Soluble oligosaccharides and galactosyl cyclitols in maturing soybean seeds in planta and in vitro.
Mature soybean seeds contain free D-pinitol, Dchiroinositol, D-ononitol, and myo-inositol and their galactosyl derivatives including galactopinitol A (O-[Alpha]-D-galactopyranosyl-(1 [right arrow] 2)-4-O-methyl-D-chiro-inositol), ciceritol (O-[Alpha]-D-galactopyranosyl-(1 [right arrow] 6)-O-[Alpha]-D-galactopyranosyl-(1 [right arrow] 2)-4-O-methyl-D-chiro-inositol), galactopinitol B (O-[Alpha]-D-galactopyranosyl-(1 [right arrow] 2)-3-O-methyl-D-chiro-inositol), fagopyritol B1 (O-[Alpha]-D-galactopyranosyl-(1 right arrow] 2)-D-chiro-inositol), and galactinol (O-[Alpha]-D-galactopyranosyl-(1 [right arrow] 1)-L-myo-inositol; a.k.a. O-[Alpha]-D-galactopyranosyl-(1 [right arrow] 3)-D-myo-inositol) (Schweizer and Horman, 1981; Quemener and Brillouet, 1983; Horbowicz and Obendorf, 1994). Galactinol, myo-inositol, and the raffinose series of oligosaccharides have been characterized during soybean seed development (Amuti and Pollard, 1977; Yazdi-Samadi et al., 1977; Dornbos and McDonald, 1986; Saravitz et al., 1987; Lowell and Kuo, 1989; Blackman et al., 1992; Sun et al., 1994; Kuo et al., 1997a, b). However, except for galactinol, the developmental pattern for accumulation of galactosyl cyclitols in developing soybean seeds has not been reported. The objective of the current study was to determine if galactosyl cyclitols accumulate in axis and cotyledon tissues of developing soybean seeds in association with the onset of desiccation tolerance and if the pattern of galactosyl cyclitol accumulation during in vitro culture of zygotic embryos is similar to that in planta. Galactosyl oligomers of the raffinose series, galactinol series, galactopinitol A series, galactopinitol B series, and fagopyritol B series (Horbowicz and Obendorf, 1994) were determined in axis and cotyledons of seeds at 17 stages of seed growth in planta, seeds matured in planta at 18 and 25 [degrees] C, and zygotic embryos matured in vitro at 15 and 25 [degrees] C. Changes in soluble carbohydrates were compared with the onset of desiccation tolerance and other seed maturation events.
MATERIALS AND METHODS
Soybean (cv. `Chippewa 64') plants were grown in a greenhouse at 27 [degrees] C day (14 h) and 21 [degrees] C night (10 h) as previously described (Obendorf et al., 1983). Nodulated plants were watered thoroughly as needed and supplemented with 1 g/pot of complete fertilizer (20-20-20, per cent as N-[P.sub.2][O.sub.5][K.sub.2]O equivalent) in water at weekly intervals. Seeds harvested at 17 growth stages during seed growth and maturation in planta were analyzed for fresh and dry weights, water content and concentration, and changes in color. Freshly harvested axis and cotyledon tissues from in planta grown seeds were frozen in liquid nitrogen and stored at -80 [degrees] C until analyzed for soluble carbohydrates. Greenhouse-grown soybean plants at stage R4 were grown to seed maturity at 18 or 25 [degrees] C constant temperature in growth chambers with approximately 600 [micro]mol [m.sup.-2] [s.sup.-1] fluorescent light for 14 h daily. Axis and cotyledon tissues from mature dry seeds were analyzed for soluble carbohydrates.
Seeds were selected and cultured as previously described (Obendorf et al., 1984) at 15 and 25 [degrees] C. Immature seeds were harvested approximately 35 d after flowering (DAF) at 280 to 320 mg fresh weight per seed and individual seeds without pods were transferred aseptically into presterilized 125-mL Erlenmeyer flasks containing 20 mL of liquid medium as previously described (Obendorf et al., 1983) except that [Na.sub.2][MoO.sub.4] was omitted, sucrose was 300 mM, and the complete medium was sterilized by ultrafiltration. Seeds were removed from culture at 4-d intervals from 0 to 32 d, rinsed in sterile distilled water and blotted to remove excess water. Freshly harvested embryos (not dried) were weighed, separated into axis and cotyledon tissues and stored frozen at -80 [degrees] C until analyzed for soluble carbohydrates.
