N. Jerry Chatterton

Characterization of fructan oligomers from species of the genus Allium L.

Summary Storage tissue of leaf bases from several species of the genus Allium [ A. cepa L. var. cepa (onion, 6 cvs.), A. cepa L. var. ascalonicum (shallot, 7 cvs.), A. ampeloprasum L. var. porrum (leek, 3 cvs.), A. schoenoprasum L. (chives), A. sativum L. (garlic), A. fistulosum L. (Japanese bunching onion/Welsh onion), A. tuberosum Rottl. ex. spr. (Chinese chives/Nira), and other species] were analyzed to determine their water soluble carbohydrate composition. The Allium species analyzed can be divided into three groups according to their fructan profiles: 1. Those with relatively high amounts of larger fructan polymers, 2. Those with relatively high amounts of small fructan polymers up to a degree of polymerization of about 15, and 3. Those with both large and small fructan polymers. Four major fructan series with exclusively (2→1) fructosyl-fructose linkages have been characterized that are typical of those Allium species containing small fructan polymers. They are: 1. an inulin series with the general formula: G - 1 , 2 - F - 1 , ( 2 - F - 1 ) n , 2 - F ( G - 1 , 2 - F = s u c r o s e ) , 2. a neokestose-based series with chain elongation only at the glucose end of the original sucrose molecule: F - 2 , ( 1 - F - 2 ) m , 1 - F - 2 , 6 - G - 1 , 2 - F , 3. a neokestose-based series with elongation from both sides of the sucrose: F - 2 , ( 1 - F - 2 ) m , 1 - F - 2 , 6 - G - 1 , 2 - F - 1 , ( 2 - F - 1 ) n , 2 - F , and 4. an inulo- n -ose series without a terminal glucose F -1,(2-F-1) n ,2-F. While the first three fructan series were present in relatively high concentrations in all samples with high amounts of small fructans, the inulo- n -ose series was detectable in most samples, but in varying concentrations.

Fructan biosynthesis and fructosyltransferase evolution : Expression of the 6-SFT (sucrose : fructan 6-fructosyltransferase) gene in crested wheatgrass (Agropyron cristatum)

Summary Crested wheatgrass ( Agropyron cristatum ), an important cool-season grass, contains a complexity of fructans, including levans, inulins and graminans. Fructan types are characterized by unique combinations of carbon-carbon linkages. This study established the DNA sequence for the 6-SFT gene (sucrose : fructan 6-fructosyltransferase) in A. cristatum and compared the sequence with invertases and fructosyltransferases from various species. Detachment with illumination, as well as cool treatment of crested wheatgrass leaves induced the accumulation of fructans. All three types of fructans, β-2,1; β-2,6 and graminans (combination of both β-2,1 and β-2,6 linkages) were detected. The cDNA of 6-SFT was cloned from A. cristatum . The deduced amino acid sequences showed 85 percnt; identity with barley ( Hordeum vulgare ) 6-SFT. It also exhibited high homology with other fructosyltransferases and invertases. The expression of 6-SFT mRNA was analyzed with Northern hybridization. mRNA of the 6-SFT gene accumulated after 8 h of illumination in detached leaves and remained at a steady state for 36 hours. Under low temperature (10 °C/5 °C, day/night), the mRNA of 6-SFT began to accumulate after 8 h and reached the highest level at 2 d and then declined after 8 days of treatment. A coincidence of fructan and 6-SFT mRNA accumulation in crested wheatgrass suggests that 6-SFT has a role in fructan biosynthesis. A cladogram comparison of the amino acid sequences of most known sequences of fructosyltransferases and 23 invertases shows separations into various clades of like genes. 6-SFT does not contain the unique NDPNG amino acid sequence characteristic of invertases. Sequence analysis indicated that fructosyltransferases likely evolved separately in dicot and monocot species.

