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Kevin E. McPhee, Robert S. Zemetra, Jack Brown, and James R. Myers

Common bean (Phaseolus vulgaris L.) is a nutritionally complete food, but contains antinutritional compounds that reduce digestibility. One group of compounds includes the raffinose family oligosaccharides (RFOs) (raffinose, stachyose, and verbascose), which are partly responsible for flatulence after beans are eaten. RFOs stabilize cell membranes during seed desiccation and when the seed rehydrates during germination. While low levels of RFOs are desirable nutritionally, high levels may enhance germination and emergence, particularly in cold, wet soils. Eight landraces selected for high and low sucrose, raffinose, and stachyose content, were crossed in a diallel mating design to investigate genetic control of the RFOs. Derivatized soluble sugars were measured using gas-liquid chromatography. Fructose, sucrose, raffinose, and stachyose were detected. In the F1, fructose varied from 0.1 to 2.5 mg·g-1 dry weight (DW), sucrose from 17.2 to 56.5 mg·g-1 DW, raffinose from 0.1 to 4.1 mg·g-1 DW, and stachyose ranged from 7.6 to 43.7 mg·g-1 DW. Griffing's analysis estimates of general combining ability were on average, 16.5 times larger than specific combining ability for all the RFOs, indicating that additive genetic variance was most important. Significant reciprocal differences were detected in the F1 and F2, but not in the F3. RFO accumulation was partially dominant as indicated by Hayman's analysis. Narrow sense heritability averaged over F2 and F3 generations for sucrose, raffinose, stachyose, total sugar, and total oligosaccharides were 0.22, 0.54, 0.44, 0.17, and 0.27, respectively. Moderate heritabilities indicate that manipulation of RFO accumulation in this set of bean lines would probably need to be done on a progeny row basis with replication.

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L.E. Hinesley, D.M. Pharr, L.K. Snelling, and S.R. Funderburk

Foliar raffinose and sucrose concentrations in eastern white pine (Pinus strobus L.), eastern redcedar (Juniperus virginiana L.), Leyland cypress (×Cupressocyparis leylandii Dallim.), and Virginia pine (Pinus virginiana L.) were measured monthly over 2 years. During cold weather, foliage of white pine and redcedar contained higher concentrations of raffinose and sucrose than did Leyland cypress and Virginia pine. Rafflnose concentrations were highest during winter and were best correlated with the frequency of occurrence of daily minima ≤ 1.7C during the 30 days before sampling. Sucrose concentrations, which also reached maximum levels during the winter, were best correlated with the frequency of occurrence of daily minima ≤ 7.2C in the prior 30 days. Sucrose concentrations were relatively high during fall and spring. Raffinose and sucrose concentrations increased in response to recurring low temperature, with correlations highest for raffinose.

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Alicia M. Borowski, Vincent A. Fritz, and Luther Waters Jr.

This study was conducted to determine if changes in the raffinose: sucrose ratio in embryos of shrunken-2 sweet corn (Zea mays L.) hybrids were related to differences in seed leachate conductivity between two hybrids harvested at four maturities and artificially dried to 0.10 g H2O/g fresh weight. The ratio of raffinose: sucrose differed for `Crisp N' Sweet 710' (CNS) and `How Sweet It Is' (HSII). The mass ratio of raffinose: sucrose in CNS was >0.3 in seed harvested between 0.44 to 0.64 g H2O/g fresh weight and increased as seed dried from the initial harvest moisture to 0.10 g H2O/g fresh weight. Raffinose: sucrose ratios of HSII were <0.3 at all harvests between 0.55 to 0.72 g H2O/g fresh weight, but changes during desiccation were not as pronounced. Leachate conductivity of whole seeds of CNS and HSII decreased as seeds were harvested at progressively lower moisture contents. We suggest that a higher raffinose: sucrose ratio may be indicative of increased seed vigor in shrunken-2 hybrids.

