# Patent

## Title
Fagopyritol synthase genes and uses thereof

## Abstract

The present invention relates to an isolated DNA molecule encoding a fagopyritol synthase. A method for producing a fagopyritol, an insulin mediator, an insulin mediator analogue, an insulin mediator homologue, or an insulin mediator inhibitor is also described. The method includes providing a fagopyritol synthase, providing a substrate comprising a galactosyl donor and a galactosyl acceptor, and combining the fagopyritol synthase with the substrate under conditions effective produce a fagopyritol, an insulin mediator, an insulin mediator analogue, an insulin mediator homologue, or an insulin mediator inhibitor.

## Background
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/379,373, filed May 9, 2002, which is hereby incorporated by reference in its entirety.


The present invention was developed with support under Cooperative State Research, Education and Extension Service, U.S. Department of Agriculture Project No. NYC-125323. The U.S. Government has certain rights.


FIELD OF THE INVENTION
The present invention relates to fagopyritol synthase genes and methods of producing fagopyritols, insulin mediators, insulin mediator analogues, or insulin mediator homologues.
BACKGROUND OF THE INVENTION
Embryos of many plant seeds accumulate sucrose and the raffinose family of oligosaccharides (RSO), such as raffinose, stachyose and verbascose, as the major soluble sugars in mature seeds (Horbowicz et al., Seed Sci. Res. 4:385-405 (1994); Obendorf, See Sci. Res. 7:63-74 (1997)). Soybean (Glycine max (L.) Merrill) seeds accumulate soluble carbohydrates, primarily sucrose, raffinose, and stachyose and lesser amounts of galactopinitol A, galactopinitol B, ciceritol, and fagopyritol B1 in axis and cotyledon tissues as part of the seed maturation process (Obendorf et al., Crop Science 38:78-84 (1998)). By contrast, embryos of maturing buckwheat (Fagopyrum esculentum Moench) seeds accumulate fagopyritols, galactosyl derivatives of D-chiro-inositol, instead of raffinose and stachyose (Horbowicz et al., Planta 205:1-11 (1998)). Six fagopyritols, in two different series, are present in buckwheat embryos: fagopyritol A1 (α-D-galactopyranosyl-(1→3)-1D-chiro-inositol), fagopyritol A2 (α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→3)-1D-chiro-inositol), fagopyritol A3(α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→3)-1D-chiro-inositol), fagopyritol B1 (α-D-galactopyranosyl-(1→2)-1D-chiro-inositol), fagopyritol B2 (α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→2)-1D-chiro-inositol), and fagopyritol B3 (α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→2)-1D-chiro-inositol) (Horbowicz et al., Planta 205:1-11 (1998); Szczecinski et al., Bull. Polish Acad. Sci., Chem. 46:9-13 (1998); Obendorf et al., Carbohydr. Res. 328:623-627 (2000); Steadman et al., Carbohydr. Res. 331:19-25 (2001)). Fagopyritols are concentrated in the axis and cotyledon tissues of embryos in mature buckwheat seeds (Horbowicz et al., Planta 205:1-11 (1998)). Buckwheat bran, a commercial milling fraction (Steadman et al., J. Cereal Sci. 33:271-278 (2001)), is a rich source of fagopyritols (Steadman et al., J. Agric. Food Chem. 48:2843-2847 (2000)).
Fagopyritols are of considerable interest for the treatment of non-insulin dependent diabetes mellitus (NIDDM) and polycystic ovary syndrome (PCOS), both insulin response disorders. Fagopyritol A1 is isosteric with 2-amino-2-deoxy-α-D-galactopyranosyl-(1→3)-1D-chiro-inositol (Berlin et al., Tetrahedron Lett. 31:1109-1112 (1990)) related to a putative insulin mediator (Berlin et al., Tetrahedron Lett. 31:1109-1112 (1990); Lamer et al., Biochem. Biophys. Res. Comm. 151:1416-1426 (1988)) deficient in subjects with NIDDM (Fonteles et al., Diabetologia 39:731-734 (1996); Lamer et al., Diabetes Rev. 7:217-231 (1999)) and PCOS (Nestler et al., J. Clin. Endocrin. Metab. 83:2001-2005 (1998); Nestler et al., New England J. Med. 340:1314-1320 (1999); Nestler et al., J. Pediatric Endocrin. Metab. 13(Suppl. 5):1295-1298 (2000)).
Enzymes (fagopyritol synthases) catalyzing the biosynthesis of fagopyritols in buckwheat or other plants have not been described. The present invention is directed to overcoming this and other deficiencies in the prior art. SUMMARY OF THE INVENTION
The present invention relates to isolated nucleic acid molecules which encode a fagopyritol synthase and the amino acid sequences encoded by such nucleic acid molecules.
Another aspect of the present invention pertains to host cells, expression vectors, transgenic plants, and transgenic plant seeds containing the isolated nucleic acid molecules of the present invention.
The present invention is also directed to a method for producing a fagopyritol, an insulin mediator, an insulin mediator analogue, or an insulin mediator homologue. This method includes providing a fagopyritol synthase, providing a substrate including a galactosyl donor and a galactosyl acceptor, and combining the fagopyritol synthase with the substrate under conditions effective to produce a fagopyritol, an insulin mediator, an insulin mediator analogue, or an insulin mediator homologue.
The fagopyritol synthases of the present invention can be used to produce fagopyritols, insulin mediators, insulin mediator analogues, or insulin mediator homologues which can be used in a pharmaceutical composition which also includes a pharmaceutical carrier. This pharmaceutical composition or, alternatively, the fagopyritols, insulin mediators, insulin mediator analogues, or insulin mediator homologues can be administered to a patient to treat disorders, such as diabetes and PCOS. In addition, the fagopyritol synthases can be used to produce transgenic plants useful for nutraceutical applications.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the complete nucleotide sequence of the full-length FeGolS-1 cDNA clone (SEQ ID NO:1). The amino acid sequence deduced from the major open reading frame of the cDNA sequence is shown below (SEQ ID NO:2). The translation start (ATG) and termination (TAA) codons are underlined.
FIG. 2 shows the complete nucleotide sequence of the full-length FeGolS-2 cDNA clone (SEQ ID NO:3). The amino acid sequence deduced from the major open reading frame of the cDNA sequence is shown below (SEQ ID NO:4). The translation start (ATG) and termination (TGA) codons are underlined.
FIG. 3 shows the complete nucleotide sequence of the partial FeGolS-3 cDNA clone (SEQ ID NO:5). The amino acid sequence deduced from the major open reading frame of the cDNA sequence is shown below (SEQ ID NO:6). The termination (TGA) codon is underlined.
FIG. 4 shows the complete nucleotide sequence of the soybean GmGolS clone (SEQ ID NO:7). The amino acid sequence deduced from themajor open reading frame of the cDNA sequence is shown below (SEQ ID NO:8). The translation start (ATG) and termination (TAA) codons are underlined.
FIG. 5 shows a summary of the cloning of three FeGolS cDNAs. The full length FeGolS-1 (1269 bp) and FeGolS-2 (1326 bp) cDNA clones and the partial FeGolS-3 (986 bp) cDNA clone are diagrammed in scale with the locations of the restriction enzyme recognition sites at the top. For FeGolS-1 and FeGolS-2, the overlapping partial cDNA clones generated by 5′ and 3′ rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) are shown under the full-length clones. The translation start (ATG) and termination (TAA/TGA) codons are shown with their relative positions indicated in the parentheses. The PCR primers used in the RACE-PCR assays are shown with arrows indicating the direction of the PCR amplifications.
FIG. 6 shows a multiple sequence alignment of the three FeGolS cDNA clones (protein ID: AAM96868, AAM96870, AAM96869; SEQ ID NOS:2, 4, and 6, respectively). Amino acid sequences deduced from the three FeGolS cDNAs were aligned by the CLUSTAL W (1.81) multiple sequence alignment program. The conserved amino acid residues are shown in bold letters. The amino acid sequences corresponding to the PCR primers used in the RT-PCR assays are boxed.
FIG. 7 shows a multiple sequence alignment of GolS amino acid sequences from various plant species. The amino acid sequences deduced from three FeGolS cDNA clones (FeGolS-1 AY126718, FeGolS-2 AY126716, FeGolS-3 AY126717; protein ID AAM96868, AAM96870, AAM96869; SEQ ID NOS: 2, 4, and 6, respectively) and G. max (SEQ. ID NO.9) were aligned with those reported from various plant species, including A. thaliana (SEQ ID NOS:9 and 10), B. napus (SEQ ID NO:11), P. sativum (SEQ ID NO:12), O. sativa (SEQ ID NO:13), A. reptans GolS-1 (SEQ ID NO:14), and A. reptans GolS-2 (SEQ ID NO: 15) (indicated in the left margin), by the CLUSTAL W program. The highly conserved amino acid residues are shown in bold letters. The hypothetical manganese-binding motif, DXD, is italicized, and an asterisk marks the conserved serine phosphorylation site. The accession numbers of the sequences used in the comparison are: Glycine max, AY126715 (protein ID AAM96867) (BE330777); Arabidopsis thaliana, AC002337 and AC009323; Brassica napus, AF106954; Pisum sativum, PSA243815; Ajuga reptans GolS-1, ARE237693; and Ajuga reptans GolS-2, ARE237694.
FIG. 8 shows the bacterial expression and purification of recombinant GolS proteins. The recombinant GolS proteins expressed in E. coli and subsequently purified proteins were examined by SDS-PAGE: lane 1, protein molecular weight marker (kDa of bands indicated in the left margin); lanes 2 and 3, 10 μg each of the total soluble protein extracts from uninduced and induced bacteria cells harboring FeGolS-1 cDNA, respectively; lane 4, 0.25 μg of the purified recombinant FeGolS-1 protein; lanes 5 and 6, 10 μg each of the total soluble protein extracts from uninduced and induced bacteria cells harboring FeGolS-2 cDNA, respectively; lane 7, 0.25 μg of the purified recombinant FeGolS-2 protein; lanes 8 and 9, 10 μg each of the total soluble protein extracts from uninduced and induced bacteria cells harboring GmGolS cDNA, respectively; lane 10, 0.25 μg of the purified recombinant GmGolS protein.
FIGS. 9A-F show product accumulation with purified recombinant protein. FIGS. 9A-C show fagopyritol synthase products with 20 mM D-chiro-inositol, 20 mM UDP-galactose (“UDP-Gal”), 5 mM MnCl2, 2 mM dithiothreitol (“DTT”), and 50 mM Hepes buffer, pH 7.0. FIGS. 9D-F show galactinol synthase products with 20 mM myo-insositol, 20 mM UDP-Gal, 5 mM MnCl2, 2 mM DTT, and 50 mM Hepes buffer, pH 7.0. Reactions were run 30 to 300 minutes at 30° C. with recombinant protein FeGolS-1 (FIGS. 9A and D), FeGolS-2 (FIGS. 9B and E), and GmGolS (FIGS. 9C and F). Products were analyzed by high resolution gas chromatography. Retention times were: fagopyritol A1 (A1), 24.3 minutes; fagopyritol B1 (B1), 24.8 minutes; and galactinol (Gol), 25.3 minutes.
FIGS. 10A-F are graphs showing accumulated soluble carbohydrates in axis and cotyledon tissues after precocious maturation of immature soybean embryos as a function of myo-inositol concentration. The results are shown after feeding myo-inositol (0 to 100 mM) plus sucrose (100 to 0 mM) (100 mM total concentration) for 24 hours at 25° C. followed by 14 days precocious maturation in slow drying series relative humidities. Values are mean ±SE(n=12). FIGS. 10A-C are axis tissues. FIGS. 10D-F are cotyledon tissues. Abbreviations in FIGS. 10A-F are as follows: myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro), fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA), galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose (Suc).
FIG. 11 is a schematic of the proposed pathways for biosynthesis of fagopyritol B1, galactinol, raffinose, stachyose, and galactopinitols. Abbreviations: Glycine max galactinol synthase (GmGolS); raffinose synthase (RFS); stachyose synthase (STS).
FIGS. 12A-F are graphs showing accumulated soluble carbohydrates in axis and cotyledon tissues after precocious maturation of immature soybean embryos as a function of time of slow drying. The results are shown after feeding 30 mM myo-inositol and 100 mM sucrose for 24 hours at 25° C. followed by 0 to 14 days precocious maturation in slow drying time series relative humidities. Values are mean±SE (n=9). FIGS. 12A-C are axis tissues. FIGS. 12D-F are cotyledon tissues. Abbreviations in FIGS. 12A-F are as follows: myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro), fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA), galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose (Suc).
FIGS. 13A-F are graphs showing accumulated soluble carbohydrates in axis and cotyledon tissues after precocious maturation of immature soybean embryos as a function of D-chiro-inositol concentration. The results are shown after feeding D-chiro-inositol (0 to 100 mM) plus sucrose (100 to 0 mM) (100 mM total concentration) for 24 hours at 25° C. followed by 14 days precocious maturation in slow drying series relative humidities. Values are mean ±SE(n=18). FIGS. 13A-C are axis tissues. FIGS. 13D-F are cotyledon tissues. Abbreviations in FIGS. 13A-F are as follows: myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro), fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA), galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose (Suc).
FIGS. 14A-F are graphs showing accumulated soluble carbohydrates in axis and cotyledon tissues after precocious maturation of immature soybean embryos as a function of time of slow drying. The results are shown after feeding 100 mM D-chiro-inositol for 24 hours at 25° C. followed by 0 to 14 days precocious maturation in slow drying time series relative humidities. Values are mean±SE (n=9). FIGS. 14A-C are axis tissues. FIGS. 14D-F are cotyledon tissues. Abbreviations in FIGS. 14A-F are as follows: myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro), fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA), galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose (Suc).
FIGS. 15A-F are graphs showing accumulated soluble carbohydrates in axis and cotyledon tissues after precocious maturation of immature soybean embryos as a function of D-pinitol concentration. The results are shown after feeding D-pinitol (0 to 100 mM) plus sucrose (100 to 0 mM) (100 mM total concentration) for 24 hours at 25° C. followed by 14 days precocious maturation in slow drying series relative humidities. Values are mean±SE (n=9). FIGS. 15A-C are axis tissues. FIGS. 15D-F are cotyledon tissues. Abbreviations in FIGS. 15A-F are as follows: myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro), fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA), galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose (Suc).
FIGS. 16A-F are graphs showing accumulated soluble carbohydrates in axis and cotyledon tissues after precocious maturation of immature soybean embryos as a function of time of slow drying. The results are shown after feeding 100 mM D-pinitol for 24 hours at 25° C. followed by 0 to 14 days precocious maturation in slow drying time series relative humidities. Values are mean±SE (n=9). FIGS. 16A-C are axis tissues. FIGS. 16D-F are cotyledon tissues. Abbreviations in FIGS. 16A-F are as follows: myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro), fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA), galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose (Suc).
FIGS. 17A-F are graphs showing accumulated soluble carbohydrates in axis and cotyledon tissues after precocious maturation of immature soybean embryos as a function of sucrose concentration. The results are shown after feeding sucrose (0 to 200 mM) for 24 hours at 25° C. followed by 14 days precocious maturation in slow drying series relative humidities. Values are mean±SE (n=9). FIGS. 17A-C are axis tissues. FIGS. 17D-F are cotyledon tissues. Abbreviations in FIGS. 17A-F are as follows: myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro), fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA), galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose (Suc).
FIGS. 18A-F are graphs showing accumulated soluble carbohydrates in axis and cotyledon tissues after precocious maturation of immature soybean embryos as a function of time of slow drying. The results are shown after feeding 100 mM D-chiro-inositol and 100 mM D-pinitol for 24 hours at 25° C. followed by 0 to 14 days precocious maturation in slow drying time series relative humidities. Values are mean±SE (n=9). FIGS. 18A-C are axis tissues. FIGS. 18D-F are cotyledon tissues. Abbreviations in FIGS. 18A-F are as follows: myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro), fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA), galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose (Suc).
FIGS. 19A-B show GmGolS products. FIG. 19A shows galactinol (retention time 25.8 min) accumulation after enzyme incubation with 25 mM myo-inositol, 25 mM UDP-Gal, 5 mM MnCl2, and 2 mM DTT at 30° C. FIG. 19B shows fagopyritol B1 (retention time 25.3 min) accumulation after enzyme incubation with 25 mM D-chiro-inositol, 25 mM UDP-Gal, 5 mM MnCl2, and 2 mM DTT at 30° C. Reactions were run to near completion to emphasize products.
FIG. 20 shows pathways for biosynthesis of D-pinitol and D-chiro-inositol (from Obendorf, Seed Sci. Res. 7:63-74 (1997), which is hereby incorporated by reference in its entirety). The D-pinitol biosynthetic pathway converts myo-inositol to D-ononitol to D-pinitol in legume leaves. The illustration of D-pinitol on the bottom left is intentionally incorrectly numbered for clarity. 1L-myo-inositol 6-O-methyltransferase (EC 2.1.1.129; also known as 1D-myo-inositol 4-O-methyltransferase; reaction d) catalyzes the conversion of myo-inositol to D-ononitol. The conversion of D-ononitol to D-pinitol (e,f) may involve a two-step oxidoreductase reaction in soybean and other legumes: step 1, D-ononitol+NAD+→4-O-methyl-1 D-myo-1-inosose+NADH; step 2, 4-O-methyl-1 D-myo-1-inosose+NADPH→D-pinitol+NADP+. It is believed that D-chiro-inositol is formed by demethylation of D-pinitol (g,h), but neither the enzyme nor the gene have been identified. Prokaryotes, algae, insects, and animals appear to make D-chiro-inositol from myo-inositol (i,j). For details see Obendorf, Seed Sci. Res. 7:63-74 (1997), which is hereby incorporated by reference in its entirety. Earlier literature proposed that myo-inositol was converted to D-pinitol via sequoyitol (a,b,c) but the identity of sequoyitol is in doubt and may have been D-ononitol.
FIG. 21 shows the raffinose family oligosaccharides (RFO) and galactosyl cyclitol biosynthetic pathways. GAS (or GolS), galactinol synthase; RFS, raffinose synthase; STS, stachyose synthase; VBS, verbascose synthase; GGT, galactan:galactan galactosyltransferase. Cyclitol may stand for D-ononitol, D-pinitol, or D-chiro-inositol, respectively. All reactions are reversible (after Peterbauer et al., Seed Sci. Res. 11:185-198 (2001), which is hereby incorporated by reference in its entirety).
FIG. 22 shows the revised RFO and galactosyl cyclitol biosynthetic pathways. GAS (or GolS), galactinol synthase; RFS, raffinose synthase; STS, stachyose synthase; VBS, verbascose synthase; GGT, galactan:galactan galactosyltransferase. All reactions are reversible (modified from Peterbauer et al., Seed Sci. Res. 11:185-198 (2001), which is hereby incorporated by reference in its entirety).
FIGS. 23A-C are graphs showing accumulation of major carbohydrates during maturation of buckwheat embryos at 15, 22, and 30° C. Values (μg/embryo) are the mean±SE of the mean for three replicate samples. DAP=days after pollination.
FIGS. 24A-C are graphs showing the accumulation of D-chiro-inositol and its digalactosides, fagopyritol A2 and fagopyritol B2, during maturation of buckwheat embryos at 15, 22, and 30° C. Values (μg/embryo) are the mean±SE of the mean for three replicate samples. DAP=days after pollination.
FIGS. 25A-C are graphs showing the accumulation of myo-inositol and its galactosides, galactinol and digalactosyl myo-inositol, during maturation of buckwheat embryos at 15, 22, and 30° C. Values (μg/embryo) are the mean±SE of the mean for three replicate samples. DAP=days after pollination.
FIGS. 26A-B are graphs showing seed germination rate (%) and seedling hypocotyls length (mm) of buckwheat seeds matured at 15, 22, and 30° C. Values are the mean±SE of the mean for three replicate samples.
FIG. 27 shows the biosynthesis UDP-galactosamine from α-D-galactose-1-phosphate and UDP-glucose using UDP-glucose:α-D-galactose-1-phosphate uridylyltransferase (EC 2.7.7.9).
FIG. 28 shows biosynthesis of the putative insulin mediator (2-amino-2-deoxy-α-D-galactosamine-(1-3)-1D-chiro-inositol) and an isomer (2-amino-2-deoxy-α-D-galactosamine-(1-2)-1D-chiro-inositol) plus UDP, using UDP-galactosamine and D-chiro-inositol as substrates. The reaction is catalyzed by the enzyme FeGolS-2.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to nucleic acid molecules encoding fagopyritol synthase enzymes. Fagopyritol is a general term used herein to mean an unspecified α-galactosyl D-chiro-inositol or its salt or derivative. More particularly, the present invention relates to an isolated nucleic acid molecule encoding a fagopyritol synthase. In accordance with the present invention, the fagopyritol synthase catalyzes the biosynthesis of a fagopyritol. Suitable fagopyritols include fagopyritol A1, particularly fagopyritol A1s have the following Formula I:




fagopyritol A2, particularly fagopyritol A2s having the following Formula II:





fagopyritol A3, particularly fagopyritol A3s having the following Formula III:





fagopyritol B1, particularly fagopyritol B1s having the following Formula IV:





fagopyritol B2, particularly fagopyritol B2s having the following Formula V:





and fagopyritol B3, particularly fagopyritol B3s having the following Formula VI:





One suitable source of a nucleic acid molecule encoding a fagopyritol synthase enzyme is Fagopyrum esculentum. 
In a first embodiment, the fagopyritol synthase from Fagopyrum esculentum is identified herein as FeGolS-1 and is encoded by a nucleic acid molecule having a nucleotide sequence of SEQ ID NO:1 as follows:









gagcacccaa agctctgcta gcaccatatt caaatcctca agaatcatca aatcttccaa
60



 


ccaatcctca agttccaacc aaatggcacc agaactcatc acaatcggag ccgatcactc
120


 


gattttgcca gcggaatcgt tgattccggt tgaccgagct tacgtgacgt ttctcgccgg
180


 


gaacggagac tatgtcaagg gagttgtcgg attagcaaag ggactgagga aagtgaaggc
240


 


tgcttatcct cttgttgtag cggttttacc ggacgttccg ctagagcatc gccgactcct
300


 


ggaggcgcag ggttgtatcg taagggaaat cgagccgata tacccgccgg aaaacaattg
360


 


cgagttcgct cacgcatact atgtcatcaa ctactccaag cttcgcatct gggagtttgt
420


 


ggagtacagt aagatgatat acttggacgg ggacatacag gtgtaccaga acattgacca
480


 


cctgtttgac cagccggacg gctactttta cgcggtgatg gactgttttt gtgagccatc
540


 


atggagcaag acgattcagt acaagatcgg atactgccaa cagtgcccgg agaaggtagc
600


 


gtggccgttg gaggctggcc cgaagccttc tctgtacttc aatgccggat tctttgttta
660


 


cgagccgagc cttgagactt acaaggatct cattgacact ctcaaagtca cgactcctac
720


 


ctcctttgcc gagcaggact tcttgaacat gtacttcaag gacaagttca agccactccc
780


 


catagactac aacttagtct tagccttcct gtggaggcat ccggagaaag ttgaccttaa
840


 


ccgagtgaag gtagttcact actgtgcggc ggggtctaag ccatggaggt acacgggcaa
900


 


ggaagagaac atggacagag aagacatcaa attgcttgtg aaaaaatggt gggatatcta
960


 


caacgacgag tcattggacc tcaagaaacc ggtccattta gtgcagcagc ccacggaggt
1020


 


gctcaaggcg gcgctctcgg aggctaggcc tgttaaatat gtggctgctc cttccgcagc
1080


 


ttaagtatcg gcttgtattt ggtaatggtt tttgtttttg cgaatgtaaa gtagaaagaa
1140


 


ggggcgagag tttgtgatat tggggcaatg gggaatggtg cgtataaatg tgtgttgtaa
1200


 


tggcaactgt ttttacttgg aattatatgt aagaagtaag aatatatgta taaaaaaaaa
1260


 


aaaaaaaaa
1269






The nucleic acid sequence corresponding to SEQ ID NO:1 encodes an isoform of fagopyritol synthase isolated from Fagopyrum esculentum, identified herein as FeGolS-1, which has a deduced amino acid sequence corresponding to SEQ ID NO:2, as follows:










Met Ala Pro Glu Leu Ile Thr Ile Gly Ala Asp His




  1               5                  10



 



Ser Ile Leu Pro Ala Glu Ser Leu Ile Pro Val Asp



         15                  20



 



Arg Ala Tyr Val Thr Phe Leu Ala Gly Asn Gly Asp



 25                  30                  35



 



Tyr Val Lys Gly Val Val Gly Leu Ala Lys Gly Leu



             40                  45



 



Arg Lys Val Lys Ala Ala Tyr Pro Leu Val Val Ala



     50                  55                  60



 



Val Leu Pro Asp Val Pro Leu Glu His Arg Arg Leu



                 65                  70



 



Leu Glu Ala Gln Gly Cys Ile Val Arg Glu Ile Glu



         75                  80



 



Pro Ile Tyr Pro Pro Glu Asn Asn Cys Glu Phe Ala



 85                  90                  95



 



His Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu Arg



            100                 105



 



Ile Trp Glu Phe Val Glu Tyr Ser Lys Met Ile Tyr



    110                 115                 120



 



Leu Asp Gly Asp Ile Gln Val Tyr Gln Asn Ile Asp



                125                 130



 



His Leu Phe Asp Gln Pro Asp Gly Tyr Phe Tyr Ala



        135                 140



 



Val Met Asp Cys Phe Cys Glu Pro Ser Trp Ser Lys



145                 150                 155



 



Thr Ile Gln Tyr Lys Ile Gly Tyr Cys Gln Gln Cys



            160                 165



 



Pro Glu Lys Val Ala Trp Pro Leu Glu Ala Gly Pro



    170                 175                 180



 



Lys Pro Ser Leu Tyr Phe Asn Ala Gly Phe Phe Val



                105                 190



 



Tyr Glu Pro Ser Leu Glu Thr Tyr Lys Asp Leu Ile



        195                 200



 



Asp Thr Leu Lys Val Thr Thr Pro Thr Ser Phe Ala



205                 210                 215



 



Glu Gln Asp Phe Leu Asn Met Tyr Phe Lys Asp Lys



            220                 225



 



Phe Lys Pro Leu Pro Ile Asp Tyr Asn Leu Val Leu



    230                 235                 240



 



Ala Phe Leu Trp Arg His Pro Glu Lys Val Asp Leu



                245                 250



 



Asn Arg Val Lys Val Val His Tyr Cys Ala Ala Gly



        255                 260



 



Ser Lys Pro Trp Arg Tyr Thr Gly Lys Glu Glu Asn



265                 270                 275



 



Met Asp Arg Glu Asp Ile Lys Leu Leu Val Lys Lys



            280                 285



 



Trp Trp Asp Ile Tyr Asn Asp Glu Ser Leu Asp Leu



    290                 295                 300



 



Lys Lys Pro Val His Leu Val Gln Gln Pro Thr Glu



                305                 310



 



Val Leu Lys Ala Ala Leu Ser Glu Ala Arg Pro Val



        315                 320



 



Lys Tyr Val Ala Ala Pro Ser Ala Ala



325                 330






The fagopyritol synthase has a molecular mass of from 38 to 41 kDa, and preferably 38.3 kDa. FeGoS-1, isolated from Fagopyrum esculentum (“buckwheat”), has a single open reading frame (“ORF”) of 1002 bp, extending between nucleotides 83-1084. The starting codon “ATG” is identified at 83-85 bp, with the stop codon “TAA” found between nucleotides 1082-1084, as shown in FIG. 1.