Characterization of fructan biosynthesis in big bluegrass (Poa secunda)

Summary Most cool-season grasses contain multiple types of fructans. One exception is big bluegrass (Poa secunda Presl.). When grown under specific controlled environmental conditions it synthesizes only β-2,6-linked fructans. This study analyzed fructan accumulations, enzyme activities and gene expression in big bluegrass. Detachment/illumination and cool treatments effectively induced the accumulation of fructans in leaf tissues. Enzyme assays indicated that 6-SST (sucrose : sucrose 6-fruc tosyltransferase)- and 6-SFT (sucrose : fructan 6-fructosyltransferase)-like activities were the major enzyme activities involved in fructan biosynthesis in big bluegrass leaves. A full-length cDNA of the putative 6-SFT gene was cloned using RT-PCR and RACE techniques. The deduced amino acid sequence showed 69 percnt; identity with barley 6-SFT. Homology was also high with other fructosyltransferases and some invertases. The abundance of putative 6-SFT mRNA showed a coincidence with fructan accumulation and 6-SFT activity. We suggest that 6-SFT is the major enzyme involved in fructan biosynthesis in big bluegrass but it may also exhibit limited 6-SST activity.

Fructan and total carbohydrate accumulation in leaves of two cultivars of timothy (Phleum pratenseVega and Climax) as affected by temperature

Fructans are thought to have a role in physiological responses and growth under low temperatures in cool-season grasses. Two cultivars of timothy (Phleum pratense), Vega, a Scandinavian cultivar, and Climax, introduced from England and currently grown in the U.S., were compared in relation to their response to different growth temperatures. Leaf blades of neither of the two cultivars accumulated any fructan of high degree of polymerisation (DP) in leaves when grown at constant day/night temperature of 20 °C. At temperatures lower than 20 °C, content of total carbohydrate increased, especially in Vega. At 10/5 °C (day/night), a pronounced accumulation of total carbohydrate and high DP fructans occurred. A pronounced accumulation occurred also when plants grown at 20 °C were transferred to 10 °C for several days before sampling. A decrease in temperature is thought to induce a temporary imbalance in source and sink activities. Accumulated fructan was in the 12-80 DP range. Only very small amounts of low DP oligosaccharides (DP 4-12) were detected. The two timothy cultivars showed no apparent qualitative difference in fructan accumulation. Generally, Vega had significantly higher total carbohydrate and fructan content than Climax, suggesting possible differences in genetic adaptation to cool growth temperatures.

Proton and carbon chemical-shift assignments for 1-kestose, from two-dimensional n.m.r.-spectral measurements

Abstract The proton chemical-shift assignment of a simple relative of inulin, namely, 1-kestose [O-β- d -fructofuranosyl-(2→1)-β- d -fructofuranosyl a- d -glucopyranoside], was determined by using two-dimensional (2D) homonuclear and heteronuclear n.m.r.-spectral methods, and corrections of, and additions to the 13C-chemical-shift assignments were made. The 1H peak of H-1 of the d -glucosyl group was determined by its chemical shift. Using H-H coupling information for this proton, the chemical shift of most of the proton signals of the β- d -glucosyl group was determined. Although the signals from the two d -fructosyl units were very similar, long-range C-H coupling allowed their complete C and H assignment. In particular, the coupling of the H-1 atom of d -glucose to C-2′ of d -fructose 1 allowed distinction between the two d -fructose units.

Carbohydrate changes in chicory (Cichorium intybus L. var. foliosum) during growth and storage

Abstract The levels of sucrose and fructose in chicory ( Cichorium intybus L. var. foliosum ) roots remain about the same over the growing season while levels of total non-structural carbohydrates (TNC) and fructans increase. At the onset of cooler fall temperatures and especially during storage, sucrose and fructose contents increase. Concurrently, a second fructan series that contains no glucose (inulo- n -ose; n = 2–18) accumulates, while at the same time, fructans (inulin) with a high degree of polymerization (DP) and TNC decrease. Chicory leaves contain low concentrations of fructans. Only trace amounts were found in the leaf lamina but leaf petioles, especially in the basal region, contained higher levels. The inulo- n -ose series up to DP 4 (inulotetraose) is also detectable in leaves.