Analysis of Sugars and Cyclitols
For each of three to four replications per treatment, one frozen cotyledon was pulverized in a mortar over dry ice, homogenized in a ground-glass homogenizer with 2 mL of ethanol:water, 1:1, v:v, containing 200 [micro]g of phenyl [Alpha]-D-glucoside as internal standard, heated at 80 [degrees] C for 45 min, and centrifuged at 23 700 X g for 20 min. For each of three to five replications per treatment, one frozen axis was pulverized in a mortar over dry ice, homogenized in a ground-glass homogenizer with 1 mL of ethanol:water, 1:1, v:v, containing 100 [micro]g of phenyl [Alpha]-D-glucoside as internal standard, heated and centrifuged. The pellet was re-extracted and supernatants pooled. Aliquots (0.5 mL) of clear supernatants were passed through a 10 000 MW cutoff filter and evaporated to dryness in a stream of nitrogen. Residues were stored overnight in a desiccator with phosphorus pentoxide to remove traces of water and derivatized with trimethylsilylimidazole:pyridine (1:1, v/v) for analysis of soluble carbohydrates by high resolution gas chromatography on a DB-1 capillary column as previously described (Horbowicz and Obendorf, 1994).
High-purity solvents were used in all analyses. Ethanol was obtained from Mallinckrodt (St. Louis, MO), and sugar standards, trimethylsilylimidazole, and pyridine were purchased from Sigma Chemical Company (St. Louis, MO). Galactinol was a gift from T.M. Kuo (Peoria, IL); D-chiro-inositol and D-pinitol were a gift from S.J. Angyal (Kensington, New South Wales, Australia); galactopinitol A, galactopinitol B, D-pinitol, and D-chiro-inositol were a gift from J.G. Streeter (Wooster, OH); verbascose, D-chiro-inositol, and D-pinitol were a gift from P. Wursch (Lausanne, Switzerland); verbascose and D-chiro-inositol were a gift from P. Adams and R.G. Jensen (Tucson, AZ); D-ononitol, sequoyitol and L-(+)-bornesitol were a gift from F.A. Loewus (Pullman, WA), and scyllo-inositol was a gift from P.P.N. Murthy (Houghton, WI). Fagopyritol B1 was isolated from buckwheat (Fagopyrum esculentum Moench) seeds. Ciceritol was identified by retention time identical to that for ciceritol in extracts of axes from chickpea (Cicer arietinum L.) seeds (Quemener and Brillouet, 1983). Structures of the raffinose series of oligosaccharides and several series of galactosyl cyclitols in seeds have been illustrated (Horbowicz: and Obendorf, 1994; Obendorf, 1997).
Peaks identified as [Alpha]-galactosides disappeared after incubation of extracts with green coffee bean [Alpha]-galactosidase (EC 126.96.36.199) (Boehringer Mannheim Corporation, Indianapolis, IN), and compounds were hydrolyzed to their monomeric components with 2 M trifluoroacetic acid. Peaks identified as D-pinitol, D-chiro-inositol, and myo-inositol were verified by GC-MS. Amounts of unknown carbohydrates were estimated by calculation with nearest known standard. Quantities are expressed as mean [+ or -] SE of the mean for three to four replications.