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V. Esensee, R. Remmele, C. Stushnoff, and M. McNeil

Woody plants can be induced to cold-acclimate by exposure to sublethal low temperatures, but only after the onset of vegetative maturity. We monitored seven woody plant taxa, at monthly intervals, to determine the date of vegetative maturity, freeze-killing temperature, cell membrane electrolyte leakage, and the quantity and diversity of endogenous oligosaccharides. The freeze-killing temperature changed from -5 to -7C before vegetative maturity to -15 to -20C after vegetative maturity. There was a 10-fold increase in raffinose and about a 3-fold increase in endogenous stachyose in samples that were cold-acclimated under controlled conditions. In field samples, endogenous raffinose increased from <0.02% in August to 2% to 11% in cortical stem tissues of all cold-acclimated taxa. The tetrasaccharide stachyose increased from <0.02% to 0.25% to 2.5% for similar comparisons. None of the other sugars or polyols showed similar, consistent patterns during the onset of cold acclimation. In response to low temperature, raffinose family oligosaccharides (RFOs) have previously been shown to increase substantially in cabbage, soybean, kidney bean, and Chlorella. RFOs also possess high water-binding characteristics and tend to enhance aqueous glass transitions. Accordingly, we hypothesize that the endogenous production of these oligosaccharides may play an important role in metabolic events associated with cryoprotection of critical cellular functions during low-temperature stress.

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Yu-Jen Chiang, C. Stushnoff, A.E. McSay, M.L. Jones, and H.J. Bohnert

Petunia ×hybrida (Hook) Vilm. cv. Mitchell was transformed with an E. coli gene encoding mannitol-1-phosphate dehydrogenase (mtlD). Four plant lines that grew on kanamycin and contained the mtlD transgene were identified. Two of these lines contained high levels of mannitol [high-mannitol lines M3 and M8; mean mannitol = 3.39 μmol·g-1 dry weight (DW)] compared to nontransformed wild-type plants (0.86 μmol·g-1 DW), while two lines had mannitol levels similar to wild-type plants (low-mannitol lines M2 and M9; mean mannitol = 1.05 μmol·g-1 DW). Transgenic and control plants were subjected to chilling stress (3 ± 0.5 °C day/0 ± 0.5 °C night, 12-hour photoperiod and 75% relative humidity) to evaluate the role of mannitol in chilling tolerance. Based upon foliage symptoms and membrane leakage after a 3-week chilling treatment, the high-mannitol containing lines, M3 and M8, were more tolerant of chilling stress than the low-mannitol containing transgenic lines, M2 and M9, and wild-type. Under nonchilling conditions mannitol was the only carbohydrate that differed among transgenic lines, but all carbohydrates were present. When subjected to chilling stress, mannitol levels dropped by 75%, sucrose by 52%, and inositol by 54% in the low-mannitol lines (M2 and M9). In M3 and M8, the high-mannitol lines, mannitol levels decreased by 36%, sucrose by 25%, and inositol by 56%, respectively. Raffinose increased 2- to 3-fold in all lines following exposure to low-temperature chilling stress. In the higher mannitol lines only 0.04% to 0.06% of the total osmotic potential generated from all solutes could be attributed to mannitol, thus its action is more like that of an osmoprotectant rather than an osmoregulator. This study demonstrates that metabolic engineering of osmoprotectant synthesis pathways can be used to improve stress tolerance in horticultural crops.

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S. Ball, Y.L. Qian, and C. Stushnoff

