Cyclitol production in transgenic tobacco

High levels of cyclic sugar alcohols (cyclitols) correlate with tolerance to osmotic stress in a number of plant species. A gene encoding a cyclitol biosynthesis enzyme from a halophyte, Mesembryanthemum crystallinum has been introduced into tobacco. The gene, lmt1 , encodes a myo -inositol O -methyl transferase that, in M. crystallinum , catalyzes the first step in the stress-induced accumulation of the cyclitol pinitol. Tobacco transformed with the lmt1 cDNA under the control of the CaMV 35S promoter appeared phenotypically normal and exhibited IMT1 enzyme activity. Transformants accumulated a carbohydrate product not detectable in non-transformed control plants. This product was identified by HPLC and NMR as ononitol (1- d -4- O -methyl myo -inositol). Ononitol was a major carbohydrate constituent in leaf tissue of plants expressing the lmt1 gene, accumulating to up to 25% the level of sucrose in transformant seedlings. The identification of ononitol as the IMT1 product and the specific accumulation of this compound in transformed tobacco support a role for ononitol as a stable intermediate in pinitol biosynthesis and indicate that an epimerization activity lacking in tobacco is responsible for the conversion of ononitol to pinitol in M. crystallinum . The production of ononitol in tobacco indicates that plant carbohydrate metabolism is flexible and can accommodate the synthesis and accumulation of non-endogenous metabolites. The transgenic system described here will serve as a useful model to test the ability of cyclitols such as ononitol to confer tolerance to environmental stress in a normally glycophytic plant.     

Carbohydrate Metabolism in Drought-Stressed Leaves of Pigeonpea (Cajanus cajan)

Pigeonpea is a tropical grain-legume, which is highly dehydrationtolerant. The effect of drought stress on the carbohydrate metabolismin mature pigeonpea leaves was investigated by withholding waterfrom plants grown in very large pots (50 kg of soil). The moststriking feature of drought-stressed plants was the pronouncedaccumulation of D-pinitol (1D-3-methyl-chiro-inositol), whichincreased from 14 to 85 mg g^–1 dry weight during a 27d stress period. Concomitantly, the levels of starch, sucroseand the pinitol precursors myo-inositol and ononitol all decreasedrapidly to zero or near-zero in response to drought. The levelsof glucose and fructose increased moderately. Drought stressinduced a pronounced increase of the activities of enzymes hydrolysingsoluble starch (amylases) and sucrose (invertase and sucrosesynthase). The two anabolic enzymes sucrose phosphate synthase(sucrose synthetic pathway) and myo-inositol methyl transferase(pinitol synthetic pathway) also showed an increase of activityduring stress. These results indicate that pinitol accumulatedin pigeonpea leaves, because the carbon flux was diverted fromstarch and sucrose into polyols. Key words: Drought, polyols, pinitol, sucrose, starch, pigeonpea     

Reduced inositol content and altered morphology in transgenic potato plants inhibited for 1D- myo -inositol 3-phosphate synthase

Myo -inositol is a precursor of many plant metabolites, including polyols, cell wall components and phosphoinositides. The first committed step in the de novo myo -inositol synthetic pathway is catalysed by the enzyme 1D- myo -inositol 3-phosphate synthase (MIPS; EC 5.5.1.4 ), which converts D-glucose 6-phosphate to 1D- myo -inositol 3-phosphate. Suppression of MIPS activity by an antisense RNA approach in transgenic potato ( Solanum tuberosum L.) plants to below 20% of the wild-type level in leaves resulted in strongly reduced levels of inositol, galactinol and raffinose (approximately 7%, 5% and 12%, respectively, of wild-type values). In contrast, increases were observed for concentrations of hexose phosphates (up to 1.7-fold), sucrose (twofold) and starch (two- to fourfold). Transgenic plants exhibited reduced apical dominance, altered leaf morphology, precocious leaf senescence and a decrease in overall tuber yield. These observations indicate a crucial role for myo -inositol in plant physiology and development.     

