Purification and molecular structure of two digalactosyl D-chiro-inositols and two trigalactosyl D-chiro-inositols from buckwheat seeds.

Two digalactosyl D-chiro-inositols and two trigalactosyl D-chiro-inositols, members of the fagopyritol A series and fagopyritol B series, were isolated from buckwheat (Fagopyrum esculentum Moench) seeds. Structures of the first three were determined by 1H and 13C NMR. Fagopyritol B2 is alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->2) -1D-chiro-inositol, and fagopyritol A2 is alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->3)- 1D-chiro-inositol. Fagopyritol A3, a trigalactosyl D-chiro-inositol, is alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1 -->6) -alpha-D-galactopyranosyl-(1-->3)- 1 D-chiro-inositol. From analysis of hydrolysis products, the second trigalactosyl D-chiro-inositol, fagopyritol B3, isalpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->6) -alpha-D-galactopyranosyl-(1-->2)-1D-chiro-inositol.

Molecular structure of fagopyritol A1 (O-alpha-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.

Fagopyritols, D-chiro-inositol, and other soluble carbohydrates in buckwheat seed milling fractions.

Fagopyritols are mono-, di-, and trigalactosyl derivatives of D-chiro-inositol that accumulate in seeds of common buckwheat (Fagopyrum esculentum Moench) and may be important for seed maturation and as a dietary supplement. Fagopyritols and other soluble carbohydrates were assayed in mature groats and 11 milling fractions of common buckwheat seed. Because fagopyritols are in embryo and aleurone tissues, differences in fagopyritol concentrations reflect varying proportions of these tissues in each milling fraction. Bran milling fractions contained 6.4 g of total soluble carbohydrates per 100 g of dry weight, 55% of which was sucrose and 40% fagopyritols. Flour milling fractions had reduced fagopyritol concentration [0.7 g/100 g of dry weight total fagopyritols in the dark (Supreme) flour and 0.3 g/100 g in the light (Fancy) flours]. Fagopyritol B1 was 70% of total fagopyritols in all milling fractions. Fagopyritols were 40% of total soluble carbohydrates in groats of two cultivars of common buckwheat but 21% in groats of tartary buckwheat [Fagopyrum tataricum (L.) Gaertn.], probably a reflection of environment and genetics. A rhamnoglucoside present in tartary buckwheat was not detected in common buckwheat.

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

O-alpha-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 alpha-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 alpha-galactosidase (EC 3.2.1.22) from green coffee bean, demonstrating alpha-galactosyl linkage. Retention time of fagopyritol B1 was identical to the retention time of O-alpha-D-galactopyranosyl-(1-->2)-D-chiro-inositol from soybean (Glycine max (L.) Merrill, Leguminosae), suggesting that the alpha-galactosyl 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 degrees 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 degrees C. We propose that fagopyritol B1 facilitates desiccation tolerance and storability of buckwheat seeds.

Maturation proteins and sugars in desiccation tolerance of developing soybean seeds.

The desiccation-tolerant state in seeds is associated with high levels of certain sugars and maturation proteins. The aim of this work was to evaluate the contributions of these components to desiccation tolerance in soybean (Glycine max [L.] Merrill cv Chippewa 64). When axes of immature seeds (34 d after flowering) were excised and gradually dried (6 d), desiccation tolerance was induced. By contrast, seeds held at high relative humidity for the same period were destroyed by desiccation. Maturation proteins rapidly accumulated in the axes whether the seeds were slowly dried or maintained at high relative humidity. During slow drying, sucrose content increased to five times the level present in the axes of seeds held at high relative humidity (128 versus 25 mug/axis, respectively). Stachyose content increased dramatically from barely detectable levels upon excision to 483 mug/axis during slow drying but did not increase significantly when seeds were incubated at high relative humidity. Galactinol was the only saccharide that accumulated to higher levels in axes from seeds incubated at high relative humidity relative to axes from seeds that were slowly dried. This suggests that slow drying serves to induce the accumulation of the raffinose series sugars at a point after galactinol biosynthesis. We conclude that stachyose plays an important role in conferring desiccation tolerance.

Maturation proteins associated with desiccation tolerance in soybean.

