Biosynthesis and accumulation of D-ononitol in Vigna umbellata in response to drought stress

Metabolic responses to water deficit that lead to an accumulation of cyclitols, have been examined in rice bean ( Vigna umbellata [Thunb.] Ohwi et Ohashi). Imposition of drought stress by withholding water from the soil for 9 days led to an accumulation of D-ononitol (lD-4- O -methyl- myo -inositol) which was most pronounced in leaves (from 33 to 88 umol g⁻¹ dry mass). However, the activity of the enzyme myo-inositol 6-O -methyltransferase (m6OMT, EC 2.1.1.X), which catalyzes the synthesis of ononitol from myo -inositol and S -adenosyl-L-methionine (AdoMet), increased in stems but not in leaves during the drought stress experiment. Detailed analysis of different plant parts revealed that the accumulation of ononitol in leaves was linearly related to stem m6OMT activity during drought stress, indicating that m6OMT may control the in vivo biosynthetic rate of this cyclitol. The availability of myo -inositol, required for enhanced rates of ononitol synthesis by m6OMT, increased during the stress experiment, while the capacity to synthezise AdoMet by S -adenosyl-L-methionine synthetase (SMS, EC 2.5.1.6) decreased. However, the high capacity for degradation of S -adenosyl-L-homocysteine (AdoHcy; a potent competitive inhibitor of m6OMT) by the enzyme S -adenosyl-L-homocysteine hydrolase (SHH, EC 3.3.1.1) provided favourable conditions for ononitol biosynthesis during the whole stress treatment.     

Alfalfa cyclitols in the honeydew of an aphid

The free cyclitols pinitol, ononitol and myo-inositol occur in the honeydew (excreta) of pea aphids (Acyrthosiphon pisum) which feed on pea aphid-susceptible alfalfa (Medicago sativa cv Caliverde). These cyclitols also occur in the leaves and stems of alfalfa. Aphids were incapable of de novo synthesis of these cyclitols. Honeydew production by the pea aphid results from ingesting phloem-sap, so the occurrence of cyclitols in honeydew results from their translocation in the phloem. The relatively high content of myo-inositol in honeydew indicates that it is selectively translocated. The most abundant alfalfa cyclitol, pinitol, had no effect on aphid feeding behavior at concentrations up to 1% (w/v; artificial diet).     

D-chiro-inositol affects accumulation of raffinose family oligosaccharides in developing embryos of Pisum sativum

Developing garden pea embryos are able to take up exogenously applied cyclitols: myo-inositol, which naturally occurs in pea, and two cyclitols absent in pea plants: d-chiro-inositol and d-pinitol. The competition in the uptake of cyclitols by pea embryo, insensitivity to glucose and sucrose, and susceptibility to inhibitor(s) of H⁺-symporters (e.g. CCCP and antimycin A) suggest that a common cyclitol transporter is involved. Both d-chiro-inositol and d-pinitol can be translocated through the pea plant to developing embryos. During seed maturation drying, they are used for synthesis of mainly mono-galactosides, such as fagopyritol B1 and galactosyl pinitol A. Accumulation of d-chiro-inositol (and formation of fagopyritols), but not d-pinitol, strongly reduces accumulation of verbascose, the main raffinose oligosaccharide in pea seeds. The reasons for the observed changes are discussed.     

Two‐step d‐ononitol epimerization pathway in Medicago truncatula

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

Identification of ononitol and O-methyl-scyllo-inositol in pea root nodules

Ononitol (4-O-methyl-myo-inositol) and O-methyl-scyllo-inositol were identified in pea (Pisum sativum L.) root nodules formed by twoRhizobium leguminosarum strains. Ononitol was the major soluble carbohydrate in nodules formed by strain 1045 while O-methyl-scyllo-inositol and two unidentified components were dominant in the carbohydrate pattern of the nodules formed by strain 1 a. The cyclitols were also present in the denodulated roots, but to a much smaller extent; in the above-ground plant parts only traces were found. The identification of ononitol and O-methyl-scyllo-inositol was established by gas chromatography and gas chromatography-mass spectrometry utilizing trimethylsilyl- and acetyl-derivatives.Abbreviations GC-MS gas chromatography-mass spectroscopy - TLC thin-layer chromatography     

