Biosynthesis of raffinose family oligosaccharides and galactosyl pinitols in developing and maturing seeds of winter vetch [Vicia villosa Roth.]

Changes in the accumulation of two types of α-D-galactosides: raffinose family oligosaccharides and galactosyl pinitols were compared with changes in the activities of galactosyltransferases during winter vetch (Vicia villosa Roth.) seed development and maturation. Occurrence of galactinol and raffinose in young seeds and changes in activities of galactinol synthase and raffinose synthase during seed development indicated that formation of raffinose oligosaccharides (RFOs) preceded synthesis of galactopinitols. Although transfer of galactose residues into raffinose oligosaccharides increased as seeds were maturing, at late stages of seed maturation the accumulation of galactopinitols was preferred to that of RFOs. In the present study, activities of enzymes transferring galactose moieties from galactinol to D-pinitol forming galactopinitol A, and further transfer of galactose moieties from galactinol to mono- and di-galactopinitol A were detected throughout seed development and maturation. This is a new observation, indicating biological potential of winter vetch seeds to synthesize mono-, di- and tri-galactosides of D-pinitol in a pathway similar to RFOs. The pattern of changes in activities of stachyose synthase and enzymes synthesizing galactopinitols (named galactopinitol A synthase and ciceritol synthase) suggests that formation of stachyose, mono- and di-galactopinitol A (ciceritol) is catalyzed by one enzyme. High correlation between activities of verbascose synthase and enzyme catalyzing synthesis of tri-galactopinitol A from galactinol and ciceritol (named tri-galactopinitol A synthase) also suggests that biosynthesis of both types of tri-galactosides was catalyzed by one enzyme, but distinct from stachyose synthase. Changes in concentrations of galactosyl acceptors (sucrose and D-pinitol) can be a factor which regulates splitting of galactose moieties between both types of galactosides in winter vetch seeds

Inhibition of raffinose family oligosaccharides and galactosyl pinitols breakdown delays germination of winter vetch [Vicia villosa Roth.] seeds

Beside RFOs, which are commonly present in legume seeds, seeds of some species contain galactosyl pinitols (GPs). These carbohydrates, like RFOs, have been hypothesized to constitute an important energy and carbon skeletal source during germination. To test this hypothesis we have applied a specific α-galactosidase inhibitor (1-deoxygalactonojirimycin, DGJ) to germinating winter vetch (Vicia villosa Roth.) seeds, containing more galactosyl pinitols than RFOs. The breakdown of RFOs but not that of GPs was completely blocked in both embryonic axes and cotyledons tissues, during the first 18 h of imbibition in DGJ. The inhibitor only decreased the rate of GPs degradation. The inhibitory effect of DGJ on GPs degradation was partially alleviated by addition of sucrose or galactose to DGJ solutions. After three days of germination in water, RFOs and GPs disappeared in axial tissues of seeds imbibed in water, galactose or sucrose. Eighteen-hour imbibition of seeds in DGJ drastically reduced germination, by ca 50%, during the first three days. The inhibitory effect of DGJ decreased during the next seven days of germination. The presence of galactose or sucrose in imbibition solution initially stimulated seed germination, but later this effect was not statistically significant. Our study provides clear evidence that galactosyl pinitols play an important role in early winter vetch seeds germination. Additionally, we suggest that galactosyl pinitols can replace RFOs as reserve material necessary for early germination

Seed alpha-D-galactosides of selected Vicia species and enzymes involved in their biosynthesis

We compared the soluble carbohydrate composition of seeds of ten wild and cultivated species of the genus Vicia. In some Vicia species (V. angustifolia, V. grandiflora, V. sativa, V. sepium) they contained only raffinose family oligosaccharides (RFOs) and in others also D-pinitol and its α-D-galactosides. In terms of galactosyl pinitol composition they were divided into three groups: those accumulating small amounts of mono-, di-, tri-galactosyl pinitol A (GPA, ciceritol and TGPA, respectively) and unknown compound (V. sylvatica and V. hirsuta); those accumulating more ciceritol than TGPA (V. tetrasperma and V. villosa); and those accumulating more TGPA than ciceritol (V. cracca and V. tenuifolia). The differences in the activity of galactosyltransferases engaged in RFOs and galactosyl pinitol synthesis confirmed this classification. Seeds of V. angustifolia, naturally accumulating only RFOs, showed an ability to accumulate exogenously applied D-pinitol or D-chiro-inositol and to form the respective α-D-galactosyl cyclitols. Levels of synthesized galactosides depended on the type and concentration of cyclitol in the feeding solution, and seed maturation stage. However, even a high level of D-pinitol or D-chiro-inositol in the feeding solution caused accumulation of only small amounts of mono- and di-galactosyl pinitols, or tri-galactosyl D-chiro-inositol in seeds of V. angustifolia. Enhanced synthesis of galactosyl cyclitols, mainly mono- and di-galactosides of D-chiro-inositol (fagopyritols), clearly reduced production of verbascose. We suggest that exogenously applied free cyclitols inhibit biosynthesis of tri- and di-galactosides and/or cause substrate competition in enzymes of Vicia species

