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myo-inositol, D-chiro-inositol, and D-pinitol synthesis, transport, and galactoside formation in soybean explants.

THE FREE CYCLITOLS D-pinitol (1D-3-O-methyl-chiro-inositol), D-chiro-inositol, and myo-inositol are present in soybean nodules and detected in xylem sap, indicating that they are transported in soybean plants (Streeter, 1981). D-Pinitol is synthesized in leaves of some legumes (Dittrich and Brandl, 1987; Streeter, 2001) and its accumulation in soybean leaves is associated with drought stress (Streeter et al., 2001). myo-Inositol is common in plant seeds because of its role in the synthesis of raffinose family oligosaccharides (RFOs) (Peterbauer and Richter, 2001), while D-pinitol and D-chiro-inositol are less widely found in seeds and plants (Horbowicz and Obendorf, 1994). During soybean seed maturation, when yellowing of the seed coat and embryo indicate cessation of dry matter accumulation (TeKrony et al., 1981; VerNooy et al., 1986), embryos accumulate galactosyl derivatives of these free cyclitols (Schweizer and Horman, 1981; Obendorf et al., 1998a, 1998b, 2004; Odorcic and Obendorf, 2003). Sucrose, raffinose, stachyose, galacto-pinitol A [[alpha]-D-galactopyranosyl-(1 [right arrow] 2)-4-O-methyl-1D-chiro-inositol], galactopinitol B [[alpha]-D-galactopyranosyl(1 [right arrow] 2)-3-O-methyl-1D-chiro-inositol], and fagopyritol B1 [[alpha]-D-galactopyranosyl-(1 [right arrow] 2)-1D-chiro-inositol] are the major soluble carbohydrates accumulating in maturing soybean seeds (Schweizer and Horman, 1981; Horbowicz and Obendorf, 1994; Obendorf et al., 1998a, 1998b). Accumulation of raffinose, stachyose, and the cyclitol galactosides begins when axis and cotyledon fresh weights approach maximum and continues through maximum dry matter accumulation and into seed desiccation in planta (Obendorf et al., 1998b). In immature seeds or excised embryos, accumulation of these soluble galactosides occurs during precocious maturation induced by slow drying, but these galactosides do not accumulate if drying is prevented by high humidity (Blackman et al., 1992).

myo-Inositol is important in the synthesis of phytin, galactinol, raffinose, and stachyose in soybean seeds and is synthesized by myo-inositol phosphate synthase (MIPS; EC 5.5.1.4; Hegeman et al., 2001; Hitz et al., 2002) and myo-inositol monophosphatase (IMP1; EC 3.1.3.25; Ishitani et al., 1996; Styer et al., 2004). Soybean galactinol synthase (GmGolS, UDP-galactose:myo-inositol galactosyltransferase, EC 2.4.1.123) forms galactinol ([alpha]-D-galactopyranosyl-(1 [right arrow] 1)-1L-myo-inositol) (Saravitz et al., 1987; Kerr et al., 1997; Obendorf et al., 2004) and catalyzes a galactosyl transfer from UDP-galactose to D-chiro-inositol for the synthesis of fagopyritol B1, but GmGolS does not catalyze the galactosyl transfer from UDP-galactose to D-pinitol to form galactopinitols (Obendorf et al., 2004). Soybean seed stachyose synthase (STS, galactinol:raffinose galactosyltransferase, EC 2.4.1.67) (T.P. Lin and R.L. Obendorf, unpublished, 1998) and raffinose synthase (RFS, galactinol: sucrose galactosyltransferase, EC 2.4.1.82) and STS from seeds of other species (Hoch et al., 1999; Peterbauer et al., 2002) catalyze the formation of galactopinitols from galactinol and D-pinitol.

Feeding myo-inositol, D-chiro-inositol, or D-pinitol to isolated immature soybean embryos promoted the accumulation of galactinol, fagopyritol B1, or galactopinitols, respectively, during precocious maturation induced by slow drying (Odorcic and Obendorf, 2003; Obendorf et al., 2004). As stachyose accumulated, galactopinitols and fagopyritol B1 increased during maturation and desiccation of soybean seeds in planta (Obendorf et al., 1998b) and during precocious maturation of isolated immature embryos in vitro (Obendorf et al., 1998a, 1998b, 2004).

Several studies provided evidence in support of the hypothesis that D-pinitol and D-chiro-inositol are synthesized in leaves (Dittrich and Brandl, 1987; Streeter, 2001; Streeter et al., 2001). Soybean leaves accumulated mostly D-pinitol with small amounts of D-chiro-inositol, myo-inositol, and D-ononitol (1D-4-O-methyl-myo-inositol) (Streeter, 2001). myo-Inositol was the precursor to D-ononitol and D-pinitol in leaves (Dittrich and Brandl, 1987) and also to D-chiro-inositol, either directly or indirectly through D-ononitol and D-pinitol (see review by Obendorf, 1997). D-Pinitol concentration was highest in seed coats and lower in axis and cotyledon tissues (Kuo et al., 1997), suggesting that D-pinitol is synthesized in maternal tissues and transported to soybean embryos.

There is no evidence for the synthesis of D-pinitol or D-chiro-inositol in the embryo. Total D-pinitol and D-chiro-inositol in isolated soybean zygotic embryos matured in vitro did not exceed that present in embryos before culture (Obendorf et al., 1998a, 1998b, 2004; Odorcic and Obendorf, 2003). 1D-myo-Inositol 4-O-methyltransferase (IMT, S-adenosyl-methionine:myo-inositol methyltransferase, EC 2.1.1.129) catalyzed the formation of D-ononitol and was located in leaves and stems (Wanek and Richter, 1997; Streeter et al., 2001). Soybean somatic embryos transformed with IMT formed D-ononitol but not D-pinitol (J.J. Finer and J.G. Streeter, 2002, personal communication), indicating that soybean somatic embryos do not express the genes that encode the enzymes for D-pinitol synthesis. Additionally, soybean and alfalfa (Medicago sativa L.) somatic embryos appeared to be deficient in D-pinitol and galactopinitols (Horbowicz et al., 1995; Obendorf et al., 1996; Chanprame et al., 1998).

