Exposure of spinach (Spinacia oleracea L.) plants to a constant 50C induced a greater tolerance to extracellular freezing. The metabolic basis of this cold acclimation response in plants is not understood. In this study we tested the hypothesis that cold acclimation derives from altered gene transcription. We found that exposure of plants to low temperature resulted in a rapid and stable change in the translatable poly(A)+ RNA populations extracted from leaves, as determined by a cell-free in vitro translation assay. The initial appearance of mRNAs for two high molecular weight translation products correlated with an increase in freezing tolerance. Cold acclimation of plants for 8 days resulted in further qualitative changes in mRNA populations. At least four additional mRNAs increased in concentration upon continued exposure of spinach to 50C, whereas three other mRNAs present in 20'C-grown leaves decreased. We also tested the possibility that the low temperature-induced mRNAs might encode heat shock proteins. We studied heat shock-induced protein synthesis by in vivo labeling techniques and found that spinach synthesized at least eight distinctive heat shock proteins during exposure to 400C. Most polypeptides induced by exposure to low temperature, however, appeared not to be heat shock proteins. Thus, the change in mRNAs induced by low temperature is a separate response from that induced by high temperature.The increase in freezing tolerance of plants during cold acclimation has been proposed to result from a combination of physiological changes and metabolic alterations that depend on altered gene expression (1). A number of earlier studies described the changes in physiology, metabolism, or enzyme activity associated with the induction of greater freezing tolerance (2). Several of these studies demonstrated an appearance and disappearance of certain membrane proteins (3, 4) and changes in the isozyme composition of a number of enzymes (5-10). Although evidence does exist for altered gene expression in other types of plant stress responses (11, 12), direct evidence of altered gene expression during cold acclimation has not been presented.We report here data that indicate a qualitative alteration in the population of translatable mRNA during cold acclimation in spinach. We have extracted RNA from nonacclimated and cold-acclimated spinach leaves, prepared and translated in vitro the mRNA fraction, and by electrophoresing the translation products found that development of increased freezing tolerance is correlated with the rapid appearance of two new, continuously expressed species of mRNA. MATERIALS AND METHODSPlant Material and Cold Acclimation. Leaves of spinach, Spinacia oleracea L. cv. Bloomsdale (Northrup King), were used for all studies. Plants were grown in a controlled environment growth chamber with an air temperature at plant height of 20 ± 10C in the light and 17 ± 10C in the dark. Illumination (12 hr/day) was provided by cool white fluorescent tubes and supplemented with incandescent lighti...

Cold‐acclimated stems of red‐osier dogwood (Cornus sericea L.) were sampled in midwinter and early spring and subjected to the following low temperature treatments: (a)0 →−40 → 0°C; (b) 0 →−40 →− 196 → 0°C; (c) 0 →−40 →−196 →−269 →−196 → 0°C; (d) 0 →−40 →−269 →−196 → 0°C; (e) 0 →−196 → 0°C; (f) 0 →−269 →−196 →0°C. The cortical parenchyma cells of the outer stem layers survived exposure to −269°C when pre‐frozen to −40°C and either transferred directly to −269°C or to −196°C and then to −269°C (treatments c and d). Acclimated stems transferred to a greenhouse (22°C) 2 weeks prior to the low temperature treatments deacclimated and were not able to survive freezing to −10°C. Cortical cells of stem samples taken in March, near the time when dogwood naturally deacclimates, survived −196°C (treatment b), but not −269°C (treatment cord). Thus, the freezing tolerance of dogwood varies seasonally from near −10°C to below −269°C.

The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with (3H-methyl]-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. One is a polypepfide with a molecular mass of 64 kD, a second has an Mr of 48 kD, and the third has a molecular mass of less than 10 kD. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide, having a molecular mass of 24 kD, is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. One methyllinkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile [3Hjmethyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo tumover of the [3H]methyl group.Many types of posttranslational modifications of proteins occur in both prokaryotic and eukaryotic organisms. Examples include phosphorylation, glycosylation, and methylation.Protein methylation in prokaryotes is exemplified by Escherichia coli and Halobacterium halobium. In E. coli chemotaxis, attractant or repellent chemicals interact with cytoplasmic-membrane receptor proteins that signal to the flagella to create the desired movement ofthe bacterium either toward or away from the chemicals; then this signal is terminated by glutamate-carboxyl methylation of these receptor proteins in the case of attractants and demethylation in the case of repellents (7,14,15,22,29,31). The methylating agent is AdoMet2 (23), and the demethylation product is methanol