In a second embodiment, the fagopyritol synthase from Fagopyrum esculentum is identified herein as FeGolS-2 and is encoded by a nucleic acid molecule having a nucleotide sequence of SEQ ID NO:3 as follows:









ttggtttcga acttgatcaa aacctcacaa aaacacgtaa gcaaaatgac ttccgagatg
60



 


gcgccacaga acataacgaa tgcagaaaga ggagccgagc aagtgaagcc gtcgagccag
120


 


ccaagccgag cctacgtgac attcttagcc gggaacggtg actacgtgaa gggagttata
180


 


gggctcgcca aaggcctgag gaaaactcag agcggttacc cgcttgtggt ggcggttctc
240


 


cctgacgttc cgcaggagca ccgccgtatg ctggtggcgc aaggctgtat aataaaggaa
300


 


atccagcccg ttaacccgcc cgataaccag actcagtttg ccatggctta ttacgtcatc
360


 


aactactcca agctccgtat atgggagttt atcgagtata gtaagatgat atatcttgat
420


 


ggagacatcc aagtttacga caacatcgac cacctcttcg cactaccaga cgggtacttg
480


 


tacggtgcca tggattgctt ttgcgagaag acttggagtc attcgcttcc atataagatt
540


 


gggtattgcc aacagtgccc ggacagggtc cagtggcccg aaaggctcgg cccaaaacca
600


 


acactctact tcaatgcagg gatgttcatc ttcgagccta gcgtttctac ttataatgat
660


 


ctccttcata cactcgagat cacccctcct acaccttttg ctgagcagga ctttttgaat
720


 


atgtacttca aggatgtgta cagaccaatt ccgaacgttt acaacttggt attggctttg
780


 


ttgtggtatc atcctgggtt aatgaagctt gatgaggtta aagtcgttca ctattgtgcc
840


 


gatggttcaa aaccatggcg gtatacaggg aagggggata acatggacag ggaagacgtt
900


 


aggatgctag tgaagaagtg gtgggagatt tacgatgatc agtctctcga ccctcagcct
960


 


aagatggtcg agggcaagaa gttcgacaaa ttagaggagt acagcgagtc cctcgaccac
1020


 


ccgcccaagg tggcagagga agataagcta gagaagccca tggcagcgat gacaggcttc
1080


 


agctacgtac acgccccgtc tgctgcctga tttgttgaaa caaggccaag gttccacaaa
1140


 


tgagggaatc aaaaacctcc tatagtatta tagatcgtat atttctgtta ttgctttcca
1200


 


attaagcaac taagatgttc atatagtagt tctggaaaat gaatacgggc atagttgtga
1260


 


acttgtaatc tcattttgtt tttcggaatg ttcaagtatt tcttctaaaa aaaaaaaaaa
1320


 


aaaaaa
1326






The nucleic acid sequence corresponding to SEQ ID NO:3 encodes an isoform of fagopyritol synthase isolated from Fagopyrum esculentum, identified herein as FeGolS-2, which has a deduced amino acid sequence corresponding to SEQ ID NO:4, as follows:










Met Thr Ser Glu Met Ala Pro Gln Asn Ile Thr Asn




  1               5                  10



 



Ala Glu Arg Gly Ala Glu Gln Val Lys Pro Ser Ser



         15                 20



 



Gln Pro Ser Arg Ala Tyr Val Thr Phe Leu Ala Gly



 25                  30                  35



 



Asn Gly Asp Tyr Val Lys Gly Val Ile Gly Leu Ala



             40                  45



 



Lys Gly Leu Arg Lys Thr Gln Ser Gly Tyr Pro Leu



     50                  55                  60



 



Val Val Ala Val Leu Pro Asp Val Pro Gln Glu His



                 65                  70



 



Arg Arg Met Leu Val Ala Gln Gly Cys Ile Ile Lys



         75                  80



 



Glu Ile Gln Pro Val Asn Pro Pro Asp Asn Gln Thr



 85                  90                  95



 



Gln Phe Ala Met Ala Tyr Tyr Val Ile Asn Tyr Ser



            100                 105



 



Lys Leu Arg Ile Trp Glu Phe Ile Glu Tyr Ser Lys



    110                115                  120



 



Met Ile Tyr Leu Asp Gly Asp Ile Gln Val Tyr Asp



                125                 130



 



Asn Ile Asp His Leu Phe Asp Leu Pro Asp Gly Tyr



        135                 140



 



Leu Tyr Gly Ala Met Asp Cys Phe Cys Glu Lys Thr



145                 150                 155



 



Trp Ser His Ser Leu Pro Tyr Lys Ile Gly Tyr Cys



            160                 165



 



Gln Gln Cys Pro Asp Arg Val Gln Trp Pro Glu Arg



    170                 175                 180



 



Leu Gly Pro Lys Pro Thr Leu Tyr Phe Asn Ala Gly



                185                 190



 



Met Phe Ile Phe Glu Pro Ser Val Ser Thr Tyr Asn



        195                 200



 



Asp Leu Leu His Thr Leu Glu Ile Thr Pro Pro Thr



205                 210                 215



 



Pro Phe Ala Glu Gln Asp Phe Leu Asn Met Tyr Phe



            220                 225



 



Lys Asp Val Tyr Arg Pro Ile Pro Asn Val Tyr Asn



    230                 235                 240



 



Leu Val Leu Ala Leu Leu Trp Tyr His Pro Gly Leu



                245                 250



 



Met Lys Leu Asp Glu Val Lys Val Val His Tyr Cys



        255                 260



 



Ala Asp Gly Ser Lys Pro Trp Arg Tyr Thr Gly Lys



265                 270                 275



 



Gly Asp Asn Met Asp Arg Gln Asp Val Arg Met Leu



            280                 285



 



Val Lys Lys Trp Trp Glu Ile Tyr Asp Asp Glu Ser



    290                 295                 300



 



Leu Asp Pro Gln Pro Lys Met Val Glu Gly Lys Lys



                305                 310



 



Phe Asp Lys Leu Glu Glu Tyr Ser Glu Ser Leu Asp



        315                 320



 



His Pro Pro Lys Val Ala Glu Glu Asp Lys Leu Glu



325                 330                 335



 



Lys Pro Met Ala Ala Met Thr Gly Phe Ser Tyr Val



            340                 345



 



His Ala Pro Ser Ala Ala



    350






The fagopyritol synthase has a molecular mass of from 38 to 41 kDa, and preferably 40.7 kDa. FeGoS-2, isolated from Fagopyrum esculentum, has a single ORF of 1065 bp, extending between nucleotides 46-1110. The starting codon “ATG” is identified at 46-48 bp, with the stop codon “TGA” found between nucleotides 1108-1110, as shown in FIG. 2.

In a third embodiment, the fagopyritol synthase from Fagopyrum esculentum is identified herein as FeGolS-3 and comprises a nucleic acid molecule having a nucleotide sequence of SEQ ID NO:5 (see FIG. 3) as follows:









gctcacgcat actatgtcat caactactcc aagctccgta tatgggagtt tatcgagtat
60



 


agtaagatga tatatcttga tggagacatc caagtttacg acaacatcga ccacctcttc
120


 


gacctaccag acgggtactt gtacggtgcc atggattgct tttgcgagaa gacttggagt
180


 


cattcgcttc catataagat tgggtattgc caacagtgcc cggacagggt ccagtggccc
240


 


gaaaggctcg gcccaaaacc aacactctac ttcaatgcag ggatgttcat cttcgagcct
300


 


agcgtttcta cttataatga tctccttcat acactcgaga tcacccctcc tacacctttt
360


 


gctgagcagg actttttgaa tatgtacttc aaggatgtgt acagaccaat tccgaacgtg
420


 


tacaacttgg tattggcttt gttgtggtat catcctgggt taatgaatct tgatgaggtt
480


 


aaagtcgttc actattgtgc cgatggttca aaaccatggc ggtatacagg gaagggggat
540


 


aacatggaca gggaagacgt taggatgcta gtgaagaagt ggtgggagat ctacgatgat
600


 


cagtctctcg accctcagcc taaggtggtc gagggcaaga agttcgacaa attagagtac
660


 


agcgagtccc tcgaccaccc gcctaaggtg gcagaggaag ataagttaga gaagcccatg
720


 


gcggcgatga cagggttcag ctacgtacac gccccgtctg ctgcctgact tgttgaaaca
780


 


aggccaaggt tccacaaatg agggaatcaa aaacctccta tagtattata gatcgtatat
840


 


ttctgttatt gctttccaat taagcaacta agatgttcat atagtagttc tggaaaatga
900


 


aaacgggcat agttgtgaac ttgtaatctc attttgtttt tcggaatgtg caagtatttc
960


 


ttctaaataa aaaaaaaaaa aaaaaa
986






The nucleic acid sequence corresponding to SEQ ID NO:5 encodes an isoform of fagopyritol synthase isolated from Fagopyrum esculentum, identified herein as FeGolS-3, which comprises a deduced amino acid sequence corresponding to SEQ ID NO:6, as follows:








Ala His Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu



  1               5                  10


 


Arg Ile Trp Glu Phe Ile Glu Tyr Ser Lys Met Ile


         15                  20


 


Tyr Leu Asp Gly Asp Ile Gln Val Tyr Asp Asn Ile


 25                  30                  35


 


Asp His Leu Phe Asp Leu Pro Asp Gly Tyr Leu Tyr


             40                  45


 


Gly Ala Met Asp Cys Phe Cys Glu Lys Thr Trp Ser


     50                  55                  60


 


His Ser Leu Pro Tyr Lys Ile Gly Tyr Cys Gln Gln


                 65                  70


 


Cys Pro Asp Arg Val Gln Trp Pro Gln Arg Leu Gly


         75                  80


 


Pro Lys Pro Thr Leu Tyr Phe Asn Ala Gly Met Phe


 85                  90                  95


 


Ile Phe Glu Pro Ser Val Ser Thr Tyr Asn Asp Leu


            100                 105


 


Leu His Thr Leu Glu Ile Thr Pro Pro Thr Pro Phe


    110                 115                 120


 


Ala Glu Gln Asp Phe Leu Asn Met Tyr Phe Lys Asp


                125                 130


 


Val Tyr Arg Pro Ile Pro Asn Val Tyr Asn Leu Val


        135                 140


 


Leu Ala Leu Leu Trp Tyr His Pro Gly Leu Met Asn


145                 150                 155


 


Leu Asp Glu Val Lys Val Val His Tyr Cys Ala Asp


            160                 165


 


Gly Ser Lys Pro Trp Arg Tyr Thr Gly Lys Gly Asp


    170                 175                 180


 


Asn Met Asp Arg Glu Asp Val Arg Met Leu Val Lys


                185                 190


 


Lys Trp Trp Glu Ile Tyr Asp Asp Gln Ser Leu Asp


        195                 200


 


Pro Gln Pro Lys Val Val Glu Gly Lys Lys Phe Asp


205                 210                 215


 


Lys Leu Glu Tyr Ser Glu Ser Leu Asp His Pro Pro


            220                 225


 


Lys Val Ala Glu Glu Asp Lys Leu Glu Lys Pro Met


    230                 235                 240


 


Ala Ala Met Thr Gly Phe Ser Tyr Val His Ala Pro


                245                 250


 


Ser Ala Ala


        255






Another suitable source of a nucleic acid molecule encoding a fagopyritol synthase enzyme is Glycine max. A fagopyritol synthase from Glycine max is identified herein as GmGolS and is encoded by a nucleic acid molecule having a nucleotide sequence of SEQ ID NO:7 as follows:









agccaaaagt ttgttttcat agtgtgtttt gtttcccaaa tcctactctt gtgaccacaa
60



 


cccttcctcc tctttctttt gaaacctctt tttttctatt ccccaaccaa acaagcaaac
120


 


gctactcact catcatcact gagatcatgg ctcctaatat caccactgtc aaaaccacca
180


 


tcaccgacgc tcaagccaag gtcgccaccg atcatggtcg tgcctacgtc accttcctcg
240


 


ccggaaacgg tgactatgtg aaaggtgtcg ttggcttggc aaaaggtctg agaaaagtga
300


 


agagcatgta ccctctggtg gttgcagtgc tacccgatgt tccccaagat caccgcaaca
360


 


ttctcacctc ccaaggttgc attgttagag agattgagcc cgtgtacccc ccagagaatc
420


 


aaacccagtt tgccatggca tattacgtca tcaactattc caagctacgt atttgggagt
480


 


ttgtggagta cagcaagatg atatacctag acggtgatat ccaagttttt gacaacattg
540


 


accacttgtt tgacttgcct gataactact tctatgcggt gatggactgt ttctgtgagc
600


 


caacttgggg ccacactaaa caatatcaga tcggttactg ccagcagtgc ccccataagg
660


 


ttcagtggcc cactcacttt gggcccaaac ctcctctcta tttcaatgct ggcatgtttg
720


 


tgtatgagcc caatttggct acttaccgtg acctccttca aacagtccaa gtcacccagc
780


 


ccacttcctt tgctgaacag gattttttga acatttactt caaggacaaa tataggccaa
840


 


ttcctaatgt ctacaatctt gtgctggcca tgctgtggcg tcaccctgag aacgttgagc
900


 


ttgacaaagt taaagtggtt cactactgtg ctgctgggtc taagccttgg aggtacactg
960


 


ggaaggagga gaatatggag agagaagata tcaagatgtt agtgaaaaag tggtgggata
1020


 


tatatgagga tgagactttg gactacaaca atccactcaa tgtggataag ttcactgcgg
1080


 


cacttatgga ggttggtgaa gtcaagttcg tccgtgcccc atctgctgct taagagtgtc
1140


 


tttggaaatc aagtgtgatc caagtacatg tacaaagtca tacatcatta cattaacttt
1200


 


tatgtatttc taaaagtcat acatcattac attaagtttt atgtatttct aaagtcttaa
1260


 


gacttaagag gacctttttt atgtgtcccg gcttttcttt ttttcttttt ccaattctgt
1320


 


cattgtaaag caggtgaata ccggtatcct taattttata aatggatatg aattttattt
1380


 


tgcaaaaaaa aaaaaaaaaa aaaaaa
1406






The nucleic acid sequence corresponding to SEQ ID NO:7 encodes an isoform of fagopyritol synthase isolated from Glycine max, identified herein as GmGolS, which has a deduced amino acid sequence corresponding to SEQ ID NO: 8, as follows:










Met Ala Pro Asn Ile Thr Thr Val Lys Thr Thr Ile




  1               5                  10



 



Thr Asp Ala Gln Ala Lys Val Ala Thr Asp His Gly



         15                  20



 



Arg Ala Tyr Val Thr Phe Leu Ala Gly Asn Gly Asp



 25                  30                  35



 



Tyr Val Lys Gly Val Val Gly Leu Ala Lys Gly Leu



             40                  45



 



Arg Lys Val Lys Ser Met Tyr Pro Leu Val Val Ala



     50                  55                  60



 



Val Leu Pro Asp Val Pro Gln Asp His Arg Asn Ile



                 65                  70



 



Leu Thr Ser Gln Gly Cys Ile Val Arg Glu Ile Glu



         75                  80



 



Pro Val Tyr Pro Pro Glu Asn Gln Thr Gln Phe Ala



 85                  90                  95



 



Met Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu Arg



            100                 105



 



Ile Trp Glu Phe Val Glu Tyr Ser Lys Met Ile Tyr



    110                 115                 120



 



Leu Asp Gly Asp Ile Gln Val Phe Asp Asn Ile Asp



                125                 130



 



His Leu Phe Asp Leu Pro Asp Asn Tyr Phe Tyr Ala



        135                 140



 



Val Met Asp Cys Phe Cys Glu Pro Thr Trp Gly His



145                 150                 155



 



Thr Lys Gln Tyr Gln Ile Gly Tyr Cys Gln Gln Cys



            160                 165



 



Pro His Lys Val Gln Trp Pro Thr His Phe Gly Pro



    170                 175                 180



 



Lys Pro Pro Leu Tyr Phe Asn Ala Gly Met Phe Val



                185                 190



 



Tyr Glu Pro Asn Leu Ala Thr Tyr Arg Asp Leu Leu



        195                 200



 



Gln Thr Val Gln Val Thr Gln Pro Thr Ser Phe Ala



205                 210                 215



 



Glu Glu Asp Phe Leu Asn Ile Tyr Phe Lys Asp Lys



            220                 225



 



Tyr Arg Pro Ile Pro Asn Val Tyr Asn Leu Val Leu



    230                 235                 240



 



Ala Met Leu Trp Arg His Pro Glu Asn Val Glu Leu



                245                 250



 



Asp Lys Val Lys Val Val His Tyr Cys Ala Ala Gly



        255                 260



 



Ser Lys Pro Trp Arg Tyr Thr Gly Lys Glu Glu Asn



265                 270                 275



 



Met Glu Arg Glu Asp Ile Lys Met Leu Val Lys Lys



            280                 285



 



Trp Trp Asp Ile Tyr Glu Asp Glu Thr Leu Asp Tyr



    290                 295                 300



 



Asn Asn Pro Leu Asn Val Asp Lys Phe Thr Ala Ala



                305                 310



 



Leu Met Glu Val Gly Glu Val Lys Phe Val Arg Ala



        315                 320



 



Pro Ser Ala Ala



325






(see FIG. 4). The fagopyritol synthase has a molecular mass of approximately 38.0 kDa.

