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Uemura, M., G. Warren and P. L. Steponkus (2003)
Freezing sensitivity in the sfr4 mutant of Arabidopsis thaliana is due to sucrose deficiency, and is manifested by loss of osmotic responsiveness.
Plant Physiology 131: 1800-1807.
Protoplasts were tested to determine whether the freezing-sensitivity of the sfr4 (sensitive to freezing) mutant of Arabidopsis thaliana was due to the mutant's deficiency in soluble sugars after cold acclimation. When grown under non-acclimated conditions, sfr4 protoplasts possessed freezing tolerance similar to that of wild type, with an LT₅₀ (the temperature at which 50% of protoplasts are injured) of -4.5℃. In both wild type and sfr4 protoplasts, at temperatures over the range of -2 to -4℃, expansion-induced lysis was the predominant lesion between -2 and -4℃, but its incidence was low (10%); below -5℃, loss of osmotic responsiveness was the predominant lesion. After cold acclimation, the LT₅₀ was decreased to only -5.6℃ for sfr4 protoplasts, compared to -9.1℃ for wild type protoplasts. Although expansion-induced lysis was precluded in both types of protoplasts, the sfr4 protoplasts remained susceptible to loss of osmotic responsiveness. After incubation of seedlings in Suc solution in the dark at 2℃, freezing tolerance and the incidence of freeze-induced lesions in sfr4 protoplasts were examined. The freezing tolerance of isolated protoplasts (LT₅₀ of -9℃) and the incidence of loss of osmotic responsiveness were now similar for wild type and sfr4. Thus, manipulation of sugar content by incubation of seedlings in a sucrose solution restored the phenotype of freezing tolerance of protoplasts isolated from the sfr4 mutant to that attained by the wild type after cold acclimation. These results indicate that the freezing-sensitivity of cold-acclimated sfr4 is due to its continued susceptibility to loss of osmotic responsiveness (presumably associated with lyotropic formation of the hexagonal II phase) and associated with the low sugar content of its cells.

Uemura, M. and P. L. Steponkus (2003)
Modification of the intracellular sugar content alters the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves.
Plant, Cell and Environment 26: 1083-1096.
Sugar content and freezing tolerance of protoplasts of Arabidopsis thaliana leaves were manipulated by incubating seedlings in a sucrose solution before protoplast isolation. Incubation in a 400 mM sucrose solution at 2℃ in the dark increased their freezing tolerance equivalent to that achieved after a conventional cold acclimation at 2℃. The increased freezing tolerance was due to a decrease in the incidence of freeze-induced lesions: expansion- induced lysis (EIL) between -2 and -4℃ and loss of osmotic responsiveness (LOR) between -5 and -12℃. The concentration of sucrose in the incubation medium required to minimize the incidence of the lesions was substantially different: 10 to 35 mM for EIL and 30 to 400 mM for LOR. Incubation in the sucrose solution at 23℃ decreased LOR only at -5 and -6℃ but less than that incubated at 2℃, and there was no effect on EIL. Incubation in sorbitol solutions at 2℃ also decreased LOR at -5 and -6℃ but much less than in the sucrose solution. These results suggest that low concentrations of sucrose are acted as a metabolic substrate for low-temperature-induced alterations required for the amelioration of EIL and, at higher concentrations, sucrose has a direct cryoprotective effect to minimize LOR.

Kawamura, Y. and M. Uemura (2003)
Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation.
Plant Journal 36: 141-154
Although enhancement of freezing tolerance in plants during cold acclimation is closely associated with an increase in the cryostability of plasma membrane, the molecular mechanism for the increased cryostability of plasma membrane is still to be elucidated. In Arabidopsis, enhanced freezing tolerance was detectable after cold acclimation at 2℃ for as short as 1 day and maximum freezing tolerance was attained after 1 week. To identify the plasma membrane proteins that change in quantity in response to cold acclimation, a highly purified plasma membrane fraction was isolated from leaves before and during cold acclimation, and the proteins in the fraction were separated with gel electrophoresis. We found that there were substantial changes in the protein profiles after as short as 1 day of cold acclimation. Subsequently, using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry, we identified 38 proteins that changed in quantity during cold acclimation. The proteins that changed in quantity during the first day of cold acclimation include those associated with membrane repair by membrane fusion, protection of the membrane against osmotic stress, enhancement of CO₂ fixation and proteolysis. To the best of our knowledge, this is the first report in which identification of cold-acclimation-associated proteins in a highly purified plasma membrane fraction was systematically performed.