A set of proteins that accumulates late in embryogenesis (Lea proteins) has been hypothesized to have a role in protecting the mature seed against desiccation damage. A possible correlation between their presence and the desiccation tolerant state in soybean seeds (Glycine max L. Chippewa) was tested. Proteins that showed the same temporal pattern of expression as that reported for Lea proteins were identified in the axes of soybean. They were distinct from the known storage proteins and were resistant to heat coagulation. The level of these "maturation" proteins was closely correlated with desiccation tolerance both in the naturally developing and in the germinating seed: increasing at 44 days after flowering, when desiccation tolerance was achieved, and decreasing after 18 hours of imbibition, when desiccation tolerance was lost. During imbibition, 100 micromolar abscisic acid or Polyethylene glycol-6000 (-0.6 megapascals) delayed disappearance of the maturation proteins, loss of desiccation tolerance, and germination. During maturation, desiccation tolerance was prematurely induced when excised seeds were dried slowly but not when seeds were held for an equivalent time at high relative humidity. In contrast, maturation proteins were induced under both conditions. We conclude that maturation proteins may contribute to desiccation tolerance of soybean seeds, though they may not be sufficient to induce tolerance by themselves.

Soybean Seed Water Relations during in Situ and in Vitro Growth and Maturation.

Water, osmotic, and pressure potentials of soybean (Glycine max [L.] Merrill) embryos and related maternal tissues were measured during periods of seed growth and maturation to test the involvement of embryo water relations in seed maturation. Seeds were matured in situ or in an in vitro liquid culture medium in detached pods or as isolated seeds. Changes in water relations of embryo tissues were independent of maternal tissues. During seed maturation in situ, water and osmotic potentials in both embryo and maternal tissues declined sharply near the time of maximum dry weight. During in vitro seed culture with and without pods, water and osmotic potentials in axis and cotyledon tissues declined continuously during growth. Water and osmotic potentials of the seed coat, which was present only during in vitro seed culture with pods, changed little during the culture period. Positive turgor in the embryo was maintained beyond maximum dry weight and the loss of green color during in vitro culture but declined to zero at maturity in situ. The osmotic potential in embryo tissues declined from -1.1 megapascals at early pod fill to between -1.65 and -2.2 megapascals at maximum seed dry weight across all maturation environments. It is suggested that the decreasing osmotic potential in the growing soybean embryo reaches a threshold level that is associated with cessation of growth and onset of seed maturation.

Precocious Germination during In Vitro Growth of Soybean Seeds.

Immature Glycine max (L.) Merrill seeds were grown and matured in liquid medium at 25 degrees C under fluorescent light. In standard medium containing minerals, 146 millimolar sucrose and 62.5 millimolar glutamine (osmolality 0.24), precocious germination seldom occurred with a starting seed size of less than 300 milligrams fresh weight. Frequency of precocious germination increased with increased starting seed size. Sucrose concentration strongly affected precocious germination while glutamine concentration had no effect. Starting with 300 to 350 milligrams fresh weight seeds, treatments which reduced the sucrose concentration or lowered the osmolality of the culture medium stimulated precocious germination, and increased the fresh weight growth but not the dry weight growth of seeds. Increasing the osmolality to 0.38 with sucrose or mannitol prevented precocious germination without reducing dry weight accumulation in seeds. In medium with initially low osmolality, precocious germination was inhibited by addition of 1 to 100 micromolar abscisic acid to the medium without a reduction in seed growth. During growth and maturation of large soybean seeds in vitro, precocious germination and other abnormal tissue growth can be prevented by high sucrose or mannitol concentrations in the medium or by addition of abscisic acid.

Photoperiod effects on growth rate of in vitro cultured soybean embryos.

The growth rate of excised soybean (Glycine max [L.] Merrill) embryos grown in liquid culture increased linearly as photoperiod was increased from 0 to 20 h at an irradiance of 9 W m-2 measured between wavelengths of 700-850 nm from clear incandescent lamps. When irradiance levels were varied between 0.1 and 1.7 W m-2, the maximum growth rates of embryos occurred at ca. 0.5 W m-2 at both 10- and 16-h photoperiods. When the light source was changed from clear incandescent lamps, with a red (600-700 nm) to far-red (700-770 nm) ratio of ca. 1.07, to a BCJ incandescent lamp (Corning Glass dark red, transparent envelope and a red to far-red ratio of ca. 0.19), the growth rate of embryos slowed. These results are consistent with a high irradiance response for growth of soybean embryos.

Use of Tetraethylthiuram Disulfide to Discriminate between Alternative Respiration and Lipoxygenase.