Other suitable sources of nucleic acid molecules encoding fagopyritol synthases include any plant that expresses galactinol synthase (i.e., any plant that accumulates raffinose series of oligosaccharides), including, but not limited to, sugar beet, vetch, beans, legumes, cereals and grasses, cucurbits, and Brassicas (see, e.g., Kuo et al., J. Agricul. Food Chem. 36:32-36 (1988), which is hereby incorporated by reference in its entirety).
Fragments of the above fagopyritol synthase enzymes are encompassed by the present invention.
Suitable fragments can be produced by several means. In one method, subclones of the genes encoding the fagopyritol synthase enzymes of the present invention are produced by conventional molecular genetic manipulation by subeloning gene fragments. The subelones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or peptide.
In another approach, based on knowledge of the primary structure of the protein, fragments of a fagopyritol synthase enzyme encoding gene may be synthesized by using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. These then would be cloned into an appropriate vector for increased expression of a truncated peptide or protein.
Chemical synthesis can also be used to make suitable fragments. Such a synthesis is carried out using known amino acid sequences for a fagopyritol synthase enzyme being produced. Alternatively, subjecting a full length fagopyritol synthase enzyme to high temperatures and pressures will produce fragments. These fragments can then be separated by conventional procedures (e.g., chromatography, SDS-PAGE).
Another example of suitable fragments of the nucleic acids of the present invention are fragments of the genes which have been identified as conserved (“con”) regions of the proteins, or alternatively, those portions of nucleotide sequences that have been identified as variable (“var”) regions. Sequences identified using DNAStar Mega alignment program as either variable or conserved in a gene can be amplified using standard PCR methods using forward and reverse primers designed to amplify the region of choice and which include a restriction enzyme sequence to allow ligation of the PCR product into a vector of choice. Combinations of amplified conserved and variable region sequences can be ligated into a single vector to create a “cassette” which contains a plurality of DNA molecules in one vector.
Mutations or variants of the above polypeptides or proteins are encompassed by the present invention. Variants may be made by, for example, the deletion or addition of amino acids that have minimal influence on the properties, secondary structure, and hydropathic nature of an enzyme. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.
Also suitable as an isolated nucleic acid molecule according to the present invention is a nucleic acid molecule having a nucleotide sequence that is at least 55% similar, preferably at least 80% similar, and most preferably, at least 90% similar, to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 by basic BLAST using default parameters analysis.
Suitable nucleic acid molecules are those that hybridize to a nucleic acid molecule comprising a nucleotide sequence of SEQ ID No:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 under stringent conditions. For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press, at 11.45 (1989). An example of low stringency conditions is 4-6×SSC/0.1-0.5% w/v SDS at 37°-45° C. for 2-3 hours. Depending on the source and concentration of the nucleic acid involved in the hybridization, alternative conditions of stringency may be employed such as medium stringent conditions. Examples of medium stringent conditions include 1-4×SSC/0.25% w/v SDS at ≧45° C. for 2-3 hours. An example of high stringency conditions includes 0.1-1×SSC/0.1% w/v SDS at 60° C. for 1-3 hours. The skilled artisan is aware of various parameters which may be altered during hybridization and washing and which will either maintain or change the stringency conditions. Other examples of high stringency conditions include: 4-5×SSC/0.1% w/v SDS at 54° C. for 1-3 hours and 4×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C. for about one hour. Alternatively, an exemplary stringent hybridization condition is in 50% formamide, 4×SSC, at 42° C. Still another example of stringent conditions include hybridization at 62° C. in 6×SSC, 0.05×BLOTTO, and washing at 2×SSC, 0.1% SDS at 62° C.
The precise conditions for any particular hybridization are left to those skilled in the art because there are variables involved in nucleic acid hybridizations beyond those of the specific nucleic acid molecules to be hybridized that affect the choice of hybridization conditions. These variables include: the substrate used for nucleic acid hybridization (e.g., charged vs. non-charged membrane); the detection method used (e.g., radioactive vs. chemiluminescent); and the source and concentration of the nucleic acid involved in the hybridization. All of these variables are routinely taken into account by those skilled in the art prior to undertaking a nucleic acid hybridization procedure.
A fagopyritol synthase enzyme of the present invention is preferably produced in purified form (e.g., at least about 80%, more preferably 90% pure) by conventional techniques. One example of a suitable technique is set forth in the Examples herein. Alternatively, a fagopyritol synthase enzyme of the present invention is secreted into the growth medium of recombinant host cells. To isolate the fagopyritol synthase enzyme, a protocol involving a host cell such as Escherichia coli may be used, in which protocol the E. coli host cell carrying a recombinant plasmid is propagated, homogenized, and the homogenate is centrifuged to remove bacterial debris. The supernatant is then subjected to sequential ammonium sulfate precipitation. The fraction containing the fagopyritol synthase enzyme of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins or polypeptides. If necessary, the protein fraction may be further purified by high performance liquid chromatography (“HPLC”).
The nucleic acid molecule encoding the fagopyritol synthase enzyme of the present invention, or a suitable portion thereof, can be incorporated into host cells using conventional recombinant DNA technology. Generally, this involves inserting the nucleic acid molecule into an expression system to which the nucleic acid molecule is heterologous (i.e. not normally present). The expression system contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
The present invention also relates to an expression vector containing a nucleic acid molecule encoding a fagopyritol synthase enzyme of the present invention. The nucleic acid molecules of the present invention may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art. In preparing a DNA vector for expression, the various DNA sequences may normally be inserted or substituted into a bacterial plasmid. Any convenient plasmid may be employed, which will be characterized by having a bacterial replication system, a marker which allows for selection in a bacterium, and generally one or more unique, conveniently located restriction sites. Numerous plasmids, referred to as transformation vectors, are available for transformation. The selection of a vector will depend on the preferred transformation technique and target cells for transfection.
Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC11, SV 40, pBluescript II SK+/− or KS+/− (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference in its entirety), pQE, pIH821, pGEX, pET series (see F. W. Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology vol. 185 (1990), which is hereby incorporated by reference in its entirety), and any derivatives thereof. Any appropriate vectors now known or later described for genetic transformation are suitable for use with the present invention. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., which are hereby incorporated by reference in their entirety.
U.S. Pat. No. 4,237,224 issued to Cohen and Boyer, which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture.
A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
Thus, certain “control elements” or “regulatory sequences” are also incorporated into the plasmid-vector constructs of the present invention. These include non-transcribed regions of the vector and 5′ and 3′ untranslated regions, which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and/or translation elements, including constitutive, inducible, and repressible promoters, as well as minimal 5′ promoter elements may be used. A constitutive promoter is a promoter that directs expression of a gene throughout the development and life of an organism. An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequences or genes will not be transcribed or will only be minimally transcribed.
The DNA sequences of eukaryotic promoters differ from those of prokaryotic promoters. Furthermore, eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a prokaryotic system, and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells.
Promotors vary in their “strength” (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the PR and PL promotors of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
Other examples of some constitutive promoters that are widely used for inducing expression of transgenes include the nopoline synthase (NOS) gene promoter, from Agrobacterium tumefaciens, (U.S. Pat. No. 5,034,322 issued to Rogers et al., which is hereby incorporated by reference in its entirety), the cauliflower mosaic virus (CaMV) 35S and 19S promoters (U.S. Pat. No. 5,352,605 issued to Fraley et al., which is hereby incorporated by reference in its entirety), the enhanced CaMV35S promoter (“enh CaMV35S”), the figwort mosaic virus full-length transcript promoter (“FMV35S”), those derived from any of the several actin genes, which are known to be expressed in most cells types (U.S. Pat. No. 6,002,068 issued to Privalle et al., which is hereby incorporated by reference in its entirety), and the ubiquitin promoter, which is a gene product known to accumulate in many cell types. Examples of constitutive promoters for use in mammalian cells include the RSV promoter derived from Rous sarcoma virus, the CMV promoter derived from cytomegalovirus, β-actin and other actin promoters, and the EF1α promoter derived from the cellular elongation factor 1α gene.
Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operations, the addition of specific inducers is necessary for efficient transcription of the inserted nucleic acid. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.
Other examples of some inducible promoters, induced, for examples by a chemical agent, such as a metabolite, growth regulator, herbicide or phenolic compound, or a physiological stress/physical means, such as cold, heat, salt, toxins, or through the action of a pathogen or disease agent such as a virus or fungus, include a glucocorticoid-inducible promoter (Schena et al., Proc. Natl. Acad. Sci. 88:10421-5 (1991), which is hereby incorporated by reference in its entirety), the heat shock promoter (“Hsp”), IPTG or tetracycline (“Tet on” system), the metallothionine promoter, which is activated by heavy metal ions, and hormone-responsive promoters, which are activated by treatment of certain hormones. A host cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell. In addition, “tissue-specific” promoters can be used, which are promoters that function in a tissue specific manner to regulate the gene of interest within selected tissues of the host. Examples of such tissue specific promoters include seed, flower, or root specific promoters as are well known in the field (e.g., U.S. Pat. No. 5,750,385 to Shewmaker et al., which is hereby incorporated by reference in its entirety). Promoters of the nucleic acid construct of the present invention may be either homologous (derived from the same species as the host cell) or heterologous (derived from a different species than the host cell.
Specific initiation signals are also required for efficient gene transcription and translation in prokaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires an SD sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
The constructs of the present invention also include an operable 3′ regulatory region, selected from among those which are capable of providing correct transcription termination and polyadenylation of mRNA for expression in the host cell of choice, operably linked to a DNA molecule which encodes for a protein of choice. A number of 3′ regulatory regions are known in the art. Virtually any 3′ regulatory region known to be operable in the host cell of choice would suffice for proper expression of the coding sequence of the nucleic acid of the present invention.
In one aspect of the present invention, the nucleic acid molecule of the present invention is incorporated into an appropriate vector in the sense direction, such that the open reading frame is properly oriented for the expression of the encoded protein under control of a promoter of choice. This involves the inclusion of the appropriate regulatory elements into the DNA-vector construct. These include non-translated regions of the vector, useful promoters, and 5′ and 3′ untranslated regions which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.
A nucleic acid molecule of the preset invention, promoter of choice, an appropriate 3′ regulatory region, and, if desired, a reporter gene, can be incorporated into a vector-expression system which contains the nucleic acids of the present invention, or suitable fragments thereof, using standard cloning techniques as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), and Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., which are hereby incorporated by reference in their entirety. The transcriptional and translational elements are operably linked to the nucleic acid molecule of the present invention or a fragment thereof, meaning that the resulting vector expresses the fagopyritol synthase when placed in a suitable host cell.
Once an isolated DNA molecule encoding a fagopyritol synthase enzyme has been cloned into an expression vector, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The nucleic acid sequences are cloned into the host cell using standard cloning procedures known in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
Thus, the present invention also relates to a host cell incorporating one or more of the isolated nucleic acid molecules of the present invention. In one embodiment, the isolated nucleic acid molecule is heterologous to the host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host system, and using the various host cells described above.
Methods of transformation may result in transient or stable expression of the DNA under control of the promoter. Preferably, the nucleic acid of the present invention is stably inserted into the genome of the host cell as a result of the transformation, although transient expression can serve an important purpose.
One approach to transforming host cells with a nucleic acid molecule of the present invention is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which are hereby incorporated by reference in their entirety. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells. Other variations of particle bombardment, now known or hereafter developed, can also be used.
Transient expression in protoplasts allows quantitative studies of gene expression, because the population of cells is very high (on the order of 106). To deliver DNA inside protoplasts, several methodologies have been proposed, but the most common are electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824-5828 (1985), which is hereby incorporated by reference in its entirety) and polyethylene glycol (PEG) mediated DNA uptake (Krens et al., Nature 296:72-74 (1982), which is hereby incorporated by reference in its entirety). During electroporation, the DNA is introduced into the cell by means of a reversible change in the permeability of the cell membrane due to exposure to an electric field. PEG transformation introduces the DNA by changing the elasticity of the membranes. Unlike electroporation, PEG transformation does not require any special equipment and transformation efficiencies can be equally high. Another appropriate method of introducing the nucleic acid molecule of the present invention into a host cell is fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies that contain the chimeric gene (Fraley, et al., Proc. Natl. Acad. Sci. USA 76:3348-52 (1979), which is hereby incorporated by reference in its entirety).
Stable transformants are preferable for the methods of the present invention. An appropriate method of stably introducing the nucleic acid molecule into plant cells is to infect a plant cell with Agrobacterium tumefaciens or Agrobacterium rhizogenes previously transformed with a DNA construct of the present invention. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots or roots, and develop further into plants.
Plant tissues suitable for transformation include without limitation, floral buds, leaf tissue, root tissue, meristems, zygotic and somatic embryos, megaspores, callus, protoplasts, tassels, pollen, embryos, anthers, and the like. The means of transformation chosen is that most suited to the tissue to be transformed.
Suitable plants include dicots and monocots. Monocots suitable for the present invention include Gramineae (e.g., grass, corn, grains, bamboo, sugar cane), Liliaceae (e.g., onion, garlic, asparagus, tulips, hyacinths, day lily, and aloes), Iridaceae (e.g., iris, gladioli, freesia, crocus, and watsonia), and Orchidacea (e.g., orchid). Examples of dicots suitable for the present invention include Salicaceae (e.g., willow, and poplar), Ranunculaceae (e.g., Delphinium, Paeonia, Ranunculus, Anemone, Clematis, columbine, and marsh marigold), Magnoliaceae (e.g., tulip tree and Magnolia), Cruciferae (e.g., mustards, cabbage, cauliflower, broccoli, brussel sprouts, kale, kohlrabi, turnip, and radish), Rosaceae (e.g., strawberry, blackberry, peach, apple, pear, quince, cherry, almond, plum, apricot, and rose), Leguminosae (e.g., pea, bean, peanut, alfalfa, clover, vetch, redbud, broom, wisteria, lupine, black locust, and acacia), Malvaceae (e.g., cotton, okra, and mallow), Umbelliferac (e.g., carrot, parsley, parsnips, and hemlock), Labiatae (e.g., mint, peppermints, spearmint, thyme, sage, and lavender), Solanaceae (e.g., potato, tomato, pepper, eggplant, and Petunia), Cucurbitaceae (e.g., melon, squash, pumpkin, and cucumber), Compositae (e.g., sunflower, endive, artichoke, lettuce, safflower, aster, marigold, dandelions, sage brush, Dalia, Chrysanthemum, and Zinna), and Rubiaceae (e.g., coffee).
After transformation, the transformed plant cells can be selected and regenerated. Preferably, transformed cells are first identified using a selection marker simultaneously introduced into the host cells along with the DNA construct of the present invention. Suitable selection markers include, without limitation, markers encoding for antibiotic resistance, such as the nptII gene which confers kanamycin resistance (Fraley, et al., Proc. Natl. Acad. Sci. USA 80:4803-4807 (1983), which is hereby incorporated by reference in its entirety), and the genes which confer resistance to gentamycin, G418, hygromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol, and the like. Any known antibiotic-resistance marker can be used to transform and select transformed host cells in accordance with the present invention. Cells or tissues are grown on a selection medium containing the appropriate antibiotic, whereby generally only those transformants expressing the antibiotic resistance marker continue to grow. Other types of markers are also suitable for inclusion in the expression cassette of the present invention. For example, a gene encoding for herbicide tolerance, such as tolerance to sulfonylurea is useful, or the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2:1099-1104 (1983), which is hereby incorporated by reference in its entirety). Similarly, “reporter genes,” which encode for enzymes providing for production of a compound identifiable are suitable. The most widely used reporter gene for gene fusion experiments has been uidA, a gene from Escherichia coli that encodes the β-glucuronidase protein, also known as GUS (Jefferson et al., EMBO J. 6:3901-3907 (1987), which is hereby incorporated by reference in its entirety). Similarly, enzymes providing for production of a compound identifiable by luminescence, such as luciferase, are useful. The selection marker employed will depend on the target species; for certain target species, different antibiotics, herbicide, or biosynthesis selection markers are preferred.
Once a recombinant plant cell or tissue has been obtained, it is possible to regenerate a full-grown plant therefrom. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beets, cotton, fruit trees, and legumes. Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
Plant regeneration from cultured protoplasts is described in Evans, et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III (1986), which are hereby incorporated by reference in their entirety.
After the DNA construct is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing or by preparing cultivars. With respect to sexual crossing, any of a number of standard breeding techniques can be used depending upon the species to be crossed. Cultivars can be propagated in accord with common agricultural procedures known to those in the field. Alternatively, transgenic seeds or propagules (e.g., cuttings) are recovered from the transgenic plants. The seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.
Another aspect of the present invention relates to a method for producing a fagopyritol, an insulin mediator, an insulin mediator analogue, an insulin mediator homologue, or an insulin mediator inhibitor. As used herein, fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, and insulin mediator inhibitors include salts and derivatives thereof.
Studies have been completed that link Type II diabetes and PCOS to deficiencies in insulin mediators composed of galactosamine D-chiro-inositol. Although their functions have yet to be fully characterized, it is known that insulin mediators act as second messengers of insulin action, and they are believed to be inositol phosphoglycans bound to cell membranes (Lamer et al., Diabetes Reviews 7:217-231 (1999), which is hereby incorporated by reference in its entirety). In the presence of insulin, these mediators are released and may activate glycogen synthesis. It has been found that feeding D-chiro-inositol to women with PCOS increased insulin response and ovulatory function (Nestler et al., N. Engl. J. Med. 340:1314-1320 (1999), which is hereby incorporated by reference in its entirety). Another study has also shown that insulin resistance has been associated with abnormal D-chiro-inositol metabolism (Ortmeyer et al., Endocrinology 132:640-645 (1993), which is hereby incorporated by reference in its entirety). Thus, synthesis of insulin mediators containing D-chiro-inositol is of importance in order to determine a treatment for Type II diabetes and PCOS.
This method of the present invention includes providing a fagopyritol synthase, providing a substrate including a galactosyl donor and a galactosyl acceptor, and combining the fagopyritol synthase with the substrate under conditions effective to produce a fagopyritol, an insulin mediator, an insulin mediator analogue, an insulin mediator homologue, or an insulin mediator inhibitor.
Suitable fagopyritols which can be produced by the above method of the present invention are described above.
Suitable insulin mediators, insulin mediator analogues, insulin mediator homologues, and insulin mediator inhibitors which can be produced by the above method of the present invention include, but are not limited to, galactosamine-D-chiro-inositols, galactosamine L-chiro-inositols, galactosamine-myo-inositols, galactosamine-scyllo-inositols, galactosamine-bornesitols, galactose-D-chiro-inositols, galactose L-chiro-inositols, galactose-myo-inositols, galactose-scyllo-inositols, galactose-bornesitols, glucose-D-chiro-inositols, glucose L-chiro-inositols, glucose-myo-inositols, glucose-scyllo-inositols, glucose-bornesitols, glucosamine-D-chiro-inositols, glucosamine L-chiro-inositols, glucosamine-myo-inositols, glucosamine-scyllo-inositols, and glucoseamine-bornesitols.
Suitable galactosyl donors include, but are not limited to, UDP-galactose, UDP-galactosamine, UDP-glucose, and UDP-glucosamine, which may be used with the enzymes described herein or enzyme mutants.
Suitable galactosyl acceptors include, but are not limited to, D-chiro-inositol, L-chiro-inositol, myo-inositol, bornesitol, and scyllo-inositol.
The fagopyritol synthase and substrate are combined to produce a fagopyritol, an insulin mediator, an insulin mediator analogue, or an insulin mediator homologue. Suitable conditions are determined by the fagopyritol synthase and substrate used, and include suitable amounts of Mn2+ (e.g., approximately 1-15 mM MnCl2, preferably 5 mM MnCl2) and suitable amounts of reducing agents, such as DTT and mercaptoethanol. One example of suitable conditions is disclosed in the enzyme assays described in the Examples, below.
Separation of the resulting fagopyritol, insulin mediator, insulin mediator analogue, or insulin mediator homologue from any other components may be achieved by methods known to one of ordinary skill in the art, such as with carbon-Celite, BioRad P2 gel, TLC, HPLC, or Dowex columns.
Thus, the method of the present invention can be used to produce an isolated or substantially pure fagopyritol, insulin mediator, insulin mediator analogue, insulin mediator homologue, insulin mediator inhibitor, or salts or derivatives thereof. As used herein, an isolated fagopyritol, insulin mediator, insulin mediator analogue, insulin mediator homologue, or insulin mediator inhibitor, is one which is substantially free of other components with which it naturally occurs. As referred to herein, substantially pure means substantially free of other compounds or materials, such as galactinol, myo-inositol, digalactosyl myo-inositol, phytin, aromatic materials (e.g. polyphenols and pigments and other colored aromatic materials), cell wall particles, proteins, and acids (e.g. organic acids, nucleic acids, and amino acids) and their salts. Typically, substantially pure fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, or insulin mediator inhibitors are those having greater than about 95% purity, such as greater than about 98% purity or from about 95% to about 99% purity.
Salts of the fagopyritols can be the reaction product of a base having a pKa (i.e., −log Ka) greater than the pKa of one or more of the fagopyritols' hydroxyl groups, such as a metal hydroxide or alkoxide, an amonium hydroxide, or an amine (e.g. a tertiary amine, like triethyl amine). Exemplary salts are alkali metal salts, such as lithium salts, sodium salts, and potassium salts, alkali earth metal salts, such as calcium salts and barium salts, ammonium salts, sufonium salts, and phosphonium salts.
Derivatives of the fagopyritols, include, for example, the reaction products of the fagopyritols with compounds bearing a carbon having a positive charge, such as an alkyl halide, in which case the derivative is an ether of the fagopyritol, or a carboxylic acid halide (e.g., acetyl chloride) or anhydride (e.g., acetic anhydride), in which case the derivative is an ester of the fagopyritol (e.g., the acetate).
The fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, and insulin mediator inhibitors produced with the fagopyritol synthase genes of the present invention can be used in a composition which includes one or more of fagopyritol A1, fagopyritol A2, fagopyritol A3, fagopyritol B1, fagopyritol B2, fagopyritol B3, D-chiro-inositol, an insulin mediator, an insulin mediator analogue, an insulin mediator homologue, or an insulin mediator inhibitor. Preferably, the composition is substantially free of one or more of galactinol, myo-inositol, digalactosyl myo-inositol, phytin, aromatic materials (e.g. polyphenols and pigments and other colored aromatic materials), cell wall particles, proteins, and acids (e.g. organic acids, nucleic acids, and amino acids) and their salts. It was observed that a mixture of fagopyritols was degraded within six hours in the presence of human fecal bacteria under in vitro conditions in the laboratory. Therefore, it is believed that the fagopyritols are digested by bacteria in the digestive tract to release free D-chiro-inositol for uptake, or in the case of monomers or dimers, may be taken up by cells of the digestive tract.
The aforementioned fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, insulin mediator inhibitors, and compositions are useful in treating diabetes in patients, such as mammals, including dogs, cats, rats, mice, and humans, by administering an effective amount of isolated or substantially pure fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, insulin mediator inhibitors, or compositions to such patients. The aforementioned fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, insulin mediator inhibitors, and compositions may also be useful in treating polycystic ovary syndrome (see Nestler et al., New England J. of Med., 340:1314-1320 (1999), which is hereby incorporated by reference in its entirety). For example, the substantially pure fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, and insulin mediator inhibitors, the compositions, or one or more isolated fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, and insulin mediator inhibitors can be administered alone, or in combination with suitable pharmaceutical carriers or diluents. The diluent or carrier ingredients should be selected so that they do not diminish the therapeutic effects of the fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, insulin mediator inhibitors, or compositions. Suitable pharmaceutical compositions include those which include a pharmaceutical carrier and, for example, one or more of an isolated fagopyritol A1, an isolated fagopyritol A2, an isolated fagopyritol A3, an isolated fagopyritol B1, an isolated fagopyritol B2, an isolated fagopyritol B3, an insulin mediator, an insulin mediator analogue, an insulin mediator homologue, or an insulin mediator inhibitor.
The fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, insulin mediator inhibitors, and compositions herein can be made up in any suitable form appropriate for the desired use; e.g., oral, parenteral, or topical administration. Examples of parenteral administration are intraventricular, intracerebral, intramuscular, intravenous, intraperitoneal, rectal, and subcutaneous administration. The preferred route for administration is oral. In cases where the fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, or insulin mediator inhibitors, are administered topically or parenterally, it is preferred that they be pre-hydrolyzed.
Suitable dosage forms for oral use include tablets, dispersible powders, granules, capsules, suspensions, syrups, and elixirs. Inert diluents and carriers for tablets include, for example, calcium carbonate, sodium carbonate, lactose, and talc. Tablets may also contain granulating and disintegrating agents, such as starch and alginic acid; binding agents, such as starch, gelatin, and acacia; and lubricating agents, such as magnesium stearate, stearic acid, and talc. Tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption. Inert diluents and carriers which may be used in capsules include, for example, calcium carbonate, calcium phosphate, and kaolin. Suspensions, syrups, and elixirs may contain conventional excipients, such as methyl cellulose, tragacanth, sodium alginate; wetting agents, such as lecithin and polyoxyethylene stearate; and preservatives, such as ethyl-p-hydroxybenzoate. Dosage forms suitable for parenteral administration include solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain suspending or dispersing agents known in the art.
For oral administration either solid or fluid unit dosage forms can be prepared. For preparing solid compositions, such as tablets, a suitable fagopyritol, insulin mediator, insulin mediator analogue, insulin mediator homologue, insulin mediator inhibitor, or composition, as disclosed above, is mixed with conventional ingredients, such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia methylcellulose, and functionally similar materials as pharmaceutical diluents or carriers. Capsules are prepared by mixing the disclosed fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, insulin mediator inhibitors, or compositions with an inert pharmaceutical diluent and filling the fixture into a hard gelatin capsule of appropriate size. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the fagopyritol, insulin mediator, insulin mediator analogue, insulin mediator homologue, insulin mediator inhibitor, or composition with an acceptable vegetable oil, light liquid petrolatum, or other inert oil.
Fluid unit dosage forms for oral administration such as syrups, elixirs, and suspensions can be prepared. The water-soluble forms can be dissolved in an aqueous vehicle together with sugar, aromatic flavoring agents, and preservatives to form a syrup. An elixir is prepared by using a hydro-alcoholic (ethanol) vehicle with suitable sweeteners, such as sugar and saccharin, together with an aromatic flavoring agent. Suspensions can be prepared with a syrup vehicle with the aid of a suspending agent, such as acacia, tragacanth, methylcellulose, and the like.
When the fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, insulin mediator inhibitors, or compositions are administered orally, suitable daily dosages can be based on suitable doses of free D-chiro-inositol, such as those described in U.S. Pat. No. 5,124,360 to Lamer et al., which is hereby incorporated by reference in its entirety. It is believed that about half of the fagopyritols as extracted is D-chiro-inositol, mostly as bound D-chiro-inositol with small amounts of free D-chiro-inositol. Therefore, suitable doses of fagopyritol are about twice the suitable doses of D-chiro-inositol. Typically, for oral administration, suitable daily doses are from about 5 mg to about 200 mg of the fagopyritol or composition per kilogram of the subject's body weight.
Alternatively, the fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, or insulin mediator inhibitors, can be administered orally in foodstuffs. For example, fagopyritols can be incorporated in purified form or in the form of buckwheat bran in bread, bread rolls, or other foodstuffs to form an edible product for consumption of fagopyritols. Fortification of breads, bread rolls, and other foodstuffs with synthesized fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, or insulin mediator inhibitors can provide a way to incorporate larger quantities of fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, or insulin mediator inhibitors into a daily diet. Suitable procedures for bread preparation can be found, for example, in Brown, The Tassajara Bread Book, Boston: Shambhala Publications (1986), which is hereby incorporated by reference.
