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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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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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Abstract

Seasonal changes in soluble carbohydrates of Fraser fir [Abies fraseri (Pursh) Poir.] needles were monitored in Fall 1984, Spring 1985, and Fall 1985 through Spring 1986. Raffinose concentration increased in the fall and decreased in the spring. There was a 23-fold increase in raffinose concentration from Aug. 1985 to Jan. 1986. Sucrose concentration varied from fall to spring with the lowest concentration occurring in February. Postharvest needle abscission from harvested branches held 6 weeks without water was inversely correlated with raffinose concentration at the time of harvest. Diurnal fluctuations in soluble carbohydrates were monitored on 12 July and 26 Oct. 1985. Raffinose concentration fluctuated slightly on both dates with a decrease during the dark period. On 12 July, sucrose increased during the day and decreased at night, whereas hexoses decreased in the day and increased at night. No significant diurnal changes in sucrose or hexose were evident on 26 Oct. Controlled-environment studies at 24° (day)/18°C (night), 18°/12°, and 12°/6° showed that most of the raffinose accumulation was due to low temperature; the remainder to short days. Postharvest needle loss was lowest in plants with high needle raffinose concentrations resulting from the 12°/6° temperature. Storage without water resulted in significant postharvest needle loss for shoots from plants preconditioned with 24°/18° and 18°/12°, but not for those exposed to 12°/76°. Compared to long days, plants preconditioned with short days lost fewer needles following harvest.

Open Access

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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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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Abstract

Nonstructural carbohydrates of sweet cherry (Prunus avium L. ‘Bing’) changed dramatically both qualitatively and quantitatively during the year. In perennial tissues, total nonstructural carbohydrates (TNC) were highest at leaf abscission. TNC increased sharply in spurs at budbreak, but, in other perennial tissues, reserves decreased with or before budbreak. TNC in all but spurs were least, e.g., 2% to 4% of fall levels, shortly after full bloom, but then immediately began to increase. Accumulations slowed during the last 4 to 6 weeks of fruit growth and then increased after harvest. Prebloom decreases and postbloom increases occurred earlier in 1- and 2-year-old shoots when compared to trunk or root tissues. Starch was the most common storage material. During winter, interconversion of starch and soluble carbohydrates in wood of the trunk and 1- and 2-year-old shoots was apparent. Sucrose was the predominant soluble carbohydrate during dormancy, but sorbitol dominated during active growth. Raffinose was present only during dormancy, and inositol only when leaves were present. Because sweet cherry flowers and fruits early, carbohydrate reserves could critically affect productivity.

Open Access

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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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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Raffinose family oligosaccharides (RFOs) perform several physiological functions in plants. In addition to accumulating during seed formation, raffinose and stachyose are translocated in the phloem and may accumulate in response to low temperatures, drought, or salt stress. Although the synthesis of galactinol, as mediated by galactinol synthase (GAS), is the first committed step in RFO formation, its expression patterns are poorly understood in most species. We have cloned and characterized the expression of two galactinol synthase gene family members in melon (Cucumis melo L. Cantalupensis Group). Both CmGAS1 and CmGAS2 are highly expressed in mature leaves. Galactinol synthase transcription in leaves was not upregulated by either water or low temperature stresses. Transcripts of CmGAS1 were present in developing melon seeds at a time coincident with the formation of raffinose and stachyose. Based on the GAS expression and RFO accumulation patterns, we propose that RFOs in melon function in carbon translocation and seed desiccation tolerance.

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Previous studies of plant tolerance to low temperature have focused primarily on the cold acclimation response, the process by which plants increase their tolerance to freezing in response to low nonfreezing temperatures, while studies on the deacclimation process have been largely neglected. In some plants, cold acclimation is accompanied by an increase in raffinose family oligosaccharides (RFO). The enzyme α-galactosidase (EC 3.2.1.22) breaks down RFO during deacclimation by hydrolyzing the terminal galactose moieties. Here we describe the isolation of PhGAL, an α-galactosidase cDNA clone from Petunia (Petunia ×hybrida `Mitchell'). The putative α-galactosidase cDNA has high nucleotide sequence homology (>80%) to other known plant α-galactosidases. PhGAL expression increased in response to increased temperature and there was no evidence of developmental regulation or tissue specific expression. Increases in α-galactosidase transcript 1 hour into deacclimation corresponded with increases in α-galactosidase activity and a concomitant decrease in raffinose content, suggesting that warm temperature may regulate RFO catabolism by increasing the transcription of the α-galactosidase gene. This information has potential practical applications whereby α-galactosidase may be targeted to modify endogenous raffinose accumulation in tissues needed for freezing stress tolerance.

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