Mitochondria from axes of Glycine max (L.) Merr. cv. Chippewa 64 seedlings purified on discontinuous Percoll gradients exhibited classical cyanide-resistant respiration. These mitochondria also possessed lipoxygenase activity, as determined by O(2) uptake in the presence of 0.8 millimolar linoleic acid. This activity is inhibited by most known inhibitors of alternative respiration (i.e. hydroxamates and propyl gallate). Tetraethylthiuram disulfide (disulfiram) at 50 micromolar inhibited cyanide-resistant succinate oxidation by 90 per cent, whereas concentrations as high as 100 micromolar had no effect on lipoxygenase activity. Use of tetraethylthiuram disulfide allows discrimination between alternative respiration and lipoxygenase activity in mitochondria.

Transcription of Ribosomal and Messenger RNAs in Early Wheat Embryo Germination.

Germinating wheat embryos (Triticum aestivum L). synthesize both ribosomal and messenger RNA at the earliest times after the onset of germination. The rates of synthesis of these two RNAs are determined at various stages in germination by an analysis of newly synthesized radioactive RNA on oligo(dT)-cellulose. The rate of messenger RNA synthesis is essentially constant throughout 18 hours of germination, while that of ribosomal RNA synthesis increases steadily, particularly after the onset of cell expansion (6 hours), reaching at 16 to 18 hours, a rate of synthesis between 5- and 20-fold greater than that observed at the earliest stages. The net effect is a relative decrease in the fraction of transcribed high molecular weight RNA that is mRNA. Throughout the first 7 hours of germination, mRNA is 25 to 30% of the transcribed fraction, whereas by 16 to 18 hours it has declined to a level of 4 to 8%.

Rapid Increase in Adenosine 5'-Triphosphate during Early Wheat Embryo Germination.

The ATP content of isolated wheat (Triticum aestivum L. var. Polk) embryos increases 5-fold during the first 30 minutes and 10-fold during the first hour of germination to 80% of maximum. The ATP level remains at approximately 800 nanomoles per gram of tissue during the next 15 hours. ADP, AMP, and total adenosine phosphates decrease between 1 and 6.5 hours, while adenylate energy charge increases from 0.6 to 0.8 and remains constant. The rapid increase in ATP during imbibition is consistent with the energy requirement for polyribosome formation and protein synthesis during the first hours of germination. A method for determining nanomole quantities of ATP in tissue extracts by isotopic dilution of gamma-(32)P-ATP in the hexokinase reaction is outlined.

Influence of age and illumination on distribution of several calvin cycle enzymes in greening barley leaves.

Activities of phosphoriboisomerase, phosphoribulokinase, and ribulose 1,5-diphosphate carboxylase, protein content, and chlorophyll accumulation in dark-grown barley seedlings were measured before and after illumination. Enzymatic activities, levels of soluble protein, and accumulation (upon illumination) of chlorophyll in leaves declined from tips toward the base. In response to increasing time of illumination, chlorophyll accumulation and activities of phosphoribulokinase and ribulose 1,5-diphosphate carboxylase (enzymes located in chloroplasts) increased most in tip portions whereas activity of phosphoriboisomerase and levels of soluble protein (constituents not confined to chloroplasts) increased similarly in all sections of the leaf. Maximum activity of phosphoribulokinase and maximum accumulation of chlorophyll shifted toward median portions of the leaf blade with increased age of seedling before illumination. Maximum activity of ribulose 1,5-diphosphate carboxylase and maximum level of soluble protein occurred in all leaf sections when the seedlings were 7 days of age before illumination.

Effects of Light Intensity on Photosynthetic Carboxylative Phase Enzymes and Chlorophyll Synthesis in Greening Leaves of Hordeum vulgare L.

The effects of various light intensities on in vivo increases in activities of phosphoriboisomerase, phosphoribulokinase and ribulose-1, 5-diP carboxylase and on synthesis of chlorophyll were studied in greening leaves of Hordeum vulgare L.Each enzyme was already present in dark-grown plants, but further increases in activities required both a light treatment of the intact plant and a favorable temperature. The amount of enzymatic activity and chlorophyll developed was governed by light intensity.Measured activities of phosphoriboisomerase and ribulose 1,5-diP carboxylase were highly correlated with synthesis of chlorophyll at all intensities studied. Measured activity of phosphoribulokinase was correlated with synthesis of chlorophyll only at saturating or near saturating light intensities. At decreasing light intensities the response curves of this enzyme differed from those of chlorophyll and of phosphoriboisomerase and ribulose-1, 5-diP carboxylase. A lag period of phosphoribulokinase increased with decreasing light intensity. After the lag period a rapid rate of increase occurred which did not level off during 48 hours of illumination. Thus, a different control mechanism may be operative in inducing increased activity of this enzyme.