Axis Tissues In Planta at 27/21 [degrees] C (day/night)
During soybean seed development in planta, axis tissues had maximum water content between 38 and 46 d after flowering (DAF), corresponding to maximum fresh weight (Fig. 1A). The axis reached maximum dry weight (physiological maturity or mass maturity) at 44 DAF (Fig. 1A) when axis water concentration was 0.59 to 0.60 g [g.sup.-1] dry weight (Fig. 1E). Sucrose in axis tissues increased early in seed development and followed the increase in axis dry weight with only minor changes after maximum dry weight at 44 DAF (Fig. 1B). By contrast, stachyose and raffinose increased rapidly between 40 and 46 DAF and continued to increase to 58 DAF (Fig. 1B) after maximum dry weight and during the early stages of water loss (Fig. 1A). The level of stachyose exceeded that of sucrose in axes of maturing seeds in planta. The rapid increase in stachyose in axis tissues (Fig. 1B) was associated with the onset of desiccation tolerance (measured as germinability after rapid drying) of seeds in planta (Fig. 1A). Galactinol increased rapidly between 40 and 44 DAF, consistent with its role as galactosyl donor to sucrose to form raffinose and to raffinose to form stachyose (Fig. 1C). myo-Inositol decreased to low amounts between 40 and 46 DAF, consistent with its role as precursor to galactinol biosynthesis (Fig. 1C). Galactopinitol A, galactopinitol B, and fagopyritol B1 accumulated between 40 and 50 DAF in axis tissues (Fig. 1D), and in general followed the profiles of desiccation tolerance (Fig. 1A), stachyose accumulation (Fig. 1B), and loss of green color (yellowing) in axis tissues (Fig. 1E). Ciceritol, in the galactopinitol A series, accumulated in small amounts between 48 and 56 DAF (Fig. 1D). Free D-pinitol was present at 10 to 15 [micro]g per axis but declined slightly during biosynthesis of galactopinitol A and galactopinitol B, consistent with its precursor role (Fig. 1D). Free D-chiro-inositol was low throughout seed development and maturation with a relative decline during rapid accumulation of fagopyritol B1.
[Figure 1 ILLUSTRATION OMITTED]
Cotyledon Tissues In Planta at 27/21 [degrees] C (day/night)
Accumulation of soluble carbohydrates in cotyledons followed similar patterns to those in axis tissues, but about 4 d later. Maximum dry weight of cotyledons occurred at 48 DAF (Fig. 2A) when cotyledon water concentration was 0.55 g [g.sup.-1] dry weight (Fig. 2E). Thereafter, fresh weight and water content declined. In contrast to the pattern in axis tissues, cotyledon sucrose increased to 62 DAF (Fig. 2B), 14 d after maximum dry weight. Stachyose accumulated rapidly between 44 and 54 DAF, continuing past maximum dry weight and into the period of slow drying in planta. The decrease in myo-inositol and a simultaneous increase in galactinol at 44 to 46 DAF (Fig. 2C) were coincident with the start of stachyose accumulation (Fig. 2B) and the accumulation of galactopinitol A, galactopinitol B, and fagopyritol B1 (Fig. 2D). Ciceritol did not accumulate to measurable levels in cotyledons. Free pinitol increased to 46 DAF (Fig. 2D). Total pinitol (free plus that in galactosyl pinitols) in cotyledons continued to accumulate after maximum dry weight, reaching [is greater than] 1000 [micro]g of pinitol per seed at 52 DAF. Free D-chiro-inositol in cotyledons also increased to 46 DAF, but to lesser amounts than pinitol, and decreased during accumulation of fagopyritol B1 (Fig. 2D). Loss of green color in cotyledons also occurred during accumulation of stachyose and galactosyl cyclitols (Fig. 2E).