Ito, K., Y. Onda, T. Sato, Y. Abe and M. Uemura (2003)
Structural requirements for the perception of ambient tempereture signals in homeothermic heat production of skunk cabbage (Symplocarpus foetidus).
Plant, Cell and Environment 26: 783-789.
The spadix of skunk cabbage, Symplocarpus foetidus, is capable of maintaining an internal temperature of around 20℃ even when the ambient temperature drops to around 0℃. To determine the crucial structure that is required for detection of ambient temperature signals, detailed measurements of the temperatures of the spadix were made under field conditions. The spadix temperature was well regulated even when the spathe or the leaf of the plant was removed. Furthermore, maintenance of the temperature of the central stalk at either 10 or 20℃ had no effect on the thermoregulation when the ambient temperature increased from 10 to 25℃ or decreased from 20 to 8℃. Therefore, it seemed that the heat production in the spadix required neither the spathe, the leaf, nor the central stalk for perception of the external temperature signals. Finally, analysis of sugar composition in xylem exudates showed that the concentrations of sucrose, glucose, and fructose, all of which are potential energy sources of thermogenesis, did not change significantly at different ambient temperatures. It is concluded that the spadix is a unique organ in which the perception of ambient temperature signals and heat production occurs in S. foetidus.

Ito, K., Y. Abe, S. Johnston and R. Seymour (2003)
Ubiquitous expression of a gene encoding for uncoupling protein isolated from the thermogenic inflorescence of the dead horse arum Helicodiceros muscivorus.
Journal of Experimental Botany 54: 1113-1114.
Uncoupling proteins (UCPs) are a family of mitochondrial inner membrane proteins that have been implicated in heat production in mammalian cells. The inflorescences of several members of the arum lily family (Araceae) have also been shown to produce heat during flowering, but the involvement of UCP- mediated heat production in plants is not known. Here we have isolated a gene termed HmUCPa that encodes for a putative uncoupling protein from Helicodiceros muscivorus, a highly thermogenic arum lily. RT-PCR analysis revealed that the expression of HmUCPa was ubiquitously found, both in thermogenic male florets and appendix, and the non-thermogenic female florets, spathe and club-shaped organs of the spadix. These results suggest that HmUCPa is not primarily involved in organ-specific heat production in H. muscivorus.

Seymour, M., M. Gibernau and K. Ito (2003)
Thermogenesis and respiration of inflorescences of the dead horse lily Helicodiceros muscivorus, a pseudo-thermoregulatory aroid associated with fly pollination.
Functional Ecology 17: 886-894.

1. In central Corsica, Helicodiceros muscivorus (Schott ex. K. Koch) produces a protogynous inflorescence that resembles the anal area of a dead mammal and produces a foetid scent during the few hours after sunrise. Flies enter the floral chamber, pollinate the female florets and become trapped until the next morning, when pollen is shed from the male florets and the flies are released.
2. The exposed appendix exhibits a strong, unimodal episode of thermogenesis associated with scent production, reaching a maximum of 30℃ at 15℃ ambient temperature. The male florets in the floral chamber are highly thermogenic throughout the second night and generally maintain stable floret temperatures of about 24℃ at ambient temperatures down to 13℃.
3. Maximum respiration rates of the appendix (0.45 μmol CO₂ s^-1 g^-1) and the male florets (0.82 μmol s^-1 g^-1) may be the highest recorded for plant tissue.
4. Thermogenesis of the appendix does not depend on ambient temperature, but that of the male florets increases with decreasing ambient temperature in most cases. However, the pattern of heat production by the males appears related more to time than to ambient temperature, hence the term 'pseudo- thermoregulation'.
5. The behaviour and thoracic temperatures of flies emerging from captivity suggests that male floral warming does not enhance their activity.

Niino, T., D. Tanaka, S. Ichikawa, J. Takano, S. Ivette, K. Shirata and M. Uemura (2003)
Cryopreservation of in vitro-grown apical shoot tips of strawberry by vitrification.
Plant Biotechnology 20: 75-80.
After the strawberry shoots on culture medium were cold-hardened at 5℃ for 20-30 days, shoot tips were dissected and precultured at 5℃ for 1 day on MS medium containing 2 M glycerol and 0.3 M sucrose. Precultured 10 shoot tips were placed in 2.0 ml cryotubes each, and treated with a mixture of 2 M glycerol and 0.3 M sucrose for 20 min at 25℃. They were then treated with PVS2 solution for 50 min at 25℃, and immersed in LN. After rapid rewarming in water at 35℃, they were transferred onto the MS medium containing 1 g l^-1 PVP and 0.2 mg l^-1 BA and grown under standard conditions. By this protocol, survival rate of cryopreserved "Donner" shoot tips reached 93%. This protocol was successfully applied to 7 other races or lines of strawberry with high survival rates. These results prompt us to setting up large-scale strawberry LN storage system.