In planta techniques were previously used to study phloem unloading in seed coats of developing soybean (Thorne and Rainbird, 1983; Rainbird et al., 1984; Ellis and Spanswick, 1987) and the transfer of assimilates between tissues that are not symplastically joined (mother to embryo) (Thorne, 1981; Wolswinkel, 1990). Soybean stem-leaf-pod explants have been used to study transport of exogenously fed substances to the seed in vitro (Neumann et al., 1983; Nooden and Letham, 1984). The detection of free cyclitols in xylem sap (Streeter, 1981) indicated the potential for adapting the soybean explant technique to study transport of free cyclitols in feeding experiments.

The objective of this research was to determine the location and transport of cyclitols and their incorporation into galactosyl cyclitols in soybean plants. We reached this objective by analyzing compounds unloaded directly from seed coats in planta and by studying the transport of free cyclitols through soybean stem-leaf-pod explants and their incorporation into galactosyl cyclitols in maturing seeds. We hoped to determine if cyclitols were directly unloaded from maternal tissues to soybean embryos, if free cyclitol concentrations were higher in maternal tissues than they were in embryonic tissues, and if galactosyl cyclitol concentrations in seeds increased as free cyclitol concentrations in maternal tissues increased.

MATERIALS AND METHODS

Three experiments were conducted. A seed coat unloading in planta experiment was designed to demonstrate the unloading of free cyclitols from maternal tissues to the developing embryo in planta. A preliminary time sequence experiment was designed to demonstrate the mass movement of free cyclitols in soybean explants through the stem to the leaf and seed coat and then to the embryo during exogenous feeding and synchronous precocious maturation of immature seeds on the explants. The major explant feeding experiment was designed to quantify the accumulation of cyclitol galactosides in mature, dry seeds after feeding solutions containing free cyclitol substrates to soybean explants.

Plant Materials

Soybean plants (cv. Chippewa 64) were grown in a greenhouse at 27[degrees]C days (14 h) and 22[degrees]C nights (10 h) with natural light supplemented with 640 [micro]mol [m.sup.-2] [s.sup.-1](PAR) incandescent light from Sylvania 1000-W metal halide lamps at Ithaca, NY, 42[degrees]N latitude. Seeds were inoculated with Bradyrhizobium japonicum (purchased from a local Agway store) and seeded in moist greenhouse soil-mix consisting of equal parts of sterilized silty loam soil (pH 7.0) and an artificial medium (0.1 [m.sup.3] sphagnum moss, 0.1 [m.sup.3] vermiculite, 0.5 kg of ferrous sulfate, and 1 kg of commercially blended fertilizer, 13-13-13) in 4-L pots. Forty pots were seeded weekly to provide a constant supply of pods and explants throughout the year. Nodulated plants (one per pot) were watered thoroughly as needed, and complete fertilizer (1 g pot 1, 20-20-20, percent as N-[P.sub.2][O.sub.5]-[K.sub.2]O equivalent) was added in water at weekly intervals.

Standards

Fructose, glucose, maltose, sucrose, raffinose, stachyose, and myo-inositol were purchased from Sigma-Aldrich (St. Louis, MO, USA). D-Pinitol and D-chiro-inositol were purchased from Industrial Research Limited (Lower Hutt, New Zealand). Fagopyritols and digalactosyl myo-inositol were extracted from buckwheat (Fagopyrum esculentum L.) seeds and purified by carbon (Mallinckrodt Baker Inc., Phillipsburg, NJ, USA)-Celite (Supelco, Bellefonte, PA, USA) column chromatography (Whistler and Durso, 1950) as described by Obendorf et al. (2000). Galactinol was extracted from lemon balm (Melissa officinalis L.) leaves and galactopinitols were extracted from seeds of hairy vetch (Vicia villosa L.) or chickpea (Cicer arietinum L.) and purified following the same general procedures.

Seed Coat Unloading In Planta

To test for compounds unloaded from seed coat cups to developing embryos in planta, soybean plants were selected at growth stages R5 (three replications) and R6 (four replications) (Fehr and Caviness, 1977). The experimental unit was one seed coat cup per plant and one plant per replication with plants replicated over time (May to August) using a completely random design. Green pods 7.2 mm in width (from both R5 and R6 plants) were selected at mid podfill (35 d after flowering) from nodes 6 to 8 and containing three seeds, each weighing 250 mg flesh weight (70 mg dry matter). Soybean embryos were surgically removed forming seed coat cups (maternal tissues) (Thorne and Rainbird, 1983). An incision was made through the pod and central seed which removed the distal half of the embryo and seed coat. Remaining embryo tissues were carefully removed from the proximal half of the seed coat, forming an empty seed coat cup. Because buffer, salts, and mannitol (Thorne and Rainbird, 1983) interfered with cyclitol analysis, unloaded compounds were collected in water. The seed coat cup was flushed with distilled water (Ellis and Spanswick, 1987) for 10 min and refilled with distilled water (200 [micro]L) to trap unloading solutes (Thorne and Rainbird, 1983). During the 2-h sampling period, distilled water in seed coat cups was replenished at 30-min intervals when samples were taken for analysis and diluted with ethanol, 1:1 (v/v). After adding 100 [micro]g of phenyl [alpha]-D-glucoside as internal standard, samples containing unloaded compounds were passed through a 10 000 MW cutoff filter (NANOSEP 10K Omega, Pall Corp., East Hills, NY, USA) by centrifugation, dried in silylation vials under nitrogen gas, and stored over P205 overnight to remove traces of water. Soluble carbohydrate residues were derivatized with trimethylsilylsylimidazole: pyridine (1:1, v/v; 200 [micro]L) for 45 min at 80[degrees]C and analyzed by high resolution gas chromatography on an HP1-MS (Agilent Technologies, Palo Alto, CA, USA) capillary column (15-m length, 0.25-mm i.d., 0.25-[micro]m film thickness) as described by Horbowicz and Obendorf (1994). Samples from plant materials that became damaged during the experimental protocol or were not collected for 2 h were excluded from analysis. Soluble carbohydrate composition was calculated as mean mg [g.sup.-1] dry matter ([+ or -] SE of the mean) for three replicate samples from R5 plants and four replicate samples from R6 plants for each 30-min interval. Significant differences (P < 0.05) were verified by t test.