No information is available regarding endogenous soluble carbohydrate accumulation in buffalograss [Buchloe dactyloides (Nutt.) Engelm.] during cold acclimation. The objective of this study was to determine composition of soluble carbohydrates and their relationship to freezing tolerance in two buffalograss cultivars, 609 and NE 91-118, with different freezing tolerances. The experiment was conducted under natural cold acclimation conditions in two consecutive years in Fort Collins, Colo. Based upon average LT50 (subfreezing temperature resulting in 50% mortality) from seven sampling intervals in 1998-99 and six sampling intervals in 1999-2000, `NE 91-118' survived 4.5 °C and 4.9 °C colder temperatures than `609', during the 1998-1999 and 1999-2000 winter seasons, respectively. Glucose, fructose, sucrose, and raffinose were found in both cultivars in both years, and were generally higher in acclimated than pre- and post-acclimated stolons. Stachyose was not present in sufficient quantities for quantification. Cultivar NE 91-118 contained 63% to 77% more glucose and 41% to 51% more raffinose than `609' in the 1998-99 and 1999-2000 winter seasons, respectively. In 1999-2000, fructose content in `NE 91-118' was significantly higher than that of `609'. A significant negative correlation was found between LT50 vs. all carbohydrates in 1999-2000, and LT50 vs. sucrose and raffinose in 1998-99. Results suggest that soluble carbohydrates are important in freezing tolerance of buffalograss.

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Manfredo J. Seufferheld, Cecil Stushnoff, Philip L. Forsline, and Gerardo H. Terrazas Gonzalez

Unlike cold-hardy apple germplasm, dormant vegetative buds from cold-tender accessions require stabilization of meristematic tissue to protect against injury during desiccation and cryopreservation. Dormant buds of six apple cultivars [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf. `Cox's Orange Pippin', `Einshemer', `Golden Delicious', `Jonagold', `K-14', and `Mutsu'] collected at specific intervals in 1993, 1994, and 1995 at Geneva, N.Y., were stabilized by encapsulation in 5% alginate, treated with step-wise imbibition of 0.5 to 1.0 m sucrose and 0.2 m raffinose solution, and desiccated with forced air at 0 °C. Sugar-alginate stabilization reduced injury during desiccation, increased cold-hardiness of the six cold-tender cultivars frozen to -30 °C, and improved recovery following cryopreservation of buds collected before optimal cold acclimation was attained. Sucrose tissue levels did not increase following stabilization treatment, but levels of glucose and fructose, and of an unknown disaccharide increased. This procedure used nontoxic cryoprotectants, and has potential to expand the scope of dormant bud cryopreservation to include cold-tender apple germplasm.

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Cindy L. Flinn and Edward N. Ashworth

The accumulation of total soluble sugars (TSS) and starch and their relationship to flower bud hardiness were studied in three Forsythia taxa: Forsythia ×intermedia `Spectabilis', Forsythia ×intermedia `Lynwood', and F. suspensa. Taxon hardiness was based on the mean temperature at which low temperature exotherms (LTEs) occurred during thermal analysis. Ethanol-extracted soluble sugars were quantified with anthrone, and starch was enzymatically digested and quantified with Trinder reagent. Qualitative changes in sugar content were determined with high-performance liquid chromatography and co-chromatography of authentic standards. Quantitative and qualitative changes in sugar content, similar for the three taxa, were observed in conjunction with fluctuations in flower bud hardiness, although neither TSS nor starch were correlated with mean LTE temperature. TSS was higher in acclimated than nonacclimated buds. However, after deacclimation began, sugars continued to increase with mean LTE temperature. Buds lacked starch except for a brief period during deacclimation. Galactose, stachyose, raffinose, and an unidentified carbohydrate were positively correlated with hardiness (P = 0.005, 0.001, 0.005, and 0.001, respectively).

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Nathan Phillips, Andrew Reynolds, and Frederick Di Profio

amounts of soluble sugars appear during winter depending on temperature ( Zapata et al., 2004 ). Soluble sugars consist primarily of sucrose, glucose, and fructose; however, oligosaccharides raffinose and stachyose are also present in winter ( Jones et al

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Yansheng Li, Ming Du, Qiuying Zhang, Guanghua Wang, Jian Jin, Stephen Herbert, and Xiaobing Liu

(250 mm × 4 mm i.d.) at a flow rate of 0.5 mL·min −1 . The mobile phase was distilled water. The detection was accomplished using a RI2414 detector. Commercial sugars, including glucose, fructose, sucrose, raffinose, and stachyose, purchased from Sigma