Myo-Inositol Turnover in the Intact Rat Brain: Increased Production after d-Amphetamine

Abstract: Apparent turnover of myo -inositol in the brain of urethane-anesthetized rats was estimated in vivo from the rate of appearance of endogenous myo -inositol in the cerebroventricular compartment. Ventricular-cisternal perfusion technique combined with isotope dilution of [¹⁴C] myo -inositol was used to determine the rate of appearance of brain-produced myo -inositol and its modification by d -amphetamine. A mean value of 0.75 nmol/min was obtained for the rate of appearance in the cerebroventricular system. A dose dependent increase in this rate was seen after the administration of d -ampheta-mine. The endogenous removal of myo -inositol from the perfusate was also studied and found to be mediated in part by a saturable transport system that was not influenced by d-amphetamine. The rate of entry of myo -inositol from blood to the erebroventricular system was very low and accounted for only 2% of the total rate of appearance, indicating that the majority of myo -inositol in the rat cerebroventricular fluid originates in the brain.     

Regulation of enzymes involved in lignin biosynthesis: induction of O-methyltransferase mRNAs during the hypersensitive reaction of tobacco to tobacco mosaic virus.

The mRNAs encoding orthodiphenol-O-methyltransferases (OMTs; EC 2.1.1.6), which are involved in the biosynthesis of lignin precursors, are highly induced in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus (TMV). OMT messengers were fractionated on a sucrose gradient and translated in vitro. Protein A-Sepharose columns adsorbed with specific antisera raised against purified OMTs were used to select translation products, and the translatable activity of OMT mRNA was measured at different stages of infection. Oligonucleotides derived from peptide sequences of purified OMT I were used to prime polymerase chain reactions; total RNA was used as template to allow the isolation of an OMT I clone. RNA blots, hybridized with the OMT I probe, revealed a unique messenger of 1.7 kb. The kinetics of accumulation of OMT I mRNAs during the hypersensitive reaction to TMV parallels the kinetics of translation and suggests that an increase in mRNA controls the increase in the rate of enzyme synthesis. In healthy plants, RNA blot hybridization showed that the steady-state level of OMT I mRNA is very high in vascular tissue compared to the level measured in leaves.     

干旱胁迫对苗期大豆异黄酮合成的影响

以不同耐旱性的2个大豆品种(高耐旱JP-6、低耐旱JP-16)为研究材料,采用高效液相色谱和实时荧光定量PCR技术,分析不同时间持续干旱胁迫下,大豆叶片和根系中异黄酮的积累变化及关键酶基因的表达情况.结果表明:大豆根部异黄酮含量显著高于叶部,而异黄酮关键酶基因的表达量则在叶片中更高,耐旱品种JP-6根部的异黄酮积累量更大.随着干旱胁迫持续时间的增加,不同耐旱品种的异黄酮合成与积累变化规律存在显著差异:强耐旱品种JP-6的根和叶中,异黄酮积累量均呈现先下降后升高的趋势;而弱耐旱品种JP-16则相反,异黄酮积累量在不同部位中均呈现先上升后降低的趋势;除JP-6叶中C4H、4CL、IFS2等异黄酮合成上游基因外,其他不同品种、不同部位的关键酶基因表达量均随着干旱胁迫持续时间的增加,呈现先下降后上升的趋势.大豆叶片是异黄酮的主要合成部位,大豆根部也存在少量的异黄酮合成.弱耐旱大豆根部的异黄酮合成和最终积累量均较低,强耐旱品种则较高.根部异黄酮积累量高的大豆品种,其耐旱性更强.     

Pre-steady state kinetics of bacteriophage T4 dam DNA-[N(6)-adenine] methyltransferase: interaction with native (GATC) or modified sites

The DNA methyltransferase of bacteriophage T4 (T4 Dam MTase) recognizes the palindromic sequence GATC, and catalyzes transfer of the methyl group from S:-adenosyl-L-methionine (AdoMet) to the N(6)-position of adenine [generating N(6)-methyladenine and S:-adenosyl-L-homocysteine (AdoHcy)]. Pre-steady state kinetic analysis revealed that the methylation rate constant k(meth) for unmethylated and hemimethylated substrates (0.56 and 0.47 s(-1), respectively) was at least 20-fold larger than the overall reaction rate constant k(cat) (0.023 s(-1)). This indicates that the release of products is the rate-limiting step in the reaction. Destabilization of the target-base pair did not alter the methylation rate, indicating that the rate of target nucleoside flipping does not limit k(meth). Preformed T4 Dam MTase-DNA complexes are less efficient than preformed T4 Dam MTase-AdoMet complexes in the first round of catalysis. Thus, this data is consistent with a preferred route of reaction for T4 Dam MTase in which AdoMet is bound first; this preferred reaction route is not observed with the DNA-[C5-cytosine]-MTases.     