For parenteral administration, fluid unit dosage forms are prepared utilizing the aforementioned fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, insulin mediator inhibitors, or compositions and a sterile vehicle, water being preferred. The fagopyritol, insulin mediator, insulin mediator analogue, insulin mediator homologue, insulin mediator inhibitor, or composition, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions, the fagopyritol, insulin mediator, insulin mediator analogue, insulin mediator homologue, insulin mediator inhibitor, or composition can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampule and sealing. Advantageously, adjuvants, such as a local anesthetic, preservative, and buffering agents, can be dissolved in the vehicle. To enhance the stability, the fluid unit dosage form can be frozen after filling into the vial, and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial, and an accompanying vial of water for injection is supplied to reconstitute the liquid prior to use. Parenteral suspensions are prepared in substantially the same manner except that the fagopyritol, insulin mediator, insulin mediator analogue, insulin mediator homologue, insulin mediator inhibitor, or composition is suspended in the vehicle instead of being dissolved, and sterilization cannot be accomplished by filtration. The fagopyritol, insulin mediator, insulin mediator analogue, insulin mediator homologue, insulin mediator inhibitor, or composition can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the parenteral suspension to facilitate uniform distribution of the fagopyritol, insulin mediator, insulin mediator analogue, insulin mediator homologue, insulin mediator inhibitor, or composition. Parenteral dosages can range from about 5 mg to about 200 mg of fagopyritol, insulin mediator, insulin mediator analogue, insulin mediator homologue, insulin mediator inhibitor, or composition per kilogram of the subject's body weight per day. Preferably, the daily parenteral dosage would be considerably less than the dose per kilogram of subject body weight, considering that, in oral administration, the galactose from the fagopyritols would be consumed by microbes in the digestive tract whereas, in parenteral administration the galactose would contribute to blood sugar levels.
Alternatively, the fagopyritol, insulin mediator, insulin mediator analogue, insulin mediator homologue, insulin mediator inhibitor, or composition can be incorporated into a sustained release formulation and surgically implanted using conventional methods. Suitable sustained release matricies include those made of ethylene vinyl acetate and other bicompatible polymers.
For topical administration, carriers, such as phospholipid vesicles, which contain the aforementioned fagopyritols, insulin mediators, insulin mediator analogues, insulin mediator homologues, or insulin mediator inhibitors, may facilitate uptake through the skin.
As indicated above, it is believed that the fagopyritols are digested in the digestive tract by bacteria to release free D-chiro-inositol for uptake. It is known that D-chiro-inositol is an anti-oxidant and, more particularly, a hydroxyl radical scavenger. Accordingly, the fagopyritol and compositions can also be used as a source of the antioxidant D-chiro-inositol, for example, by administering, preferably orally, the subject fagopyritols and compositions to a subject.
The present invention is further illustrated by the following examples.
EXAMPLES
Example 1
Fagopyritol Synthase, A Novel Multi-Functional Galactinol Synthase Homologue, Catalyzes the Biosynthesis of Fagopyritol A1 and Fagopyritol B1 in Buckwheat Seeds
Nucleotide and Amino Acid Sequence Analyses
The nucleotide sequences of galactinol synthase genes identified to date and their corresponding amino acid sequences were obtained from the nucleotide and protein databases (http://www.ncbi.nlm.nih.gov). Nucleotide and amino acid sequences were compared using a multiple sequence alignment program, CLUSTAL W (http://workbench.sdsc.edu). The identities of buckwheat cDNA fragments amplified from RT-PCR and RACE-PCR assays were examined by BLASTN and BLASTX programs (http://www.ncbi.nlm.nih.gov and http://workbench.sdsc.edu).
Isolation of FeGolS cDNA
The synthesis of PCR-directed cDNA from the poly(A)+ RNA isolated from developing buckwheat seeds (harvested at 20 to 25 days after pollination) was described previously (Lewis et al., Gene 246:81-91 (2000), which is hereby incorporated by reference in its entirety). Briefly, it involved the synthesis of the first strand cDNA using an oligo-dT primer (primer A, 5′-GCGGCCGCTTTTTTTTTTTTTTTTT-3′ (SEQ ID NO:16), FIG. 5) and reverse transcriptase, followed by oligo-dG-homopolymer-tailing of the first-strand cDNA with terminal transferase. Buckwheat FeGolS cDNAs were isolated by 5′ and 3′ RACE-PCR assays which were typically performed in either 25 or 50 μl reaction volume containing 100 pmol primers, 200 μM dNTPs, diluted G-tailed first strand cDNA (2 to 20 ng), 2 mM MgCl2 in 1×PCR reaction buffer (50 mM Tris/HCl, 10 mM KCl, 5 mM (NH4)2SO4, pH 8.3) with 1 to 2 units of FastStart Taq DNA Polymerase (Roche Applied Science, Indianapolis, Ind.). In the PCR assays, after the initial 4 minute denaturation step at 94° C., 38 to 40 cycles of amplification were carried out with each cycle consisting of the three consecutive incubations at 94° C. for 45 seconds, at 50 to 58° C. for 45 seconds, and at 72° C. for 45 seconds. Finally, the assays were terminated after a 10 minute final extension cycle at 72° C. All PCR products were cloned into pCRII-TOPO vector (Invitrogen, Carlsbad, Calif.) and propagated in Escherichia coli. For the isolation of cDNAs corresponding to buckwheat GolS genes, the initial amplification was carried out using the G-tailed cDNA preparation in combination with GS1 primer (5′-GGGCCACTGAACCTTATGGGGGCACTGCTGGC-3′) (SEQ ID NO:17) representing an internal protein coding sequence highly conserved in most GolS genes, and primer B (5′-AAGGAATTCCCCCCCCCCCCCC-3′) (SEQ ID NO:18) partially complementary to the G-tailed 5′-end of the first strand cDNAs (FIG. 5). One of the amplified cDNA fragments, 469 bp in length, was shown to represent a GolS homolog in buckwheat when its nucleotide sequence was analyzed by BLASTN and BLASTX programs. The gene represented by this partial cDNA clone was designated as FeGolS-1 for Fagopyrum esculentum GolS-1. The overlapping cDNA fragments containing the 5′-end region of FeGolS-1 cDNA were further amplified in 5′ RACE-PCR assays using an upstream internal primer, GS2 (5′-GCTCCATGATGGCTCACAGAAACAGTCC-3′) (SEQ ID NO:19) and primer B (FIG. 5). This PCR amplification yielded a cDNA fragment of 548 bp in length which contained the complete 5′-end of the protein coding sequence and 82 bp long 5′ untranslated region (5′UTR). An overlapping cDNA fragment of about 900 bp in length containing the complete 3′-end region of FeGolS-1 was also obtained in 3′ RACE-PCR assays, using an internal primer, GS3 (5′-GCTCACGCATACTATGTCATCAACTACTCC-3′) (SEQ ID NO:20) and primer A (FIG. 5). In addition, two additional cDNA fragments of about 960 bp in length exhibiting nucleotide sequences that were nearly identical to each other but clearly distinct from the 3′-end region of FeGolS-1 cDNA were obtained. Analyses of their nucleotide sequences by BLASTN and BLASTX programs also identified them as GolS homologues. Thus, the genes corresponding to these two additional cDNAs were designated as FeGolS-2 and FeGolS-3. In an attempt to amplify the 5′-end regions of the FeGolS-2 and FeGolS-3 cDNAs, 5′ RACE-PCR assays were performed using primer A and an internal primer, GS4 (5′-GAACTTCTTGCCCTCGACCATCTTAGGCTGAG-3′) (SEQ ID NO:21) representing the nucleotide sequence that was common to FeGolS-2 and FeGolS-3 cDNA's but not shared by FeGolS-1 cDNA (FIG. 5). An overlapping cDNA fragment of 984 bp in length was obtained from the assays (FIG. 5). The nucleotide sequence of the cDNA fragment confirmed that it was a part of FeGolS-2 cDNA. Finally, an intact FeGolS-1 cDNA containing the complete protein coding sequence as well as 5′ and 3′ UTRs was reconstituted by joining the 398 bp long 5′-end region of the 5′ RACE-PCR clone with the 871 bp long 3′-end region of the 3′ RACE-PCR clone at the unique HindIII site (FIG. 5). Similarly, an intact FeGolS-2 cDNA was reconstituted by joining the 700-bp 5′-end region of the 5′ RACE-PCR clone with the 650-bp long 3′-end region of the 3′ RACE-PCR clone at the unique XhoI site (FIG. 5).
DNA Sequencing
All PCR-generated cDNA clones were sequenced at the DNA Sequencing Facility, BioResource Center, Cornell University (http://brcweb.biotech.cornell.edu).
Bacterial Expression and Purification of Recombinant GolS Proteins
The entire 1002 bp long protein coding sequence of FeGolS-1 cDNA was amplified from the reconstituted FeGolS-1 cDNA using two oligonucleotide primers, FG1-5 (5′-GTTCCAACCATATGGCACCAGAACTC-3′) (SEQ ID NO:22) and FG1-3 (5′-GGATCCGATACTTAAGCTGCGGAAGGAGC-3′) (SEQ ID NO:23) (FIG. 5). FG1-5 and FG1-3 primers contained the restriction enzyme recognition sites for NdeI and BamHI, respectively, to allow easy cloning of the amplified coding sequence into a bacterial expression vector, pET-14b (Novagen, Madison, Wis.), in frame with the preceding poly-histidine codons in the vector. After initial cloning into pCRIITOPO vector and amplification of the plasmid in E. coli, the protein coding sequence was excised from the plasmid by digestion with NdeI and BamHI, and cloned into pET-14b vector at the corresponding cloning sites. Similarly, the 1065 bp long entire protein coding sequence from the reconstituted FeGolS-2 cDNA was inserted into pET14b vector after amplifying it with FG2-5 (5′-CATATGACTTCCGAGATGGCGCCACAG-3′) (SEQ ID NO:24) and FG2-3 (5′-GGATCCTCAGGCAGCAGACGGGGCGTGTACG-3′) (SEQ ID NO:25) primers which also contained NdeI and BamHI sites, respectively (FIG. 5). In addition, the 987 bp long entire coding sequence was isolated from a soybean EST clone (GenBank accession no. BE330777) presumed to encode soybean galactinol synthase (GmGolS) in leaf tissues (INCYTE GENOMICS, cat. no. Gm-c1041), and it was cloned into pET14-b vector. Since only partial cDNA sequence data were available in GenBank, the whole cDNA insert was re-sequenced (GenBank Accession No. AY126715). Two primers, GG-5 (5′-CATCACTGAGCATATGGCTGG-3′) (SEQ ID NO:26) and GG-3 (5′-GGATCCAAAGACACTCTTAAGCAGCAGATGGGG-3′) (SEQ ID NO:27), containing NdeI and BamHI restriction enzyme recognition sites, respectively, were used for the amplification of the protein coding sequence. After cloning into pCRIITOPO vector and amplification in E. coli, the NdeI/BamHI fragment containing the entire protein coding sequence was isolated and cloned into pET-14b vector. The pET14b plasmids containing the buckwheat and soybean GolS cDNAs were mobilized into E. coli strain BL21 (DE3) (Novagen, Madison, Wis.). Expression of the recombinant GolS proteins in E. coli were induced with 1 mM isopropyl β-D-thiogalactoside (IPTG) according to the manufacture's recommended protocol (Novagen, Madison, Wis.). The bacterial cells were collected by centrifugation, and resuspended in 10 mM Tris-HCl buffer (pH 8.0). The soluble protein fraction was extracted from the bacterial cells by the gentle disruption of their cell walls with BugBuster Protein Extraction Reagent (Novagen, Madison, Wis.) containing Benzonase (Novagen, Madison, Wis.). In some experiments, the soluble protein fraction was extracted from bacterial cells through disruption of bacterial cells by sonic oscillation (at 50% level, twice for 10 seconds each, at 4° C.) with a sonicator (Fisher Scientific Sonic Dismembrator Model 500). Poly-histidine tagged recombinant proteins were purified from the extracts using His.Bind Quick 300 Cartridges (Novagen, Madison, Wis.) according to the manufacture's recommended protocol. Purified recombinant proteins were dialyzed against 50 mM Hepes buffer, pH 7.0, containing 5 mM MnCl2, immediately after elution from the His.Bind Quick 300 Cartridges and before enzyme assay. Aliquots (0.25 to 0.5 μg) of samples of the purified proteins were checked by SDS-PAGE using a 12% resolving gel and a 5% stacking gel. Protein samples (10 μg each) extracted from uninduced and induced bacterial cells prior to protein purification were also included in the SDS-PAGE analysis. Proteins in the gels were visualized by staining with Coomassie Brilliant Blue R250 solution (25 g/liter in methanol:acetic acid:water, 45:10:45, v/v/v) and destained in methanol:acetic acid:water (30:10:60, v/v/v).
Enzyme Assays
Both the crude soluble protein extracts from E. coli and the purified GolS recombinant proteins were used in enzyme assays. Fagopyritol synthase assays included 20 mM UDP-Gal as the galactosyl donor, 20 mM D-chiro-inositol as the galactosyl acceptor, 50 mM Hepes buffer, pH 7.0, 2 mM dithiothreitol, 5 mM MnCl2, and 1 to 5 μg of crude protein extract or purified enzyme protein (estimated by the Bio-Rad Protein Assay, BIO-RAD) in 50 μL total volume. In galactinol synthase assays, UDP-Gal was substituted with 20 mM galactinol as the galactosyl donor. Assays were run at 30° C. for 30 to 300 minutes. Reactions were stopped by addition of 50 μL of 100% ethanol. After addition of 25 μg of phenyl α-D-glucoside as internal standard, the reaction mixture was heated at 80° C. for 30 minutes, passed through a 10,000 MW cutoff filter (NANOSEPTM Microconcentrators, Pall Filtron Co.), and evaporated to dryness under a stream of nitrogen gas. Residues were stored overnight in a desiccator with phosphorus pentoxide to remove traces of water, derivatized with trimethylsilylimidazole:pyridine (1:1, v/v) at 80° C. for 45 minutes, and analyzed for fagopyritols or other soluble carbohydrate products by high resolution gas chromatography on a HP1-MS (Agilent Technologies) capillary column (15 m length, 0.25 mm i.d., 0.25 μm film thickness) as previously described (Horbowicz et al., Seed Sci. Res. 4:385-405 (1994); Horbowicz et al., Planta 205:1-11 (1994), which are hereby incorporated by reference in their entirety).
Results
Cloning of cDNAs Encoding Two Distinct Types of GolS Enzymes in Buckwheat Seeds
Initially, several GolS gene sequences reported from various plant species, either derived from genomic or cDNA clones, were compiled and compared to identify stretches of highly conserved nucleotide sequences corresponding to the conserved amino acid domains of GolS enzymes. By using oligonucleotide primers representing these conserved nucleotide sequences and the first-strand cDNA synthesized from polyA+ RNA extracted from developing seeds in our PCR assays, a total of three different GolS cDNAs from buckwheat were isolated (FIG. 5). The genes corresponding to these three buckwheat cDNA clones were designated as FeGolS-1, -2, and -3 for Fagopyrum esculentum GolS-1, -2, and -3.
FeGolS-1 cDNA was initially obtained as a partial clone of 469 bp in length, using an internal GolS gene-specific primer (GS1) and primer B corresponding to the dG homopolymer tail present at the 5′ end of the cDNA (FIG. 5). Subsequently, the missing 5′-end region of FeGolS-1 cDNA was obtained by 5′ RACE-PCR using the second internal primer (GS2) and primer B (FIG. 5). One of the 5′ RACE-PCR clones contained a complete 5′-end of the protein coding region together with 82 bp long 5′ untranslated region (5′UTR) (FIG. 5). The missing 3′-end region of FeGolS-1 cDNA was obtained by 3′ RACE-PCR using an internal primer (GS3) and primer A complementary to the polyA tail present in all cDNAs (FIG. 5). In the 3′ RACE-PCR assays, two additional clones (FeGolS-2 and FeGolS-3) were obtained. They were longer (987 bp and 986 bp for FeGolS-2 and FeGolS-3, respectively) than the FeGolS-1 cDNA clone (901 bp) and exhibited restriction patterns clearly distinct from that of FeGolS-1. No obvious polyadenylation signals were found upstream of the polyadenylation sites in any of the three genes. The 5′-end region of FeGolS-2 cDNA containing the complete 5′-end of the protein coding region was obtained by 5′ RACE-PCR using a gene-specific primer, GS4 and primer B (FIG. 5). Cloning of cDNA fragments containing the 5′-end of the FeGolS-3 gene was not successful.
Intact FeGolS-1 and FeGolS-2 cDNAs containing the complete protein coding sequences with 5′ and 3′ UTRs were reconstituted by joining the overlapping 5′ and 3′ RACE-PCR clones for each gene (FIG. 5). The reconstituted FeGolS-1 cDNA is 1269 bp long containing a single open reading frame (ORF) (GenBank accession no. AY126718). On the other hand, the reconstituted FeGolS-2 cDNA is 1326 bp long; it also contains a single ORF (GenBank Accession No. AY126716). The partial FeGolS-3 cDNA clone is 986 bp long and contains the complete 3′-end of the cDNA (GenBank accession no. AY126717). According to the nucleotide sequence comparison, FeGolS-1 is distinct from FeGolS-2 sharing only 62.2% sequence identity. On the other hand, FeGolS-2 and FeGolS-3 share a nearly identical nucleotide sequence in their 3′ regions. Whereas the FeGolS-2 cDNA clone differs from FeGolS-3 only by 15 nucleotides within the 986/987 bp long 3′ region, FeGolS-2 differs from FeGolS-1 by 385 nucleotides at the corresponding 3′ region. These results suggest that FeGolS-1 and FeGolS-2 represent two different members of a GolS gene family in buckwheat. The complete 1406 bp nucleotide sequence of the soybean galactinol synthase (GmGolS) cDNA (assigned GenBank Accession No. AY126715) had a high degree of sequence similarity to FeGolS-1.
Primary Structures of GolS Polypeptides Deduced From cDNA Sequences
The amino acid sequence deduced from the reconstituted FeGolS-1 cDNA indicated that it is capable of encoding a polypeptide of 333 amino acid residues with a predicted molecular mass of 38.3 kDa (FIG. 1). On the other hand, FeGolS-2 cDNA is capable of encoding a polypeptide of 354 amino acids with a predicted molecular mass of 40.7 kDa (FIG. 2). Predicted FeGolS-2 and -3 differ from each other only by three amino acid residues in the carboxyl half of the polypeptide whereas each differs from FeGolS-1 by 96 amino acid residues in the corresponding region. The presence of a longer stretch (additional 17 residues) of amino acid sequence was identified near the carboxyl termini in FeGolS-2 (and also in FeGolS-3), mainly accounting for its larger predicted molecular mass than that for FeGolS-1 (FIG. 6). The amino acid sequence deduced from the 987 bp long coding sequence of the soybean GmGolS cDNA indicated that it is capable of encoding a polypeptide of 328 amino acid residues with a predicted molecular mass of 38.0 kDa (FIG. 4).
Both FeGolS-1 and FeGolS-2 polypeptides share a high degree of amino acid sequence similarity with other GolSs identified from a wide variety of plant species (FIG. 7). The highly conserved serine phosphorylation site and the carboxyl terminal pentapeptide, APSAA (SEQ ID NO:28) (Sprenger et al., Plant J. 21:249-258 (2000), which is hereby incorporated by reference in its entirety) are also present in all three FeGolS proteins. In addition, a putative manganese binding motif, DXD, believed to be conserved in most galactosyl transferases (Breton et al., J. Biochem. 123:1000-1009 (1998); Busch et al., J. Biol. Chem. 273:19566-19572 (1998); Wiggins et al., Proc. Natl. Acad. Sci. USA 95:7945-7950 (1998), which are hereby incorporated by reference in their entirety) is also present in all GolSs examined, including the three FeGolSs. A phylogenetic analysis indicated that both FeGolS-1 and FeGolS-2 are evolutionarily most closely related to a Brassica napus GolS.
Recombinant Protein Expression and Purification
FIG. 8 shows an SDS-PAGE gel used to monitor the protein expression and purification steps. Total soluble protein extracts from uninduced and induced bacteria cells harboring FeGolS-1 cDNA are shown in lanes 2 and 3, respectively. The purified recombinant FeGolS-1 protein fraction (lane 4) contained a single prominent polypeptide with an apparent molecular mass of 43 kDa. Total soluble protein extracts from uninduced and induced bacteria cells harboring FeGolS-2 cDNA are shown in lanes 5 and 6, respectively. The purified recombinant FeGolS-2 protein fraction (lane 7) contained a single prominent polypeptide with an apparent molecular mass of 45.5 kDa. Total soluble protein extracts from uninduced and induced bacteria cells harboring GmGolS cDNA are shown in lanes 8 and 9, respectively. A single polypeptide of with an apparent molecular mass of 43 kDa was found in the purified recombinant GmGolS protein fraction (lane 10). No polypeptide with its molecular mass corresponding to any of the recombinant GolS proteins described above was found after purification of histidine-tagged protein from the total soluble protein extract from control bacteria which had been transformed with the pET-14b vector alone. These results indicated that the purified recombinant FeGolS-1, FeGolS-2, and GmGolS proteins were derived from the expression of their corresponding genes.
Substrate Specificity of FeGolS-1 and FeGolS-2
Both purified recombinant FeGolS-1 and FeGolS-2 proteins exhibited fagopyritol synthase activities. FeGolS-1 catalyzed the biosynthesis of fagopyritol B1 with UDP-Gal as the galactosyl donor and D-chiro-inositol as the galactosyl receptor (FIG. 9A). However, only FeGolS-2 catalyzed the biosynthesis of both fagopyritol A1 and fagopyritol B1 in a ratio of 1:4 demonstrating the unique product specificity of FeGolS-2 (FIG. 9B). Both FeGolS-1 and FeGolS-2 catalyzed the biosynthesis of galactinol with UDP-Gal as galactosyl donor and myo-inositol as galactosyl receptor (FIGS. 9D and 9E), consistent with the structural homology of these enzymes to galactinol synthase. No products were biosynthesized using protein extracts from control bacteria transformed with the vector only, confirming that FeGolS-1 and FeGolS-2 catalyzed the biosynthesis of fagopyritols and galactinol. Neither FeGolS-1 nor FeGolS-2 was active with galactinol as the galactosyl donor, demonstrating that both enzymes had substrate specificity for UDP-Gal. Neither FeGolS-1 nor FeGolS-2 biosynthesized fagopyritol A1 from fagopyritol B1 (as both donor and receptor) indicating that FeGolS-2 catalyzes the biosynthesis of fagopyritol A1 directly by transfer of the galactosyl residue from UDP-Gal. As a control, soybean galactinol synthase (GmGolS) catalyzed the biosynthesis of galactinol with UDP-Gal and myo-inositol as substrates (FIG. 9F), but also catalyzed the biosynthesis of fagopyritol B1, but not fagopyritol A1, with UDP-Gal and D-chiro-inositol as substrates (FIG. 9C). Activity of FeGolS-1 was similar to that for GmGolS, whereas FeGolS-2, by catalyzing the biosynthesis of fagopyritol A1, was uniquely different from the soybean enzyme.
Discussion
The FeGolS-1 gene encodes an enzyme that catalyzes fagopyritol B1 biosynthesis using UDP-Gal as galactosyl donor and D-chiro-inositol as galactosyl acceptor. The FeGolS-2 gene, a unique member of the buckwheat galactinol synthase gene family, encodes a fagopyritol synthase that catalyzes the biosynthesis of both fagopyritol A1 and fagopyritol B1 using UDP-Gal as galactosyl donor and D-chiro-inositol as galactosyl acceptor. Based on the molecular structure and absolute configuration of fagopyritol A1 and fagopyritol B1 determined by NMR (Obendorf et al., Carbohydr. Res. 328:623-627 (2000), which is hereby incorporated by reference in its entirety), FeGolS-2 catalyzes the formation of the α-(1→3)-linkage unique to fagopyritol A1 and other members of the fagopyritol A series found only in buckwheat, as well as the α-(1→2)-linkage of fagopyritol B1 and other members of the fagopyritol B series (Obendorf et al., Carbohydr. Res. 328:623627 (2000); Steadman et al., Carbohydr. Res. 331:19-25 (2001), which are hereby incorporated by reference in their entirety). FeGolS-1, FeGolS-2, and GmGolS all biosynthesize galactinol using UDP-Gal as galactosyl donor and myo-inositol as galactosyl acceptor. However, buckwheat FeGolS-1 and soybean GmGolS do not form fagopyritol A1. Thus, the novel buckwheat FeGolS-2 gene and its protein product are distinctly different in both structure and function from the buckwheat FeGolS-1 gene and the soybean GmGolS gene and their corresponding proteins. The longer amino acid sequence (13 to 23 amino acids) near the carboxyl end of buckwheat FeGolS-2 (and also FeGolS-3) is unique among known GolS sequences from various species and may be related to the property of FeGolS-2 to form the unique α-(1→3)-linkage.
Retention of fagopyritol synthase activity by purified recombinant FeGolS-1, FeGolS-2, and GmGolS protein required Mn+2 (5 mM optimal) as a cofactor, as it has been reported with galactinol synthase from other sources (Saravitz et al., Plant Physiol. 83:185-189 (1987); Castillo et al., J. Agric. Food Chem. 38:351-355 (1990); Smith et al., Plant Physiol. 96:693698 (1991); Liu et al., Plant Physiol. 109:505-511 (1995); Kuo et al., Plant Sci. 125:1-11 (1997), which are hereby incorporated by reference in their entirety). One to 10 mM Mn+2 was most commonly used for the retention of galactinol synthase activity. Interestingly, the antihyperglycemic effects of D-chiro-inositol were associated with manganese (Fonteles et al., Hormone Metab. Res. 32:129 132 (2000), which is hereby incorporated by reference in its entirety) in subjects with non-insulin dependent diabetes melitus. Buckwheat seeds are a rich source of manganese (Steadman et al., J. Sci. Food Agric. 81:1094-1100 (2001), which is hereby incorporated by reference in its entirety), and buckwheat has been used for the treatment of diabetes (Lu et al., in Proceedings of the 5th International Symposium on Buckwheat, eds. Lin et al., Agriculture Publishing House, Beijing, pp 458-464 (1992); Wang et al., in Proceedings of the 5th International Symposium on Buckwheat, eds. Lin et al., Agriculture Publishing House, Beijing, pp 465-467 (1992), which are hereby incorporated by reference in their entirety).
Pea (Pisum sativum L.) seed galactinol synthase (Frydman et al., Biochem. Biophys. Res. Comm. 12:121-125 (1963), which is hereby incorporated by reference in its entirety) and lentil (Lens culinaris Medik.) stachyose synthase (Hoch et al., Arch. Biochem. Biophys. 366:75-81 (1999), which is hereby incorporated by reference in its entirety) have been reported to form a product with D-chiro-inositol as substrate, but the product was not confirmed to be a fagopyritol. The lack of activity of Adzuki bean (Vigna angularis Ohwi and Ohashi) stachyose synthase with D-chiro-inositol (Peterbauer et al., Plant Physiol. 117:165-172 (1998), which is hereby incorporated by reference in its entirety) and the very limited accumulation of stachyose in buckwheat seeds (Horbowicz et al., Planta 205:1-11 (1998), which is hereby incorporated by reference in its entirety) suggest that stachyose synthase is not involved in the biosynthesis of fagopyritols. The results reported herein clearly demonstrate that FeGolS-2, a galactinol synthase homologue, catalyzes the biosynthesis of both fagopyritol A1 and fagopyritol B1.
Among seven GolS genes identified in Arabidopsis thaliana, three were identified as stress responsive (Taji et al., Plant J. 29:417-426 (2002), which is hereby incorporated by reference in its entirety). AtGolS-1 and AtGolS-2 were induced by drought and high-salinity stresses but not by cold stress. In contrast, AtGolS-3 was induced by cold stress by not by drought or high-salinity stress. Buckwheat seeds matured at 18° C. accumulated more fagopyritol A1 and fagopyritol B1 than seeds matured at 25° C. (Horbowicz et al., Planta 205:1-11 (1998), which is hereby incorporated by reference in its entirety), indicating that FeGolS genes may be cold-responsive.
The nucleotide sequence of the soybean EST clone, BE330777, isolated by a public source (Shoemaker et al., Public soybean EST project; GenBank BE33077; Genome Systems Clone ID: Gm-c1041-80 (5′), Genome Systems, Inc., 4633 World Parkway Circle, St. Louis, Mo. 63134 (1999), which is hereby incorporated by reference in its entirety) with the full sequence first reported herein, demonstrated a very high homology to the soybean seed galactinol synthase gene (I55634), sequence 6 (U.S. Pat. No. 5,648,210 to Kerr et al., which is hereby incorporated by reference in its entirety). The deduced amino acid sequence (328 amino acids) differed by only one amino acid, Ile 223 in GmGolS (AY126715) rather than Met 223 (155634) (U.S. Pat. No. 5,648,210 to Kerr et al., which is hereby incorporated by reference in its entirety). Of the multiple genes for galactinol synthase, some are specifically expressed in seeds. Modification of galactinol biosynthesis is of commercial interest (U.S. Pat. No. 5,648,210 to Kerr et al.; U.S. Pat. No. 5,710,365 to Kerr, which are hereby incorporated by reference in their entirety) for producing soybean seeds with lower stachyose concentrations for the poultry and pig feed industry (Sebastian et al., in Soy in Animal Nutrition, ed. Drackley, Federation of Animal Science Societies, Savoy, Ill., pp 56-73 (2000), which is hereby incorporated by reference in its entirety). A mutant with a single base change in a seed-expressed myo-inositol l-phosphate synthase (MIPS, EC 5.5.1.4) gene coupled with appropriate modifiers resulted in soybean seeds with both reduced phytic acid and reduced stachyose (Hitz et al., Plant Physiol. 128:650-660 (2002), which is hereby incorporated by reference in its entirety) for use in the feed industry.
Fagopyritol A1 is isosteric with 2-amino-2-deoxy-α-D-galactopyranosyl-(1→3)-D-chiro-inositol (Berlin et al., Tetrahedron Lett. 31:1109-1112 (1990), which is hereby incorporated by reference in its entirety) related to a putative insulin mediator (Lamer et al., Biochem. Biophys. Res. Comm. 151:1416-1426 (1988), which is hereby incorporated by reference in its entirety) deficient in subjects with NIDDM and PCOS. The novel FeGolS-2 gene and FeGolS-2 enzyme described herein may be used to form the unique α-(1→3)-linkage between galactose and D-chiro-inositol.
Example 2
Seed Galactosyl Cyclitols Enhanced by Substrate Feeding
Materials and Methods
Plant Materials
Soybean (Glycine max (L.) Merrill) plants were grown in the greenhouse (Obendorf et al., Crop Sci. 20:483-486 (1980); Obendorf et al., Crop Sci. 38:78-84 (1998), which are hereby incorporated by reference in their entirety) at 27° C. during the day (14 hours) and 22° C. at night (10 hours) under natural sunlight supplemented 14 hours daily with 640 μmol m−2 s−1 incandescent light from metal halide lamps (Sylvania 1000 watt BU). Three embryos isolated from immature seeds (250±20 mg fresh weight, approximately 35 DPA) by removal of the seed coat and nucellus remnants were incubated in 20 mL screw-capped vials containing 3 mL of substrate (cyclitol and/or sucrose) solutions for 24 hours at 25° C. and 200 μmol m−2 s−1 fluorescent light. Embryos were blotted, placed in small plastic Petri dishes, and subjected to slow drying at 22° C. by daily transfer to successive lower relative humidity (RH) controlled by saturated salt solutions (Blackman et al., Plant Physiol. 100:225-230 (1992), which is hereby incorporated by reference in its entirety): day 1, 92% RH; day 2, 87% RH; day 3, 75% RH; day 4, 54% RH; day 5, 45% RH; day 6, 32% RH; day 7, 12% RH; and remained at 12% RH days 8-14.
Embryo Feeding Experiments—Substrate Concentration Series
Four substrate concentration experiments were conducted. Embryos for each experiment were incubated in each of the substrate solutions for 24 hours, blotted, and slow dried for 14 days. Axis and cotyledon tissues were separated and analyzed for soluble carbohydrates. Four replications of three embryos each (total of 12 embryos/treatment) were incubated in the myo-inositol-sucrose concentration series: A) 0 mM myo-inositol+100 mM sucrose, B) 10 mM myo-inositol+90 mM sucrose, C) 25 mM myo-inositol+75 mM sucrose, D) 50 mM myo-inositol+50 mM sucrose, E) 100 mM myo-inositol+0 mM sucrose, and F) 0 mM myo-inositol+0 mM sucrose. Six replications of three embryos each (total of 18 embryos/treatment) were incubated in the D-chiro-inositol-sucrose concentration series, and three replications of three embryos each (total of 9 embryos/treatment) were incubated in the D-pinitol-sucrose concentration series. Treatments A) through F) were identical in both concentration series, except for the substitution of D-chiro-inositol or D-pinitol instead of myo-inositol. In the sucrose concentration series, three replications of three embryos (total of 9 embryos/treatment) were incubated with 0, 25, 50, 75, 100, and 200 mM sucrose.
Embryo Feeding Experiments—Drying Time Series
Six slow drying time experiments were conducted. In each experiment, three replications of three embryos each (total of 9 embryos per treatment) were incubated in a different sucrose and/or cyclitol substrate solution for 24 hours, blotted, and slow dried for 0, 1, 2, 3, 4, or 14 days. Axis and cotyledon tissues were separated and analyzed for soluble carbohydrates. The substrate solutions for the six experiments were as follows: 30 mM myo-inositol plus 100 mM sucrose; 100 mM D-chiro-inositol; 100 mM D-pinitol; 100 mM D-pinitol plus 100 mM D-chiro-inositol; 50 mM D-pinitol plus 50 mM D-chiro-inositol; and 100 mM D-pinitol plus 100 mM D-chiro-inositol plus 100 mM sucrose.
Substrates
Sucrose, myo-inositol, scyllo-inositol, epi-inositol, and UDP-Gal were purchased from Sigma-Aldrich (St. Louis, Mo.). D-Pinitol, D-chiro-inositol, L-chiro-inositol, D-ononitol, and L-quebrachitol were purchased from Industrial Research Limited (Lower Hutt, New Zealand). Sequoyitol was purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Bornesitol was purified from seeds of Lathyrus odoratus L. Galactinol was purified from lemon balm (Melissa officinalis L.) leaves. When needed, substrates were purified by carbon-Celite column chromatography (Whistler et al., J. Amer. Chem. Soc. 72:677-679 (1950), which is hereby incorporated by reference in its entirety) before use. Carbon was purchased from Mallinckrodt Baker Inc (Phillipsburg, N.J.). Celite was purchased from Supelco (Bellefonte, Pa.).
Carbohydrate Analysis
Soluble carbohydrates were extracted from 2 cotyledons or 1 axis for each embryo. Two cotyledons were extracted with 2.0 mL of ethanol:water (1:1, v/v) containing 300 μg of phenyl α-D-glucoside as internal standard. One axis was extracted with 1.0 mL of ethanol:water (1:1, v/v) containing 100 μg of phenyl α-D-glucoside as internal standard. Extracts were passed through a 10,000 molecular weight cut-off filter (NANOSEP 10K Omega, Paul Filton Co., Northborough, Mass.) by centrifugation, and 200 μL were dried in silylation vials under nitrogen gas, derivatized with 200 μL of trimethylsilylsylimidazole:pyridine (1:1, v/v), and analyzed by high resolution gas chromatography on a HP1-MS (Agilent Technologies, Palo Alto, Calif.) capillary column (15 m length, 0.25 mm i.d., 0.25 μm film thickness) as previously described (Horbowicz et al., Seed Sci. Res. 4:385-405 (1994), which is hereby incorporated by reference in its entirety).
Results
Cyclitols, including myo-inositol, D-chiro-inositol, and D-pinitol, were fed to immature soybean embryos followed by precocious maturation induced by slow-drying of the embryos and analysis of soluble carbohydrates in axis and cotyledon tissues. Exogenously fed free cyclitols were taken up by embryo tissues. In 250 mg fresh weight embryos, initial concentrations of cyclitols in axis and cotyledon tissues, respectively, were myo-inositol 10.9 and 11.0 mg/g dry weight, D-chiro-inositol 1.4 and 1.2 mg/g dry weight, and D-pinitol 6.0 and 4.0 mg/g dry weight. After incubation with 30 mM myo-inositol, 100 mM D-chiro-inositol, or 100 mM D-pinitol for 24 hours at 22° C., concentrations of myo-inositol increased 1.8 fold in axis and 2 fold in cotyledon tissues, D-chiro-inositol increased 18 fold and 40 fold, and D-pinitol increased 6 fold and 11 fold, respectively.
Both embryonic axis and cotyledon tissues were assayed for experiments reported herein. Embryonic axes mature earlier than cotyledons and accumulate higher concentrations of soluble carbohydrates (up to 25% of dry weight) (Horbowicz et al., Seed Sci. Res. 4:385-405 (1994); Obendorf et al., Crop Sci. 38:78-84 (1998), which are hereby incorporated by reference in their entirety). Accumulation of products in axis tissues generally precedes accumulation of products in cotyledons, reflecting the differential in progression toward maturation. In general, data were more variable for axis tissues than for cotyledon tissues, mainly because of the small mass of axis tissues, about 1 mg dry weight for experiments reported herein.