Cyclitols as cryoprotectants for spinach and chickpea thylakoids

Thylakoid membranes isolated from either spinach or chickpea leaves were used as a model system for evaluating the capacity of cyclitols to act as cryoprotectants. The effect of freezing for 3 h at -18 degrees C on cyclic photophosphorylation and electron transport was measured. The cyclitols, ononitol, O-methyl-muco-inositol, pinitol, quebrachitol and quercitol at 50-150 mol m(-3) decreased membrane damage by freezing and thawing to a similar degree as the well known cryoprotectants sucrose and trehalose. On addition of the cryotoxic solute NaCl (100 mol m(-3)) to the test system these methylated cyclohexanhexols again provided a protection comparable to that of the two disaccharides. Quercitol (cyclohexanpentol) was not effective when added in lower concentrations (50-100 mol m(-3)) and in case of this cyclitol a ratio of membrane toxic to membrane compatible solute of 0.66 was apparently needed to prevent a loss of cyclic photophosphorylation. Little difference was observed in the results from spinach or chickpea thylakoids although these plants naturally accumulate different cyto-solutes (spinach: glycinebetaine; chickpea: pinitol).     

Patterns of pinitol accumulation in soybean plants and relationships to drought tolerance

Previous studies indicate that methylated cyclitols are potentially important osmolytes in plants. In a search for genetic diversity for pinitol (D -3-O-methyl-chiro-inositol) accumulation in soybean (Glycine max (L.) Merr.), two- to three-fold differences in pinitol accumulation in leaf blades were found among Chinese plant introductions. Furthermore, it was found that genotypes that accumulated high concentrations of pinitol, when grown under well-watered conditions, had been selected for performance in regions of China having low rainfall. Among the carbohydrates analysed, pinitol accumulation was uniquely associated with adaptation to dry areas of China. A detailed study of pinitol accumulation in the soybean plant showed two- to three-fold gradients in pinitol concentration from the bottom to the top of the plant. The gradient shifted during plant development, with consistently higher concentrations of pinitol in the uppermost leaves. Pinitol accumulation was not correlated with activity of the key biosynthetic enzyme, inositol methyl transferase. This result and other lines of evidence indicated that shifting patterns of pinitol accumulation were due to translocation of the cyclitol from lower to upper nodes. Pinitol, proline, and sugars accumulated in leaf blades on soybean plants subjected to drought, but the molar concentration of pinitol in stressed plants was greater than the concentrations of proline or sugars. Although the mechanism by which pinitol participates in drought tolerance is not fully known, our results provide additional correlative evidence linking pinitol and drought tolerance in soybean.     

High concentrations of d-pinitol or d-chiro-inositol inhibit the biosynthesis of raffinose family oligosaccharides in maturing smooth tare (Vicia tetrasperma [L.] Schreb.) seeds

In the present study we have investigated the effect of exogenous cyclitols on accumulation of their galactosides and raffinose family oligosaccharides (RFOs) in maturing smooth tare (Vicia tetrasperma [L.] Schreb) seeds. Feeding d-pinitol to pods of smooth tare increased the amount of free d-pinitol and its galactosides: galactopinitol A, galactopinitol B, di- and trigalactopinitol A in seeds. Similarly, feeding d-chiro-inositol, which does not occur naturally in Vicia seeds, resulted in the transport of this cyclitol in the seed, and caused accumulation of high levels of d-chiro-inositol galactosides (fagopyritol B1, B2 and B3). Accumulation of both cyclitols and their galactosides drastically reduced accumulation of verbascose and, to a lesser extent, stachyose and di-galactosyl- myo-inositol. Feeding d-chiro-inositol also decreased accumulation of di- and tri-galactosyl pinitols, naturally occurring in seeds. Inhibition of RFOs accumulation by elevated levels of free cyclitols indicates competition between biosynthesis of both types galactosides, and similarity of both biosynthetic pathways in smooth tare seeds.     

