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Vanessa S. Gordon and Jack E. Staub

these studies were found to perform similarly in response to chilling stress, mean damage ratings were, on average, slightly less in Chung et al. (2003) . Likewise, because the conditions in our study are those set forth by Chung et al. (2003) , the

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Eleazar Reyes and Paul H. Jennings

The effect of chilling stress on induction of the cyanide-resistant pathway was investigated using roots of 3-day-old cucumber (Cucumis sativus L.) grown at 26C and then chilled at 2C, 10C, or 15C for 24, 48, 72, and 96 h. A 24-h post-chilling treatment was imposed on different sets of chilled cucumber roots at 26C. Exposing seedlings to 2C, 10C, and 15C, as well as to a post-chilling treatment, induced differential responses in the activity of the cyanide-resistant pathway. Cucumber seedling roots exhibited an increase in the cyanide-resistant pathway after a 96-h chilling treatment at 2C. The involvement of the cyanide-resistant pathway in the chilling stress response will be discussed.

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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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Windy Boyd and Paul H. Jennings

Previous experiments have shown that chilling-stressed cucumber seedlings treated with ethanol have greater chilling tolerance when compared to untreated seedlings. To determine whether this increased chilling tolerance would diminish with time after treatment, cucumber seedlings were treated with ethanol and placed at chilling temperatures for 0, 2, 4, 6, or 8 hours after ethanol treatment. Ethanol-induced chilling tolerance declined as the time interval between treatment and chilling exposure increased. A second ethanol treatment was given 3 hours after the first treatment in an attempt to extend the enhanced chilling tolerance response. Ethanol has been reported to function as an anesthetic in some systems, interacting with cellular membranes. To determine the effect of ethanol and chilling on membrane integrity, a malondialdehyde assay was used. Since chilling stress effects may result from accumulation of active oxygen species, the activity of one radical scavenging enzyme (catalase) has been assayed. Ethanol treatment resulted in a rapid increase in catalase activity, and was associated with increased chilling tolerance. The effect of a second ethanol treatment will be discussed as related to induced chilling tolerance, membrane effects, and catalase activity.

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Julián Miralles-Crespo, Juan Antonio Martínez-López, José Antonio Franco-Leemhuis, and Sebastián Bañón-Arias

Physiological and biochemical indicators that reflect the responses of plants to chilling stress could be useful for identifying plant damage caused by freezing or other stresses. The objective of this study was to determine any relationship between changes in chlorophyll fluorescence and the appearance of visual symptoms resulting from freezing temperatures in two cultivars of oleander. In the least frost-sensitive cultivar (yellow oleander), freezing temperatures (–4 °C for 3 h) did not produce changes in the photochemical parameters. In the more frost-sensitive cultivar (pink oleander), non-photochemical quenching (NPQ) and the maximum photochemical efficiency of photosystem II (Fv/Fm) decreased after the same freezing treatment. The first of these potential indicators remained low, whereas the second steadily recovered during the 4 months after freezing simulation. The results suggest that measuring chlorophyll fluorescence may provide a rapid method for assessing freezing injury in oleander.

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Stephen P. Lee, Paul M. Chen, Tony H.H. Chen, Diane M. Varga, and Eugene A. Mielke

A proportion of `d'Anjou' pear fruit (Pyrus communis L.) developed a disorder, “black speck” or “skin speckling”, after prolonged controlled atmosphere (CA) storage (1% O2, - 0.5 C). A comparative study of biochemical components revealed that there was no significant difference in succinic, citric, fumaric, and pyruvic acids between the speckled' and normal skin tissues. The content of malic acid in the affected tissue was almost three times lower than that in the normal tissue. The specific activity of NADP-malic enzyme (EC 1.1.1.40) in the affected tissue was also lower, but the total activities were similar. The affected tissue contained higher percentages of dry matter and soluble proteins than the normal tissue. Two-dimensional gel electrophoresis of proteins showed that two groups of novel polypeptides appeared only in the affected skin tissue. This study indicated that a certain proportion of `d'Anjou' pear fruit might have been exposed to unfavorable preharvest environmental stresses, and, therefore, could no longer tolerate the subsequent semi-anaerobic and chilling stresses during prolonged CA storage.

