Ralph L. Obendorf

Seed desiccation tolerance and storability: Dependence on flatulence-producing oligosaccharides and cyclitols—review and survey

Stachyose, raffinose and related flatulence-producing oligosaccharides (α-galactosyl derivatives of sucrose) are associated with desiccation tolerance and storability of seed germplasm. Orthodox seeds of species with a sucrose-to-oligosaccharide ratio of 10 years while those >1.0 have a storability half-viability period myo -inositol are universally present and galactinol is present in seeds accumulating stachyose series oligosaccharides. Seeds of some species of Leguminosae accumulate mostly stachyose series oligosaccharides, whereas seeds of other species accumulate varying levels of galactosyl derivatives of cyclitols in addition. Castor bean (Euphorbiaceae) seeds accumulate galactinol and buckwheat (Polygonaceae) embryos accumulate galacto- chiro -inositol instead of the stachyose series oligosaccharides. The mass ratio of sucrose:non-sucrose is related to storability and is applicable to seeds accumulating cyclitol derivatives. Galactinol and galacto- chiro -inositol are proposed to function in the same role as raffinose and stachyose in facilitating desiccation tolerance and storability.

Oligosaccharides and galactosyl cyclitols in seed desiccation tolerance

Soluble carbohydrates are one of multiple components required for the acquisition of desiccation tolerance during seed development and maturation. Sucrose and the raffinose series of oligosaccharides have been extensively studied in relation to seed desiccation tolerance. These galactosyl sucrose oligosaccharides are present in viable tissues of many edible seeds, especially the legumes, and contribute to flatulence after ingestion. A reduction in oligosaccharides of the raffinose series is desired by nutritionists but, if present at less than a threshold level, this this may result in reduced desiccation tolerance and storability of seeds. Some seeds that have very low amounts of raffinose and stachyose accumulate galactosyl cyclitols and small amounts of free cyclitols. Galactosyl cyclitols present in various seeds include galactosyl or methyl derivatives of myo -inositol, D-pinitol, D- chiro -inositol, D-ononitol, and scyllo -inositol. Castor bean seeds accumulate galactinol and buck-wheat seeds accumulate fagopyritol in addition to sucrose. Fagopyritol accumulation is associated with the acquisition of desiccation tolerance in buckwheat seeds. The galactosyl sucrose and galactosyl cyclitol soluble carbohydrates in maturing seeds have been proposed to have various roles in desiccation tolerance including non-toxic and non-reducing forms of seed storage products and intracellular osmotic agents contributing to the structural stability of organelles, membranes, enzymes and proteins, other macromolecules, and the glassy state. Ether derivatives of cyclitols may form liquid crystals. Methyl ether derivatives of cyclitols may have roles as cryoprotectants, desiccation protectants, and hydroxyl radical scavengers.

Fagopyritol B1, O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol, a galactosyl cyclitol in maturing buckwheat seeds associated with desiccation tolerance

Abstract.O-α-D-Galactopyranosyl-(1→2)-D-chiro-inositol, herein named fagopyritol B1, was identified as a major soluble carbohydrate (40% of total) in buckwheat (Fagopyrum esculentum Moench, Polygonaceae) embryos. Analysis of hydrolysis products of purified compounds and of the crude extract led to the conclusion that buckwheat embryos have five α-galactosyl D-chiro-inositols: fagopyritol A1 and fagopyritol B1 (mono-galactosyl D-chiro-inositol isomers), fagopyritol A2 and fagopyritol B2 (di-galactosyl D-chiro-inositol isomers), and fagopyritol B3 (tri-galactosyl D-chiro-inositol). Other soluble carbohydrates analyzed by high-resolution gas chromatography included sucrose (42% of total), D-chiro-inositol, myo-inositol, galactinol, raffinose and stachyose (1% of total), but no reducing sugars. All fagopyritols were readily hydrolyzed by α-galactosidase (EC 3.2.1.22) from green coffee bean, demonstrating α-galactosyl linkage. Retention time of fagopyritol B1 was identical to the retention time of O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol from soybean (Glycine max (L.) Merrill, Leguminosae), suggesting that the α-ga-lactosyl linkage is to the 2-position of D-chiro-inositol. Accumulation of fagopyritol B1 was associated with acquisition of desiccation tolerance during seed development and maturation in planta, and loss of fagopyritol B1 correlated with loss of desiccation tolerance during germination. Embryos of seeds grown at 18 °C, a condition that favors enhanced seed vigor and storability, had a sucrose-to-fagopyritol B1 ratio of 0.8 compared to a ratio of 2.46 for seeds grown at 25 °C. We propose that fagopyritol B1 facilitates desiccation tolerance and storability of buckwheat seeds.

