Lesław B. Lahuta

The effect of soil drought on the composition of carbohydrates in yellow lupin seeds and triticale kernels

Carbohydrate analysis was made of yellow lupin seeds (cv. Juno) and triticale kernels (cv. Dagro), produced by plants exposed to drought stress for 21 days after the initial flowering of the first node of lupin and initial earing of triticale. The seeds of all experimental variants were harvest at full maturity, dried and stored in linen bags at 18–20 °C. Soluble carbohydrates were extracted and analysed as described by Horbowicz and Obendorf (1994).Gas chromatographic separation of carbohydrates showed that raffinose family oligosaccharides (RFO) were dominant in lupin seeds. The other carbohydrates present were sucrose (10 %), cyclitols and galactosyl cyclitols (12–13 %). Soil drought resulted in higher levels of verbascose, but decreased the quantities of the other carbohydrates in lupine seeds.In triticale kernels, over 50 % of soluble sugars were composed of sucrose and maltose, while 17.7 % were raffinose and stachyose. In response to drought the content of mono- and oligosaccharides declined. The decrease of soluble carbohydrates content in seeds of lupin and triticale kernels has no effect on the seed germination and vigour. It is assumed that the changes in the concentration of soluble sugars observed under drought may impair the storability of triticale kernels, but improve it for lupine seeds.

Effect of jasmonic acid-methyl ester on the composition of carbohydrates and germination of yellow lupine (Lupinus luteus L.) seeds.

Mature seeds of yellow lupine contained sucrose, raffinose family oligosaccharides (RFOs), and galactosyl cyclitols as major soluble carbohydrates. The study showed that RFOs dominated in lupine seeds (16% DW). The disappearance of both types of alpha-d-galactosides in germinating lupine seeds was strongly inhibited by the presence of jasmonic acid-methyl ester (JA-Me) at a concentration of 10(-3)M in the incubation medium. JA-Me inhibited the activity of alpha-D-galactosidase (fraction I) during seed germination. Anatomical studies of lupine roots have shown certain cell structure differences between control and JA-Me-treated seedlings. The cross-sections of plant roots treated with JA-Me showed a characteristic folding of the cell walls in all root tissues, starting from the rhyzodermis, cortex and vascular cylinder. In water-treated (control) plants, the cell walls were rounded with no folding.

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.

Differences in accumulation of soluble α-galactosides during seed maturation of several Vicia species

Composition and levels of soluble α-galactosides: raffinose family oligosaccharides (RFOs) and galactosyl cyclitols (Gal-C) in developing seeds were measured by high resolution gas chromatography (HRGC) method. The studies were performed on maturing seeds of several wild and cultivated Vicia species: Vicia angustifolia L. (common vetch), Vicia cracca L. (bird vetch), Vicia grandiflora Scop. (large yellow vetch), Vicia hirsuta (L.) S.F.Gray (tiny vetch), Vicia sativa L. (garden vetch, spring-growing cultivar Kwarta), and Vicia villosa Roth (winter vetch).In all Vicia species similar patterns in the accumulation of RFOs were observed. Galactinol — the donor of galactosyl moieties in α-galactosides biosynthesis was present in the middle stage of seed development, before appearing measurable levels of RFOs. Accumulation of RFOs started parallel with seed desiccation process. At first accumulation of the raffinose, then few days later stachyose and finally verbascose was noticed. In the final stage of seed maturation the verbascose was the main soluble α-galactoside (up to 3% of dry weight, V. sativa). Besides the RFOs seeds of three Vicia species (V. cracca, V. hirsuta, and V. villosa) accumulated d-pinitol and its α-galactosides (Gal-C). Mono-galactosylpinitols (similar to raffinose) appeared in these species 2–4 days after galactinol, di-galactosyl pinitol A (common name: ciceritol) and di-galactosyl myo-inositol were present several days later than raffinose, and accumulation of tri-galactosyl pinitol A (TGPA) began after accumulation of stachyose. Matured seeds of V. hirsuta contained much more RFOs than Gal-C, opposite to seeds of V. villosa, and V. cracca where concentration of Gal-C was 4–8-fold higher than RFOs. In V. cracca seeds RFOs were almost replaced by Gal-C.In seeds of V. cracca and V. villosa the level of d-pinitol was significantly higher, than the level of myo-inositol. Contents of both cyclitols declined rapidly at the beginning of seed desiccation, when accumulation of RFOs and Gal-C quickly increased. We suggest that α-galactosides of d-pinitol can substitute raffinose family oligosaccharides and play similar role during seed maturation and storage.

Cyclitols affect accumulation of α-D-galactosides in developing Vicia seeds.

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 α-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 α-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.

Dehydration induces expression of GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE in seedlings of pea (Pisum sativum L.)