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Kanogwan Kerdnaimongkol and William R. Woodson

Transgenic tomatoes (Lycopersicon esculentum Mill. `Ohio 8245') expressing an antisense catalase gene (ASTOMCAT1) were used to test the hypothesis that modification of the reactive oxygen species scavenging mechanism in plants can lead to changes in oxidative stress tolerance. A 2- to 8-fold reduction in total catalase activity was detected in the leaf extracts of transformants. A 2-fold increase in levels of H2O2 was observed in the transgenic plants with reduced catalase activity. Electrophoretic characterization of multiple catalase isoforms revealed the specific suppression of CAT1 in transgenic plants. Homozygous plants carrying the antisense catalase transgene were used to study the effect of alteration in the expression of catalase on stress tolerance. Transgenic plants treated with 3% H2O2 showed visible damage within 24 hours and subsequently died. In contrast, wild-type and azygous control plants recovered from the treatment. Transgenic plants did not survive 4 °C chilling stress compared to control wild-type and azygous lines. Physiological analysis of these plants indicated that suppression of catalase activity in transgenic tomato led to enhanced sensitivity to oxidative stress. Our data support a role for catalase in oxidative stress defense system in tomato.

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Eleazar Reyes and Paul H. Jennings

The effect of chilling stress on respiration and induction of the cyanide-resistant pathway was investigated using roots of cucumber and pea grown at 26°C and 23°C and exposed to 2°C,. 10°C or 15°C for either 24 or 96h. Oxygen uptake of 2°C treated cucumber roots decreased between 24 and 96 h of chilling and then dramatically increased between 96 and 192h. The cyanide-resistant pathway did not change in cucumber roots at 10°C or 15°C for 24h or 96h, nor after a 24h recovery period at 26°C. At 2°C, cyanide resistant O2 uptake increased during the 24h recovery period following a 24h chilling but not after 96h chilling. Cyanide resistant oxygen uptake in pea roots was unaffected by 10°C and 15°C for 24h or 96h and a 24h recovery at 23°C. At 2°C, no effect in cyanide resistant O2 uptake was observed by 24h chilling but a significant increase occurred after 96h which returned to pre-stress levels with a 24h recovery at 23°C.

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Eleazar Reyes and Paul H. Jennings

The effects of chilling stress on respiration and induction of the alternative pathway (AP) were investigated with roots of 3-day-old cucumber seedlings (Cucumis sativus L.) grown at 26 °C and then chilled at 2, 10, or 15 °C for 24, 48, 96, 144, or 192 hours. Oxygen uptake by roots exposed to 2 °C was significantly lower than by 10 or 15 °C-treated roots, and the inhibition of oxygen uptake in the presence of SHAM (salicylhydroxamic acid) increased from 29% in unchilled tissue to 60% after 96 hours of treatment. At 10 and 15 °C, the capacity of the AP was nearly double that of the unchilled control and the 2 °C-treated seedlings. A 24 hours, postchilling treatment at 26 °C resulted in greater oxygen uptake at all temperatures as treatment time increased up to 96 hours. At 2 °C, the capacity of the AP was significantly reduced below the level of the 10 and 15 °C-treated tissue and the untreated control. The activity of the AP became fully activated by 96 hours in roots chilled at 2 °C. Results suggest that the capacity of the AP can be affected by low-temperature treatments, and exposure to 2 °C for up to 96 hours leads to a significant loss in capacity of the AP.

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Eleazar Reyes and Paul H. Jennings

The effect of chilling stress on induction of the cyanide-resistant pathway was investigated using roots of 3-day-old cucumber (Cucumis sativus L.) and 5-day-old pea (Pisum sativum L.) grown at 26°C, and then chilled at 2°C for 48 or 96 hours for cucumber, and 72 or 192 hours for pea. A 24-hour post-chilling treatment at 26°C was imposed on different sets of chilled roots from both crops. Carbohydrate status was determined by gas chromatography with an autosampler using a 12.5-m cross-linked methyl silicone capillary column (0.1 mm). Exposing seedlings to 2°C, as well as to a postchilling treatment, induced differential responses in the activity of the cyanide-resistant pathway. Cucumber seedling roots exhibited an accumulation of fructose, glucose and sucrose during chilling, with a rapid decline observed during the post-chilling treatment at 26°C. Pea seedling roots maintained a constant level of carbohydrates throughout the chilling period, and exhibited a slight decrease by the end of 192 hours at 2°C. There was an increase in carbohydrate levels during the post-chilling treatment. The involvement of the cyanide resistant pathway and carbohydrate changes will be discussed.