Raffinose accumulation related to desiccation tolerance during maize (Zea mays L.) seed development and maturation

Summary A comprehensive analysis of soluble carbohydrates in developing embryos of field-grown maize seed was used to establish the relationship of sucrose and raffinose levels and their mass ratio to the occurrence of desiccation tolerance in immature whole kernels and in isolated embryos. Kernel, endosperm, and embryo dry mass were maximum at 56 d after pollination (DAP). Freshly harvested seeds germinated between 64 and 86 DAP, but after fast drying at 12 % relative humidity, seeds germinated between 30 and 36 DAP and between 14 and 22 DAP after slow drying on the ear. Freshly harvested isolated embryos germinated between 14 and 16 DAP, between 26 and 45 DAP after fast drying, and between 18 and 22 DAP after slow drying. Sucrose accumulated independendy of desiccation tolerance. Raffinose accumulation was not required for germination but was associated with survival after drying. Desiccation tolerance was not observed in the absence of raffinose accumulation, but the presence of small quantities of raffinose was not always associated with the onset of desiccation tolerance during early embryo development. Desiccation tolerance in maize embryos was associated with sucrose-to-raffinose mass ratios less than 20∶1 and stage 2–3 in embryo development.

Soluble carbohydrates in desiccation tolerance of yellow lupin seeds during maturation and germination

Maturing yellow lupin seeds were desiccation tolerant. Glucose, sucrose and cyclitols (mainly D-pinitol, D- chiro -inositol and myo -inositol) were predominant at the early stages of seed growth. Accumulation of the raffinose family oligosaccharides (RFOs) and the galactosyl cyclitols including galactinol, digalactosyl myo -inositol, galactopinitol A, galactopinitol B, trigalactopinitol A, ciceritol, fagopyritol B1 and fagopyritol B2 appeared during seed maturation; their increase correlated with seed germinability after desiccation. The loss of desiccation tolerance after seed germination was also studied. For the desiccation tolerance test, intact seedlings were dried rapidly or slowly followed by rehydration. Soluble carbohydrates were assayed before and after drying. Root tissues were more sensitive to desiccation than hypocotyl tissues and completely lost desiccation tolerance within 36 h of imbibition after both fast and slow-drying treatments. Survival of hypocotyls decreased gradually up to 96 h after imbibition. Loss of RFOs and galactosyl cyclitols in axis tissues preceded visible germination. Loss of desiccation tolerance was accompanied by loss of RFOs and galactosyl cyclitols and an increase in reducing sugars in cotyledon, hypocotyl and radicle tissues. Drying did not induce the accumulation of RFOs and galactosyl cyclitols in seedling tissues.

Soluble saccharides and cyclitols in alfalfa (Medicago sativa L.) somatic embryos, leaflets, and mature seeds

Abstract Soluble carbohydrates were identified and quantified during development, maturation and desiccation of somatic embryos of alfalfa ( Medicago sativa L.) and compared to soluble carbohydrates in leaflets and mature seeds, to relate changes in soluble carbohydrates to maturation events. Somatic embryos have elevated levels of sucrose. However, in contrast to mature seeds, alfalfa somatic embryos do not accumulate d -pinitol or the galactosyl derivatives of d -pinitol such as galactopinitol A, galactopinitol B and ciceritol. Lower levels of stachyose accumulate during maturation of somatic embryos, but stachyose increases to levels in mature seeds during desiccation of somatic embryos. When stachyose accumulation is limited in somatic embryos, galactinol and digalactosyl myo -inositol increase. Reducing sugars decline to low levels during desiccation of somatic embryos and sucrose: oligosaccharide ratio decreases from 2.7 to 0.9, approaching the ratio 0.2 to 0.3 in mature dry seeds. Except for the lack of pinitol and galactosyl pinitols, changes in soluble carbohydrates during the maturation and desiccation of alfalfa somatic embryos are typical of changes occurring in mature seeds that have been reported to be associated with desiccation tolerance and storability.

Soluble carbohydrates in legume seeds

Mature dry legume seeds may contain up to 30 different soluble carbohydrates. Sucrose is a major component of the total soluble carbohydrates; others include the raffinose family oligosaccharides (RFOs; raffinose, stachyose, verbascose) that are mono-, di- and tri-α-galactosyl derivatives of sucrose. Other galactosides may include α-galactosyl derivatives of the cyclitols myo -inositol (galactinol, digalactosyl myo -inositol and trigalactosyl myo -inositol), d -pinitol (galactopinitol A, digalactosyl pinitol A (ciceritol) and trigalactosyl pinitol A; and galactopinitol B; higher galactosyl oligomers of galactopintiol B have rarely been detected), d - chiro -inositol (fagopyritol B1, fagopyritol B2 and fagopyritol B3) and d -ononitol (galactosyl d -ononitol and digalactosyl d -ononitol). Small amounts of myo -inositol, d -pinitol and d - chiro -inositol may also be present. Raffinose, stachyose and verbascose increase late in seed maturation, with 70% of RFOs accumulating after maximum seed dry weight is attained. RFOs are mostly degraded during germination. Sucrose, myo -inositol, d -pinitol and d - chiro -inositol are synthesized in maternal tissues of some legumes and are transported to and unloaded by seed coats into the apoplastic space surrounding developing seed embryos. Free cyclitols may be 60% of total soluble carbohydrates in leaves and 20% in seed coat cup exudates. Increasing the supply of free cyclitols may increase the accumulation of their respective α-galactosides in mature seeds. Seeds with reduced RFO accumulation, but with normal to elevated concentrations of galactosyl cyclitols (including fagopyritols), have normal field emergence and are also tolerant to imbibitional chilling under laboratory conditions. Molecular structures, biosynthetic pathways, accumulation of soluble carbohydrates in response to seed-expressed mutations and the physiological role of galactosides are reviewed.