[Figure 2 ILLUSTRATION OMITTED]
Zygotic Embryo Growth In Vitro at 15 and 25 [degrees] C
Soluble carbohydrates in axis tissues were determined during in vitro growth of zygotic embryos at 25 [degrees] C (Fig. 3). Embryos were removed from plants at about 34 DAF. Loss of green color in axis tissues began within the first 4 d of culture and was complete after about 20 d in culture at 25 [degrees] C (Fig. 3A). Cotyledons lost their green color by 32 d in culture. Axis fresh weight increased during the first 16 d in culture (Fig. 3A) to about four times that of axes in planta. Likewise, sucrose accumulation in axes of zygotic embryos grown in vitro also increased to four to five times the amount of sucrose in axes in planta, raffinose increased two times, and stachyose accumulated to amounts similar to those in planta (Fig. 3B). Small amounts of verbascose were detected at 24 to 32 d of in vitro culture (Fig. 3B). The concentration of stachyose and raffinose was lower and the ratio of sucrose to oligosaccharides (raffinose plus stachyose) was higher in axes matured in vitro compared with axes matured in planta. Loss of green color was not a good indicator of stachyose accumulation in axes of zygotic embryos grown in vitro, because stachyose accumulated after axes were mostly yellow. While more variable than in planta, the patterns of stachyose accumulation (Fig. 3B), myo-inositol and galactinol (Fig. 3C), and galactopinitol A, galactopinitol B, and fagopyritol B1 (Fig. 3D) in axes of zygotic embryos matured in vitro followed patterns similar to those in axes of seeds matured in planta, except that myo-inositol was higher and galactopinitol A, galactopinitol B, and fagopyritol B1 were lower in vitro than in planta. D-Pinitol and D-chiro-inositol levels were similar to those in axes of seeds in planta (Fig. 3D). In addition, seven unknowns with retention times between raffinose and stachyose were observed to accumulate in axis tissues during in vitro culture (Fig. 3E).
[Figure 3 ILLUSTRATION OMITTED]
When zygotic embryos were cultured at 15 [degrees] C, fresh weight growth of axis tissues was slower and continued to 26 d in culture (Fig. 4A). Final fresh weight was about 30% less than at 25 [degrees] C. The profiles for loss of green color in axis and cotyledon tissues were similar to those at 25 [degrees] C. Sucrose accumulated rapidly during the first 4 d of culture (Fig. 4B). Stachyose and raffinose accumulated more slowly between 10 and 26 d in culture but reached amounts similar to axes cultured at 25 [degrees] C or in planta. Galactinol increased at 10 and 12 d in culture (Fig. 4C) corresponding to the start of stachyose accumulation. myo-Inositol was relatively constant throughout the 32 d of culture (Fig. 4C). D-Pinitol and D-chiro-inositol declined during culture at 15 [degrees] C, but galactopinitol A, galactopinitol B, and fagopyritol B1 accumulated rapidly at 10 d and then more slowly to 26 d (Fig. 4D), reaching amounts similar to those at 25 [degrees] C but less than in axes in planta. The seven unknowns accumulated to lesser amounts at 15 [degrees] C than they did at 25 [degrees] C (Fig. 4E).
[Figure 4 ILLUSTRATION OMITTED]
Seeds Matured at 18 and 25 [degrees] C
Fifteen soluble carbohydrates were detected in axis and cotyledons of soybean seeds matured at 18 and 25 [degrees] C (Table 1). Axes of seeds matured at 25 [degrees] C accumulated higher concentrations of sucrose, raffinose, D-pinitol, D-chiro-inositol, fagopyritol B1 and total soluble carbohydrates than those matured at 18 [degrees] C [95% confidence interval; calculated standard deviations do not overlap for marked (*) means]. Surprisingly, stachyose and galactinol concentrations were not different (95% confidence interval; calculated standard deviations overlap) in axis tissues between the maturation temperatures. Likewise, myo-inositol, galactopinitol A, ciceritol, galactopinitol B, and maltose were not significantly different (P [is greater than] 0.05; standard deviations overlap) between maturation temperatures. However, the mass ratio of sucrose to raffinose plus stachyose was lower in axes from seeds matured at 18 [degrees] C than those matured at 25 [degrees] C. Only D-chiro-inositol concentration was higher in cotyledons from seeds matured at 25 [degrees] C. Concentrations of the other soluble carbohydrates in cotyledons were not significantly different (P [is greater than] 0.05; standard deviations overlap) between seeds matured at 18 and 25 [degrees] C.
