The effects of temperature on a 58-kDa phosphoprotein (PP58) have been examined in cell-free extracts of two alfalfa (Medicago sativa L.) cultivars, Apica and Trek. In the extracts prepared without the use of Triton X-100, PP58 is present in a 12,000 x g (P12), 28,000 x g (P28) and 100,000 x g (P100) pellets but is enriched in the P28 fraction. In these fractions PP58 is substantially and equally phosphorylated at both 4° and 24°C. When extracts are prepared in the presence Triton X-100, PP58 is present in the 28,000 x g supernatant (TXS fraction) is extensively dephosphorylated at 24°C but highly phosphorylated at 4°C. The phosphorylation of this protein increased sharply as temperature declined below 12°C, and was 15 times greater at 0° than at 24°C. The phosphorylation level doubled between 12° and 8°C and again between 8° and 4°C. Thus temperature effect is not mediated by Q10 effect. Interestingly, temperature-response curve of PP58 phosphorylation is similar to that of the reported cold-induced calcium influx (Plant Cell 7: 321-331). Labeling reactions carried out in the presence of [gamma-35S]thioATP indicated that low temperature inhibited the dephosphorylation reaction. These results were not mimicked at room temperature by the protein phosphatase 1 and 2A inhibitor okadaic acid. In reactions performed at 4°C, addition of calcium caused a 2-fold increase in the phosphorylation of PP58. A decrease in phosphorylation was observed when equimolar amounts of EGTA were added in the presence of MgCl 2 or MnCl2, but not in the presence of CaCl2, suggesting that this protein is phosphorylated by a calcium-dependent protein kinase. These results are consistent with the suggestion that PP58 and its putative kinase are membrane-localized whereas the putative PP58 phosphatase is a loosely-associated membrane peripheral protein lost to the supernatant during fractionation. We suggest that PP58 could be involved in low te

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Phosphoenolpyruvate carboxylase (PEPC) was examined during cold acclimation of seedlings of the freezing-tolerant cultivar (Medicago sativa ssp falcata cv Anik) and the relatively freezing-sensitive cultivar (Medicago sativa cv Trek) of alfalfa. With four days of cold acclimation, PEPC activity increased to 3.5-fold and 2-fold the control levels in Anik and Trek, respectively. This was associated with an increase in the level of a 110 kD PEPC protein and a decrease in the amount of a 120 kD PEPC polypeptide in both cultivars. The role of reversible phosphorylation in regulating PEPC activity was demonstrated by in vitro phosphorylation and dephosphorylation, which caused partial activation and deactivation of PEPC, respectively. In vivo phosphorylation experiments revealed that the 110 kD PEPC subunit is phosphorylated on serine residue(s) during cold acclimation in Anik but not in Trek. Increased PEPC activity could account for the 70% increase in the non-autotrophic or dark fixation of carbon observed in cold acclimated Anik seedlings. A possible role for dark carbon fixation in the cold-induced development of freezing tolerance is through the production of NADPH. Such a source of reducing power may be required for the repair of cold-induced damage and restoration of normal cellular functions.

During the present studies we attempted to answer the following questions: (i) Does low temperature alter the phosphorylation level of proteins in isolated nuclei? (ii) Does the nuclear phosphoprotein population change during a prolonged exposure of seedlings to cold? (iii) Do heat-stable proteins accumulate in the nucleus during a prolonged exposure of seedlings to cold? (iv) Are the answers to the above three questions related to freezing tolerance? A possible relationship between the observed cold-induced changes in phosphoproteins and the level of freezing tolerance was explored by comparing the results of experiments conducted on two cultivars (Apica and Trek) of alfalfa (Medicago sativa L.) which markedly differ in their capacity for cold acclimation. / We show that the phosphorylation level of several nuclear proteins is subject to rapid and reversible enhancement by low temperature. Several phosphoproteins were found to be constitutively present in the nucleus of both cultivars. The cold-induced stimulation of the phosphorylation of many of these proteins was much greater in the relatively freezing tolerant cultivar Apica than in the relatively freezing sensitive cultivar Trek. Population of nuclear phosphoproteins was found to be considerably more complex in Apica than in Trek. During a prolonged exposure of the seedlings to 4$ sp circ$C, additional phosphoproteins were imported into the nucleus of Apica seedlings but not those Trek. / Some heat-stable proteins were constitutively present in the nucleus of both cultivars. However during the 4-day cold treatment, a large accumulation of several additional heat-stable proteins was observed in the tolerant, but not the sensitive, cultivar. (Abstract shortened by UMI.)

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To identify a model legume experimental system for studying low temperature signaling and cold acclimation, cold-induced expression and regulation of homologues of alfalfa (Medicago sativa) cold acclimation-specific genes cas15 and cas30 were examined in M. arborea (relatively frost tolerant) and M. truncatula (relatively frost sensitive). Both cas15 and cas30 genes are present in the genomes of both species but whereas both genes are cold-induced in M. arborea, only cas15 is induced in M. truncatula. Cold-induced expression of these genes is inhibited by calcium chelators and channel blockers and by the membrane fluidizer benzyl alcohol. Treatment of leaves with dimethylsulfoxide, a membrane rigidifier, induced both genes at 25°C. A cold-activated MAP kinase activity was expressed in both species. These results suggest that M. truncatula, an annual, self-pollinated species may be successfully used as model experimental systems in studies of cold signaling and role of cas genes in cold acclimation in legumes.

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