Major Explant Feeding Experiment

Soybean stem-leaf-pod explants were used in feeding experiments with free cyclitols. Explants were excised above the third node from the bottom and below the third node from the top of plants at growth stage R5 (Fehr and Caviness, 1977) before leaf senescence was evident (Neumann et al., 1983; Nooden and Letham, 1984). Explants were cut mid podfill (about 35 d after flowering), when green pods were 7.2 mm in width. Pod number was reduced to one containing three seeds, each weighing 250 mg fresh weight (70 mg dry weight). Each explant included one node, one leaf, one pod and one internode. The cut, basal end of the internode (stem) of each explant was placed in a 50-mM solution of free cyclitols: 50 mM myo-inositol, 50 mM D-pinitol, or 50 mM D-chiro-inositol, or a control solution without cyclitols. All solutions contained 30 mM sucrose, 10 mM asparagine, and 10 [micro]M kinetin. Each solution was loaded into an explant through the cut stem and transported to the leaf by the transpiration stream and to the embryo through the phloem. Control solution or solutions with free cyclitols were fed to explants for 7 d (25[degrees]C; 300 [micro]mol [m.sup.-2] [s.sup.-1] PAR, fluorescent) after which explants were allowed to air dry. Seeds were removed and dried to 60 mg [g.sup.-1] moisture at 12% relative humidity over a saturated solution of LiCl for 14 d. Axis and cotyledons from mature, dry seeds were analyzed for soluble carbohydrates. Two explants per treatment in a randomized complete block design were replicated 12 times during March to May and August to November. Explants with visible contamination or mechanical damage, or those failing to transport cyclitols, were excluded.

Preliminary Time Sequence Experiment

In a preliminary experiment, two replications of explants were fed solutions of free cyclitols for 3 d and then air dried at 25[degrees]C and ambient relative humidity for 0, 2, 4, or 14 d to synchronize precocious maturation within and between feeding treatments. One replication of leaf disks (1 [cm.sup.2]) harvested after 24 h of feeding and two replications of seed coats, axis, and cotyledon tissues harvested at 0, 2, 4, or 14 d of precocious maturation were analyzed for free cyclitols and sucrose.

Analysis of Soluble Carbohydrates

Cotyledon and axis tissues were separated, weighed, and pulverized in liquid nitrogen with a mortar and pestle. Each sample was homogenized in a ground glass homogenizer with 2.2 mL of ethanol:water (1:1, v/v) and phenyl [alpha]-D-glucoside (300 [micro]g for cotyledons or 100 [micro]g for axis) as the internal standard, heated at 80[degrees]C for 45 min, and centrifuged at 27 000 x g for 20 min. Clear supernatants were passed through a 10 000 MW Nanosep cutoff filter and evaporated to dryness with nitrogen gas. Residues were stored overnight in a desiccator above [P.sub.2][O.sub.5] to remove traces of water, derivatized with trimethylsilylimidazole:pyridine (1:1, v/v), and analyzed by gas chromatography (Horbowicz and Obendorf, 1994; Obendorf et al., 1998b). Soluble carbohydrate composition was calculated as mean mg [g.sup.-1] dry matter ([+ or -] SE of the mean) for 10 to 24 replicate samples of cotyledons and axes from mature dry seeds. Significant differences (P < 0.05) from the control treatment were verified by t test.

RESULTS

Seed Coat Unloading In Planta

Seed coat cup exudates from developing soybean seeds contained myo-inositol, D-chiro-inositol, D-pinitol, and sucrose (Table 1) demonstrating the transport of these compounds from maternal tissues to the developing embryo, D-Ononitol, galactinol, galactopinitols, fagopyritol B1, raffinose, stachyose, glucose, fructose, and maltose were not detected in seed coat cup exudates. Seed coat exudates sampled on plants at the R5 growth stage had a gradual decline in the amount of downloaded sucrose, myo-inositol, and D-chiro-inositol during the 2-h sampling period. Seed coat exudates sampled on plants at the R6 growth stage had less sucrose and less cyclitols but were more consistent in amount downloaded during the 2-h sampling period. Since the samples from R5 and R6 plants were taken at different dates (May-August), we cannot be certain that the differences in amounts and patterns of downloading were totally due to plant growth stage.

Preliminary Time Sequence Experiment

A preliminary experiment followed the free cyclitols and sucrose in explants from stem feeding to leaf accumulation at 24 h after feeding, to seed coat and embryo tissues after 3 d of feeding (0 d drying), and to seed coat and embryo tissues after drying for 2, 4, and 14 d of precocious maturation. Compared with the control treatment without cyclitols, feeding myo-inositol, D-chiro-inositol, or D-pinitol to soybean explants resulted in 10-fold more free myo-inositol, 35-fold more free D-chiroinositol, and fivefold more D-pinitol in leaf tissues at 24 h after the start of the feeding experiments (data not shown). Raffinose, stachyose, galactinol, galactopinitols, and fagopyritol B1 were not detected in leaf disks from soybean explants. Total soluble carbohydrates were twofold higher in leaf disks after feeding myoinositol and threefold higher after feeding D-chiro-inositol or D-pinitol compared with the control treatment without cyclitols.