The exposition of 7-day-old pea seedlings to dehydration induced sudden changes in the concentration of monosaccharides and sucrose in epicotyl and roots tissues. During 24h of dehydration, the concentration of glucose and, to a lesser extent, fructose in seedling tissues decreased. The accumulation of sucrose was observed in roots after 4h and in epicotyls after 8h of stress. Epicotyls and roots also began to accumulate galactinol and raffinose after 8h of stress, when small changes in the water content of tissues occurred. The accumulation of galactinol and raffinose progressed parallel to water withdrawal from tissues, but after seedling rehydration both galactosides disappeared. The synthesis of galactinol and raffinose by an early induction (during the first hour of treatment) of galactinol synthase (PsGolS) and raffinose synthase (PsRS) gene expression as well as a later increase in the activity of both enzymes was noted. Signals possibly triggering the induction of PsGolS and PsRS gene expression and accumulation of galactinol and raffinose in seedlings are discussed.

The acquisition of desiccation tolerance in developing Vicia hirsuta seeds coincides with an increase in galactinol synthase expression and soluble α-D-galactosides accumulation.

Galactinol is the galactosyl donor for the biosynthesis of both the raffinose family oligosaccharides (RFOs) and galactosyl cyclitols (Gal-C). Its synthesis by galactinol synthase (GolS, EC 2.4.1.123) is the first committed step of the soluble α-D-galactosides biosynthetic pathway in orthodox seeds. The deposition of galactosides in seeds is suggested to be associated with desiccation tolerance (DT). In this work, for the first time, we cloned and characterized two Vicia hirsuta (L.) S.F. Gray galactinol synthase genes (VhGolS1, VhGolS2), analyzed galactinol synthase activity and measured the accumulation of galactosides of both sucrose and D-pinitol in relation to the acquisition of DT in developing seeds of this wild species. A developmentally induced increase of VhGolS1 expression preceded the rise of GolS activity in crude protein extract from maturing seeds, while the expression of the VhGolS2 gene remained low. GolS activity peaked just after the beginning of the maturation drying phase. The increase of GolS activity was not followed by galactinol accumulation, instead the high enzyme activity was related to high levels of galactose bound in soluble galactosides of the RFO and galactosyl pinitol series. Acquisition of DT coincided with an increase of VhGolS1 expression, high galactinol synthase activity and the accumulation of oligogalactosides in seeds. DT was positively correlated with the high content of soluble α-D-galactosides of both the RFO and galactosyl pinitol series as well as with the amount of galactose bound in these galactosides.

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.

Sorbitol accumulation during natural and accelerated ageing of pea (Pisum sativum L.) seeds

The aim of the present study was to determine the effect of accelerated ageing on the composition and content of the soluble carbohydrates in pea seeds of six genotypes differing in the composition of raffinose family oligosaccharides. A gradual decrease in the concentration of higher homologues of raffinose was observed along with seed ageing. At the same time the seeds lost vigor, viability and germination capacity. No increase in the concentration of reducing sugars was recorded, but sorbitol accumulated in pea embryos. Sorbitol accumulation may indicate seed quality deterioration during storage.

Soluble carbohydrates in developing and mature diaspores of polar Caryophyllaceae and Poaceae

The accumulation of soluble carbohydrates in maturing diaspores of flowering plants comprising Arctic populations of Cerastium alpinum, indigenous Antarctic species Colobanthus quitensis and Deschampsia antarctica, and cosmopolitan Poa annua from the Antarctic was investigated. For comparative purposes, the diaspores of two species of flowering plants growing in the area of Olsztyn (Poland), Poa annua (Poaceae) and Cerastium arvense (Caryophyllaceae) were used. A qualitative and quantitative analysis of soluble carbohydrates conducted by means of high-resolution gas chromatography showed that monosaccharides (glucose and fructose), maltose and sucrose, raffinose, myo-inositol and galactinol are ubiquitous in developing and mature diaspores among investigated species. Moreover, D. antarctica and P. annua caryopses additionally contained stachyose and 1-kestose; the seeds of Caryophyllaceae studied were found to contain d-pinitol and d-ononitol. The development and maturation of the seeds of polar Caryophyllaceae and Poaceae were accompanied by the changes in the concentration of their soluble carbohydrates. During maturation, seeds accumulated galactinol and raffinose family of oligosaccharides (RFOs), except C. quitensis. Although seeds of the studied Caryophyllaceae contained d-pinitol and lower amounts of d-ononitol, they did not accumulate α-d-galactoside derivatives of mentioned cyclitols. P. annua caryopses, occurring in the Antarctic, were found to accumulate considerably higher amounts of sucrose and 1-kestose than those developed in Olsztyn.