Concentration series experiments were adjusted to be a constant 100 mM (cyclitol plus sucrose) excluding the sucrose concentration series experiment. Feeding myo-inositol up to 50 mM doubled free myo-inositol concentration in dry axis and cotyledon tissues after precocious maturation with small increases in D-pinitol and D-chiro-inositol (FIGS. 10A and D). Galactinol accumulation doubled in cotyledons after feeding 25 to 50 mM myo-inositol while fagopyritol B1 accumulation was reduced (FIG. 10E), demonstrating a competition between the biosynthesis of galactinol and fagopyritol B1. There was little change in galactopinitol A, galactopinitol B, raffinose, or stachyose concentrations in either axis or cotyledon tissues after feeding myo-inositol (FIGS. 10B, C, E, and F). In the absence of exogenous sucrose, sucrose concentration in axis tissues was reduced to 50%, but sucrose concentration in cotyledons remained constant (FIGS. 10C and F). These results are consistent with the role of myo-inositol as a substrate in galactinol biosynthesis and a product in the biosynthesis of raffinose and stachyose in seeds (FIG. 11). Feeding 30 mM myo-inositol and 100 mM sucrose together resulted in elevated amounts of free myo-inositol during day 1 of slow drying and then a decline in myo-inositol (FIGS. 12A and D), a transient increase in galactinol during days 2 and 3 (FIGS. 12B and E), and then a decline in galactinol as raffinose and stachyose accumulated (FIGS. 12C and F). The decrease in total myo-inositol indicates metabolism of myo-inositol to other products, including phytin and cell walls, within the embryo (Loewus et al., Plant Sci. 150:1-19 (2000); Hegeman et al., Plant Physiol. 125:1941 1948 (2001); Hitz et al., Plant Physiol. 128:650-660 (2002), which are hereby incorporated by reference in their entirety).
Feeding D-chiro-inositol resulted in a 40- to 50-fold increase in free D-chiro-inositol concentration in axis and cotyledons (FIGS. 13A and D), a 17-fold increase in fagopyritol B1 concentration in axis tissues and a 7-fold increase in cotyledons (FIGS. 13B and E), but did not increase D-pinitol, myo-inositol, galactopinitol A, galactopinitol B, galactinol, raffinose, or stachyose concentrations (FIG. 13). The high concentrations of free D-chiro-inositol declined (FIGS. 14A and D) and a large increase in fagopyritol B1 occurred between day 2 and day 4 of slow drying accompanied by the decrease in concentration of free D-chiro-inositol in axis and cotyledon tissues (FIGS. 14A, B, D, and E). A transient accumulation of galactinol signaled an accumulation of raffinose and stachyose and modest accumulation of galactopinitol A and galactopinitol B (FIGS. 14B, C, E, and F, compared to FIG. 12). These results suggest that D-chiro-inositol does not serve as precursor to myo-inositol or D-pinitol in soybean embryos, and that fagopyritol B1 does not serve as an alternate galactosyl donor for the biosynthesis of raffinose and stachyose. The large increase in fagopyritol B1 from externally applied D-chiro-inositol suggests that D-chiro-inositol is not biosynthesized within the embryo but is transported to the embryo from maternal tissues. The increase in sucrose during slow drying (FIGS. 14C and F) probably reflects starch degradation within the embryo.
Feeding D-pinitol resulted in an 8-fold increase in free D-pinitol concentration (FIGS. 15A and D) and a more than 4-fold increase in both galactopinitol A and galactopinitol B concentrations (FIGS. 15B and E). Concentrations of D-chiro-inositol, myo-inositol, fagopyritol B1, galactinol, raffinose, and stachyose were not increased (FIG. 15). Feeding 100 mM D-pinitol resulted in high concentrations of free D-pinitol and a substantial increase in galactopinitol A and galactopinitol B between day 2 and day 4 of slow drying (FIGS. 16A, B, D, and E). A transient increase in galactinol occurred as raffinose and stachyose accumulated (FIGS. 16B, C, E, and F). The larger increase in stachyose in cotyledons, compared to feeding D-chiro-inositol (FIG. 14F), suggests that galactopinitol A may be effective as a galactosyl donor for stachyose biosynthesis as suggested by Hoch et al., Arch. Biochem. Biophys. 366:75-81 (1999) and Peterbauer et al., Seed Sci. Res. 11:185-198 (2001), which are hereby incorporated by reference in their entirety. The large increase in galactopinitols from externally applied D-pinitol suggests that D-pinitol is not biosynthesized within the embryo but is transported to the embryo from maternal tissues. Sucrose concentration increased through day 3 of slow drying (FIGS. 16C and F).
Feeding sucrose at 0 to 200 mM resulted in a small decrease in galactinol (FIG. 17B) and an increase in sucrose in axis tissues (FIG. 17C) but little change in concentrations of soluble carbohydrates in cotyledon tissues (FIG. 17). These results suggest that osmotic concentrations, per se, have little effect on soluble carbohydrate concentrations under the experimental conditions used in these experiments.
Feeding a combination of 100 mM D-pinitol and 100 mM D-chiro-inositol resulted in high concentrations of both free D-pinitol and free D-chiro-inositol; free D-chiro-inositol declined with elevated concentrations of fagopyritol B1, D-pinitol decreased less but with increases in galactopinitol A, galactopinitol B, stachyose, and raffinose between day 2 and day 3 in embryo cotyledon tissues (FIG. 18). Galactinol concentration peaked by day 1 (axis) or day 2 (cotyledons) and declined as raffinose, stachyose, and galactopinitols accumulated.
Accumulation of fagopyritol B1 appeared to be independent of accumulation of galactopinitols, raffinose, and stachyose, indicating fagopyritol B1 biosynthesis is independent of galactopinitol biosynthesis. Feeding a combination of D-pinitol and D-chiro-inositol (FIG. 18) resulted in a 50% decrease (14 days in steady state galactinol concentration and a 50% decrease (14 days) in galactopinitol A plus galactopinitol B concentration in cotyledons (FIG. 18E), compared to feeding D-pinitol alone (FIG. 16E), but only a 10 to 15% decrease (4 days) in fagopyritol B1 concentration (FIG. 18E) compared to feeding D-chiro-inositol alone (FIG. 14E) (14 versus 16 mg/g DW at day 4 of slow drying). In axis tissues, galactinol, galactopinitol A, galactopinitol B, raffinose, and stachyose were not decreased by feeding a combination of D-pinitol and D-chiro-inositol (FIGS. 18C and D) compared to feeding D-pinitol alone (FIGS. 16C and D). Fagopyritol B1 in axis tissues was reduced about 50% (14 days) after feeding a combination of D-pinitol and D-chiro-inositol (FIG. 18B) compared to feeding D-chiro-inositol alone (FIG. 14B). In all cases, fagopyritol B1 was maximum in axis tissues on day 3 of slow drying while in cotyledons fagopyritol B1 continued to increase during day 4 of slow drying. The small mass of the axis tissues (approximately 1 mg dry weight) may have hastened the cessation of galactosyl cyclitol accumulation in axis tissues compared to cotyledons during precocious maturation. In addition, axis tissues yellowed 1 to 2 days sooner during precocious maturation after feeding D-pinitol or combinations of D-pinitol and D-chiro-inositol than after feeding D-chiro-inositol alone. Feeding a combination of 50 mM D-pinitol plus 50 mM D-chiro-inositol resulted in patterns identical to those with 100 mM (FIG. 18), indicating the cyclitol substrates were at saturating concentrations. Feeding a combination of 100 mM D-pinitol, 100 mM D-chiro-inositol, and 100 mM sucrose resulted in patterns identical to those without sucrose (FIG. 18), except that sucrose concentrations were higher initially.
Cyclitols detected in soybean embryos include myo-inositol, D-pinitol, and D-chiro-inositol (Horbowicz et al., Seed Sci. Res. 4:385-405 (1994); Obendorfet al., Plant Sci. 132:1-12 (1998); Obendorfet al., Crop Sci. 38:78-84 (1998), which are hereby incorporated by reference in their entirety). If present, other cyclitols were below the level of detection. myo-Inositol is biosynthesized in soybean embryos, and inhibition of myo-inositol biosynthesis results in reduced accumulation of phytic acid, galactinol, raffinose, and stachyose (Hegeman et al., Plant Physiol. 125:1941-1948 (2001); Hitz et al., Plant Physiol. 128:650-660 (2002), which are hereby incorporated by reference in their entirety). Total D-chiro-inositol or total D-pinitol did not increase in the absence of exogenous feeding of the corresponding cyclitols, consistent with our previous results with soybean zygotic embryos matured in vitro (Obendorf et al., Plant Sci. 132:1-12 (1998); Obendorf et al., Crop Sci. 38:78-84 (1998), which are hereby incorporated by reference in their entirety) and indicating a lack of biosynthesis of both D-chiro-inositol and D-pinitol during precocious maturation of soybean zygotic embryos.
Axis tissues accumulate higher concentrations of soluble carbohydrate products than cotyledons, suggesting that biosynthetic enzymes may be more active in axis tissues. Yellowing of axis and cotyledon tissues is a visual indicator of the cessation of growth and tissue maturation; axis tissues mature earlier than cotyledon tissues in planta (Obendorf et al., Crop Sci. 38:78-84 (1998), which is hereby incorporated by reference in its entirety). This difference in maturation must be considered when assaying gene expression in whole embryos or seeds in contrast to assaying axis and cotyledon tissues separately. Feeding D-pinitol or combinations of D-pinitol and D-chiro-inositol resulted in yellowing of axis tissues 1 to 2 days earlier during precocious maturation than feeding D-chiro-inositol alone. Because of their small size and more rapid maturation rate after feeding D-pinitol, precociously matured axis tissues may not reflect product accumulation patterns as accurately as cotyledons. Therefore, more emphasis should be placed on the product accumulation patterns in precociously matured cotyledons.
Feeding both D-pinitol and D-chiro-inositol reduced galactinol concentration in cotyledons by 50% compared to feeding D-pinitol alone, indicating a competition between the biosynthesis of fagopyritol B1 and galactinol by GolS. The 50% reduction in the biosynthesis of galactopinitols reflects the 50% reduction in galactinol, the galactosyl donor for galactopinitol biosynthesis by stachyose synthase (Peterbauer et al., Seed Sci. Res. 11:185-198 (2001), which is hereby incorporated by reference in its entirety). The small decrease in fagopyritol B1 biosynthesis after feeding both D-pinitol and D-chiro-inositol compared to feeding D-chiro-inositol alone, probably reflects competition for available UDP-Gal between galactinol and fagopyritol B1 biosynthesis. Results of substrate feeding experiments are consistent with the interpretation that D-pinitol and D-chiro-inositol are transported from maternal tissues and not biosynthesized in the embryo tissues. In addition, galactopinitols and fagopyritol B1 are biosynthesized by different pathways, fagopyritols are biosynthesized by GolS, galactopinitols are biosynthesized by stachyose synthase/raffinose synthase, and galactopinitols may serve as galactosyl donors for stachyose biosynthesis.
Example 3
Soybean EST Clone Corresponding to Galactinol Synthase (GolS) Gene
Gene or cDNA sequences corresponding to the GolS gene in soybean were searched in the nucleotide and protein databases using the BLAST programs (http://www.ncbi.nlm.nih.gov) and a multiple sequence alignment program, CLUSTAL W (http://workbench.sdsc.edu). A soybean EST clone (GenBank accession number BE330777) sharing a very high level of DNA sequence identity with the GolS genes reported from other plant species was identified, and obtained from INCYTE GENOMICS, Palo Alto, Calif. (cat. no. Gm-c1041). Since only partial DNA sequence data were available for this EST clone in GenBank, the whole EST insert was re-sequenced (nucleotide sequence assigned to GenBank Accession Number AY126715) at the DNA Sequencing Facility at BioResource Center at Cornell University (http://brcweb.biotech.cornell.edu).
The 987 bp long entire protein coding sequence of GmGolS was amplified from the soybean EST clone by PCR. Two primers, 5′-CATCACTGAGCATATGGCTGG-3′ (SEQ ID NO:29) and 5′-GGATCCAAAGACACTCTTAAGCAGCAGATGGGG-3′ (SEQ ID NO:30), containing NdeI and BamHI restriction enzyme recognition sites respectively, were used in the PCR assays. After cloning into the pCRII-TOPO vector (Invitrogen, Carlsbad, Calif.) and amplification in Escherichia coli, the NdeI/BamHI fragment containing the entire protein coding sequence was isolated and cloned into the corresponding sites in pET-14b vector (Novagen, Madison, Wis.). This insertion resulted in the placement of the GmGolS protein coding sequence in frame with the preceding poly-histidine codons in the pET-14b vector. The pET14b plasmid containing soybean GmGolS cDNA was mobilized into E. coli strain BL21 (DE3) (Novagen, Madison, Wis.). Expression of the recombinant GmGolS protein was induced in E. coli with 1 mM isopropylthio-β-D-galactoside (IPTG) according to the manufacturer's recommended protocol (Novagen, Madison, Wis.). The bacterial cells were collected by centrifugation, and resuspended in 10 mM Tris-HCl buffer (pH 8.0). The soluble protein fraction was extracted from the bacterial cells by the gentle disruption of their cell walls with BugBuster Protein Extraction Reagent (Novagen, Madison, Wis.) containing Benzonase (Novagen, Madison, Wis.). Poly-histidine tagged recombinant proteins were purified from the extracts using His.Bind Quick 900 Cartridges (Novagen, Madison, Wis.) according to the manufacturer's recommended protocol. Purification of proteins was verified by SDS-PAGE. Purified recombinant proteins were dialyzed against 50 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]-NaOH buffer, pH 7.0, containing 5 mM MnCl2, immediately after elution from the His.Bind Quick 900 Cartridges and prior to enzyme assay.
Both crude soluble protein extracts from E. coli containing the recombinant GmGolS protein and purified recombinant GmGolS protein were used in enzyme assays. GolS activity assays included 20 mM UDP-Gal as the galactosyl donor, 20 mM myo-inositol as the galactosyl acceptor, 50 mM HEPES buffer, pH 7.0, 2 mM DTT, 5 mM MnCl2 and 1 to 5 μg of crude protein extract or purified GmGolS protein in 50 μL total volume. In fagopyritol synthase assays, myo-inositol was substituted with 20 mM D-chiro-inositol as the galactosyl acceptor. Assays were run at 30° C. for 30 to 300 minutes. Reactions were stopped by addition of 50 μL of 100% ethanol. After addition of 25 μg of phenyl α-D-glucoside as internal standard, the reaction mixture was heated at 80° C. for 30 minutes, passed through a 10,000 MW cutoff filter (NANOSEP), and evaporated to dryness under a stream of nitrogen gas. Residues were stored overnight in a desiccator with phosphorus pentoxide to remove traces of water, derivatized with trimethylsilylimidazole:pyridine (1:1, v/v) at 80° C. for 45 minutes, and analyzed for fagopyritols or other soluble carbohydrate products by high resolution gas chromatography on a HP1-MS (Agilent Technologies) capillary column as previously described (Horbowicz et al., Seed Sci. Res. 4:385-405 (1994), which is hereby incorporated by reference in its entirety).
To confirm that GolS catalyzes the biosynthesis of fagopyritol B1, a soybean galactinol synthase (GmGolS) gene was cloned (GenBank accession number AY126715) and heterologously expressed in Escherichia coli. The purified recombinant protein was assayed for fagopyritol synthase activity. Recombinant GmGolS catalyzed the biosynthesis of galactinol with UDP-Gal as the galactosyl donor and myo-inositol as the galactosyl acceptor (FIG. 19A), but also catalyzed the biosynthesis of fagopyritol B1 with UDP-Gal as the galactosyl donor and D-chiro-inositol as the galactosyl receptor (FIG. 19B). GmGolS was not active with galactinol as the galactosyl donor. Using UDP-Gal as the galactosyl donor, GmGolS was not active with O-methylated cyclitols including D-pinitol (1D-3-O-methyl-chiro-inositol), D-ononitol (1D-4-O-methyl-myo-inositol), sequoyitol (5-O-methyl-myo-inositol), or L-quebrachitol (1L-2-O-methyl-chiro-inositol) as galactosyl acceptors, except for reduced activity with D-bornesitol (1D-1-O-methyl-myo-inositol). GmGolS was active with L-chiro-inositol as the galactosyl acceptor, but had reduced activity with scyllo-inositol and no activity with epi-inositol using UDP-Gal as galactosyl donor.
The recombinant soybean galactinol synthase (GmGolS) is a multi-functional enzyme with both GolS activity and fagopyritol synthase activity, but GmGolS does not exhibit galactopinitol synthase activity. GolS activity in developing and maturing soybean seeds is associated with stachyose accumulation and remained high through seed maturity (Handley et al., J. Amer. Soc. Hort. Sci. 108:600-605 (1983); Saravitz et al., Plant Physiol. 83:185-189 (1987); Lowell et al., Crop Sci. 29:459-465 (1989); Kuo et al., Plant Sci. 125:1-11 (1997), which are hereby incorporated by reference in their entirety). During soybean seed development in planta, GolS mRNA was first detected in axis tissues at 44 days post anthesis (DPA) and in cotyledons at 46 to 48 DPA (Volk, Ph.D. Dissertation, Cornell University, Ithaca, N.Y., pp 176-187 (1998), which is hereby incorporated by reference in its entirety), coincident with galactinol accumulation and at the onset of stachyose accumulation (Obendorf et al., Crop Sci. 38:78-84 (1998), which is hereby incorporated by reference in its entirety). GolS transcripts remained high during seed desiccation (Volk, Ph.D. Dissertation, Cornell University, Ithaca, N.Y., pp 176-187 (1998), which is hereby incorporated by reference in its entirety). GolS enzyme activity and mRNA increase in response to cold or desiccation (Castillo et al., J. Agric. Food Chem. 38:351-355 (1990); Liu et al., Plant Sci. 134:11-20 (1998), which are hereby incorporated by reference in their entirety). Among seven Arabidopsis thaliana GolS genes, three were stress responsive (Taji et al., Plant J. 29:417-426 (2002), which is hereby incorporated by reference in its entirety). AtGolS1 and AtGolS2 were induced by water-deficit stress and high-salinity stress but not by cold stress. AtGolS3 was induced by cold stress but not by drought or salt stress. Soybean seeds matured at 25° C. had increased D-chiro-inositol and fagopyritol B1 compared to seeds matured at 18° C., but galactinol remained unchanged (Obendorf et al., Crop Sci. 38:78-84 (1998), which is hereby incorporated by reference in its entirety), indicating a lack of response to a lower temperature. Similarly, tomato (Lycopersicon esculentum Mill.) seed GolS (LeGolS-1) mRNA increased in maturing seeds before desiccation, was concentrated in the radicle tip of mature dry seeds, was induced by desiccation but not cold in germinating seeds, and was induced by both desiccation and cold in seedling leaves (Downie et al., Plant Physiol. 131:1347-1359 (2003), which is hereby incorporated by reference in its entirety).
Substrate specificities of soybean GolS and stachyose synthase are different. The lack of soybean GolS activity with D-pinitol, D-ononitol, and sequoyitol as galactosyl acceptors contrasts with the activity of stachyose synthase with these O-methylated cyclitols (Peterbauer et al., Plant Physiol. 117:165-172 (1998); Hoch et al., Arch. Biochem. Biophys. 366:75-81 (1999); Peterbauer et al., J. Biol. Chem. 277:194-200 (2002), which are hereby incorporated by reference in their entirety). Likewise, activity of GmGolS with D-bornesitol contrasts with the lack of activity of stachyose synthase with D-bornesitol or L-bornesitol (Peterbauer et al., Plant Physiol. 117:165-172 (1998); Hoch et al., Arch. Biochem. Biophys. 366:75-81 (1999), which are hereby incorporated by reference in their entirety). Lentil (Lens culinaris Medic.) stachyose synthase has been demonstrated to catalyze the biosynthesis of galactopinitols (Hoch et al., Arch. Biochem. Biophys. 366:75-81 (1999), which is hereby incorporated by reference in its entirety); this enzyme had low activity with D-chiro-inositol and no activity with L-chiro-inositol. By contrast, adzuki bean (Vigna angularis Ohwi and Ohashi) stachyose synthase had only a trace of activity with D-pinitol and no activity with D-chiro-inositol or L-chiro-inositol (Peterbauer et al., Plant Physiol. 117:165-172 (1998), which is hereby incorporated by reference in its entirety). A recombinant raffinose synthase from pea (Pisum sativum L.) seeds was active with D-ononitol and D-pinitol to form galactosyl ononitol and galactosyl pinitol using galactinol as the galactosyl donor (Peterbauer et al., Planta 215:839-846 (2002), which is hereby incorporated by reference in its entirety). This Pisum sativum raffinose synthase also exhibited a neutral α-galactosidase activity (Peterbauer et al., Planta 215:839-846 (2002), which is hereby incorporated by reference in its entirety), consistent with its amino acid sequence similarity to a family of alkaline α-galactosidases (Seed Imbibition Proteins, SIPs) (Carmi et al., Plant J. 33:97-106 (2003), which is hereby incorporated by reference in its entirety). A multi-functional pea seed stachyose synuthase had low activities for biosynthesis of galactopinitol and verbascose (Peterbauer et al., J. Biol. Chem. 277:194-200 (2002), which is hereby incorporated by reference in its entirety). Collectively, these observations demonstrate substrate specificity of these multi-functional enzymes to be species-specific and product accumulation to be dependent upon the availability of specific cyclitol substrates to the embryo tissues. Clearly, GmGolS can catalyze the biosynthesis of fagopyritol B1, but not galactopinitols, in maturing soybean embryos.
Example 4
Biosynthesis of Fagopyritol B1 and Galactopinitols in Soybean Explants Following Feeding With Free Cyclitols
Soybean is a leguminous plant that bears monocarpic fruit only once before death. During maturation, tissues become yellow starting with radical tips, leaf blades, pod walls, hypocotyls, and cotyledons (Benner et al., Biochemie und Physiologie der Pflanzen 179:269-275 (1984), which is incorporated herein by reference in its entirety). Yellowing of the seed coat and embryo indicate cessation of dry matter accumulation in the seed (TeKrony et al., Agronomy Journal 73:553-556 (1981); VerNooy et al., Plant Physiology 82:222-225 (1986), which are hereby incorporated by reference in their entirety). Leaf yellowing, however, is not always a good indicator of when a given soybean seed has stopped growing (Neumann et al., Plant Physiology 72:182-185 (1983), which is incorporated herein by reference in its entirety). Because there is transport from the leaves to the pod, seed weight may continue to increase as long as leaves are still alive. Consequently, pod yellowing is the indicator that is often used to determine the time at which maximum dry weight is reached (Benner et al., Biochemie und Physiologie der Pflanzen 179:269-275 (1984), which is incorporated herein by reference in its entirety). The onset of this yellowing/desiccation is what brings about galactosyl cyclitol accumulation in axis and cotyledon tissue (Obendorf et al., Plant Science 132:1-12 (1998); Obendorf et al., Crop Science 38:78-84 (1998), which are incorporated herein by reference in their entirety).
Soybean seeds accumulate galactosyl cyclitols as opposed to free cyclitols (Horbowicz et al., Seed Science Research 4:385-405 (1994), which is incorporated herein by reference in its entirety). These include galactosyl derivatives of D-pinitol, D-chiro-inositol, and myo-inositol in soybean seeds (Obendorf et al., Crop Science 38:78-84 (1998), which is incorporated herein by reference in its entirety). Among the fifteen soluble carbohydrates or maturation sugars are sucrose, raffinose and stachyose (raffinose oligosaccharides series), galactopinitol A and galactopinitol B (galactopinitol series), and fagopyritol B1 (fagopyritol series) (Schweizer et al., Carb. Res. 95:61-71 (1981); Obendorf et al., Plant Science 132:1-12 (1998); Obendorf et al., Crop Science 38:78-84 (1998), which are incorporated herein by reference in their entirety). Soluble carbohydrates of this type may have multiple functions in the desiccation tolerance of maturing seeds. They are harmless forms of seed storage products and intracellular osmotic agents contributing to the structural stability of organelles, membranes, enzymes, proteins, and other macromolecules (Obendorf, Seed Science Research 7:63-74 (1997), which is hereby incorporated by reference in its entirety).
Upon being fed to soybean, free cyclitols undergo biosynthetic reactions to form galactosyl cyclitols. Several important reactions of myo-inositol, D-chiro-inositol, and D-pinitol will be discussed hereafter. Firstly, myo-inositol is encountered in all living cells and is the primary source for the biosynthesis of various cyclitols. Feeding myo-inositol to soybean promotes the production of galactinol. The three components of the galactinol series are myo-inositol, galactinol, and digalactosyl myo-inositol. Galactinol is far-reaching in its ability to donate galactose for the formation of stachyose, raffinose, and verbascose (Peterbauer et al., Seed Science Research 11:185-198 (2001); Taji et al., Plant Journal 29:417-426 (2002), which are incorporated herein by reference in their entirety). If galactose is donated to another galactinol molecule, digalactosyl myo-inositol is formed. Secondly, feeding D-pinitol enhances accumulation of galactopinitol A and galactopinitol B common in legume seeds (Odorcic et al., The Biology of Seeds: Recent Research Advances. Wallingford, UK, CABI Publishing (2003), which is incorporated herein by reference in its entirety). As stachyose accumulates during soybean seed maturation, galactopinitols also increase (Obendorf et al., Crop Science 38:78-84 (1998), which is incorporated herein by reference in its entirety). In addition to this, galactopinitols accumulate during precocious maturation of immature seeds. Lastly, feeding of D-chiro-inositol results in enhanced accumulation of fagopyritol B1 (Odorcic et al., The Biology of Seeds: Recent Research Advances. Wallingford, UK, CABI Publishing (2003), which is incorporated herein by reference in its entirety). The fagopyritol B series enhanced through feeding consists of fagopyritol B1 (first identified in soybean seeds), D-chiro-inositol, fagopyritol B2, and fagopyritol B3, which accumulate in buckwheat seeds (Obendorf, Seed Science Research 7:63-74 (1997); Horbowicz et al., Planta 205:1-11 (1998), which are incorporated herein by reference in their entirety). A novel series of fagopyritols, fagopyritol A1, fagopyritol A2, and fagopyritol A3, also accumulate in buckwheat seeds (Horbowicz et al., Planta 205:1-11 (1998); Obendorf et al., Carbohydrate Research 328:623-627 (2000); Steadman et al., Carbohydrate Research 331:19-25 (2001), which are incorporated herein by reference in their entirety).
Knowledge of the translocation patterns of cyclitols is indispensable in understanding their function (Noodén et al., Journal of Plant Growth Regulation 2:265-279 (1984), which is incorporated herein by reference in its entirety). Previous studies used labeled chemicals, hormones, or sugars in order to observe these very translocation patterns within plants of interest. In an experiment done by Noodén and Letham in 1983, for example, 3H (ring-labeled) zeatin riboside was used to trace the production of the hormone cytokinin. The hormone was fed to soybean explants and transported via the transpiration stream. This biological marker allowed for the clear observation of transport from the xylem to the leaf and embryo of the explant. This experiment also resulted in leaves retaining their green color longer, which is important in experimentation with soybean explants (Noodén et al., Journal of Plant Growth Regulation 2:265-279 (1984), which is incorporated herein by reference in its entirety). A previous study by Quebedeaux and Chollet (Quebedeaux et al., Plant Physiology 55:745-748 (1975), which is hereby incorporated by reference in its entirety) used radioactive tracers to demonstrate that the pods (and seeds contained therein) of soybeans are the main sinks for the photosynthetic assimilates from the leaf, indicating that the decrease in the production of photosynthate is therefore due to the decrease in photosynthetic activity of the plant, which accompanies senescence (Benner et al., Biochemie und Physiologie der Pflanzen 179:269-275 (1984), which is incorporated herein by reference in its entirety). In addition to these methods, translocation patterns can also be observed through analysis of products formed following exogenous feeding of large quantities of the compound(s) of interest.
It is known that myo-inositol is biosynthesized in soybean embryos. Johnson and Wang (Johnson et al., J. Biol. Chem. 271:17215-17218 (1996), which is hereby incorporated by reference in its entirety) demonstrated that 1L-myo-inositol 1-phosphate synthase (also known as 1D-myo-inositol 3-phosphate synthase, MIPS) catalyzes the transformation of Glc-6-P to 1L-myo-inositol 1-phosphate in embryos of developing legume seeds. However, it remains unknown whether D-pinitol or D-chiro-inositol are biosynthesized in the embryo. In order to understand the function of cyclitols, it is necessary to first understand how they are transported and from where they are transported. Therefore, one objective of this Example was to determine which cyclitols are biosynthesized in soybean embryos and which are transported to the embryo from the leaves.
Several studies provide evidence in support of the hypothesis that D-pinitol and D-chiro-inositol are biosynthesized in the leaves of soybean plants. Labeling studies done by Diettrich and Brandl (Diettrich et al., Phytochemistry 26:1925-1926 (1987), which is hereby incorporated by reference in its entirety), for example, showed that myo-inositol goes to D-ononitol (FIG. 20, reaction d) and afterwards to D-pinitol (FIG. 20, reaction e,f), and then presumably to D-chiro-inositol (FIG. 20, reaction g) in legume leaves. Kuo (Kuo et al., Phytochemistry 45:29-35 (1997), which is hereby incorporated by reference in its entirety) demonstrated that the concentration of D-pinitol was highest in seed coats and lower in axis and cotyledon tissues, suggesting that D-pinitol is biosynthesized in maternal tissue and transported to soybean embryos. In addition to this, soybean and alfalfa (Medicago sativa L.) somatic embryos also appear to be deficient in D-pinitol and galactopinitols (Horbowicz et al., Plant Science 109:191-198 (1995); Obendorf et al., Mol. Cell. Biol. Soybean 6:40 (1996); Chanprameet al., in Vitro Cell Developmental Biology—Plant 34:64-68 (1998), which are incorporated herein by reference in their entirety), and total D-pinitol or total D-chiro-inositol in soybean zygotic embryos matured in vitro did not exceed that present in embryos before culture (Obendorf et al., Plant Science 132:1-12 (1998); Obendorf et al., Crop Science 38:78-84 (1998), which are incorporated herein by reference in their entirety), indicating a lack of D-pinitol and D-chiro-inositol biosynthesis by embryo tissues. myo-inositol 6-O-methyltransferase (mI6OMT or IMT, S-adenosyl-L-methionine:myo-inositol O-methyltransferase, EC 2.1.1.129) that forms D-ononitol, is located in leaves and stems (Wanek et al., Physiologia Plantarum 101:416-424 (1997); Streeter et al., Plant, Cell and Environment 24:429-438 (2001), which are incorporated herein by reference in their entirety). Soybean somatic embryos transformed with a gene for this enzyme form D-ononitol but not D-pinitol indicating that soybean somatic embryos do not express the enzymes that form D-pinitol. Soybean leaves accumulate mostly D-pinitol with small amounts of D-chiro-inositol, myo-inositol and D-ononitol (Streeter, Crop Sci. 41:1985-1987 (2001), which is hereby incorporated by reference in its entirety). Using this background information in conjunction with the knowledge that D-pinitol is a proposed precursor to D-chiro-inositol, it was hypothesized that though myo-inositol is biosynthesized in soybean embryos, D-chiro-inositol and D-pinitol are biosynthesized in the leaves and afterwards transported to the seeds. If this hypothesis is correct, then increasing the concentration of D-pinitol and D-chiro-inositol in soybean explants via exogenous feeding should result in a dramatic increase in the accumulation of fagopyritol B1 and galactopinitols in the embryo. However, if D-pinitol and D-chiro-inositol are biosynthesized in the embryo, then exogenous feeding of free cyclitols should have a less pronounced effect on galactosyl cyclitol concentrations in the seed.
Materials and Methods
Soybean plants [Glycine max (L.) Merrill cv. Chippewa 64] were grown in a greenhouse at 27° C. days (14 hours) and 22° C. nights (10 hours) with natural light supplemented by 640-μmol m−2 s−1 artificial light from Sylvania 1000-watt metal halide lamps.
Plants were excised above the third node from the bottom and below the third node from the top before leaf senescence was evident as was done by Neumann et al. Plant Physiology 72:182-185 (1983), which is hereby incorporated by reference in its entirety. Explants were cut mid podfill (about 35 days after flowering), when the pods were still green and approximately 7.2 mm in width, and the seeds weighed about 250 mg fresh weight. Pod number was reduced to one, containing three seeds. Each explant included one node, one leaf, one pod, and one internode. The cut basal end of the internode (stem) of the explants was placed in 50 mM solutions of cyclitols: 50 mM myo-inositol, 50 mM D-pinitol, 50 mM D-chiro-inositol, and a control without cyclitols, all in 1% sucrose by weight, and all containing 10 mM asparagine and kinetin, a cytokinin. These solutions were loaded into the explant through the cut stem and transported to the leaf by the transpiration stream and to the embryo through the phloem. A fourth solution consisting of 10 mM asparagine and kinetin in 1% sucrose (by weight) served as the control. Solutions were fed to explants for one week, and explants were allowed to dry, after which seeds were moved to the desiccators and fully dried (to 6% moisture) during a period of 14 days at 12% relative humidity over a saturated solution of LiCl.
After the seeds had slow dried, extraction and analysis of soluble carbohydrates was performed. Cotyledon and axis tissues were separated, weighed, pulverized in liquid nitrogen with a mortar and pestle, and homogenized in a ground glass homogenizer with 2.2 ml of ethanol:water (1:1, v/v), containing 300 μg (cots) or 100 μg (axis) of phenyl α-D-glucoside as the internal standard, heated at 80° C. for 45 minutes, and centrifuged at 27,000×g for 20 minutes. Clear supernatants were passed through a 10,000 MW cutoff filter and evaporated to dryness with nitrogen gas. Residues were stored overnight in a desiccator with P2O5 to remove traces of water and afterwards derivatized with trimethylsilylimidazole:pyridine (1:1, v/v). Analysis of soluble carbohydrates was done using a Hewlett Packard 5890 Series II gas chromatograph equipped with a flame ionization detector and ChemStation software as previously described (Horbowicz et al., Seed Science Research 4:385-405 (1994); Obendorf et al., Crop Science 38:78-84 (1998), which are incorporated herein by reference in their entirety). The amounts of each soluble carbohydrate present in the samples was determined by regression equations calculated from gas chromatograms of known standards, allowing the relative amounts of cyclitols present in the leaf and embryo as a result of the feeding of excess cyclitols to be determined. Soluble carbohydrate composition is reported as mean±SE of the mean on a dry weight basis for six replicate samples of cotyledons from mature seeds.
Results
Overall, none of the feeding experiments resulted in large changes in sucrose, raffinose, or stachyose except for some low values observed in explants fed with D-chiro-inositol. Results for the experiments were consistent with the results and interpretations of feeding experiments where cyclitols were fed to immature soybean embryos (Odorcic et al., The Biology of Seeds: Recent Research Advances. Wallingford, UK, CABI Publishing (2003), which is incorporated herein by reference in its entirety).
myo-inositol
Feeding 50 mM myo-inositol to soybean explants slightly increased free myo-inositol and caused a 50% increase in galactinol in axis and cotyledon tissue (Tables 1 and 2).