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     

AUXIN-CYTOKININ CONTROL OF SECONDARY VASCULAR TISSUE FORMATION IN ISOLATED ROOTS OF RAPHANUS

A comparative study was made of the effectiveness of various hormone and metabolite mixtures in inducing vascular cambium initiation and secondary vascular tissue formation in isolated first-transfer roots of the radish, Raphanus sativus L. ‘White Icicle,‘ when provided to the cut basal end of the root grown in sterile culture. An auxin, such as indoleacetic acid (IAA) at 10^–5 m , a cytokinin, such as 6-benzylamino purine at 5 × 10^–6 m , a cyclitol, such as myo-inositol at 5 × 10^–4 M and sucrose at 8% were all required for maximum response. Requirements for auxin and cytokinin were absolute; in their absence no cambium was formed. The addition of cyclitol, while not an absolute requirement for cambium initiation, increased the magnitude of the response markedly. Alternative auxins such as α-naphthaleneaeetic acid and 2,4-dichlorophenoxyacetic acid were equally as effective as IAA. Alternative effective cytokinins included 6-furfurylaminopurine, 6-phenylaminopurine and 6-(γ,γ-dimethylallylamino)purine. Alternative cyclitols equivalent to myo-inositol were seyllitol and pinitol. Other related cyclitols tested were much less effective or totally inactive.     

Gas chromatographic analysis of in situ cyclitol utilization by Klebsielleae growing in redwood extracts.

Gas chromatographic analysis was employed to demonstrate in situ cyclitol utilization in aqueous extracts of redwood by isolates of Klebsiella, Enterobacter, and several other genera of gram-negative bacteria. In aqueous redwood extracts, all but one of the Klebsiella and Enterobacter isolates tested reached densities exceeding 5.0 x 10(6) cells/ml within 4 days, and all utilized pinitol and sequoyitol. Other enteric bacteria did not utilize cyclitols in this extract. A defined minimal medium, containing the carbohydrates and cyclitols (including myo-inositol) in redwood, was used to determine which carbon sources are preferentially utilized by Klebsielleae and other bacteria. It was found that D-glucose and L-arabinose were consumed by Klebsiella before the three cyclitols were utilized. Pinitol utilization proceeded in more slowly than that of sequoyitol and myo-inositol. Cyclitol utilization in the defined medium was also observed for Yersinia, Erwinia, and Salmonella. Escherichia coli isolates did not utilize cyclitol compounds. The ability to use cyclitols as a sole source of carbon can explain the high cell densities of Klebsielleae in redwood water reservoirs and in redwood lumber.     

Gas chromatographic analysis of in situ cyclitol utilization by Klebsielleae growing in redwood extracts

Gas chromatographic analysis was employed to demonstrate in situ cyclitol utilization in aqueous extracts of redwood by isolates of Klebsiella, Enterobacter, and several other genera of gram-negative bacteria. In aqueous redwood extracts, all but one of the Klebsiella and Enterobacter isolates tested reached densities exceeding 5.0 x 10(6) cells/ml within 4 days, and all utilized pinitol and sequoyitol. Other enteric bacteria did not utilize cyclitols in this extract. A defined minimal medium, containing the carbohydrates and cyclitols (including myo-inositol) in redwood, was used to determine which carbon sources are preferentially utilized by Klebsielleae and other bacteria. It was found that D-glucose and L-arabinose were consumed by Klebsiella before the three cyclitols were utilized. Pinitol utilization proceeded in more slowly than that of sequoyitol and myo-inositol. Cyclitol utilization in the defined medium was also observed for Yersinia, Erwinia, and Salmonella. Escherichia coli isolates did not utilize cyclitol compounds. The ability to use cyclitols as a sole source of carbon can explain the high cell densities of Klebsielleae in redwood water reservoirs and in redwood lumber.     