After feeding myo-inositol to soybean explants for 3 d, concentrations of free myo-inositol in seed coats (Table 2), cotyledons (Table 3), and axis tissues (data not shown) were double those in the control treatment at 0 to 2 d of air drying of explants. In the same myoinositol feeding treatment, free D-chiro-inositol was twofold to fivefold higher in seed coats (Table 2), twofold higher in cotyledons (Table 3), and fourfold to sixfold higher in axis tissues (data not shown) than in the control treatment, at 2 to 4 d of drying, and provided evidence for the synthesis of D-chiro-inositol directly from myoinositol in maternal tissues. After feeding D-chiro-inositol to soybean explants for 3 d, free D-chiro-inositol was 36-fold to 57-fold higher in seed coats (Table 2), sixfold to 12-fold higher in cotyledons (Table 3), and 11-fold to 25-fold higher in axis tissues (data not shown) at 2 to 14 d of drying than in the control treatment without cyclitols. After feeding free D-pinitol to soybean explants for 3 d, free D-pinitol was threefold to fivefold higher in seed coats (Table 2), twofold higher in cotyledons (Table 3), and twofold to sixfold higher in axis tissues (data not shown), compared with the control treatment, at 2 to 4 d of air drying. The lack of a significant difference (P > 0.05) or only small differences, compared with the control treatment, in amount of D-chiro-inositol after feeding D-pinitol to explants indicated that D-pinitol may not be the precursor to D-chiro-inositol synthesis in soybean explants.

This preliminary experiment demonstrated that free cyclitols fed to explants were in leaf tissues by 24 h after feeding and in the seed coat by 3 d after feeding. Compared with the control treatment without cyclitols, large amounts of D-chiro-inositol appeared in seeds after 3 d of feeding D-chiro-inositol to explants. Compared with the control treatment without cyclitols, smaller differences in amounts of myo-inositol or D-pinitol appeared in seeds after 3 d of feeding myo-inositol or D-pinitol, respectively, to explants. These results indicated that longer feeding times and more replications were desirable. Feeding time was increased to 7 d in the major experiment.

Major Explant Feeding Experiment

This experiment concentrated on analysis of soluble carbohydrates in mature dry seeds. After feeding 50 mM myo-inositol to soybean explants for 7 d, there was no significant difference in free myo-inositol or galactinol in axis and cotyledon tissues of mature dry seeds compared with the control treatment (Tables 4 and 5). Free D-chiro-inositol concentration was threefold higher in cotyledons and slightly higher (P < 0.05) in axis tissues, and fagopyritol B1 was slightly higher in mature dry seeds after feeding myo-inositol to explants than in the control treatment (Tables 4 and 5). Stachyose, raffinose, D-pinitol, galactopinitols, or fagopyritol B2 concentrations were not significantly different (P > 0.05) than in the control treatment.

Feeding 50 mM D-chiro-inositol to soybean explants for 7 d resulted in ninefold higher free D-chiro-inositol, 20-fold higher fagopyritol B1, and 10-fold higher fagopyritol B2 concentrations in cotyledon tissues of mature dry seeds than in the control treatment (Table 4). Feeding D-chiro-inositol to soybean explants also resulted in a 20-fold higher concentration of D-chiro-inositol in the axis (Table 5) than in the control treatment. This corresponded to 17-fold higher fagopyritol B1 and 11-fold higher fagopyritol B2 concentrations in the axis. Ciceritol concentrations were slightly lower and galactopinitol A concentration in axis tissues was slightly higher than in the control treatment (Tables 4 and 5). Total D-pinitol (free D-pinitol plus pinitol galactosides) concentration was not higher after feeding D-chiro-inositol than in the control treatment, myo-Inositol and galactinol concentrations were not different than in the control treatment after feeding D-chiro-inositol. Sucrose, raffinose, and stachyose concentrations in cotyledons were 43%, 35%, and 48% lower than in the control (Table 4). All explants fed D-chiro-inositol had shriveled seeds (113 mg per two cotyledons; 77% of control), while those explants that were fed myo-inositol (133 mg), D-pinitol (131 mg), or the control treatment (146 mg) had full and round seeds.

Feeding D-pinitol resulted in fourfold higher free D-pinitol, threefold higher galactopinitols and slightly higher ciceritol concentrations in both axis (Table 5) and cotyledon (Table 4) tissues than in the control, myo-Inositol and galactinol concentrations were slightly lower in cotyledons (Table 4), and fagopyritol B1 concentrations were slightly higher than in the control (Tables 4 and 5). Sucrose and stachyose concentrations in the cotyledons were lower than in the control (Table 4).

The same concentration of sucrose (30 mM) was fed to all treatments. Overall, none of the explant feeding experiments resulted in large changes in concentrations of sucrose, raffinose or stachyose in mature dry seeds except for some low values (30-50% lower) observed in cotyledons of explants fed with D-chiro-inositol or D-pinitol. Only traces of glucose, fructose, and maltose were detected in mature dry seeds.

DISCUSSION

This research demonstrated that sucrose, myo-inositol, D-chiro-inositol, and D-pinitol are unloaded from soybean seed coats to embryos in planta. D-Ononitol, galactosyl cyclitols, and RFOs were not detected. D-Ononitol has been detected in soybean nodules and leaves but only in small amounts (Streeter, 1985, 2001). Amides (glutamine and asparagine), amino acids, and ammonia also are unloaded by soybean seed coats (Rainbird et al., 1984), but these components were not analyzed in our experiments. Rates of sucrose downloading were similar to those (0.5-1.0 [micro]mole per hour) reported by Thorne and Rainbird (1983). To our knowledge, this is the first report confirming the transport of free cyclitols from maternal tissues, demonstrating their unloading from seed coats to developing soybean embryos in planta.

