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This investigation was part of a continuing effort to ascertain the role of soluble proteins in the development of cold tolerance of alfalfa, Medicago sativaL. The research was conducted to determine whether certain soluble proteins, known to be closely associated with the development of cold tolerance, exhibited hydrolytic enzyme activity. Cold‐tolerant ‘Vernal’ and cold‐sensitive ‘Sonora’ alfalfa plants grown for 66 days under simulated “summer” (nonhardening) and “winter” (hardening) conditions exhibited different levels of cold tolerance as indicated, by cold tolerance assays performed at three freezing temperatures. Sonora winter plants were only moderately tolerant to −6 C, whereas Vernal plants were well hardened to −8 C and were tolerant to −10 C. Soluble protein concentrations were higher in hardened than in unhardened plants, with differences in protein concentration greater for Sonora than Vernal. Localization of hydrolytic enzymes on polyacrylamide gels after disc electrophoresis and subsequent densitometric scanning of zymograms demonstrated that activities of amylases, leucine amino peptidases, and α‐esterases were greater in winter than summer samples of both cultivars; whereas ATPase activities were lower in winter than summer samples. Furthermore, differences between αaesterase levels of hardened and unhardened samples were greater than those of amylase or peptidase. New esterase forms and higher quantities of a heat‐stable amylase also were detected in hardened samples. Differences in enzyme solubility between distilled water and tris‐HCl extracts, related to environment, extractant, or cultivar influences, were evident for esterases and ATPase and for individual forms of amylase and peptidase. The large quantitative changes in amylases and esterases localized within gel column regions 7 and 8 of hardened Vernal zymograms implied a close relationship of these hydrolytic enzymes to the hardening process; whereas the decreased ATPase activities reflected an inverse relationship with cold tolerance. It is, therefore, reasonable to conclude that quantitative and qualitative differences in hydrolytic enzyme activity are evident between hardened and nonhardened plants. The differences among hydrolytic enzymes do not necessarily follow the same quantitative or qualitative pattern, and for this reason they should be studied collectively to understand the biochemistry of hardening.

Certain soluble proteins are believed to be closely associated with cold tolerance. The objectives of this work were to determine whether the proteins exhibited dehydrogenase activity and if there were qualitative and/or quantitative differences in dehydrogenases between hardened and nonhardened plants. Cold‐tolerant ‘Vernal’ and cold‐sensitive ‘Sonora’ alfalfa (Medicago sativaL.) plants were grown for 66 days under simulated summer (non‐hardening) and winter (hardening) conditions, using growth chambers, and were assayed for the levels of cold tolerance developed at three freezing temperatures (−6, −8, and −10 C). To determine differences in dehydrogenases, soluble proteins were separated from extracts of crown and root tissues by polyacrylamide disc gel electrophoresis. Localization of dehydrogenases on gels and densitometric scanning of zymograms indicated that activities of glutamate, lactate, isocitrate, glucose‐6‐phosphate, 6‐phosphogluconate, nicotinamide adenine dinucleotide (NAD) malate and nicotinamide adenine dinucleotide phosphate (NADP) malate dehydrogenases were high in both cultivars exposed to hardening and were generally highest in Vernal. Percent increases for winter over summer levels of glutamate, lactate, glucose‐6‐phosphate, and 6‐phosphogluconate dehydrogenases were large compared with those of isocitrate, NAD‐malate, and NADP‐malate dehydrogenases. Moreover, the magnitude of change in enzyme activity between summer and winter plants also was influenced by extractant (distilled water, pH 7 or 0.10 Mtris‐HCl, pH 7) and varied quantitatively according to the solubility of the dehydrogenases in the two extractants. Enzyme solubility differences related to cultivar or environmental influences and cold tolerance were also observed, especially for isocitrate dehydrogenase. Large quantitative differences in glucose‐6‐phosphate and lactate dehydrogenase activities and substantial increases in isocitrate and 6‐phosphogluconate dehydrogenases localized within gel column regions 7 and 8 of zymograms developed from winter samples implied a close relationship of these enzymes to cold tolerance. Forms of lactate, glucose‐6‐phosphate, and isocitrate dehydrogenases were present in hardened tissues that were not present in un‐hardened tissues. It is concluded that the observed differences in dehydrogenasees between hardened and non‐hardened plants are major indicators of the cold tolerant state. Based on these and past studies, the reactions analyzed by representative hydrolytic and dehydrogenase enzymes may serve as a working hypothesis for future studies concerning the mechanics of induction and the suspected catenary nature of the hardening process.