Molecular structure of fagopyritol A1 (O-α-d-galactopyranosyl-(1→3)-d-chiro-inositol) by NMR

The molecular structure of fagopyritol A1, a novel galactopyranosyl cyclitol from buckwheat seeds, was determined to be O-alpha-D-galactopyranosyl-(1 --> 3)-D-chiro-inositol by 1H and 13C NMR. Fagopyritol A1 is a positional isomer of fagopyritol B1 (O-alpha-D-galactopyranosyl-(1 --> 2)-D-chiro-inositol), representing a different series of fagopyritol oligomers. Trimethylsilyl derivatives of both compounds have similar mass spectra, but each may be identified by different abundance ratios of fragments with m/z 305/318 and 318/319.

Drying rate alters soluble carbohydrates, desiccation tolerance, and subsequent seedling growth of soybean (Glycine max l. Merrill) zygotic embryos during in vitro maturation

Abstract In vitro culture of soybean ( Glycine max [L.] Merr.) seeds and zygotic embryos is used to study factors affecting seed growth, maturation, desiccation tolerance, and germination. While germination of in vitro matured zygotic embryos is usually greater than 80%, seedling growth is often lower than that of seeds matured in planta. Soluble carbohydrates in axis and cotyledon tissues of soybean zygotic embryos at 0–32 days of in vitro culture were assayed before drying, after fast drying, and after slow drying and related to changes in germination and axis growth of seedlings. Germinability was improved by slow drying. A low mass ratio of sucrose to raffinose plus stachyose was associated with germinability but was not sufficient for the maintenance of high seedling growth after prolonged culture in vitro. The loss of seedling growth as a function of days in culture correlated ( r 2 =0.94) with a decline in stachyose concentration in axis tissues after slow drying. Total pinitol did not exceed that present in embryos before culture, and accumulation of galactopinitols and fagopyritol B1 was limited in embryos grown in vitro. Responses of zygotic embryos to in vitro culture may be useful in understanding problems associated with the conversion of soybean somatic embryos to plantlets and vigorous growth of plantlets.

Cyclitol galactosides in embryos of buckwheat stem–leaf–seed explants fed D-chiro-inositol, myo-inositol or D-pinitol

Crop seeds accumulate soluble carbohydrates as part of their maturation process. In legume seeds, the major soluble carbohydrates are sucrose and its galactosides raffinose, stachyose and verbascose. In buckwheat (Fagopyrum esculentum Moench) seeds, the major soluble carbohydrates are sucrose and galactosides of d-chiro-inositol, named fagopyritols. This study was conducted to determine changes in soluble carbohydrate accumulation in embryos of buckwheat seeds after feeding solutions containing the free cyclitols D-chiro-inositol, myo-inositol and d-pinitol to stem–leaf–seed explants. Feeding D-chiro-inositol to explants resulted in a fourfold to fivefold increase in the accumulation of free d-chiro-inositol, fagopyritol A1 and fagopyritol B1 in embryos of mature seeds, but resulted in 30% less embryo dry weight compared to the control treatment without cyclitols. Feeding myo-inositol to buckwheat explants increased d-chiro-inositol in leaves and increased accumulation of fagopyritol A1 and fagopyritol B1 fivefold in embryos, fagopyritol A2 and fagopyritol B2 fourfold; fagopyritol A3 and fagopyritol B3 were also detected, with no reduction in accumulated embryo dry weight. Feeding d-pinitol to buckwheat explants resulted in accumulation of free d-pinitol in mature embryos, but not galactopinitols. D-Pinitol, galactopinitol A and galactopinitol B were not detected in embryos from explants fed solutions without d-pinitol. Feeding d-pinitol also resulted in reduced D-chiro-inositol accumulation by buckwheat seeds. The results indicate that myo-inositol may be the precursor to d-chiro-inositol synthesis, and fagopyritols accumulated in response to d-chiro-inositol availability in the embryo. We suggest that increasing myo-inositol in buckwheat maternal tissues may be an effective means to enhance the accumulation of D-chiro-inositol and fagopyritols in seeds, compounds that may be beneficial for the treatment of non-insulin-dependent diabetes mellitus.