Cold acclimation in plants is associated with the expression of COR (cold-regulated) genes that encode polypeptides of unknown function. It has been widely speculated that products of these genes might have roles in freezing tolerance. Here we provide direct evidence in support of this hypothesis. We show that constitutive expression of COR15a, a cold-regulated gene of Arabidopsis thaliana that encodes a chloroplast-targeted polypeptide, enhances the in vivo freezing tolerance of chloroplasts in nonacclimated plants by almost 2؇C, nearly one-third of the increase that occurs upon cold acclimation of wild-type plants. Significantly, constitutive expression of COR15a also affects the in vitro freezing tolerance of protoplasts. At temperatures between ؊5 and ؊8؇C, the survival of protoplasts isolated from leaves of nonacclimated transgenic plants expressing COR15a was greater than that of protoplasts isolated from leaves of nonacclimated wild-type plants. At temperatures between ؊2 and ؊4؇C, constitutive expression of COR15a had a slight negative effect on survival. The implications of these data regarding possible modes of COR15a action are discussed.

The DNA sequences of cDNAs for two cor (cold-regulated) genes of Arabidopsis thaliana L. (Heyn) were determined. One cDNA (approximately 70% full-length) corresponds to a cor gene, designated cor47, that encodes a 47 kDa hydrophilic polypeptide. The data indicate that COR47 has amino acid sequence homology with Group II LEA (late embryogenesis abundant) proteins, a class of proteins that accumulate late in embryo development. DNA sequence analysis of a second cDNA (containing the complete protein coding sequence) indicates that it represents a cor gene, designated cor6.6, that encodes an alanine-rich 6.6 kDa hydrophilic polypeptide. COR6.6 is almost identical to KIN1, a cold-regulated Arabidopsis gene that has been suggested to have amino acid sequence similarities with type I fish antifreeze proteins (S. Kurkela, M. Franck, Plant Mol Biol 15: 137-144, 1990). Northern analysis indicated that transcripts for cor47 and cor6.6 do not accumulate to high levels in late-developing embryos or fresh mature seeds as is typical of lea gene transcripts. The similarities and differences between COR and LEA proteins are discussed as are their possible roles in freezing and drought tolerance.

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NAD-malic enzyme (NAD-ME) is a primary regulatory enzyme for the metabolism of malate in plant mitochondria. NAD-ME serves an anaplerotic function for the production of pyruvate, and provides CO, for retixation in the Calvin cycle in certain C, and Crassulacean acid metabolism plants. Clues regarding the mechanism of control of NAD-ME in vivo come from numerous studies on the physical and kinetic properties of the enzyme. The kinetics are complex and are altered by the pH of the assay medium as well as by serveral effecters, including divalent cations. CoA, sulfate, acetyl CoA, and fructose 1,6-bisphosphate (activators) and chloride, citrate, and bicarbonate (inhibitors). The enzyme is functional as a dimer, tetramer and octamer and the variation in kinetics is at least in part due to its association/dissociation.