from soybean

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) catalyses the first committed step of the 2C-methyl-d-erythritol-4-phosphate (MEP) pathway, which is an alternative isoprenoids biosynthetic route that has been recently discovered. In this work, a DXS1-like cDNA (GmDXS1) was isolated from soybean. The full-length cDNA of GmDXS1 encoded 708 amino acid residues with a predicted molecular mass of 76.4 KD. Sequence alignment showed that GmDXS1 had high homology to known DXS proteins from other plant species and contained the conserved N-terminal plastid transit peptide, the N-terminal thiamine binding domain and pyridine binding DRAG domain. Phylogenetic analysis indicated that GmDXS1 belonged to the plant DXS1 cluster. Southern blot analysis indicated that a single copy of GmDXS1 gene existed in soybean genome. Tissue expression analysis revealed that GmDXS1 expressed in all photosynthetic tissues except pod walls and roots. Green fluorescence analysis with the fusion protein 35S:GmDXS1:GFP suggested that GmDXS1 was localized in plastid. The relatively higher photosynthetic pigment content in transgenic tobacco leaves compared to the control implied that GmDXS1 catalyzed the first potential regulatory step in photosynthetic pigment biosynthesis via the MEP pathway.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.