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

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.K Ke ey y W Wo or rd ds s: : D-chiro-inositol, D-pinitol, raffinose family of oligosaccharides, galactosyl pinitols, enzymes, Vicia, seed.

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 a-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.KEY WORDS: germination, galactosyl pinitols, raffinose family oligosaccharides, seed, winter vetch. _______________ Abbreviations:RFOs raffinose family of oligosaccharides; GPs a-D-galactosides of D-pinitol; GPA galactosyl pinitol A; GPB galactosyl pinitol B; DGPA di-galactosyl pinitol A (ciceritol); TGPA tri-galactosyl pinitol A; DGJ 1-deoxygalactonojirimycin; DGMI di-galactosyl-myo-inositol

The mechanism preferentially regulating accumulation of raffinose family oligosaccharides (RFOs) or galactosyl cyclitols in legume seeds still remains unknown. The broad range of raffinose family oligosaccharides and galactosyl pinitols in the composition of seeds of Vicia genus gives researchers an exceptional opportunity for investigations on relationships in biosynthesis of both types of a-D-galactosides. Feeding explants of Vicia species radically different in the composition of RFOs and galactosyl pinitols with basic galactose acceptors, sucrose (for RFOs) or cyclitols (for galactosyl cyclitols) can be a helpful method for assessment of their regulatory role in accumulation of a-D-galactosides in seeds. Garden vetch (Vicia sativa L.) seeds, naturally accumulating RFOs, demonstrated an ability to take up and use exogenously applied D-pinitol and D-chiro-inositol for synthesis of their mono-, di-and tri-galactosides. Together with the accumulation of new galactosides, the concentration of RFOs decreased. In fine-leaved (Vicia tenuifolia Roth.) vetch seeds such a remarkably high concentration of galactosyl pinitols (GPs) was discovered that they nearly replaced RFOs, which is unique among legumes. If the accumulation of both types of galactosides is correlated with concentration of galactose acceptors, elevated levels of sucrose or myo-inositol should promote accumulation of RFOs, instead of GPs. Unexpectedly, feeding fine-leaved vetch raceme explants with myo-inositol or sucrose promoted accumulation of GPs, but not of RFOs. Our comparison of accumulation and biosynthesis of both types of galactosides (RFOs and GPs) throughout development and maturation of seeds from fine-leaved vetch has indicated that preferential accumulation of GPs is associated with the drying of seeds during maturation. Different patterns in activities of enzymes engaged in RFOs' biosynthetic pathway and galactosyltransferases involved in biosynthesis of GPs indicated that distinct forms of enzymes can operate in both pathways. The feeding of explants with D-chiro-inositol causes accumulation of fagopyritols B1 in seeds of both Vicia species, which suggests presence of the same or a similar form of galactinol synthase. Accumulation of fagopyritols in fine-leaved vetch seeds did not affect accumulation of RFOs or galactosyl pinitols.Keywords D-Pinitol Á D-chiro-Inositol Á Raffinose oligosaccharides Á Galactosyl cyclitols Á Seed Á Vetch Abbreviations RFOsRaffinose family oligosaccharides GPs a-D-Galactosides of D-pinitol Communicated by F. Corbineau.

In the present study, the feeding of stem-flag leaf-ear explants of wheat, triticale and barley with D-chiro-inositol and D-pinitol was used for modification of the composition of soluble carbohydrates in grains without genetic transformation of plants. Maturing grains indicated ability to uptake exogenously applied cyclitols, not occurring naturally in cereal plants, and synthesized their α-D-galactosides. The pattern of changes in soluble carbohydrates during grain maturation and germination was not disturbed by the uptake and accumulation of cyclitols. Both, D--chiro-inositol and D-pinitol as well as their α-D-galactosides can be an additional pool of soluble carbohydrates accumulated by maturing grains, without decreasing seeds viability. This is the first report indicating the possibility of introduction of cyclitols with potentially human health benefits properties into cereal grains.