Sugars,cytolitols and seagrass phylogeny

Seagrasses of the Zannichelliaceae accumulate larger amounts and a greater range of cyclitols than do other seagrasses. Amphibolis, Cymodocea, Syringodium and Thalassodendron contained up to 10% dry weight of compounds identified as 1-chiro-inositol, muco-inositol and a methyl-O-muco-inositol, in addition to traces of myo-inositol. These compounds were not utilised as carbohydrate reserves and there is some evidence that they accumulated as by-products of an unusual glucose cyclisation mechanism. Sucrose can accumulate to more than 50% of dry weight in underground rhizomes and roots of these and other seagrasses and it was also the major initial product of photosynthesis in most seagrasses leaves.A distinct phylogenetic trend, based on their cyclitols, can be distinguished within the seagrass. This is discussed in terms of seagrass origins and biogeography.     

Preferential Leaching of Pinitol from Soybeans during Imbibition

Sugars and cyclitols leached from soybeans (Glycine max var Sparks) during imbibition were assayed as a function of time. Pinitol leached many times faster than carbohydrates. During the initial 20 minutes of imibition, the pinitol/carbohydrate ratio was 3.4, declining to 0.29 for fully imbibed seeds. The value for dry soybeans was 0.14. Hypochlorite treatment of seeds more than doubled the rate at which carbohydrates leached out, but had little effect on pinitol. A role in development of soil microorganisms is postulated for pinitol.     

Pinitol, a Compatible Solute in Mesembryanthemum crystallinum L.?

The irrigation of Mesembryanthemum crystallinum L. plants with400 mol m^–3 NaCl to induce crassulacean acid metabolism(CAM) was accompanied by the accumulation of pinitol. Pinitolconstituted 71% of the soluble carbohydrate fraction and 9.7%dry weight in the CAM form. Pinitol in the C₃ form did not exceed5% of the soluble carbohydrate fraction. Pinitol appeared metabolicallyinert: it was not readily degraded during 96 h of darkness inthe CAM form or during CAM deinduction. Preparations of CAMM. crystallinum protoplasts, vacuoles and chloroplasts showedpinitol to be chloroplastic at a concentration of about 230mol m^–3 and cytosolic at about 100 mol m^–3. No pinitolwas detected in vacuoles. CAM leaf extracts possessed a highermyo-inositol phosphate synthesising capacity than C₃ extracts,revealing greater activity in the CAM form of glucose-6-phosphatecycloaldolase, an enzyme in the pathway of pinitol synthesis. Although pinitol accumulation and CAM induction could not beseparated and appeared to be specific responses to water stress,there may not be a causal link between them. Pinitol may functionas a compatible solute in the cytosol and especially the chloroplaststo counteract the presence of high concentrations of Na⁺ andCl^– ions in the vacuole. The accumulation of pinitol,though apparently not directly related to CAM may, like CAM,be viewed as an aspect of the adaptation of the plant to a reductionin water availability. Key words: pinitol, Mesembryanthemum crystallinum L, CAM, compatible solute     

(+)-Pinpollitol: A di-O-methyl d-(+)-chiro-inositol from Pinus radiata

(+)-Pinpollitol, a new cyclitol recently isolated from the pollen of Pinus radiata, was found in the needles of this species. (+)-Pinpollitol was found to be a di-O-methyl ether Of d-(+)-chiro-inositol, and tentative isomeric structures have been proposed for the cyclitol. (+)-Pinpollitol is the first di-O-methyl inositol to be found in a gymnosperm and is one of only three di-O-methyl inositols yet found in nature.