TABLE 1







Concentration of soluble carbohydrates in cotyledons


of mature soybean seeds after feeding explants 50 mM


myo-inositol, D-chiro-inositol, or D-pinitol.













B





A
D-chiro-
C
D



myo-Inositol
Inositol
D-Pinitol
Control





D-Pinitol
 7.77 ± 0.92
 6.00 ± 0.52
35.77 ± 2.50
 8.34 ± 1.12


Galactopinitol
 2.01 ± 0.16
 1.89 ± 0.14
 5.81 ± 0.38
 1.59 ± 0.15


A


Galactopinitol
 1.76 ± 0.21
 1.62 ± 0.18
 4.88 ± 3.10
 1.60 ± 0.22


B


Ciceritol
 0.63 ± 0.08
 0.34 ± 0.11
 1.13 ± 0.16
 0.85 ± 0.07


D-chiro-
 5.15 ± 0.77
15.59 ± 2.08
 1.63 ± 0.11
 1.63 ± 0.26


Inositol


Fagopyritol
 1.78 ± 0.23
21.11 ± 2.06
 1.77 ± 0.11
 1.05 ± 0.08


B1


Fagopyritol
 0.25 ± 0.07
 1.52 ± 0.47
 0.16 ± 0.02
 0.15 ± 0.04


B2


Myo-Inositol
 2.35 ± 0.79
 0.58 ± 0.05
 0.67 ± 0.07
 1.69 ± 0.44


Galactinol
 0.35 ± 0.06
 0.23 ± 0.05
 0.05 ± 0.01
 0.25 ± 0.04


Sucrose
37.73 ± 4.81
27.88 ± 4.09
32.49 ± 2.05
48.76 ± 7.62


Raffinose
11.22 ± 1.12
 7.18 ± 0.58
 9.73 ± 0.46
11.00 ± 1.47


Stachyose
23.10 ± 1.94
12.63 ± 1.40
14.60 ± 1.06
24.51 ± 3.73
















TABLE 2







Concentration of soluble carbohydrates in axis of


mature soybean seeds after feeding explants 50 mM


myo-inositol, D-chiro-inositol, or D-pinitol.













B





A
D-chiro-
C
D



myo-Inositol
Inositol
D-Pinitol
Control





D-Pinitol
 3.65 ± 0.03
 4.07 ± 0.38
21.40 ± 2.41
 4.51 ± 0.58


Galactopinitol
 4.61 ± 0.41
 4.96 ± 0.30
12.14 ± 1.06
 3.65 ± 0.35


A


Galactopinitol
 3.17 ± 0.40
 3.61 ± 0.30
 9.42 ± 0.88
 2.84 ± 0.42


B


Ciceritol
 0.65 ± 0.12
 0.37 ± 0.15
 1.58 ± 0.26
 0.79 ± 0.23


D-chiro-
 1.08 ± 0.14
12.31 ± 1.44
 1.28 ± 0.30
 0.61 ± 0.12


Inositol


Fagopyritol
 2.81 ± 0.28
30.95 ± 2.46
 2.99 ± 0.22
 1.79 ± 0.22


B1


Fagopyritol
 0.24 ± 0.09
 1.62 ± 0.50
 0.08 ± 0.02
 0.14 ± 0.06


B2


Myo-Inositol
 1.52 ± 0.15
 1.37 ± 0.13
 0.98 ± 0.13
 1.19 ± 0.20


Galactinol
 0.89 ± 0.10
 1.00 ± 0.07
 0.58 ± 0.08
 0.69 ± 0.08


Sucrose
34.02 ± 3.52
32.18 ± 4.31
30.86 ± 4.11
35.59 ± 5.63


Raffinose
 8.87 ± 0.77
 6.61 ± 0.81
 9.81 ± 1.08
1.030 ± 0.76


Stachyose
24.56 ± 2.55
17.95 ± 1.79
23.16 ± 2.50
22.02 ± 2.32









No significant changes in the amount of stachyose, raffinose, D-pinitol, or galactopinitols were observed. A 3.15-fold increase in free D-chiro-inositol was also observed in cotyledons and D-chiro-inositol concentrations were doubled in axis tissue. Still, there was no significant increase in concentrations of fagopyritol B1.

D-chiro-inositol

Feeding 50 mM D-chiro-inositol to soybean explants caused a 9.6-fold increase in free D-chiro-inositol, a 20-fold increase in fagopyritol B1, and a 10-fold increase in fagopyritol B2 in cotyledon tissues (Table 1). Free myo-inositol decreased but galactinol in cotyledons remained unchanged. Feeding D-chiro-inositol to soybean explants also resulted in a 20-fold increase in free D-chiro-inositol in axis tissues (Table 2). This corresponded with a 17-fold increase in fagopyritol B1 and an 11-fold increase in fagopyritol B2. All of the D-chiro-inositol fed explants had shriveled seeds, while those explants that were fed myo-inositol, D-pinitol, or the control treatment, had full and round seeds.
D-pinitol
Feeding D-pinitol quadrupled free D-pinitol and tripled galactopinitols in both axis and cotyledon tissues (Tables 1 and 2). Ciceritol concentrations increased 30% in cotyledon tissue and they doubled in axis tissue. myo-Inositol and galactinol were decreased 25%, and free D-chiro-inositol concentrations in axes doubled.
Discussion
Relative amounts of soluble carbohydrates observed can be attributed to biochemical pathways in soybean and the roles that D-pinitol, D-chiro-inositol, and myo-inositol play in these pathways.
In soybean explants, galactinol synthase (GolS or GAS) produces galactinol from myo-inositol and UDP-galactose (FIG. 21). Galactinol then undergoes two reactions. In the first reaction, galactinol acts as a galactosyl donor to sucrose, which reacts with raffinose synthase (RFS) to produce raffinose and myo-inositol as a by-product. Raffinose and galactinol then reacts with stachyose synthase (STS) to produce stachyose and myo-inositol as a by-product. In the second reaction, galactinol and D-pinitol react with STS to produce galactopinitol A and galactopinitol B. Subsequent reactions with STS produce ciceritol (a digalactosyl pinitol A) from galactinol and galactopinitol A, and digalactosyl pinitol B from galactinol and galactopinitol B (FIG. 21).
When feeding 50 mM myo-inositol, there were high levels of galactinol, the galactosyl donor for galactopinitol biosynthesis, present. The lack of increase in accumulation of galactopinitols may have been due to limited levels of D-pinitol in the explant. Biosynthesis of D-chiro-inositol in legumes is believed to be via myo-inositol to D-ononitol to D-pinitol to D-chiro-inositol (FIG. 20, reactions d,e,f,g; Dittrich et al., Phytochemistry 26:1925-1926 (1987) which is hereby incorporated by reference in its entirety). If the D-pinitol levels were 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 the cotyledons suggest that myo-inositol, rather than D-pinitol, is a direct precursor to production of D-chiro-inositol in the leaves (FIG. 20, reactions i,j). In the absence of D-pinitol, myo-inositol goes to D-myo-1-inosose, and then to D-chiro-inositol (FIG. 20, reactions i,j). The high levels of myo-inositol present in the soybean explant following feeding with exogenous myo-inositol may limit the accumulation of raffinose and stachyose by feedback inhibition in the cotyledons of the seed. Since myo-inositol is produced as a byproduct, exogenous myo-inositol decreased the progress of the reaction of sucrose and galactinol by RFS, explaining why raffinose and stachyose levels stayed the same with this treatment.
Soybean galactinol synthase (GmGolS or GAS) produces fagopyritol B1 from D-chiro-inositol and UDP-galactose (FIG. 22). When feeding 50 mM D-chiro-inositol to soybean explants, a decrease in myo-inositol and D-pinitol was observed. Because myo-inositol and D-pinitol are precursors to D-chiro-inositol (FIG. 20, reactions d,e,f,g; Dittrich et al., Phytochemistry 26:1925-1926 (1987), which is hereby incorporated by reference in its entirety), they may not have been needed to produce D-chiro-inositol because it was fed to the explant in excess. The reason for decreased production of raffinose and stachyose is unknown. Decreases in galactosyl and digalactosyl pinitols are due to decreases in their precursor, D-pinitol. Increased levels of D-chiro-inositol caused fagopyritol B1 and fagopyritol B2 to increase as expected.
In this experiment, feeding D-pinitol increased levels of free D-pinitol in the seed. This increase served to increase the amount of galactosyl pinitols and, after reaction with STS, production of digalactosyl pinitol B. High levels of D-pinitol may have also temporarily increased D-chiro-inositol levels (FIG. 20, reaction g), which subsequently went towards increasing fagopyritol B1 production. The increased level of digalactosyl myo-inositol accounts for the decreased levels of galactinol and myo-inositol.
myo-Inositol is biosynthesized in embryo tissues of developing legume seeds (Johnson et al., Journal of Biological Chemistry 271, 17215-17218 (1996); Hegeman et al., Plant Physiology 125:1941-1948 (2001); Hitz et al., Plant Physiology 128:650-660 (2002), which are incorporated by reference in their entirety). D-Pinitol is biosynthesized in leaves from myo-inositol through D-ononitol as precursor (FIG. 20, reactions d,e,f; Dittrich et al., Phytochemistry, 26:1925-1926 (1987), which is hereby incorporated by reference in its entirety) and D-chiro-inositol is believed to be biosynthesized by demethylation of D-pinitol (FIG. 20, reaction g; see review by Obendorf, Seed Sci. Res. 7:63-74 (1997), which is hereby incorporated by reference in its entirety). It is not known if D-pinitol and D-chiro-inositol are biosynthesized in cotyledons of seeds. Further, the enzymes and genes responsible for the biosynthesis of D-pinitol (FIG. 20, reactions e,f) and D-chiro-inositol (FIG. 20, reaction g or FIG. 20, reactions i,j) are unknown (Obendorf, Seed Science Research 7:63-74 (1997), which is hereby incorporated by reference in its entirety). The results herein are consistent with the interpretation that both of D-pinitol and D-chiro-inositol are biosynthesized in leaves and transported to seeds. Of special interest is the evidence presented herein that D-chiro-inositol may be biosynthesized directly from myo-inositol, either instead of or in addition to demethylation of D-pinitol.
The results in this Example are consistent with the following interpretations: myo-inositol is formed in maternal tissues and in embryos of seeds, D-pinitol and D-chiro-inositol are biosynthesized in maternal tissues (leaves) and transported to seeds, D-chiro-inositol may be biosynthesized directly from myo-inositol, galactinol synthase utilizes D-chiro-inositol to form fagopyritol B1, stachyose synthase utilizes D-pinitol to form galactopinitols, and feeding free cyclitols to soybean explants does not increase raffinose and stachyose accumulation in cotyledons of soybean seeds.
Example 5
Soybean Explant Feeding Experiments
Soybean explants, consisting of a stem segment with attached leaf and pod, were cultured as the soybean explants described in Example 4. In this example, the soybean explant system was used to study the timing of transport of cyclitols, fed through the stem, to the developing soybean seed and the timing of their incorporation into galactosyl cyclitols in axis, cotyledons, and seed coat of developing and maturing soybean seeds. myo-inositol, D-pinitol and D-chiro-inositol were fed to soybean explants as described in Example 4, except that 50 mM cyclitol in 1% sucrose solution was fed to stems of soybean explants for three days followed by slow drying. Soluble carbohydrates were extracted and assayed by high resolution gas chromatography after slow drying of seeds (as described in Example 4).







TABLE 3







Accumulation of soluble carbohydrates in soybean axis (μg/axis) after 3


days transport of sucrose (1% solution) and myo-inositol (50 mM) into


the stem of soybean explants and after slow drying of seeds for


2, 4, or 14 days (micrograms/1 axis)












Before
After 2 days
After 4 days
After 14 days



slow drying
slow drying
slow drying
slow drying















Soluble Carbohydrate
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2


















D-Pinitol
5.89
5.97
5.05
5.72
2.74
2.86
6.70
5.72


Unknown
0.91
0.67
0.57
0.49
0.52
0.38
1.25
1.46


D-chiro-Inositol
1.98
2.17
3.65
4.69
3.40
1.75
9.39
5.12


myo-Inositol
37.08
37.50
3.34
1.96
1.74
1.69
4.22
3.84


Sucrose
143.88
139.88
38.38
76.58
50.48
82.93
77.01
63.96


Galactopinitol A
4.31
0
5.86
8.20
9.93
11.97
13.99
10.23


Galactopinitol B
0.72
0
1.29
2.20
3.06
4.60
5.50
3.91


Fagopyritol B1
0
0
3.15
5.91
6.25
7.43
11.14
6.13


Galactinol
3.01
3.59
12.80
10.83
7.31
4.07
6.76
4.14


Raffinose
Tr
0
5.44
1.83
6.37
7.57
14.51
11.98


Ciceritol
Tr
0
0
0
0.39
0.42
0.56
0.29


Fagopyritol B2
0
0
0
0
0
0
0
0


Stachyose
0
0
9.92
6.33
62.18
55.60
89.46
57.18
















TABLE 4







Accumulation of soluble carbohydrates in soybean axis (μg/axis) after 3


days transport of sucrose (1% solution) and D-chiro-inositol (50 mM)


into the stem of soybean explants and after slow drying of


seeds for 2, 4, or 14 days (micrograms/1 axis)












Before
After 2 days
After 4 days
After 14 days



slow drying
slow drying
slow drying
slow drying















Soluble Carbohydrate
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2


















D-Pinitol
6.94
6.11
6.18
7.87
6.21
3.86
7.68
4.13


Unknown
0.47
0
0.39
0.46
0.65
0.42
0
0.92


D-chiro-Inositol
25.26
22.53
20.29
25.33
11.74
7.67
39.99
26.32


myo-Inositol
18.22
16.89
1.93
2.75
1.27
1.03
0.92
1.01


Sucrose
111.55
149.75
22.44
28.32
35.75
23.35
59.50
30.63


Galactopinitol A
0
0
6.35
6.24
13.54
12.18
15.50
10.18


Galactopinitol B
0
0
1.47
1.56
6.35
4.73
9.35
5.03


Fagopyritol B1
2.04
0
32.08
25.93
81.94
73.53
78.96
55.76


Galactinol
0
2.71
6.29
10.19
3.46
2.25
4.23
2.27


Raffinose
0.23
0
2.57
3.37
2.61
3.24
6.68
5.73


Ciceritol
0
0
0
0
0
0.27
0
2.69


Fagopyritol B2
0
0
0
0
0
1.72
2.07
0


Stachyose
0
0
11.56
8.94
49.55
22.07
47.82
39.47
















TABLE 5







Accumulation of soluble carbohydrates in soybean axis (μg/axis) after 3


days transport of sucrose (1% solution) and D-pinitol (50 mM)


into the stem of soybean explants and after slow drying of seeds


for 2, 4, or 14 days (micrograms/1 axis)












Before
After 2 days
After 4 days
After 14 days



slow drying
slow drying
slow drying
slow drying















Soluble Carbohydrate
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2


















D-Pinitol
16.89
18.34
22.93
20.86
14.00
20.05
26.07
56.18


Unknown
1.30
1.47
0
0
0.69
0.45
0.95
1.53


D-chiro-Inositol
1.30
2.20
2.40
1.39
0.59
0.48
1.83
1.32


myo-Inositol
13.58
15.43
1.58
2.01
1.12
0
2.22
3.62


Sucrose
130.10
160.58
32.16
37.53
66.76
27.38
48.95
56.62


Galactopinitol A
0
0
7.62
9.41
25.04
19.32
24.72
32.49


Galactopinitol B
0
0
3.09
3.49
9.62
7.25
10.61
12.71


Fagopyritol B1
0
0
4.30
2.75
5.79
4.01
6.41
8.76


Galactinol
2.27
2.97
6.40
10.37
5.46
2.87
3.92
4.18


Raffinose
0
0
6.33
3.86
7.49
3.46
7.60
11.19


Ciceritol
0
0
0
0.80
0.89
0.56
1.42
1.43


Fagopyritol B2
0
0
0
0
0
0
0
1.26


Stachyose
0
0
11.91
7.69
79.54
28.64
54.54
78.56
















TABLE 6







Accumulation of soluble carbohydrates in soybean axis (μg/axis) after 3


days transport of sucrose (1% solution) without cyclitols into


the stem of soybean explants and after slow drying of seeds


for 2, 4, or 14 days (micrograms/1 axis)












Before
After 2 days
After 4 days
After 14 days



slow drying
slow drying
slow drying
slow drying















Soluble Carbohydrate
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2


















D-Pinitol
10.61
11.12
7.28
10.54
4.24
4.89
8.64
4.32


Unknown
0.32
0.98
0.85
0.48
0.69
0.41
1.58
1.68


D-chiro-Inositol
1.96
2.03
0.89
0.94
0.39
0.30
1.92
0.62


myo-Inositol
16.11
20.11
2.42
2.14
1.49
1.66
2.62
2.36


Sucrose
230.72
282.09
48.81
84.07
87.13
159.22
115.27
91.58


Galactopinitol A
0
0
7.55
9.97
17.42
16.07
16.14
12.28


Galactopinitol B
0
0
1.81
2.60
7.11
7.06
7.78
5.09


Fagopyritol B1
1.70
0
3.51
5.52
7.09
6.40
8.41
4.98


Galactinol
1.20
5.49
11.09
13.41
5.60
4.29
4.38
4.27


Raffinose
0.62
2.65
5.83
8.95
8.58
12.44
13.14
8.02


Ciceritol
0
0
0.01
0.07
0.80
0.71
0.94
0.63


Fagopyritol B2
0
0
5.67
0
0
0
0
0


Stachyose
0
7.83
0
27.73
96.03
118.05
101.39
69.71
















TABLE 7







Accumulation of soluble carbohydrates in soybean cotyledons (μg/cotyledon)


after 3 days transport of sucrose (1% solution) and myo-inositol (50 mM)


into the stem of soybean explants and after slow drying of seeds


for 2, 4, or 14 days (micrograms/1 cot)












Before
After 2 days
After 4 days
After 14 days



slow drying
slow drying
slow drying
slow drying















Soluble Carbohydrate
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2


















D-Pinitol
99.88
127.21
121.52
132.23
101.97
128.68
61.67
95.93


Unknown
4.53
8.94
53.99
63.64
86.25
85.24
62.60
111.06


D-chiro-Inositol
52.20
55.90
133.52
129.44
150.98
192.49
95.36
137.86


myo-Inositol
257.87
275.47
67.54
29.93
21.34
12.69
7.82
11.96


Sucrose
1429.70
1496.60
635.91
995.62
921.47
1952.20
717.30
1228.20


Galactopinitol A
9.46
10.00
12.84
10.98
28.27
54.91
34.57
28.69


Galactopinitol B
0
0
6.27
3.19
11.92
28.11
15.29
9.70


Fagopyritol B1
0
0
7.36
14.06
29.15
86.71
50.31
49.29


Galactinol
0
0
85.06
104.07
57.03
25.34
14.65
14.10


Raffinose
0
0
29.20
89.65
122.10
351.33
165.12
218.44


Ciceritol
0
0
0
0
0
0
2.01
0


Fagopyritol B2
0
0
0
0
0
0
1.29
5.27


Stachyose
0
0
0
50.41
224.95
774.04
399.25
323.00
















TABLE 8







Accumulation of soluble carbohydrates in soybean cotyledon (μg/cotyledon)


after 3 days transport of sucrose (1% solution) and D-chiro-inositol


(50 mM) into the stem of soybean explants and after slow drying


of seeds for 2, 4, or 14 days (micrograms/1 cot)












Before
After 2 days
After 4 days
After 14 days



slow drying
slow drying
slow drying
slow drying















Soluble Carbohydrate
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2


















D-Pinitol
175.37
149.51
140.00
177.82
131.32
171.09
91.50
90.76


Unknown
49.44
36.70
65.35
59.28
74.11
69.17
39.34
56.75


D-chiro-Inositol
518.30
476.04
635.01
719.52
388.25
589.84
335.21
286.00


myo-Inositol
138.61
142.47
21.83
55.39
4.25
10.39
5.77
3.80


Sucrose
1978.90
2102.30
360.00
363.10
395.07
579.04
580.31
736.23


Galactopinitol A
15.98
0
12.41
16.08
47.20
42.69
48.41
47.44


Galactopinitol B
0
0
0
0
22.34
20.06
25.58
23.82


Fagopyritol B1
10.55
0
45.25
25.67
711.00
535.23
612.60
626.65


Galactinol
14.29
0
42.99
42.35
15.89
32.53
11.02
9.34


Raffinose
7.28
10.73
21.08
15.23
122.16
124.39
198.46
211.32


Ciceritol
0
6.06
13.35
0
12.69
0
3.82
2.83


Fagopyritol B2
0
0
0
4.57
12.10
11.12
23.98
22.70


Stachyose
0
0
44.86
0
308.62
277.15
357.02
349.60
















TABLE 9







Accumulation of soluble carbohydrates in soybean cotyledon (μg/cotyledon)


after 3 days transport of sucrose (1% solution) and D-pinitol (50 mM)


into the stem of soybean explants and after slow drying of seeds


for 2, 4, or 14 days (micrograms/1 cot)












Before
After 2 days
After 4 days
After 14 days



slow drying
slow drying
slow drying
slow drying















Soluble Carbohydrate
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2


















D-Pinitol
351.23
497.66
428.40
431.73
472.26
438.53
160.89
303.14


Unknown
62.38
62.50
46.56
38.08
76.63
85.85
24.57
45.01


D-chiro-Inositol
33.59
27.90
46.09
46.18
25.69
68.75
14.91
32.16


myo-Inositol
116.75
167.27
20.25
28.65
2.77
7.67
4.02
6.03


Sucrose
1364.90
2193.00
322.30
431.68
463.59
849.80
369.28
614.41


Galactopinitol A
9.09
9.60
12.29
8.49
94.29
117.43
86.03
104.22


Galactopinitol B
0
0
0
1.48
41.39
50.76
29.37
40.39


Fagopyritol B1
0
3.39
4.99
4.57
51.96
69.76
29.66
51.85


Galactinol
0
0
28.42
40.81
20.87
37.07
11.23
10.39


Raffinose
0
4.09
10.85
15.22
72.18
187.91
77.60
162.90


Ciceritol
0
0
0
2.47
0
3.32
4.88
4.85


Fagopyritol B2
0
0
4.91
0
15.04
11.92
1.77
0


Stachyose
0
0
0
0
211.21
552.89
268.51
427.70
















TABLE 10







Accumulation of soluble carbohydrates in soybean cotyledon (μg/cotyledon)


after 3 days transport of sucrose (1% solution) without cyclitols


into the stem of soybean explants and after slow drying of seeds


for 2, 4, or 14 days (micrograms/1 cot)












Before
After 2 days
After 4 days
After 14 days



slow drying
slow drying
slow drying
slow drying















Soluble Carbohydrate
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2


















D-Pinitol
199.64
176.34
162.30
222.80
187.65
166.14
98.90
153.50


Unknown
11.90
12.07
22.73
20.87
60.13
5840
14.10
8.01


D-chiro-Inositol
45.57
37.40
50.36
57.42
69.00
73.60
54.07
46.46


myo-Inositol
127.35
113.66
23.50
29.70
12.71
15.03
7.16
16.01


Sucrose
2188.80
2395.60
731.21
923.69
1035.10
2211.00
1281.70
1787.10


Galactopinitol A
9.90
9.21
9.17
9.30
114.04
84.24
102.80
68.60


Galactopinitol B
4.55
1.73
1.56
1.54
40.67
36.28
49.91
30.24


Fagopyritol B1
1.89
2.10
4.72
13.12
72.21
62.04
83.93
57.10


Galactinol
1.88
2.16
83.71
166.27
52.22
25.06
28.60
26.57


Raffinose
4.97
2.41
34.55
85.98
200.22
312.69
273.49
444.34


Ciceritol
0
0
1.63
0
1.79
2.22
10.22
4.63


Fagopyritol B2
0
0
2.69
0
11.94
2.08
4.52
4.65


Stachyose
0
0
12.20
18.15
773.51
516.66
1331.00
904.20
















TABLE 11







Accumulation of soluble carbohydrates in soybean seed coats


(μg/seed coat) after 3 days transport of sucrose


(1% solution) and myo-inositol (50 mM) into stem of


soybean explants and after slow drying of seeds for 2,


4, or 14 days (micrograms/1 seed coat)











After 2 days
After 4 days
After 14 days



slow drying
slow drying
slow drying













Soluble Carbohydrate
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2
















D-Pinitol
11.24
9.35

6.81
12.13
15.41


D-chiro-Inositol
15.00
17.15

7.20
13.32
22.63


myo-Inositol
38.71
30.23

4.83
6.94
9.10


Sucrose
21.35
23.43

14.28
78.62
138.83


Galactopinitol A
0
0

4.87
6.98
7.08


Galactopinitol B
0
0

1.07
0
2.22


Fagopyritol B1
3.04
1.97

1.90
5.51
6.47


Galactinol
0
0

0
2.61
2.91


Raffinose
1.58
0

0
7.44
24.24


Ciceritol
0
0

0
0
0


Fagopyritol B2
0
0

0
0
0


Stachyose
0
0

0
20.21
35.12
















TABLE 12







Accumulation of soluble carbohydrates in soybean seed


coat (μg/seed coat) after 3 days transport of sucrose


(1% solution) and D-chiro-inositol (50 mM) into the stem of


soybean explants and after slow drying of seeds for 2, 4, or


14 days (micrograms/1 seed coat)