Information about cyclitol transport in stem-leaf-pod explants was gathered from analysis of leaf discs, seed coats, cotyledon tissues, and axis tissues. Large accumulations of free cyclitols in leaf discs at 24 h of feeding explants demonstrated that uptake of cyclitols occurred through the stem to the leaf, presumably via the transpiration stream. Similarly, feeding of free cyclitols to soybean explants increased free cyclitol accumulation in seed coats, cotyledons, and axis. This demonstrated movement of free cyclitols to the seed coat (maternal tissues), presumably via the phloem (Thorne, 1981), and subsequent transport to the embryo. Galactosyl cyclitols, raffinose, or stachyose were not detected in leaf tissue disks or in seed coat cup exudates indicating the absence of galactosyl cyclitols in leaves and suggesting that free cyclitols are transported from maternal tissues to soybean embryos where they are converted to their respective galactosides.

Support for the hypothesis that myo-inositol is a precursor to D-chiro-inositol in maternal tissues was provided by seed coat analysis where feeding of myo-inositol to soybean stem-leaf-pod explants resulted in twofold to fivefold more D-chiro-inositol compared with seed coats from the control treatment without cyclitols, while D-pinitol was not different. In the absence of maternal tissues, feeding myo-inositol and/or sucrose to isolated zygotic embryos did not increase total D-chiro-inositol nor total D-pinitol indicating that these cyclitols are not synthesized in soybean embryos (Obendorf et al., 2004). When feeding 50 mM myo-inositol to explants, concentrations of galactinol, the galactosyl donor for galactopinitol synthesis, and galactopinitols in embryos of mature seeds were not significantly different from control embryos. The lack of enhanced accumulation of galactopinitols in the embryo may have been due to limited levels of D-pinitol in explants fed myo-inositol. Synthesis of D-chiro-inositol in legumes is commonly believed to be via myo-inositol to D-ononitol to D-pinitol (Dittrich and Brandl, 1987) and then to D-chiro-inositol by demethylation, but neither genes nor enzymes for demethylation have been identified (reviewed by Obendorf, 1997). If the D-pinitol levels remained low, it follows that D-chiro-inositol should also have been low, but this was not the case. High levels of D-chiro-inositol in maternal tissues and in the cotyledons suggest that myo-inositol, rather than D-pinitol, is a direct precursor to the synthesis of D-chiro-inositol in soybean stem-leaf-pod explants. The high levels of free myo-inositol present in cotyledons following the feeding of soybean explants with exogenous myo-inositol may have limited the accumulation of raffinose and stachyose by feedback inhibition in the embryo. Since myo-inositol is a reaction byproduct (Peterbauer and Richter, 2001), exogenously fed myo-inositol decreased the progress of the RFS and STS reactions, explaining why raffinose and stachyose levels in the embryo were unchanged or reduced. Metabolism of myo-inositol to other products, including phytic acid and cell walls, may explain why myo-inositol concentrations were low in embryos of mature seeds.

GmGolS catalyzes the synthesis of fagopyritol B1 from o-chiro-inositol and UDP-galactose (Obendorf et al., 2004). Feeding 50 mM D-chiro-inositol to soybean explants increased the amount of free D-chiro-inositol transported to the seed resulting in large accumulations of fagopyritol B1 and fagopyritol B2 in embryos as expected. Similarly, feeding D-pinitol increased the amount of free D-pinitol transported to the seed and increased galactopinitol accumulation in the embryo. These results confirmed that accumulations of fagopyritols and galactopinitols in soybean seeds are limited by the supply of free D-chiro-inositol and D-pinitol, respectively, transported to the embryo. Excess accumulation of free D-chiro-inositol in seed coats and D-chiro-inositol and D-pinitol in the embryo tissues may alter normal transport and seed maturation processes. This could result in shriveled seeds and less dry matter accumulation in cotyledons, as observed after feeding o-chiro-inositol, or variably less accumulation of sucrose and RFOs in cotyledons of dry seeds, as observed after feeding D-chiroinositol or D-pinitol. The effect of feeding D-chiro-inositol on sucrose transport remains inconclusive due to the variable results for sucrose in seed coat tissues.

Expression of MIPS genes have been studied in developing legume seeds (Johnson and Wang, 1996; Hegeman et al., 2001; Hitz et al., 2002), but there is no evidence for the synthesis of D-pinitol or D-chiro-inositol in embryos of soybean seeds (Obendorf et al., 2004). The results presented herein are consistent with the following interpretations: (i) D-pinitol and D-chiro-inositol are synthesized in maternal tissues and transported to the soybean embryo where they accumulate as their respective galactosyl derivatives, galactopinitols, and fagopyritol B1; (ii) D-pinitol is synthesized in maternal tissues from myo-inositol through D-ononitol as an intermediate (Dittrich and Brandl, 1987); and (iii) D-chiroinositol is synthesized in maternal tissues directly from myo-inositol. Except for IMT that converts myo-inositol to D-ononitol (Wanek and Richter, 1997; Streeter et al., 2001), the enzymes and their genes responsible for the synthesis of D-pinitol and D-chiro-inositol in soybean are unknown (Obendorf, 1997). Of special interest is the evidence presented herein that D-chiro-inositol may be synthesized directly from myo-inositol and that free cyclitols were downloaded from seed coats to developing soybean embryos in planta.
Table 1. Unloading of myo-inositol, D-chiro-inositol, D-pinitol and
sucrose by soybean seed coat cups in planta. Means are in italic type.