The effects of drought stress on free amino acid accumulation and protein synthesis in Brassica napus

Changes in the concentrations of free amino acids and specific organic acids were analysed during the induction of drought stress in Brassica napus . Most of the amino acids showed a characteristic linear increase with the induction of drought stress in Brassica leaves, increasing an average of 5.9-fold over control levels, followed by a reduction in concentration upon rehydration of the plants. Pyruvate concentrations doubled after 4 days of drought stress whereas 2-oxoglutarate concentrations remained relatively constant. The activities of two of the enzymes involved in amino acid biosynthesis, alanine aminotransferase (EC 2.6.1.2) and aspartate aminotransferase (EC 2.6.1.1), were also measured. Neither enzyme showed any increase in activity, except when the plants were rehydrated. This suggests that the increase in both alanine and aspartate levels results from the increase in their precursors pyruvate and glutamate and may not require increased enzyme activity. The effect of drought stress upon changes in protein synthesis was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We found that there was an overall decrease in protein synthesis with the induction of drought stress, followed by a resumption of synthesis upon rehydration. In addition, the synthesis of a number of specific polypeptides was found to decrease upon water loss in the leaves.     

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).     

Purification of tobacco O-methyltransferases by affinity chromatography and estimation of the rate of synthesis of the enzymes during hypersensitive reaction to virus infection

The three tobacco (Nicotiana tabacum L.) S-adenosyl-L-methionine: o-diphenol-O-methyltransferases (OMTs; EC 2.1.1.6) were purified to homogeneity by affinity chromatography on adenosine-agarose. Amounts and catalytic actities of the enzymes were measured in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus. The drastic increase in activity of each enzyme upon infection was shown to arise from the accumulation of enzymatic protein with constant specific enzymatic activity. Rates of OMT synthesis were determined from pulse-labeling experiments with L-[¹⁴C]leucine injected into the leaves. The specific radioactivities of the homogenous enzymes were compared in healthy and tobacco mosaic virus-infected tobacco. The results demonstrated that increase in OMT amounts is a consequence of de novo synthesis of the enzymes.Abbreviations DEAE diethylaminoethyl - OMT O-methyltransferase - SAM S-adenosyl-L-methionine - TMV tobacco mosaic virus     

Effect of Sorbinil and Ascorbic Acid on myo-Inositol Transport in Cultured Rat Schwann Cells Exposed to Elevated Extracellular Glucose

Abstract: The effect of long-term (2 weeks) exposure to 0–50 m M glucose and 0–1 m M sorbitol on myo -inositol metabolism was studied in cultured rat Schwann cells. Experiments were carried out to determine the effect of sorbinil and ascorbic acid on myo -inositol uptake in rat Schwann cells cultured in the presence of increased extracellular glucose or sorbitol. myo -Inositol uptake and its incorporation into phospholipids decreased significantly when cells were grown in ≥30 m M glucose for a period of 2 weeks. This inhibitory effect was partly blocked by sorbinil, an aldose reductase inhibitor, in a dose-dependent fashion. Significant prevention was achieved with 0.5 and 1 m M sorbinil. Ascorbic acid also prevented the reduction in myo -inositol uptake due to excess extracellular glucose, at 3 and 30 µ M concentrations, but not at 300 µ M . Neither sorbinil nor ascorbic acid could prevent the alterations in myo -inositol transport in cells exposed to high sorbitol levels for the same period of time. These data suggest that glucose-induced alteration of myo -inositol transport in Schwann cells is mediated, at least in part, via sorbitol accumulation. This myo -inositol transport impairment is prevented by sorbinil and also by ascorbic acid. Ascorbic acid may hold a fresh promise for the treatment/prevention of diabetic neuropathy/complications, at least as an adjunct therapy along with known aldose reductase inhibitors.     