Proton and carbon chemical-shift assignments for 6-kestose and neokestose from two-dimensional n.m.r. measurements☆

The proton and carbon chemical shift assignments of the simplest levan, 6-kestose, [O-beta-D-fructofuranosyl-(2----6)-beta-D-fructofuranosyl-(2 in equilibrium with 1)-alpha-D-glucopyranoside], along with another trisaccharide, neokestose, [O-beta-D-fructofuranosyl-(2----6)-alpha-D-glucopyranosyl-(1 in equilibrium with 2)-beta-D-fructofuranoside], were determined using two-dimensional (2D) homonuclear and heteronuclear n.m.r. methods. The 1H peak of H-1 of the glucose residue was determined by its chemical shift. Using H-H coupling information to this proton, the chemical shifts of most of the proton signals of the glucose moiety were determined. Though the signals from the two fructose residues were very close, a NOESY experiment and long-range C-H correlation experiments allowed their complete carbon and proton assignment. This work completes and corrects literature assignments.

Fructans in crested wheatgrass leaves

Crested wheatgrass is an important cool-season grass that has become naturalized in many semiarid regions of the western U.S. It provides ground cover and reduces soil erosion caused by water and wind. Additionally, crested wheatgrass produces important forage for livestock and wildlife on 6 to 8 million hectars of western rangeland. It is well adapted to semiarid cold desert regions because of its cool temperature growth and drought tolerance. Understanding the biosynthesis of fructans in crested wheatgrass is important because of their likely role in both cool temperature growth and drought tolerance. Recent research described a major gene (6-SFT) in crested wheatgrass that is involved in fructan biosynthesis. 1-kestotriose, the major DP3 fructan in crested wheatgrass, serves as the substrate for the two major DP4 fructans, 1&6-kestotetraose and 1,1-kestotetraose. The three major DP5 fructans are 1&6,1-kestopentaose, 1,1&6-kestopentaose and 1,1,1-kestopentaose. The major DP6 fructan is 1&6, 1&6-kestohexaose. We postulate that 1&6,1&6-kestohexaose is synthesized from the addition of a fructose to 1&6, 1-kestopentaose. This paper provides structures of the various DP 3, 4, 5 and 6 fructan types produced by crested wheatgrass and provides suggested biosynthetic pathways for all major fructan linkage types present.

Selection response for molecular markers associated with anthocyanin coloration and low-temperature growth traits in crested wheatgrasses

Hycrest is an outcrossing tetraploid cultivar of crested wheatgrass developed from a hybrid between an induced tetraploid form of Fairway (Agropyron cristatum) and natural tetraploid Standard (A. desertorum). The CD-II cultivar was selected from cv. Hycrest on the basis of vigorous vegetative growth and green leaf coloration during early spring. This study examines the selection response of molecular markers associated with anthocyanin coloration (AC), growth habit (GH) and other traits in Hycrest, CD-II, and two second-generation polycross (PX2) populations derived from three purple-leaf selections and three green-leaf selections of Hycrest. AC was positively correlated with prostrate GH and inversely correlated with plant height and leaf width in the experimental PX2 populations. Of the 578 AFLP markers surveyed, 13 showed pleiotropic effects on GH and AC in the PX2 populations. In all cases, marker alleles associated with prostrate GH also enhanced AC. Four of these 13 markers also showed large selecti...

Do Inulin Oligomers Adopt a Regular Helical Form in Solution

Abstract The 13C NMR spectra of inulin oligomers in D2O with degree of polymerization (DP) of 3 through 9, along with two other inulin oligomer mixtures of average DP = 17 and DP = 31 were recorded. Significant variations in the chemical shift of some fructofuranose carbon signals indicates that unlike glucans, simple helical structures are not the predominant conformation for inulin oligomers—at least up to DP = 9. Models of the DP = 5 oligomer show that it should prefer a single helical conformation which however, would not be accessible to longer DP oligomers due to severe steric interactions.