Time (min)              myo-Inositol            D-chiro-Inositol

                  [micro]g per seed coat cup per 30-min interval

R5 (3 plants)
0-30             4.1 [+ or -] 0.3 ([dagger])    2.2 [+ or -] 0.8
30-60            2.7 [+ or -] 0.4               1.5 [+ or -] 0.7
60-90            2.7 [+ or -] 0.5               1.5 [+ or -] 0.4
90-120           2.4 [+ or -] 0.5               0.9 [+ or -] 0.3
Mean (N = 11)    3.0 [+ or -] 0.3#              1.6 [+ or -] 0.3#

R6 (4 plants)
0-30             2.1 [+ or -] 0.6               0.9 [+ or -] 0.4
30-60            1.7 [+ or -] 0.3               0.7 [+ or -] 0.3
60-90            2.1 [+ or -] 0.5               0.8 [+ or -] 0.2
90-120           2.2 [+ or -] 0.5               0.8 [+ or -] 0.3
Mean (N = 16)    2.0 [+ or -] 0.2               0.8 [+ or -] 0.1#
Mean (N = 27)    2.4 [+ or -] 0.2               1.1 [+ or -] 0.2

Time (min)             D-Pinitol               Sucrose

                 [micro]g per seed coat cup per 30-min interval

R5 (3 plants)
0-30               12.1 [+ or -] 2.1     219.3 [+ or -] 9.0
30-60              11.2 [+ or -] 3.6     149.4 [+ or -] 11.1
60-90              14.8 [+ or -] 2.4      94.2 [+ or -] 20.2
90-120             11.6 [+ or -] 3.3      69.3 [+ or -] 0.4
Mean (N = 11)      12.5 [+ or -] 1.3#    138.8 [+ or -] 18.9#

R6 (4 plants)
0-30               3.8 [+ or -] 1.0       98.8 [+ or -] 18.2
30-60              3.0 [+ or -] 0.7       78.1 [+ or -] 14.3
60-90              4.9 [+ or -] 1.3       83.6 [+ or -] 12.0
90-120             5.5 [+ or -] 2.2       90.9 [+ or -] 28.3
Mean (N = 16)      4.3 [+ or -] 0.6#      87.4 [+ or -] 7.9#
Mean (N = 27)      7.6 [+ or -] 1.0      108.3 [+ or -] 10.1

([dagger]) Mean [+ or -] SE of the mean for three replicates of R5
plants and four replicates of R6 plants.

Note: Values wit # are unloading of myo-inositol, D-chiro-inositol,
D-pinitol and sucrose by soybean seed coat cups in planta.

Table 2. Free cyclitols and sucrose in soybean seed coats at 2, 4, and
14 d of air drying after feeding leaf-stem-pod explants for 3 d with
solutions containing 50 mM myo-inositol, 50 nM D-chiro-Inositol, or 50
mM D-pinitol (A-C), or a control without cyclitols (D), each in 30 mM
sucrose, 10 mM asparagine, and 10 [micro]M kinetin.

                                 A                        B

Soluble carbohydrate        myo-Inositol          D-chiro-Inositol

                                    [micro]g per seed coat

2 d drying
  myo-Inositol           34.5 [+ or -] 4.2       12.1 [+ or -] 0.2
                            ([dagger]) *
  D-chiro-Inositol       16.1 [+ or -] 1.1 *    183.2 [+ or -] 10.1 *
  D-Pinitol              10.3 [+ or -] 1.0        6.3 [+ or -] 0.3
  Sucrose                22.4 [+ or -] 1.0       16.9 [+ or -] 0.3
4 d drying
  myo-Inositol            4.8 [+ or -] 0.0        5.5 [+ or -] 0.1
  D-chiro-Inositol        7.2 [+ or -] 0.0      159.7 [+ or -] 10.1 *
  D-Pinitol               6.8 [+ or -] 0.0       12.8 [+ or -] 1.9
  Sucrose                14.3 [+ or -] 0.0        2.5 [+ or -] 0.2
14 d drying
  myo-Inositol            8.0 [+ or -] 1.1        4.1 [+ or -] 0.4
  D-chiro-Inositol       18.0 [+ or -] 4.7      110.7 [+ or -] 23.5 *
  D-Pinitol              13.8 [+ or -] 1.6       14.6 [+ or -] 1.8
  Sucrose               108.7 [+ or -] 30.1      34.6 [+ or -] 24.8

                                 C                        D

Soluble carbohydrate         D-Pinitol                 Control

                                    [micro]g per seed coat

2 d drying
  myo-Inositol          12.4 [+ or -] 1.8       12.6 [+ or -] 0.3
  D-chiro-Inositol       4.5 [+ or -] 0.2 *      3.2 [+ or -] 0.1
  D-Pinitol             75.7 [+ or -] 2.4 *     14.2 [+ or -] 2.0
  Sucrose               19.5 [+ or -] 3.9       26.8 [+ or -] 5.1
4 d drying
  myo-Inositol           2.7 [+ or -] 0.1 *      5.7 [+ or -] 0.1
  D-chiro-Inositol       4.9 [+ or -] 0.9        2.8 [+ or -] 1.0
  D-Pinitol             74.4 [+ or -] 15.8 *    19.8 [+ or -] 0.4
  Sucrose                3.6 [+ or -] 0.9       11.8 [+ or -] 3.5
14 d drying
  myo-Inositol           3.9 [+ or -] 0.3        4.3 [+ or -] 0.4
  D-chiro-Inositol       6.3 [+ or -] 1.8        3.0 [+ or -] 0.4
  D-Pinitol             73.7 [+ or -] 22.9      21.0 [+ or -] 4.7
  Sucrose               56.1 [+ or -] 22.8      50.8 [+ or -] 31.4

* Significantly different (P < 0.05; t test) from the control
treatment.

([dagger]) Mean [+ or -] SE of the mean for two replications.

