Hydrolytic Enzyme Differences in Cold‐Tolerant and Cold‐Sensitive Alfalfa1,2

Abstract

AbstractThis investigation was part of a continuing effort to ascertain the role of soluble proteins in the development of cold tolerance of alfalfa, Medicago sativa L. 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.