After 2 days
After 4 days
After 14 days


Soluble
slow drying
slow drying
slow drying













Carbohydrate
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2
















D-Pinitol
6.55
5.97
14.69
10.95
12.79
16.30


D-chiro-Inositol
193.33
173.15
169.87
149.60
87.18
134.18


myo-Inositol
12.32
11.96
5.53
5.38
3.76
4.51


Sucrose
16.60
17.20
2.65
2.33
9.81
59.33


Galactopinitol A
0
4.13
4.72
4.29
0
7.33


Galactopinitol B
0
0.79
1.76
0.78
0
3.14


Fagopyritol B1
3.52
4.88
9.14
8.69
14.54
51.81


Galactinol
0
1.30
0
1.19
0
1.60


Raffinose
0
0
0.36
0.18
0
10.08


Ciceritol
0
0
0
0
0
0


Fagopyritol B2
0
0
0
0
0
1.35


Stachyose
0
0
0
0
0
15.45
















TABLE 13







Accumulation of soluble carbohydrates in soybean seed


coat (μg/seed coat) after 3 days transport of sucrose


(1% solution) and D-pinitol (50 mM) into the stem of


soybean explants and after slow drying of seeds for


2, 4, or 14 days (micrograms/1 seed coat)











After 2 days
After 4 days
After 14 days


Soluble
slow drying
slow drying
slow drying













Carbohydrate
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2
















D-Pinitol
78.06
73.32
58.50
90.19
50.78
96.60


D-chiro-Inositol
4.66
4.29
4.05
5.81
4.52
8.11


myo-Inositol
10.61
14.11
2.68
2.62
4.13
3.58


Sucrose
15.56
23.40
2.73
4.47
33.23
78.92


Galactopinitol A
0
5.55
4.81
5.72
10.65
19.70


Galactopinitol B
0
3.09
1.44
2.10
3.57
6.98


Fagopyritol B1
0
2.10
1.66
1.66
4.07
10.12


Galactinol
0
0
0
0
0
2.77


Raffinose
0
0
0
0.54
0
18.53


Ciceritol
0
0
0
0
0
0


Fagopyritol B2
0
0
0
0
0
0


Stachyose
0
0
0
0
0
64.19
















TABLE 14







Accumulation of soluble carbohydrates in soybean seed coats


(μg/seed coat) after 3 days transport of sucrose (1% solution)


without cyclitols into the stem of soybean explants and after slow


drying of seeds for 2, 4, or 14 days (micrograms/1 seed coat)











After 2 days
After 4 days
After 14 days


Soluble
slow drying
slow drying
slow drying













Carbohydrate
Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2
















D-Pinitol
16.22
12.15
20.16
19.36
25.66
16.34


D-chiro-Inositol
3.27
3.10
3.77
1.74
3.38
2.68


myo-Inositol
12.27
12.84
5.67
5.65
3.88
4.63


Sucrose
21.74
31.93
8.26
15.33
19.35
82.18


Galactopinitol A
0
4.95
5.54
8.05
5.79
6.14


Galactopinitol B
0
1.22
1.45
0
0
2.18


Fagopyritol B1
3.43
3.27
1.69
5.41
2.54
3.44


Galactinol
0
0
1.50
0
0
1.66


Raffinose
3.50
5.06
1.08
0
0
8.44


Ciceritol
0
0
0
0
0
0


Fagopyritol B2
0
0
0
0
0
0


Stachyose
0
0
0
0
0
16.46
















TABLE 15







Accumulation of soluble carbohydrates (μg/cm2)


in soybean leaves (1 cm2 leaf disks) at 24 hours after


feeding 50 mM myo-inositol, D-chiro-inositol, or D-pinitol,


each in 1% sucrose solution, or 1% sucrose solution


alone to stems of soybean explants









24 hours after feeding 50 mM cyclitol in 1%



sucrose solution to explants













D-chiro-




Soluble
myo-Inositol
Inositol
D-Pinitol
Sucrose only








Carbohydrate
μg/cm2 leaf area














Fructose
88.82
121.03
62.53
24.85


Glucose
50.75
115.12
69.82
21.14


D-Pinitol
147.24
124.80
757.97
133.76


D-chiro-
18.98
439.69
23.29
12.21


Inositol


myo-Inositol
296.44
10.15
5.60
25.31


Sucrose
46.10
29.51
28.89
29.90


Maltose
9.34
9.85
4.11
11.59


Galactopinitol A
0
0
0
0


Galactopinitol B
0
0
0
0


Fagopyritol
0
0
0
0


B1


Galactinol
0
0
0
0


Raffinose
0
0
0
0


Ciceritol
0
0
0
0


Fagopyritol
0
0
0
0


B2


Stachyose
0
0
0
0









Some results and conclusions drawn from this series of experiments are as follows. Feeding myo-inositol, D-chiro-inositol, or D-pinitol to soybean explants increased free myo-inositol 10 fold, free D-chiro-inositol 35 fold, or D-pinitol 5 fold, respectively, in leaf tissues at 24 hours after the start of feeding explants demonstrating the uptake of cyclitols through the stem to the leaf via the transpiration stream. Free D-chiro-inositol in leaf tissues was increased slightly after feeding myo-inositol or D-pinitol, but there was no detection of galactosyl cyclitols, raffinose, or stachyose in leaf tissues indicating the absence of accumulation of these compounds in leaves.
Feeding myo-inositol, D-chiro-inositol, or D-pinitol to soybean explants increased free myo-inositol 2 fold, free D-chiro-inositol 20 to 40 fold, or D-pinitol 2 to 4 fold, respectively, in seed coat tissues of dry seeds (14 days slow drying) demonstrating the movement of cyclitols to the seed coat, presumably via the phloem. Feeding myo-inositol increased D-chiro-inositol 5 to 10 fold and doubled raffinose and stachyose, with no increase in D-pinitol or galactopinitols in the seed coat, suggesting that myo-inositol may directly serve as precursor for biosynthesis of D-chiro-inositol or through D-pinitol as intermediate. Feeding D-chiro-inositol also increased fagopyritol B1 5 to 15 fold in seed coats, but not other cyclitols, galactosyl cyclitols, or raffinose and stachyose. Feeding D-pinitol doubled galactopinitols and increased D-chiro-inositol, fagopyritol B1, raffinose, and stachyose indicating that D-pinitol may serve as precursor to D-chiro-inositol biosynthesis and that galactopinitols may serve as a galactosyl donor for the biosynthesis of stachyose.
Feeding myo-inositol, D-chiro-inositol, or D-pinitol to soybean explants increased free myo-inositol slightly, free D-chiro-inositol 15 to 40 fold, or D-pinitol 4 to 15 fold, respectively, in axis tissues of dry seeds (14 days slow drying) demonstrating the downloading of cyclitols from the seed coat to the embryonic axis. Feeding myo-inositol had little effect on the accumulation of other soluble carbohydrates in the embryonic axis. Feeding D-chiro-inositol also increased fagopyritol B1 10 fold in seed coats, but not other cyclitols, galactosyl cyclitols, or raffinose and stachyose. Feeding D-pinitol doubled galactopinitols in the embryonic axis, but not other cyclitols, galactosyl cyclitols, or raffinose and stachyose. These results suggest that galactopinitols and fagopyritols are biosynthesized by different pathways.
Feeding myo-inositol, D-chiro-inositol, or D-pinitol to soybean explants did not increase free myo-inositol, but increased free D-chiro-inositol 5 to 6 fold, or D-pinitol 2 fold, respectively, in cotyledon tissues of dry seeds (14 days slow drying) demonstrating the downloading of cyclitols from the seed coat to the soybean embryo. Feeding myo-inositol doubled free D-chiro-inositol but had little effect (or decreased) other soluble carbohydrates consistent with myo-inositol being a precursor for the biosynthesis of D-chiro-inositol. Feeding D-chiro-inositol also increased fagopyritol B1 6 to 10 fold in cotyledons, but not other cyclitols, galactosyl cyclitols, or raffinose and stachyose, indicating that fagopyritols do not serve as galactosyl donors for stachyose biosynthesis. Feeding D-pinitol did not increase accumulation of cyclitols (other than D-pinitol), galactosyl cyclitols, raffinose, or stachyose in cotyledons.
These results are in general agreement with the those of Examples 2 and 3.
Example 6
Buckwheat Plant Temperature Experiments
Common buckwheat (Fagopyrum esculentum Moench) belongs to Polygonoceae family. Originating in northeast Asia, southern Siberia and northern China, there are 18 recognized natural species in Fagopyrum. Among them, common buckwheat is most important from economical, agricultural, and nutritional points of view. In buckwheat, the triangular fruit (achene) forms a single seed. The buckwheat embryo is rich in lipids (Horbowicz et al., J. Agric. Food Chem. 40:745-750 (1992), which is hereby incorporated by reference in its entirety), and high quality proteins (Elpidina et al., J. Exp. Bot. 41:969-977 (1990), which is hereby incorporated by reference in its entirety), and is embedded in a starchy endosperm (Marshall et al., Adv. Cereal Sci. Tech. 5:157-210 (1982); Steadman et al., J. Cereal Sci. 33:271-278 (2001), which are hereby incorporated by reference in their entirety).
Common buckwheat plants are dimorphic and heterostylous. One-half of the plants have pin-type flowers with long styles and short stamens, and one-half of the plants have thrum-type flowers with short styles and long stamens (Marshall et al., Adv. Cereal Sci. Tech. 5:157-210 (1982), which is hereby incorporated by reference in its entirety). Each type is self-incompatible and cross-incompatible among plants with the same flower type. Seed set requires legitimate cross pollination, pin by thrum and thrum by pin, by insects under field conditions or by hand pollination in the greenhouse as in the present study (Horbowicz et al., J. Agric. Food Chem. 40:745-750 (1992), which is hereby incorporated by reference in its entirety).
Buckwheat plants grow best in cool, moist climates. Daytime air temperatures of 17° C. to 19° C. are optimal during flowering and seed maturation of this plant (Marshall et al., Adv. Cereal Sci. Tech. 5:157-210 (1982), which is hereby incorporated by reference in its entirety). Because the crop matures in 10 to 12 weeks, it can be grown in temperate regions and higher altitude areas. The crop is sensitive to high temperatures and dry weather, especially when the plants are flowering (Slawinska et al., Seed Sci. Res. 11:223-233 (2001); Taylor et al., Crop Sci. 41:1792-1799 (2001), which are hereby incorporated by reference in their entirety).
Recent evidence points to the importance of special types of carbohydrates in development of seed desiccation tolerance and storability (Koster et al., Plant Physiol. 88:829-832 (1998); Blackman et al., Plant Physiol. 100:225-230 (1992); Horbowicz et al., Seed Sci. Res. 4:385-405 (1994); Obendorf et al., Seed Sci. Res. 7:63-74 (1997); Obendorf et al., Crop Sci. 38:78-84 (1998), which are hereby incorporated by reference in their entirety). During development of legume seeds mainly sucrose and α-galactosides of sucrose are accumulated (Horbowicz et al., Seed Sci. Res. 4:385-405 (1994); Obendorf et al., Seed Sci. Res. 7:63-74 (1997); Brenac et al., J. Plant Physiol. 150:481-488 (1997), which are hereby incorporated by reference in their entirety). Instead, buckwheat seeds contains sucrose and α-galactosides of D-chiro-inositol (Horbowicz et al., Planta 205:1-11 (1998), which is hereby incorporated by reference in its entirety).
Six fagopyritols (galactosyl cyclitols), representing two distinct series differing in bonding positions, were found in buckwheat seeds (Horbowicz et al., Planta 205:1-11 (1998); Steadman et al., J. Cereal Sci. 33:271-278 (2001); Steadman et al., Carbohydr. Res. 331:19-25 (2001); Szczecinski et al., Bull. Pol. Acad. Sci. 46:9-13 (1998), which are hereby incorporated by reference in their entirety). Fagopyritol B1 and fagopyritol A1 are the major galactosides accumulated, and correlated to desiccation tolerance in buckwheat seeds (Horbowicz et al., Planta 205:1-11 (1998); Obendorf et al., Carbohydr. Res. 328:623-627 (2000), which are hereby incorporated by reference in their entirety). Structures of di- and trigalactosides of D-chiro-inositol have been confirmed as well (Steadman et al., Carbohydr. Res. 331:19-25 (2001), which is hereby incorporated by reference in its entirety). All fagopyritols accumulate mainly in the embryo of buckwheat seeds, and much lower amounts in endosperm (Horbowicz et al., Planta 205:1-11 (1998), which is hereby incorporated by reference in its entirety).
chiro-inositol plays a role in the biosynthesis of galactosamine-D-chiro-inositol, an insulin mediator in type II diabetes (Lamer et al., Biochem. Biophys. Res. Commun. 151:1416-1426 (1988); Romero et al., Adv. Pharmacology 24:21-50 (1993), which are hereby incorporated by reference in their entirety). In Type II (non-insulin dependent diabetes mellitus) diabetic patients have deficiency of an insulin mediator containing galactosamine-D-chiro-inositol phosphate (Asplin et al., Proc. Nat. Acad. Sci. 90:5924-5928 (1993), which is hereby incorporated by reference in its entirety). Adding D-chiro-inositol as a dietary supplement appeared to be effective in lowering symptoms of diabetes (Ortmeyer et al., Endocrinology 132:640-645 (1993), which is hereby incorporated by reference in its entirety). Several research groups are developing sources for natural and synthetic supplies of D-chiro-inositol (U.S. Pat. No. 5,091,596 to Kennigton et al; Mandel et al., J. Org. Chem. 58:2331-2333 (1993), which are hereby incorporated by reference in their entirety). One natural source of D-chiro-inositol (in free form and as galactosides) is buckwheat seed, and the bran milling fraction from buckwheat seed can be used for isolation and production of fagopyritols and free D-chiro-inositol preparations for medical purposes (Obendorf et al., Carbohydr. Res. 328:623-627 (2000); Steadman et al., J. Agric. Food Chem. 48:2843-2847 (2000); Horbowicz et al., J. Agric. Food Chem. 40:745-750 (1992), which are hereby incorporated by reference in their entirety).
Temperature during development of legume seeds had only minor effects on soluble carbohydrate biosynthesis and accumulation (Gorecki et al., Crop Sci. 36:1277-1282 (1996); Obendorf et al., Crop Sci. 38:78-84 (1998), which are hereby incorporated by reference in their entirety). However during our preliminary studies, temperature during seed maturation affected soluble carbohydrate content and composition of buckwheat embryos (Horbowicz et al., Planta 205:1-11 (1998), which is hereby incorporated by reference in its entirety). Warm temperature (25° C.) favored biosynthesis of sucrose, and embryos matured at cool temperature (18° C.) accumulated higher quantities of fagopyritol A1 and fagopyritol B1. During maturation of soybean embryos, warm temperature (25° C.) favors biosynthesis of fagopyritol B1, as well as sucrose, raffinose, D-chiro-inositol and D-pinitol (Obendorf et al., Crop Sci. 38:78-84 (1998), which is hereby incorporated by reference in its entirety). The objective of this Example was to determine if temperature (15, 22 and 30° C.) during buckwheat seed maturation in plants affects accumulation of soluble carbohydrates, dry and fresh mass, and germination of buckwheat embryos and seeds.
Materials and Methods
Buckwheat plants (cv. Mancan) were grown in the greenhouse at 24° C. day (14 hours) and 18° C. night (10 hours). Natural sunlight was supplemented 14 hours daily with 740 μmol m2 s−1 light from 1000 W Sylvania metal halide lamps. After opening first flowers, plants were separated into pin and thrum types and placed in separate growth chambers at 18° C. All plants received 14 hours of fluorescent light daily at about 300 μmol m2 s−1. After 7 to 10 days, plants were hand pollinated by legitimate cross-pollination, pin×thrum and thrum×pin. Eight days after pollination the temperature in three growth chambers was changed from 18° C. to 15° C., 22° C., and 30° C., respectively. Seeds were harvested at 8, 12, 16, 20, and 28 days after pollination (DAP) and analyzed for soluble carbohydrates. After the last harvest (28 DAP) seeds were placed in a desiccator over saturated LiCl solution (RH=12%), and dried for 14 days before analysis. Weight of each groat was measured. After drying over LiCl, seeds (four replications of 10 groats each) were germinated on wet germination papers at 25° C. in darkness. After 2, 4, and 6 days the germination rate (in %) was measured, as well as hypocotyl length.
Carbohydrates in buckwheat embryo were analyzed by high resolution gas chromatography as previously described (Horbowicz et al., Seed Sci. Res. 4:385-405 (1994); Horbowicz et al., Planta 205:1-11 (1998), which are incorporated herein by reference in their entirety). Carbohydrate standards (sucrose, myo-inositol, fructose, glucose, raffinose and stachyose), internal standard (phenyl α-D-glucoside), pyridine and trimethylsilylimidazole (TMSI) were purchased from Sigma. Fagopyritol standards were purified from buckwheat (Horbowicz et al., Planta 205:1-11 (1998); Steadman et al., Carbohydr. Res. 331:19-25 (2001), which are incorporated herein by reference in their entirety). Galactinol and D-chiro-inositol standards were a gift.
Results
Buckwheat embryos accumulated maximum fresh weight by 20 days after pollination (DAP) when matured at 15° C., by 16 DAP when matured at 22° C., and by 12 DAP when matured at 30° C. (Table 16).







TABLE 16







Dry weight (DW) and fresh weight (FW) of buckwheat embryos


(mg/embryo) from seeds matured at 15, 22, or 30° C.


as a function of days after pollination (DAP). Values are


mean ± SE for three replicate samples.











Maturation at 15° C.
Maturation at 22° C.
Maturation at 30° C.













DAP
FW (mg)
DW (mg)
FW (mg)
DW (mg)
FW (mg)
DW (mg)
















8
0.99 ± 0.08
0.24 ± 0.12
0.99 ± 0.08
0.24 ± 0.07
0.99 ± 0.08
0.24 ± 0.07


12
3.00 ± 0.71
0.70 ± 0.27
6.77 ± 1.41
1.47 ± 0.14
11.17 ± 0.95 
4.43 ± 0.43


16
11.50 ± 1.25 
4.23 ± 1.07
14.13 ± 3.06 
5.77 ± 1.62
10.57 ± 0.20 
5.17 ± 0.23


20
17.37 ± 0.64 
8.67 ± 0.32
13.43 ± 0.67 
6.97 ± 0.61
9.90 ± 0.52
7.63 ± 0.26


28
14.43 ± 1.07 
8.03 ± 0.26
8.60 ± 0.38
6.37 ± 0.13
6.67 ± 0.87
6.83 ± 0.65


28 DAP + 2 wk
7.80 ± 0.72
7.07 ± 0.64
7.40 ± 0.98
6.50 ± 0.86
9.73 ± 0.59
6.93 ± 0.54


12% RH









Highest daily increase in fresh weight occurred between 12 and 16 DAP when matured at 15 and 22° C. and between 8 and 12 DAP when matured at 30° C.

Independently of maturation temperature, the dry weight of embryos reached maximal values after 20 DAP, but fastest daily increase of DW occurred between 8 and 12 DAP at 30° C., between 12 and 16 DAP at 22° C., and at 15° C. between 16 and 20 DAP (Table 16). Although differences in the rates of dry matter accumulation occurred between all temperatures, the final dry weight of embryos matured at 15, 22 and 30° C. was similar. The slight decrease of dry weight in embryos matured at 15° C. noted after 2 weeks of drying over LiCl solution probably was the effect of difficulty in removing all remnants of cotyledons surrounded by endosperm tissue. Equal accumulation of embryo dry weight was also noted in our previous experiments, where seeds were matured in 18 and 25° C. (Horbowicz et al., Planta 205:1-11 (1998), which is hereby incorporated by reference).
Mean dry weight of groats gradually declined when maturation temperature increased. Mean dry weight of buckwheat groats matured at 15° C. was 48.17±1.75 mg, at 22° C.- 41.27±1.48 mg, and at 30° C.- 35.20±1.31 mg. Data presented here are the groat mean (±SE) dry weights from 50 seeds. Calculated average decline of buckwheat groat weight with increasing temperature was −0.86 mg/1° C.
Maturation temperature had no effect on the total amount of soluble carbohydrates in buckwheat embryos (Table 17).







TABLE 17







Soluble carbohydrates (μg/embryo) in buckwheat


embryos from seeds matured at 15, 22, or 30° C.


All seed harvested at 28 days after pollination (DAP)


and dried 2 weeks at 12% RH. Values are mean ± SE


for three replicate samples.










Sol.
Maturation
Maturation
Maturation


carbohydrate
at 15° C.
at 22° C.
at 30° C.





D-chiro-Inositol
 9.76 ± 2.86
 6.81 ± 1.07
 3.49 ± 0.66


Fagopyritol A1
45.59 ± 5.24
 34.15 ± 10.02
21.78 ± 1.55


Fagopyritol B1
256.00 ± 38.30
219.15 ± 24.20
159.60 ± 7.70 


Fagopyritol A2
 3.66 ± 0.84
11.12 ± 3.04
15.52 ± 0.68


Fagopyritol B2
 2.47 ± 0.74
12.98 ± 3.59
19.70 ± 1.74


Sub total
317.50 ± 48.00
284.60 ± 42.00
220.10 ± 12.30


myo-Inositol
 2.91 ± 0.54
 5.03 ± 0.74
 3.25 ± 0.64


Galactinol
0
 1.57 ± 0.79
 1.60 ± 0.04


Digalactosyl
 0.25 ± 0.25
 0.55 ± 0.55
 1.69 ± 0.73


myo-inositol


Sub total
 3.16 ± 0.79
 7.15 ± 2.08
 6.54 ± 1.41


Sucrose
225.00 ± 14.90
250.20 ± 22.70
376.40 ± 59.20


Total


soluble
545.70 ± 63.70
542.00 ± 66.80
603.00 ± 72.90


carbohydrates









Reducing sugars, fructose and glucose were present only in early stages of embryo development (8 and 12 DAP). Sucrose slightly decreased between 8 and 12 DAP, probably due to temperature and pollination shocks, and then during next 4 days increased dramatically reached maximal values 16 DAP (FIG. 23A). This increase was due to a rapid increase of embryo fresh weight during maturation at 15 and 22° C., but not at 30° C. (FIG. 23A and Table 16). During maturation at 30° C., the highest daily increase of fresh weight occurred between 8 and 12 DAP, but in the same time sucrose level slightly declined. After 16 DAP sucrose level in embryos matured at 15 and 22° C. decreased, and finally after drying over LiCl solution, the embryo sucrose content was 225.0 and 250.2 μg/embryo, respectively. Maturation at 30° C. and further drying over LiCl solution of buckwheat embryos did not change the level of sucrose, which remained much higher at 376.4 μg (Table 17).

Monogalactosides of D-chiro-inositol (isomers fagopyritol A1 and fagopyritol B1) were the dominant soluble carbohydrates in embryos of buckwheat seeds matured in 15° C., but not when matured at 22 or 30° C. (FIGS. 23B and C). After drying of harvested buckwheat seeds at 12% relative humidity (RH) over LiCl solution, the ratio of fagopyritol B1 to sucrose was 1.14:1 when embryos were matured at 15° C., 0.88:1 in embryos matured at 22° C., and only 0.43:1 in embryos matured at 30° C. (Table 17). A similar situation, a clear decline of sucrose in relation to increased temperature, occurred in the case of positional isomer fagopyritol A1, although level of fagopyritol B1 was 5 to 7 times higher than fagopyritol A1 (FIGS. 23B and C and Table 17).
An opposite situation occurred in the case of D-chiro-inositol digalactosides, fagopyritol A2 and fagopyritol B2 (FIGS. 24A-C); higher amounts accumulated in embryos matured at higher temperatures (22 and 30° C.) than at 15° C. After 2 weeks of drying of buckwheat seeds, embryos of seeds matured at 30° C. contained about 4 times more fagopyritol A2, and almost 8 times more fagopyritol B2 than embryos of seeds matured at 15° C. (Table 17). Similar effect of maturation temperature was found in case galactosides of myo-inositol (FIGS. 25A-C). Accumulation of myo-inositol in embryo was similar at all temperatures of buckwheat seed maturation, however the amount of its galactosides (galactinol and digalactosyl myo-inositol (DGMI)) was much less in embryos of seeds matured at 15° C. than in embryos of seeds matured at 22° C. and especially in seeds matured at 30° C. (Table 18).







TABLE 18







Minor soluble carbohydrates (μg/embryo) in buckwheat embryos


from seeds matured at 15, 22, or 30° C. as a function of days after


pollination (DAP). Values are mean ± SE for three replicate samples.












Soluble
Maturation



28 DAP + 2 


Carbohydrate
temperature
16 DAP
20 DAP
28 DAP
wk 12% RH





Digalactosyl
15° C.
0
0
0
0.25 ± 0.25


myo-inositol
22° C.
0
1.38 ± 0.84
1.12 ± 0.18
0.55 ± 0.55



30° C.
1.02 ± 1.02
3.54 ± 2.51
2.21 ± 0.32
1.69 ± 0.73


Fagopyritol A3
15° C.
0
0
0
0



22° C.

0
0



30° C.

9.80 ± 5.47
4.41 ± 4.41


Raffinose
15° C.
0
0
0
0



22° C.

0
0.78 ± 0.45



30° C.

0.70 ± 0.12
1.21 ± 0.95


Stachyose
15° C.
0
0
0
0



22° C.

0
3.04 ± 3.04



30° C.