Regulation of arginine decarboxylase by spermine in osmotically-stressed oat leaves

Biosynthesis of polyamines in plants is controlled primarily by the enzymes ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (ADC: EC 4.1.1.19), which are responsible for the production of putrescine, and S -adenosyl-L-methionine (SAM) decarboxylase (EC 4.1.1.50) that is necessary for the formation of spermidine and spermine (Spm). Little is known about the metabolic or molecular mechanisms regulating the synthesis of these enzymes. We have studied the regulation of ADC synthesis by Spm in osmotically-stressed oat ( Avena sativa L. ev. Victory) leaves, using a polyclonal antibody to oat ADC and a cDNA clone encoding oat ADC. Treatment with Spm in combination with osmotic stress resulted in increased steady-state levels of ADC mRNA, yet the levels of ADC activity decreased. This absence of correlation is explained by the fact that Spm inhibits processing of the ADC proenzyme, which results in increased levels of this inactive ADC form and a consequent decrease in the ADC-processed form. Spermine treatment leads to delayed loss of chlorophyll in dark-incubated and osmotically-treated oat leaves. Thus, post-translational regulation of ADC synthesis by Spm may be important in explaining its anti-senescence properties.     

Changes in non-structural carbohydrates in olive (Olea europaea) leaves during root zone salinity stress

Self-rooted olive ( Olea europaea L.) plants were grown in hydroponics at various NaCl concentrations (from 0 to 200m M ) for 28 to 32 days followed by 28 to 30 days of relief from salinity over two growing seasons. Olive leaves accumulated both glucose and mannitol during the period of salinity stress. The concentrations of fructose, myo -inositol, galactose, galactinol, sucrose, raffinose, and stachyose were not significantly affected by salinity. Starch content was decreased by salinity. The mannitol/glucose and mannitol/soluble carbohydrates ratios increased as the external NaCl concentration was increased, but returned to the control levels during the relief period. The increase in mannitol or glucose molar concentrations, expressed on a leaf tissue water basis, was partially due to a reduction in leaf tissue water content under salinity stress. However, an increase in mannitol concentration was also observed when expressed on a dry weight basis. The accumulation of mannitol in leaf tissue preceded any reduction in leaf area rate or net assimilation rate. The increase in leaf mannitol or glucose concentration was positively correlated with the increasing level of salinity at the root zone, but not with the accumulation of Na⁺ in the shoot. The role of mannitol. a potential osmoregulator in leaf mesophyll during salinity stress, is discussed in relation to the complex carbohydrate composition of olive leaves.     

Effect of drought on sorbitol and sucrose metabolism in sinks and sources of peach

In peach (Prunus persica [L.] Batsch.), sorbitol and sucrose are the two main forms of photosynthetic and translocated carbon and may have different functions depending on the organ of utilization and its developmental stage. The role and interaction of sorbitol and sucrose metabolism was studied in mature leaves (source) and shoot tips (sinks) of ‘Nemaguard’ peach under drought stress. Plants were irrigated daily at rates of 100, 67, and 33% of evapotranspiration (ET). The relative elongation rate (RER) of growing shoots was measured daily. In mature leaves, water potential (Ψ_w), osmotic potential (Ψ_s), sorbitol‐6‐phosphate dehydrogenase (S6PDH, EC 1.1.1.200), and sucrose‐phosphate synthase (SPS, EC 2.4.1.14) activities were measured weekly. Measurements of Ψ_s, sorbitol dehydrogenase (SDH, 1.1.1.14), sucrose synthase (SS, EC 2.4.1.13), acid invertase (AI, EC 3.2.1.26), and neutral invertase (NI, EC 3.2.1.27) activities were taken weekly in shoot tips. Drought stress reduced RER and Ψ_w of plants in proportion to water supply. Osmotic adjustment was detected by the second week of treatment in mature leaves and by the third week in shoot tips. Both SDH and S6PDH activities were reduced by drought stress within 4 days of treatment and positively correlated with overall Ψ_w levels. However, only SDH activity was correlated with Ψ_s. Among the sucrose enzymes, only SS was affected by drought, being reduced after 3 weeks. Sorbitol accumulation in both mature leaves and shoot tips of stressed plants was observed starting from the second week of treatment and reached up to 80% of total solutes involved in osmotic adjustment. Sucrose content was up to 8‐fold lower than sorbitol content and accumulated only occasionally. We conclude that a loss of SDH activity in sinks leads to osmotic adjustment via sorbitol accumulation in peach. We propose an adaptive role of sorbitol metabolism versus a maintenance role of sucrose metabolism in peach under drought stress.     