Effect of exogenous abscisic acid on accumulation of raffinose family oligosaccharides and galactosyl cyclitols in tiny vetch seeds [Vicia hirsuta [L.] S.F.Gray]

The role of the abscisic acid (ABA) in biosynthesis of raffinose family oligosaccharides (RFOs) and galactosyl cyclitols (Gal-C) in tiny vetch (Vicia hirsuta [L.] S.F. Gray) seeds was investigated. The ABA was applied through incubation of seed at various stage of its development. The level of RFOs and Gal-C was determined in seed maturing on plant and in seed maturing in vitro. In early stages of V. hirsuta seed development, the ABA activated the biosynthesis of galactinol, although the level of arisen galactinol quickly declined. In the later stages of V. hirsuta seed development ABA had stimulatory effect of RFOs and Gal-C biosynthesis. Influence of ABA on biosynthesis of a-galactosides in Vicia hirsuta seed seems to be dependent on abscisic acid concentration. Low concentration of ABA had stimulatory effect on a-galactosides biosynthesis, but high concentration of ABA inhibited the process

Exogenously applied D-pinitol and D-chiro-inositol modifies the accumulation of alpha-D-galactosides in developing tiny vetch [Vicia hirsuta [L.] S.F.Gray] seeds

In the present study we have investigated the effect of exogenous cyclitols on the accumulation of their galactosides and raffinose family oligosaccharides (RFOs), as well as on some enzymes important for their biosynthesis in seeds of tiny vetch (Vicia hirsuta [L.] S.F. Gray). Immature seeds during 6-day incubation with D-chiro-inositol (naturally does not appear in seeds of tiny vetch) were accumulated cyclitol and its galactosides (fagopyritols: B1 and B2). Short 4-hour incubation with D-chiro-inositol, and subsequent slow desiccation process caused accumulation of free cyclitol only, without biosynthesis of its galactosides. Feeding D-chiro-inositol to pods of tiny vetch induced accumulation of high levels of its galactosides (fagopyritol B1, B2 and B3) in maturing seeds. Similarly, feeding D-pinitol increased accumulation of its mono-, di- and tri-galactosides: GPA, GPB, DGPA and TGPA in tiny vetch seed. Accumulation of both cyclitols and their galactosides drastically reduced accumulation of verbascose. Inhibition of RFOs biosynthesis by elevated levels of free cyclitols suggests some competition between formation of both types of galactosides and similarity of both biosynthetic routes in tiny vetch seeds. Galactinol synthase (GolS) from tiny vetch seeds demonstrated ability to utilize D-chiro-inositol as galactosyl acceptor, instead of myo-inositol. Presence of both cyclitols, as substrates for GolS, caused synthesis of their galactosides: fagopyritol B1 and galactinol. However, formation of galactinol was more efficient than fagopyritol B1. D-chiro-Inositol and D-pinitol at concentrations several-fold higher than myo-inositol had inhibitory effect on GolS. Thus, we suggest that a level of free cyclitols can have an influence on the rate of galactinol biosynthesis and further accumulation of RFOs and galactosyl cyclitols in tiny vetch seeds

Benchmarking the prediction of dynamic derivatives: wind tunnel tests, validation, acceleration methods

The dynamic derivatives are widely used in linear aerodynamic models which are considered to determine the flying qualities of an aircraft: the ability to predict them reliably, quickly and sufficiently early in the design process is more and more important, in order to avoid late and costly component redesigns. This paper describes some experimental and computational activities dealing with the determination of dynamic derivatives. The work has been carried out within the FP6 European project SimSAC. Numerical and experimental results are compared for two aircraft configurations: the generic civil transport aircraft, wing-fuselage-tail configuration DLR-F12 and a generic Transonic CRuiser (TCR), which is a canard configuration. Static and dynamic wind tunnel tests have been carried out for both configurations and are briefly described. The data base generated for the DLR-F12 configuration includes force and pressure coefficients obtained during small amplitude pitch, roll and yaw oscillations while the data base for the TCR configuration includes force coefficients for small amplitude oscillations, dedicated to the determination of dynamic derivatives, and large amplitude oscillations, in order to investigate the dynamic effects on nonlinear aerodynamic characteristics. The influence of the canard has been investigated too. Dynamic derivatives have been determined on both configurations with a large panel of tools, from linear aerodynamic (Vortex Lattice Methods) to CFD (unsteady Reynolds-Averaged Navier-Stokes solvers). The study confirms that an increase in fidelity level enables dynamic derivatives to be better calculated. Linear aerodynamics (VLM) tools can give satisfactory results but are very sensitive to the geometry/mesh input data. Although all the quasi-steady CFD approaches give very comparable results (robustness) on steady dynamic derivatives, they do not allow the prediction of unsteady components of the dynamic derivatives (angular derivatives w.r.t. time): this can be done with either a fully unsteady approach (with a time-marching scheme) or with Frequency Domain solvers, both of them giving very comparable results for the DLR-F12 test case. As far as the canard configuration is concerned; strong limitations of linear aerodynamic tools are observed. A specific attention is paid to acceleration techniques in CFD methods, which allow the computational time to be dramatically reduced while keeping a satisfactory accuracy