2.71 ± 2.71
5.07 ± 2.53









During later stages of buckwheat embryo development (after 20 DAP and 28 DAP) at 22 and 30° C., small amounts of raffinose and stachyose were found (Table 18). In embryos matured in 30° C., fagopyritol A3 (a trigalactoside of D-chiro-inositol) was present as well. Embryos matured in 15° C. did not contain these carbohydrates in measurable quantities (Table 18). After 2 weeks dehydration of buckwheat seeds, analyzed embryo raffinose, stachyose, and fagopyritol A3 declined to levels below the limit of detection.
The germination rate of seeds matured in low temperatures (15 or 22° C.) was lower than for seeds matured at 30° C. (FIG. 26A). Differences were quite clear after 4 and 6 days of germination on moist germination paper in darkness and 25° C. Germination rate of seeds matured in 22° C. was 14, 18, and 20% lower after 2, 4, and 6 days respectively, than for seeds matured at 15° C. When compared to seeds matured at 30° C., the germination rate of seeds matured in 22° C. was 20%, 44%, and 41% lower. Germination rate of seeds matured in 30° C. was similar to those matured in 15° C. after 2 days of germination, however after 4 and 6 days, seeds matured at 30° C. germinated 90%, and seeds matured at 15° C. germinated only 66% and 71% (FIG. 26A).
Growth of hypocotyls in germinating buckwheat seeds was faster in seeds matured at 15 and 22° C., than for seeds matured at 30° C. (FIG. 26B). Such a situation occurred after 2 and 4 days of germination process, but after 6 days the differences in hypocotyl length were not significant.
Discussion
The response of plants to stress involves complex physiological and biochemical responses. Conditions during seed development and maturation can have an impact on subsequent seed quality. Soil moisture and temperature stress in that time has been suggested to have an influence on seed and seedling vigor. Factors during seed maturation such as environmental conditions also have an impact on seed viability (Baskin et al., Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination, Academic Press, New York, pp. 41-43 (1998), which is hereby incorporated by reference in its entirety). High temperatures during growth can increase biochemical reactions in plants, but it might not always be transferred to higher productivity because of heat stress constraints such as limited water supply, increase in leaf temperature, increased respiration, decline of the synthesis and/or activity of photosynthetic enzymes. In buckwheat groats matured in high temperatures (22 or 30° C.), reduced mean weight was noted, than when produced in low temperature (15° C.). Although high temperature maturation (30° C.) can change physiological reactions the buckwheat embryos obtained in such conditions have similar dry weight to those from matured in lower temperatures (15 or 22° C.). Dry weight of whole seed was lower, mainly due to decrease of endosperm deposition (Horbowicz et al., Planta 205:1-11 (1998), which is hereby incorporated by reference in its entirety). Additionally, plants growing at 25° C. produced only half as many seeds as plants at 18° C. (Slawinska et al., Seed Sci. Res. 11:223-233 (2001), which is hereby incorporated by reference in its entirety). All mentioned facts can have huge impact on buckwheat seed yield. Probably, the difference in temperature during buckwheat flowering and seed filling is the main factor influencing the large variability in seed set and seed yield among years (Slawinska et al., Seed Sci. Res. 11:223-233 (2001); Taylor et al., Crop Sci. 41:1792-1799 (2001), which is hereby incorporated by reference in its entirety).
During high temperature stressed plants make a special proteins called heat shock proteins (HSPs). Among the different HSPs produced by plants, the small (sm) HSPs appear to be particularly important because of their abundance. In addition, smHSPs are expressed during specific stages of plant development including seed maturation, indicating they also function in the absence of stress to protect components essential for seed development (Schoffl et al., Plant Physiol. 117:1135-1141 (1998), which is hereby incorporated by reference in its entirety). HSPs showing a reversible interaction with other proteins and preventing either complete denaturation or supporting proper folding of enzymes under or after protein denaturing conditions. Some HSP-like proteins are involved in the processes of targeting other proteins to organelles or to their suborganellar localization and a number of HSPs are expressed in the absence of external stressors, during embryogenesis and seed maturation in many plant species (Schoffl et al., Acta Physiol. Plantarum 19:549-556 (1997), which is hereby incorporated by reference in its entirety).
It is possible that HSPs might have an influence on biosynthesis of carbohydrates during maturation of buckwheat embryos. In buckwheat embryos matured in higher temperatures biosynthesis of fagopyritols B1 and its positional isomer fagopyritol A1 was partly inhibited (Horbowicz et al., Planta 205:1-11 (1998), which is hereby incorporated by reference in its entirety). In present studies total amounts of both fagopyritols in embryos matured at 15° C. is about twice as high as those matured at 30° C. However, sucrose level is much higher in buckwheat embryos matured at high temperatures. This observation differs from soybean embryos, where maturation at 25° C. enhanced the amount of fagopyritol B1 when compared to embryos matured at 18° C. (Obendorf et al., Crop Sci. 38:78-84 (1998), which is hereby incorporated by reference in its entirety).
D-chiro-inositol and its galactosides (fagopyritols) have potential medical importance in lowering symptoms of non-insulin dependent diabetes mellitus (Asplin et al., PNAS USA 90:5924-5928 (1993); Lamer et al., Biochem. Biophys. Res. Commun. 151:1416-1426 (1988); Ortmeyer et al., Endocrinology 132:640-645 (1993); Romero et al., Adv. Pharmacology 24:21-50 (1993), which are hereby incorporated by reference in their entirety). Buckwheat flour produced from seeds matured at low temperature (15 or 18° C.) is therefore more valuable than from seeds matured at 22 or 30° C. Buckwheat seeds can be an excellent and natural source for production of medicines used by diabetes patients (U.S. Pat. No. 6,162,795 to Obendorf et al; U.S. Pat. No. 6,492,341 to Obendorf et al., which are hereby incorporated by reference in their entirety).
High temperature during buckwheat seed maturation enhanced the biosynthesis of di-a-galactosides of D-chiro-inositol (fagopyritol A2 and fagopyritol B2) and a-galactosides of sucrose (raffinose and stachyose). This observation is opposite to our earlier results, where increased level of sucrose galactosides was noted in buckwheat embryos of seeds matured at 18° C. in comparison to embryos from seeds matured at 25° C. (Horbowicz at al., Planta 205:1-11 (1998), which is hereby incorporated by reference in its entirety). Similarly, in the present study, a higher level of galactinol, the substrate for biosynthesis of raffinose and stachyose, was found in buckwheat embryos matured at higher temperatures. Galactinol is the galactosyl donor for both raffinose and stachyose biosynthesis, as well as the digalactoside of myo-inositol. According to Castillo et al., J. Agric. Food Chem. 38:351-355 (1990), which is hereby incorporated by reference in its entirety, low temperature during soybean seed maturation promotes galactinol biosynthesis. In buckwheat is the opposite situation—high temperature promotes accumulation of galactinol, raffinose, and stachyose. Based on that it was concluded that physiological response to temperature stress during seed maturation in buckwheat is different than what occurs in legumes (Castillo et al., J. Agric. Food Chem. 38:351-355 (1990); Gorecki et al., Crop Sci. 36:1277-1282 (1996), which are hereby incorporated by reference in their entirety). In fact, for growing of legumes, high temperatures are needed, whereas for buckwheat, daily temperatures 17 to 19° C. are optimal.
Surprisingly, germination was higher in case of buckwheat seeds matured at 30° C. than for those matured at 15 or 22° C. Lowest germination rate was found in seeds matured at 22° C. Possibly during maturation of buckwheat seeds at 22° C. germination inhibitors are biosynthesized in higher concentration and they affect the proteolytic enzymes during germination (Belozersky et al., J. Plant Physiol. 46(3):330-339 (1999), which is hereby incorporated by reference in its entirety). Seeds matured at 15° C. have delayed maturation, and therefore inhibitors are probably absent or in low, insufficient quantities. At 30° C. seeds mature very fast and it is quite possible that these seeds have lower levels of germination inhibitors, due to the shorter time of maturation.
Example 7
Buckwheat Explant Feeding Experiments
Buckwheat explants, consisting of a stem segment with attached leaf and terminal floral cluster, were patterned after the soybean explants described in Example 4. This example uses the buckwheat explant system to study the transport of cyclitols, fed through the stem, to the developing buckwheat seed and their incorporation into fagopyritols. D-chiro-inositol, D-pinitol, or myo-inositol (100 mM in 1% sucrose) or 1% sucrose (without cyclitols) were fed to buckwheat explants through the stem for 5 days and then the seeds were slow dried. Soluble carbohydrates were extracted and analyzed from embryos of the seeds and from leaf disks. The results are shown in Tables 19-25, below.







TABLE 19







Soluble carbohydrates (μg/embryo) in embryos of seeds from


buckwheat explants fed 100 mM D-chiro-inositol in 1% sucrose


solution - feeding 5 days before slow drying (micrograms/embryo)














After 5
2 days
4 days
7 days


Soluble
After 1 day
days
slow
slow
slow


Carbohydrate
feeding
feeding
drying
drying
drying















D-Pinitol
0
0
0
0
0



0
0
0
0
0


D-chiro-Inositol
2.28
93.10
1.32
2.20
56.97



14.89
87.78
25.03
44.55
41.29




122.37

138.86
55.10


myo-Inositol
1.63
2.03
1.84
3.29
2.20



1.71
4.59
1.88
2.64
1.20




2.74

2.19
1.10


Sucrose
66.64
268.14
124.83
202.99
162.68



232.10
90.04
185.14
147.00
121.44




202.20

118.11
121.99


Galactopinitol A
0
0
0
0
0



0
0
0
0
0




0

0
0


Galactopinitol B
0
0
0
0
0



0
0
0
0
0




0

0
0


Fagopyritol A1
0
10.71
25.53
27.38
41.11



0
133.47
117.32
42.86
21.71




69.49

52.16
27.96


Fagopyritol B1
0
33.64
127.23
125.16
298.43



0
623.00
635.89
388.66
190.64




322.50

270.67
208.33


Galactinol
0
3.15
9.84
6.82
7.21



0
3.75
3.63
3.90
1.71




2.62

2.70
0


Fagopyritol A2
0
0
6.43
4.19
2.57



0
10.11
4.91
2.04
1.19




1.93

4.42
1.33


Fagopyritol B2
0
0
5.51
3.95
2.28



0
5.48
3.45
2.83
0.91




1.72

1.57
1.99


Digalactosyl
0
0
0
0
0


myo-inositol
0
0
0
0
0




0

0
0
















TABLE 20







Soluble carbohydrates (μg/embryo) in embryos of seeds from


buckwheat explants fed 100 mM D-pinitol in 1% sucrose


solution - feeding 5 days before slow drying (micrograms/embryo)














After 5
2 days
4 days
7 days


Soluble
After 1 day
days
slow
slow
slow


Carbohydrate
feeding
feeding
drying
drying
drying















D-Pinitol
35.53
204.10
230.83
134.07
148.04




263.02
163.47
162.97
85.34




328.27
56.90
173.51
129.52


D-chiro-Inositol
4.50
1.32
1.51
2.19
1.97




28.25
0.70
4.78
1.01




9.41
0.94
1.11
7.71


myo-Inositol
1.00
1.38
6.63
5.34
2.78




4.60
3.07
4.92
3.38




3.43
1.37
4.81
3.33


Sucrose
212.67
89.73
106.43
87.98
84.50




86.41
167.61
229.72
122.05




147.85
84.96
104.05
177.42


Galactopinitol A
0
0
0
3.39
6.41




6.18
12.91
0
9.09




5.84
6.68
0
0


Galactopinitol B
0
1.61
4.39
5.88
0




1.10
0
0
1.81




1.90
0
0
0


Fagopyritol A1
0
0
2.38
16.78
10.82




21.43
29.81
39.10
12.44




22.91
23.96
15.80
20.01


Fagopyritol B1
1.44
0.98
7.29
116.5
64.64




90.76
165.96
165.56
77.20




75.14
111.41
77.90
119.44


Galactinol
2.51
0
10.69
2.68
0




3.26
7.75
4.14
1.24




3.40
2.79
3.31
0


Fagopyritol A2
0
0
0
6.18
1.30




0
9.52
6.64
0.71




4.65
6.89
4.73
2.21


Fagopyritol B2
0
0
0
5.70
1.60




0
6.69
4.29
0.48





4.98
4.46
2.47


Digalactosyl
0
0
0
0
0


myo-inositol

0
0
1.05
0




0
0
0
0
















TABLE 21







Soluble carbohydrates (μg/embryo) in embryos of seeds from


buckwheat explants fed 100 mM myo-inositol in 1% sucrose


solution - feeding 5 days before slow drying (micrograms/embryo)














After 5
2 days
4 days
7 days


Soluble
After 1 day
days
slow
slow
slow


Carbohydrate
feeding
feeding
drying
drying
drying















D-Pinitol
0
0
0
tr
tr




tr
0
tr
tr


D-chiro-inositol
6.45
15.19
6.42
3.54
14.01




126.48
1.07
6.67
9.90


myo-Inositol
4.56
4.93
1.67
3.66
2.37




4.72
0
2.27
2.11


Sucrose
306.16
225.64
313.35
180.43
163.04




74.48
66.92
161.20
121.70


Galactopinitol A
0
0
0
0
0




0
0
0
0


Galactopinitol B
0
0
0
0
0




0
0
0
0


Fagopyritol A1
0
18.90
62.19
19.72
14.49




10.94
29.81
41.42
34.61


Fagopyritol B1
0
95.33
300.84
111.78
79.17




44.47
129.87
183.36
188.73


Galactinol
0
19.19
10.36
8.41
2.09




3.71
3.46
4.79
0


Fagopyritol A2
0
2.60
38.72
11.49
2.54




0
10.89
4.52
2.64


Fagopyritol B2
0
1.62
35.81
13.37
3.53




0
3.22
3.31
2.57


Digalactosyl
0
0.85
6.63
2.13
0.35


myo-inositol

0
8.61
0
0
















TABLE 22







Soluble carbohydrates (μg/embryo) in embryos of seeds from


buckwheat explants fed 1% sucrose (without cyclitols)


solution - feeding 5 days before slow drying (micrograms/embryo)














After 5
2 days
4 days
7 days


Soluble
After 1 day
days
slow
slow
slow


Carbohydrate
feeding
feeding
drying
drying
drying















D-Pinitol

0
0
0
0




tr
tr
0
tr





tr


D-chiro-Inositol

3.22
5.57
1.94
14.09




24.47
8.02
2.09
5.09





26.74


myo-Inositol

1.50
3.63
6.44
2.46




17.80
2.71
2.94
4.07





2.30


Sucrose

211.90
111.20
189.33
177.61




524.20
141.36
105.55
246.49





151.69


Galactopinitol A

0
0
0
0




tr
0
0
0





tr


Galactopinitol B

0
0
0
0




tr
0
0
0





0


Fagopyritol A1

0.70
12.27
16.02
23.01




27.67
51.20
14.64
15.97





76.65


Fagopyritol B1

0.87
55.08
89.92
138.32




151.69
237.27
72.98
111.37





476.65


Galactinol

1.47
15.87
26.38
0




19.03
10.02
8.45
5.93





5.03


Fagopyritol A2

0
0.58
10.60
7.35




8.12
12.29
6.87
6.34





8.09


Fagopyritol B2

0
0.66
11.05
7.65




6.72
10.83
5.72
6.34





4.39


Digalactosyl

0
0
2.98
0.82


myo-inositol

3.02
0.60
0.74
1.48





0
















TABLE 23







Soluble carbohydrates (μg/10 mg leaf disk) in


leaves from buckwheat explants fed 100 mM D-chiro-inositol


in 1% sucrose solution - leaf composition,


micrograms in 10 mg disc











After 1 hour
After 24 hours
After 72 hours


Soluble Carbohydrate
feeding
feeding
feeding













Fructose
2.05
19.96
115.25


Glucose
3.90
16.53
77.78


D-Pinitol
0
0
0


D-chiro-inositol
2.06
21.16
72.91


myo-Inositol
3.76
1.88
3.91


Sucrose
16.50
15.13
16.15
















TABLE 24







Soluble carbohydrates (μg/10 mg leaf disk) in


leaves from buckwheat explants fed 100 mM D-pinitol


in 1% sucrose solution - leaf composition,


micrograms in 10 mg disc











After 1 hour
After 24 hours
After 72 hours


Soluble Carbohydrate
feeding
feeding
feeding













Fructose
53.82
60.78
42.84


Glucose
35.78
48.92
35.53


D-Pinitol
2.45
121.45
64.73


D-chiro-lnositol
3.42
4.82
3.71


myo-Inositol
4.46
3.05
4.23


Sucrose
84.83
2.18
9.04
















TABLE 25







Soluble carbohydrates (μg/10 mg leaf disk) in


leaves from buckwheat explants fed 1% sucrose


(without cyclitols) solution - leaf composition,


micrograms in 10 mg disc











After 1 hour
After 24 hours
After 72 hours


Soluble Carbohydrate
feeding
feeding
feeding













Fructose
4.05
20.25
23.35


Glucose
4.63
10.35
8.01


D-Pinitol
0
0
0


D-chiro-Inositol
3.51
3.81
6.26


myo-Inositol
4.21
5.58
8.45


Sucrose
39.23
14.29
17.40









Based on the above data it was determined that feeding D-chiro-inositol to buckwheat explants increased free D-chiro-inositol 40 fold in leaves demonstrating the transport of cyclitols to leaves via the transpiration stream. Feeding D-pinitol to buckwheat explants increased free D-pinitol dramatically in leaves. D-Pinitol does not accumulate in buckwheat leaves or seeds of explants fed D-chiro-inositol, myo-inositol, or sucrose without cyclitols. Galactosyl cyclitols, raffinose, and sucrose do not accumulate in leaf tissues. Feeding D-chiro-inositol to buckwheat explants increased free D-chiro-inositol 3 to 10 fold and fagopyritol B1 2 fold in embryos of buckwheat seeds demonstrating the transport of D-chiro-inositol to buckwheat seeds and its incorporation into fagopyritols. Feeding D-pinitol to buckwheat explants increased free D-pinitol in buckwheat embryos demonstrating the transport of D-pinitol to seeds and embryos; these embryos did not accumulate galactopinitols, indicating that buckwheat does not have the enzymes for accumulation of galactopinitols. Signals corresponding to galactopinitol retention times were similar to background signals. Presence of galactopinitols could not be verified. If present, galactopinitols were present only in trace amounts. Results of these experiments further demonstrate that fagopyritols and galactopinitols are biosynthesized by different pathways.
Example 8
Biosynthesis of an Insulin Mediator
Growth of Recombinant E. coli and Isolation of Recombinant Proteins
cDNAs corresponding to the FeGolS-1, FeGolS-2, and GmGolS genes were inserted into pET-14B expression vectors. The vector also contained a gene for ampicillin resistance and a sequence that codes for six histidines on the N-terminal end of the expressed protein. The vectors containing the gene inserts were used to transform E. coli strain BL21, containing the bacteriophage lysogen DE3. The bacteria were then streaked on ampicillin-containing plates and incubated overnight (8-12 hours) at 37° C. One colony from each plate was then transferred to 2 mL of Luria Broth (LB) containing 0.05 mM ampicillin in 10 mL screw-capped Pyrex tubes. The tubes were then placed in an incubator at 37° C. with shaking at 175 rpm overnight (8-12 hours). One mL of the starter cultures was then transferred to 250 mL of the LB-Amp solution and grown under the same conditions for three hours. After three hours, IPTG was added to induce expression of the genes in the pET-14B vector. The bacteria were then grown for another three hours and harvested via centrifugation at 6,000 rpm. Bacteria from 500 mL of LB-Amp were lysed using 5 mL of BugBusterTM solution. Nucleic acids and non-soluble cellular matter were removed from the crude extract by centrifugation and filtration and the soluble extract was then loaded onto a Ni2+-NTA column. The target proteins with the N-terminal histidine tag bound to the column while all other soluble proteins were washed away. These enzymes were eluted from the column by the addition of imidazole containing extraction buffer. The protein solution was dialyzed against 5 mM Mn2+ solution and then used for enzyme assays.
Enzyme Assays
Assays were completed under varying conditions to begin to characterize the purified galactinol synthase enzymes. Assays were first designed to determine if the enzymes could synthesize galactinol and fagopyritols (A). The optimal concentration of Mn2+ for enzyme action was then determined (B). The enzymes were next used in assays to determine their substrate specificity (C). Finally, assays were completed to determine the reaction kinetics of the enzymes (D).
(A) Initial Assays of purified recombinant FeGolS-1, FeGolS-2, and GmGolS enzymes:
It was first determined that the purified recombinant FeGolS-1, FeGolS-2, and GmGolS enzymes could synthesize fagopyritols and galactinol. To determine galactinol synthase activity, assays were completed using myo-inositol as the galactosyl acceptor and UDP-galactose as the galactosyl donor. Approximately 1-2 μg of each enzyme was added to a 50 μL solution containing 20 mM myo-inositol, 20 mM UDP-galactose, 50 mM HEPES, pH 7.0, 2 mM DTT, and 3 mM Mn2+ (MnCl2) at 30° C. The reactions were stopped after 3 hours with the addition of 50 μL of 100% EtOH. To determine fagopyritol synthase activity, the same reaction conditions were used, except D-chiro-inositol was used as the galactosyl acceptor instead of myo-inositol.
(B) Optimal Concentration of Mn2+:
To determine the concentration of Mn2+ in which the enzymes had the greatest activity, multiple assays were completed varying the amount of Mn2+. Earlier studies of galactinol synthase enzymes from other plants reported optimal Mn2+ concentrations ranging from 1 mM to 15 mM. Two different sets of assays were completed, one using myo-inositol as the galactosyl acceptor, and the other using D-chiro-inositol. In both sets, 1-2 μg of each enzyme was added to a 50 μL solution containing 20 mM galactosyl acceptor, 20 mM UDP-galactose, 50 mM HEPES, pH 7.0, 2 mM DTT, and varying Mn2+ concentrations of 0, 1, 3, 5, 10 and 15 mM, at 30° C. After 3 hours, the reactions were stopped with the addition of 50 μL of 100% EtOH.
(C) Substrate Specificity Assays:
The substrate specificity of the three galactinol synthase enzymes was characterized through assays varying the galactosyl acceptor. myo-inositol, D-chiro-inositol, pinitol, L-chiro-inositol, ononitol, bornesitol, sequoyitol, quebrachitol, epi-inositol and scyllo-inositol were used as substrates in reactions with all three enzymes. The reactions were completed using 1-2 μg of enzyme in a 50 μL solution containing 20 mM galactosyl acceptor, 20 mM UDP-galactose, 50 mM HEPES, pH 7.0, 2 mM DTT, 5 mM Mn2+ at 30° C. After 3 hours, the reactions were stopped with the addition of 50 μL of 100% EtOH.
(D) Reaction Kinetics:
The assays to determine the Km and Vmax of the enzymes in the synthesis of galactinol from myo-inositol and UDP-galactose were set up as follows:

    
    
        Reaction A: 5 mM myo-inositol
        
            20 mM UDP-Galactose
            1 mM DTT
            50 mM Hepes, pH 7.0
            5 mM MnCl2 
        
        
        Reaction B: 10 mM myo-inositol
        
            20 mM UDP-Galactose
            1 mM DTT
            50 mM Hepes, pH 7.0
            5 mM MnCl2 
        
        
        Reaction C: 15 mM myo-inositol
        
            20 mM UDP-Galactose
            1 mM DTT
            50 mM Hepes, pH 7.0
            5 mM MnCl2 
        
        
        Reaction D: 20 mM myo-inositol
        
            20 mM UDP-Galactose
            1 mM DTT
            50 mM Hepes, pH 7.0
            5 mM MnCl2 
        
        
        Reaction E: 25 mM myo-inositol
        
            20 mM UDP-Galactose
            1 mM DTT
            50 mM Hepes, pH 7.0
            5 mM MnCl2 
        
        
    
    


The assays to determine the Km and Vmax of the enzymes in the synthesis of fagopyritol A1 and fagopyritol B1 from D-chiro-inositol and UDP-galactose were set up as follows:

    
    
        Reaction A: 5 mM D-chiro-inositol
        
            20 mM UDP-Galactose
            1 mM DTT
            50 mM Hepes, pH 7.0
            5 mM MnCl2 
        
        
        Reaction B: 10 mM D-chiro-inositol
        
            20 mM UDP-Galactose
            1 mM DTT
            50 mM Hepes, pH 7.0
            5 mM MnCl2 
        
        
        Reaction C: 15 mM D-chiro-inositol
        
            20 mM UDP-Galactose
            1 mM DTT
            50 mM Hepes, pH 7.0
            5 mM MnCl2 
        
        
        Reaction D: 20 mM D-chiro-inositol
        
            20 mM UDP-Galactose
            1 mM DTT
            50 mM Hepes, pH 7.0
            5 mM MnCl2 
        
        
        Reaction E: 25 mM D-chiro-inositol
        
            20 mM UDP-Galactose
            1 mM DTT
            50 mM Hepes, pH 7.0
            5 mM MnCl2 
        
        
    
    


To each reaction,  ̃4-5 μg of enzyme were added. Each reaction was run for 0, 3, 6, 9, and 12 minutes at 30° C. The reactions were stopped with the addition of 50 μL of 100% EtOH and 25 μL of internal standard. The reactions were then filtered through Nanosep tubes and 100 μL of each reaction added to silyation vials. Samples were dried under nitrogen and stored over P2O5 overnight. Dry residues were derivatized with 100 μL of trimethylsilylsylimadazole:pyridine (1:1, v/v) at 80° C. for 45 minutes, and 1 μL was injected for GC analysis of products as previously described (Horbowicz et al., Planta 205:1-11 (1998), which is hereby incorporated by reference in its entirety) using an HP1-MS capillary column.
All five reactions were plotted on a product concentration vs. time plot. The concentration of the enzyme had to be small enough so that the reaction was still linear after six minutes. The Vo for each reaction was determined by finding the slope of this linear portion of the curve (i.e. if its linear, use the zero point and the concentration of product after three minutes to calculate the slope of that portion of the reaction). Once this was completed, Vo (Rate) versus myo-inositol concentration was plotted. Finally, a Lineweaver-Burke Plot was made by plotting 1/Vo vs. 1/[substrate]. If the line was linear, then its slope was the Km/Vmax. The y-intercept was 1/Vmax, the x-intercept was −1/Km.
All samples from the assays were analyzed by gas chromatography. All were prepared for analysis in the same way. After addition of 50 μL of 100% EtOH, 25 μL of Internal Standard (25 μg of phenyl α-D-glucoside) was added to the reaction mixture. The solution was then filtered using NanoSep tubes and 100 μL was transferred to a silation vial. The samples were then dried under nitrogen and desiccated over P2O5 overnight. The dried samples were then derivitized with 100 μL of TMSI:pyridine (1:1, v/v) and then analyzed by gas chromatography.
Synthesis of the Putative Insulin Mediator
In order to synthesize the putative insulin mediator, it is necessary to first synthesize UDP-galactosamine. Work was completed developing a protocol for the synthesis of the compound and its purification for use in further assays. UDP-galactosamine was synthesized from galactosamine-1-phosphate (from Sigma). The synthesis was done using the procedure outlined in Heidlas et al., J. Org. Chem. 57:152-157 (1992), which is hereby incorporated by reference in its entirety. The procedure uses an uridyltransferase (EC 2.7.7.9) to transfer a UDP moiety from UDP-glucose to galactosamine-1-phosphate to make UDP-galactosamine on a gram scale (FIG. 27). The UDP-galactosamine synthesized in the reaction was purified and desalted using a Bio-Rad P-2 Gel column. The fractions containing UDP-galactosamine were analyzed by HPLC using an Alltech Econosil C18 10U column (250 mm length, 4.6 mm I.D.) and a variable-wavelength detector at 254 nm. The mobile buffer was 20 mM TEAA (triethyl ammonium acetate buffer, pH 7.0) with an increasing gradient of acetonitrile (0-4% acetonitrile) after 30 minutes to clean the column (Rabina et al., Glycoconjugate J. 18:799-805 (2001), which is hereby incorporated by reference in its entirety). Identification was based upon retention times determined earlier with known substrates and the developed separation method. Fractions containing UDP-galactosamine were concentrated by freeze drying, and the lyophilized powder containing UDP-galactosamine was resuspended in 1 mL of water. The purified UDP-galactosamine and D-chiro-inositol can now be used as substrates for the recombinant FeGolS-2 enzyme to biosynthesize the insulin mediator galactosamine D-chiro-inositol (FIG. 28). Two products are expected: 2-amino-2-deoxy-α-D-galactosamine-(1-3)-1D-chiro-inositol (a putative insulin mediator) and 2-amino-2-deoxy-α-D-galactosamine-(1-2)-1D-chiro-inositol (isomer of the putative insulin mediator) in addition to UDP. Initial determination of successful synthesis can be assayed by gas chromatography. The peaks corresponding to fagopyritol A1, fagopyritol B1, D-chiro-inositol, and many other soluble carbohydrates are known, and the two galactosamine D-chiro-inositol products should correspond to fagopyritol A1 and fagopyritol B1 with one less hydroxyl for TMS-derivatization resulting in shorter retention times. Synthesis of the insulin mediator can then be optimized in order to obtain appreciable amounts of the compound. Depending on efficiency, carbon-Celite columns, TLC, HPLC, or Dowex ion exchange columns can be used to purify the insulin mediator (and its isomeric form) from the reaction mixture. The purified insulin mediator can then be lyophilized to a white powder. The structure of the purified insulin mediator can be determined by 1H-NMR and 13C-NMR (Obendorf et al., Carbohydrate Research 328:623-627 (2000); Steadman et al., Carbohydrate Research 331:19-25 (2001), which are incorporated herein by reference in their entirety), to confirm the successful biosynthesis of the insulin mediator. Similarly, substituting L-chiro-inositol, scyllo-inositol, or bornesitol (or other cyclitols reactive with the FeGolS-2 enzyme) in the reaction (FIG. 28) would form products that may be used as inhibitors of the galactosamine D-chiro-inositol insulin mediator.
Discussion
A protocol has been developed that resulted in purification of the target enzymes from the bacterial preparation without loss of activity. Dialysis was used to remove the enzymes from the extraction buffer and into a solution of Mn2+ ions. This change retained enzyme activity throughout the purification procedure. Also, adjusting bacterial growth times and preparation methods further maximized the expression system.
Manganese concentration assays were used to determine that optimal enzyme action occurred in 5 mM Mn2+ solution. Results from the substrate specificity assays helped to identify the inositols the enzymes could use as galactosyl acceptors. myo-inositol, D-chiro-inositol, L-chiro-inositol, bornesitol and scyllo-inositol all can be used as galactosyl acceptors by all three enzymes. The Vmax and Km has been difficult to determine due to the sensitivity of the reaction. However, initial estimates of the Km for the enzyme FeGolS-2 using myo-inositol as a substrate was 7.53 mM and the Vmax 0.0817 μM/min. Determination of the Vmax and Km has proven difficult for the synthesis of fagopyritols because there are multiple products produced in the reaction.
Reactions to synthesize UDP-galactosamine and purification of the compound have been completed (FIG. 27). UDP-galactosamine can then be used as the galactosyl donor in the reaction synthesizing the putative insulin mediator (FIG. 28).
Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

## Claims

1. A method for producing a fagopyritol, an insulin mediator, an insulin mediator analogue, an insulin mediator homologue, or an insulin mediator inhibitor comprising:
expressing a nucleic acid encoding a fagopyritol synthase comprising the amino acid sequence of SEQ ID NO:4;
providing a galactosyl donor and a galactosyl acceptor; and
combining the fagopyritol synthase with the galactosyl donor and the galactosyl acceptor under conditions effective to produce a fagopyritol, an insulin mediator, an insulin mediator analogue, or an insulin mediator homologue.

2. The method according to claim 1, wherein the galactosyl donor is UDP-galactose.

3. The method according to claim 1, wherein the galactosyl donor is UDP-galactosamine.

4. The method according to claim 1, wherein the galactosyl acceptor is selected from the group consisting of D-chiro-inositol, L-chiro-inositol, myo-inositol, bornesitol, and scyllo-inositol.

5. The method according to claim 4, wherein the galactosyl acceptor is D-chiro-inositol.

6. The method according to claim 1, wherein the fagopyritol synthase is purified.