Table 3. Free cyclitols and sucrose in soybean seed cotyledons at 0, 2,
4, and 14 d of air drying after feeding leaf-stem-pod explants for 3 d
with solutions containing 50 mM myo-inositol, 50 mM D-chiro-inositol,
or 50 mM D-pinitol (A-C), or a control without cyclitols (D), each in
30 mM sucrose, 10 mM asparagine, and 10 [micro]M kinetin.

                                A                      B

Soluble carbohydrate       myo-inositol        D-chiro-inositol

                                  [micro]g per cotyledon

0 d drying
  myo-Inositol           267 [+ or -] 9        141 [+ or -] 2
                           ([dagger]) *
  D-chiro-Inositol        54 [+ or -] 2        497 [+ or -] 21 *
  D-Pinitol              114 [+ or -] 14       162 [+ or -] 13
  Sucrose               1463 [+ or -] 33 *    2041 [+ or -] 62
2 d drying
  myo-inositol            48 [+ or -] 19        39 [+ or -] 17
  D-chiro-inositol       132 [+ or -] 2 *      677 [+ or -] 42 *
  D-Pinitol              127 [+ or -] 5        159 [+ or -] 19
  Sucrose                816 [+ or -] 180      362 [+ or -] 2
4 d drying
  myo-inositol            17 [+ or -] 4          7 [+ or -] 3
  D-chiro-inositol       172 [+ or -] 21 *     489 [+ or -] 100 *
  D-Pinitol              115 [+ or -] 13       151 [+ or -] 20
  Sucrose               1437 [+ or -] 515      487 [+ or -] 92 *
14 d drying
  myo-inositol            10 [+ or -] 2          5 [+ or -] 1
  D-chiro-inositol       117 [+ or -] 21       311 [+ or -] 25 *
  D-Pinitol               79 [+ or -] 17        91 [+ or -] 1
  Sucrose                973 [+ or -] 255      658 [+ or -] 78

                                C                      D

Soluble carbohydrate        D-Pinitol               Control

                                  [micro]g per cotyledon

0 d drying
  myo-Inositol           142 [+ or -] 25       121 [+ or -] 7
  D-chiro-Inositol        31 [+ or -] 3         41 [+ or -] 4
  D-Pinitol              424 [+ or -] 73       188 [+ or -] 11
  Sucrose               1779 [+ or -] 414     2292 [+ or -] 103
2 d drying
  myo-inositol            24 [+ or -] 4         27 [+ or -] 3
  D-chiro-inositol        46 [+ or -] 0.1 *     54 [+ or -] 3
  D-Pinitol              430 [+ or -] 2 *      192 [+ or -] 30
  Sucrose                377 [+ or -] 55       827 [+ or -] 96
4 d drying
  myo-inositol             5 [+ or -] 2         14 [+ or -] 1
  D-chiro-inositol        47 [+ or -] 21        71 [+ or -] 2
  D-Pinitol              455 [+ or -] 17 *     177 [+ or -] 11
  Sucrose                657 [+ or -] 193     1623 [+ or -] 588
14 d drying
  myo-inositol             5 [+ or -] 1         11 [+ or -] 4
  D-chiro-inositol        24 [+ or -] 8         50 [+ or -] 4
  D-Pinitol              232 [+ or -] 71       126 [+ or -] 27
  Sucrose                492 [+ or -] 122     1534 [+ or -] 252

* Significantly different (P < 0.05; t test) from the control
treatment.

([dagger]) Mean [+ or -] SE of the mean for two replications.

Table 4. Soluble carbohydrates in cotyledons of mature dry soybean
seeds after feeding leaf-stem-pod explants for 7 d with solutions
containing 50 mM myo-inositol, 50 mM D-chiro-inositol, or 50 mM
D-pinitol (A-C), or a control without cyclitols (D), each containing
30 mM sucrose, 10 mM asparagine, and 10 [micro]M kinetin.

                                  A                        B

Soluble carbohydrate        myo-Inositol           D-chiro-Inositol

                                   mg [g.sup.-1] dry matter

D-Pinitol                7.77 [+ or -] 0.92       6.00 [+ or -] 0.52
                              ([dagger])
Galactopinitol A         2.01 [+ or -] 0.16       1.89 [+ or -] 0.14
Galactopinitol B         1.76 [+ or -] 0.21       1.62 [+ or -] 0.18
Ciceritol                0.63 [+ or -] 0.08 *     0.34 [+ or -] 0.11 *
D-chiro-Inositol         5.15 [+ or -] 0.77 *    15.59 [+ or -] 2.08 *
Fagopyritol B1           1.78 [+ or -] 0.23 *    21.11 [+ or -] 2.06 *
Fagopyritol B2           0.25 [+ or -] 0.07       1.52 [+ or -] 0.47 *
myo-Inositol             2.35 [+ or -] 0.79       0.58 [+ or -] 0.05
Galactinol               0.35 [+ or -] 0.06       0.23 [+ or -] 0.05
Sucrose                 37.73 [+ or -] 4.81      27.88 [+ or -] 4.09 *
Raffinose               11.22 [+ or -] 1.12       7.18 [+ or -] 0.58 *
Stachyose               23.10 [+ or -] 1.94      12.63 [+ or -] 1.40 *

