Dehydrogenase Levels in Cold-Tolerant and Cold-Sensitive Alfalfa1,2

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 sativa L.) 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 M tris-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.