Distribution of myo-inositol dehydrogenase in algae

The enzyme myo-inositol dehydrogenase (InDH; EC 1.1.1.18) catalyses the NAD-dependent oxidation of myo-inositol to scyllo-inosose (2-keto-inositol). A survey within different algal groups showed that this enzyme is present in rhodophytes, glaucocystophytes, phaeophytes, xanthophytes and haptophytes but not in green algae, euglenophytes and chrysophytes. Anion-exchange chromatography of crude homogenates resulted in two distinct peaks of activity. Both isoenzymes were specific for myo-inositol and scyllo-inosose. epi- and scyllo-inositol as well as epi-inosose were only poor substrates, while all other polyols and sugars tested did not serve as substrates. A possible role of the InDH isoenzymes is the shuttling of reducing power between the mitochondrion and the cytosol.     

Differential Effects of Lithium on Muscarinic Cholinoceptor-Stimulated CMP-Phosphatidate Accumulation in Cerebellar Granule Cells, CHO-M3 Cells, and SH-SY5Y Neuroblastoma Cells

Abstract: The ability of lithium to potentiate muscarinic cholinoceptor-stimulated CMP-phosphatidate (CMP.PA) accumulation has been examined in various cells in which muscarinic cholinoceptor agonists evoke a phosphoinositide response. Cell types examined include rat cerebellar granule cells, Chinese hamster ovary cells transfected to express the human muscarinic M3 receptor (CHO-M3 cells), and SH-SY5Y neuroblastoma cells. Neither carbachol (1 m M ) nor lithium (10 m M ) caused significant increases in CMP.PA accumulation in rat cerebellar granule cells; however, when added together for 20 min a linear 17-fold increase over basal levels was observed. The increase was dependent on the concentration of carbachol and lithium present, and the effect could be reversed by addition of exogenous myo -inositol (10 m M ). Addition of carbachol alone to CHO-M3 cells caused a five-fold increase in CMP.PA accumulation. In the presence of lithium, a 70-fold increase was observed at 20 min after carbachol plus lithium addition. This latter response was concentration dependent and could be abolished by preincubation in the presence of 10 m M myo -inositol. In contrast, whereas carbachol elicited a three-fold increase in CMP.PA accumulation in SH-SY5Y neuroblastoma cells, which reached a plateau 10 min after agonist addition, the response could neither be augmented by addition of lithium nor inhibited by addition of myo -inositol. These results emphasise that the ability of lithium to affect agonist-stimulated CMP.PA accumulation is not simply a function of stimulus strength, but is also crucially dependent on the intracellular concentration of inositol.     

Galactosylononitol and Stachyose Synthesis in Seeds of Adzuki Bean : Purification and Characterization of Stachyose Synthase

Stachyose synthase (STS) (EC 2.4.1.67) was purified to homogeneity from mature seeds of adzuki bean (Vigna angularis). Electrophoresis under denaturing conditions revealed a single polypeptide of 90 kD. Size-exclusion chromatography of the purified enzyme yielded two activity peaks with apparent molecular masses of 110 and 283 kD. By isoelectric focusing and chromatofocusing the protein was separated into several active forms with isoelectric point values between pH 4.7 and 5.0. Purified STS catalyzed the transfer of the galactosyl group from galactinol to raffinose and myo-inositol. Additionally, the enzyme catalyzed the galactinol-dependent synthesis of galactosylononitol from d-ononitol. The synthesis of a galactosylcyclitol by STS is a new oberservation. Mutual competitive inhibition was observed when the enzyme was incubated with both substrates (raffinose and ononitol) simultaneously. Galactosylononitol could also substitute for galactinol in the synthesis of stachyose from raffinose. Although galactosylononitol was the less-efficient donor, the Michaelis constant value for raffinose was lower in the presence of galactosylononitol (13.2 mm) compared with that obtained in the presence of galactinol (38.6 mm). Our results indicate that STS catalyzes the biosynthesis of galactosylononitol, but may also mediate a redistribution of galactosyl residues from galactosylononitol to stachyose.