                                    mg per two cotyledons

Dry matter                133 [+ or -] 6           113 [+ or -] 8 *

                                  C                        D

Soluble carbohydrate          D-Pinitol                 Control

                                   mg [g.sup.-1] dry matter

D-Pinitol               35.77 [+ or -] 2.50 *     8.34 [+ or -] 1.12

Galactopinitol A         5.81 [+ or -] 0.38 *     1.59 [+ or -] 0.15
Galactopinitol B         4.88 [+ or -] 0.45 *     1.60 [+ or -] 0.22
Ciceritol                1.13 [+ or -] 0.16 *     0.85 [+ or -] 0.07
D-chiro-Inositol         1.63 [+ or -] 0.11       1.63 [+ or -] 0.26
Fagopyritol B1           1.77 [+ or -] 0.11 *     1.05 [+ or -] 0.08
Fagopyritol B2           0.16 [+ or -] 0.02       0.15 [+ or -] 0.04
myo-Inositol             0.67 [+ or -] 0.07 *     1.69 [+ or -] 0.44
Galactinol               0.05 [+ or -] 0.01 *     0.25 [+ or -] 0.04
Sucrose                 32.49 [+ or -] 2.05 *    48.76 [+ or -] 7.62
Raffinose                9.73 [+ or -] 0.46      11.00 [+ or -] 1.47
Stachyose               14.60 [+ or -] 1.06 *    24.51 [+ or -] 3.73

                                    mg per two cotyledons

Dry matter                131 [+ or -] 4 *         146 [+ or -] 5

* Significantly different (P < 0.05; t test) from the control
treatment.

([dagger]) Mean + SE of the mean for 21, 10, 22, and 20 replications,
A-D respectively, of two cotyledons per seed.

Table 5. Soluble carbohydrates in axes of mature dry soybean seeds
after feeding leaf-stem-pod explants for 7 d with solutions containing
50 mM myo-inositol, 50 mM D-chiro-inositol, or 50 mM D-pinitol (A-C),
or a control without cyclitols (D), each containing 30 mM sucrose, 10
mM asparagine, and 10 [micro]M kinetin.

                                  A                        B

Soluble carbohydrate        myo-Inositol           D-chiro-Inositol

                                   mg [g.sup.-1] dry matter

D-Pinitol                3.65 [+ or -] 0.03       4.07 [+ or -] 0.38
                             ([dagger])
Galactopinitol A         4.61 [+ or -] 0.41       4.96 [+ or -] 0.30
Galactopinitol B         3.17 [+ or -] 0.40       3.61 [+ or -] 0.30
Ciceritol                0.65 [+ or -] 0.12       0.37 [+ or -] 0.15
D-chiro-Inositol         1.08 [+ or -] 0.14 *    12.31 [+ or -] 1.44 *
Fagopyritol B1           2.81 [+ or -] 0.28 *    30.95 [+ or -] 2.46 *
Fagopyritol B2           0.24 [+ or -] 0.09       1.62 [+ or -] 0.50 *
myo-Inositol             1.52 [+ or -] 0.15       1.37 [+ or -] 0.13
Galactinol               0.89 [+ or -] 0.10       1.00 [+ or -] 0.07
Sucrose                 34.02 [+ or -] 3.52      32.18 [+ or -] 4.31
Raffinose                8.87 [+ or -] 0.77       6.61 [+ or -] 0.81 *
Stachyose               24.56 [+ or -] 2.55      17.95 [+ or -] 1.79

                                        mg per one axis

Dry matter                2.6 [+ or -] 0.2         2.6 [+ or -] 0.1

                                  C                        D

Soluble carbohydrate          D-Pinitol                 Control

                                   mg [g.sup.-1] dry matter

D-Pinitol               21.40 [+ or -] 2.41 *     4.51 [+ or -] 0.58

Galactopinitol A        12.14 [+ or -] 1.06 *     3.65 [+ or -] 0.35
Galactopinitol B         9.42 [+ or -] 0.88 *     2.84 [+ or -] 0.42
Ciceritol                1.58 [+ or -] 0.26 *     0.79 [+ or -] 0.23
D-chiro-Inositol         1.28 [+ or -] 0.30       0.61 [+ or -] 0.12
Fagopyritol B1           2.99 [+ or -] 0.22 *     1.79 [+ or -] 0.22
Fagopyritol B2           0.08 [+ or -] 0.02       0.14 [+ or -] 0.06
myo-Inositol             0.98 [+ or -] 0.13       1.19 [+ or -] 0.20
Galactinol               0.58 [+ or -] 0.08       0.69 [+ or -] 0.08
Sucrose                 30.86 [+ or -] 4.11      35.59 [+ or -] 5.63
Raffinose                9.81 [+ or -] 1.08      10.30 [+ or -] 0.76
Stachyose               23.16 [+ or -] 2.50      22.02 [+ or -] 2.32

                                        mg per one axis

Dry matter                2.4 [+ or -] 0.2         3.0 [+ or -] 0.1

* Significantly different (P < 0.05; t test) from the control treatment.

([dagger]) Values represent mean [+ or -] SE for 24, 18, 20, and 17
replications, A-D respectively, of one axis per seed.


ACKNOWLEDGMENTS

We gratefully acknowledge Angela Zimmerman, Hirotoki Takemasa, Elizabeth Vasallo, and Marika Olson for assistance with experiments. This research was conducted as part of Multistate Projects W-168 (NY-C 125-423) and W-1168 (NY-C 125-802) and was supported in part by a fellowship from The Kosciuszko Foundation to M.H., a Cornell-Hughes Program Undergraduate Research Fellowship to C.I.G., Morley and Hatch-Multistate Undergraduate Research Grants (C.I.G.), and Cornell University Agricultural Experiment Station federal formula funds received from Cooperative State Research, Education and Extension Service, U.S. Department Agriculture. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.

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Carly I. Gomes, Ralph L. Obendorf, * and Marcin Horbowicz

C.I. Gomes and R.L. Obendorf, Seed Biology, Department of Crop and Soil Sciences, 617 Bradfield Hall, Cornell University Agricultural Experiment Station, Cornell University, Ithaca, NY 14853-1901; M. Horbowicz, Research Institute of Vegetable Crops, 96-100 Skierniewice, Poland. Received 19 Apr. 2004. * Corresponding author (rlo1@ cornell.edu).
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