[TABULAR DATA 1 NOT REPRODUCIBLE IN ASCII]
Galactopinitol A, galactopinitol B, and fagopyritol B1 accumulate in axis tissues in association with the onset of desiccation tolerance in soybean seeds during development in planta. Accumulation of these galactosyl cyclitols follow the profile of stachyose accumulation, but collectively, the amount of galactosyl cyclitols is [is less than] 15% of that of stachyose. Profiles for accumulation of the galactosyl cyclitols in cotyledons are similar to those in axis tissues, but their accumulation in cotyledons is about 4 d later than in axis tissues. Galactopinitol A, galactopinitol B, and fagopyritol B1 also accumulate in axis tissues of zygotic embryos during in vitro culture at 15 or 25 [degrees] C, but at slightly reduced levels, concomitantly with accumulation of stachyose. These results demonstrate that in vitro culture of soybean zygotic embryos could provide a useful tool for study of the genetic and metabolic regulation of galactopinitol and fagopyritol biosynthesis. Temperature during soybean zygotic embryo maturation in vitro and in planta had only minor effects on the accumulation of galactopinitol A, galactopinitol B, and fagopyritol B1, but sucrose accumulation was greater at 25 [degrees] C both in planta and in vitro. The lower concentration of sucrose in axis tissues from seeds matured at 18 [degrees] C contributed to the lower mass ratio of sucrose to raffinose plus stachyose at 18 [degrees] C compared with 25 [degrees] C. In contrast to soybean, the concentration of galactopinitols was increased when white lupin (Lupinus albus L.) seeds were matured at 28 [degrees] C, but overall the temperature effects on soluble carbohydrates were small (Gorecki et al., 1996).
Accumulation of galactopinitol A, galactopinitol B, and fagopyritol B1 was also associated with the loss of green color in axis and cotyledon tissues of seeds grown in planta. The lack of association between yellowing of axis tissues and galactosyl cyclitol accumulation during in vitro culture of zygotic embryos, indicates that loss of green color is independent of the biosynthesis of soluble galactosyl oligomers. The visual use of tissue yellowing to indicate the timing of accumulation of stachyose, galactopinitols, and fagopyritol B1 is valid only for embryo tissues of seeds maturing in planta.
In contrast to soybean, buckwheat seeds do not accumulate raffinose, stachyose, or galactopinitols, but produce vigorous seedlings after prolonged dry storage. During seed development and maturation, buckwheat embryos accumulate fagopyritols, primarily fagopyritol B1, in association with the onset of desiccation tolerance (Horbowicz et at., 1997). In future work, it would be of interest to determine if galactosyl cyclitols (fagopyritol B1 or galactopinitols) may substitute for stachyose in providing protection for desiccation tolerance and storability in soybean seeds.
We thank T.M. Kuo, S.J. Angyal, J.G. Streeter, P. Wursch, P. Adams, R.G. Jensen, F.A. Loewus, and P.P.N. Murthy for supplying authentic standards. We thank Mei Wong and Jacque Chang for assistance with some of the seed cultures, and Simona Gokhin for assistance with analysis of mature seeds. With special thanks, we acknowledge J.L. Koch and D.L. Linscott for preliminary experiments.
This work was conducted as part of Western Regional Research Project W-168 (NY-C 125423) and was supported in part by a grant from Pioneer Hi-Bred International, Inc. to R.L.O. and M.E.S. We gratefully acknowledge Fellowship support from The Kosciuszko Foundation to M.H. and partial support from the Cornell Tradition Program and federal work-study funds.
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Ralph L. Obendorf,(*) Marcin Horbowicz, Alexandra M. Dickerman, Patrick Brenac, and Margaret E. Smith
Ralph L. Obendorf, Alexandra M. Dickerman, and Patrick Brenac, Seed Biology, Dep. of Soil, Crop and Atmospheric Sciences, Cornell Univ. Agric. Exp. Stn, 619 Bradfield Hall, Cornell Univ., Ithaca, New York 14853-1901; Marvin Horbowicz, Res. Inst. of Vegetable Crops, Skierniewice, Poland; Margaret E. Smith, Dep. of Plant Breeding and Biometry, Cornell Univ. Agric. Exp. Stn, Cornell Univ., Ithaca, New York 14853-1901. Received March 14, 1997. (*)Corresponding author (email@example.com).
Published in Crop Sci. 38:78-84 (1998).