Study of isozyme polymorphism in spring barley (Hordeum vulgare L.) contrasting in tolerance to lead

Lead is one of the most hazardous heavy metals (HM), for which the mechanisms of plant resistance is not completely clear. One of the mechanisms for implementing toxic effect of lead is the generation of reactive oxygen species (ROS), which normally perform important regulatory function, but cause multiple violations of vital activity of cells at increased concentrations. ROS level adjustment is carried out by antioxidant enzymes such as superoxide dismutase (SOD) and peroxidase (PER). The effectiveness of most enzymes depends upon the existence of multiple isoforms, ensuring the most optimal operation under changing environment conditions. As a working hypothesis, it has been suggested that the level of plant tolerance to the HM can be determined by differences in composition of izoforms of antioxidant enzymes differently contributeing to neutralization of the ROS excess. To test this hypothesis the study of isoenzymatic spectra of superoxide dismutase and peroxidase enzyme systems and key enzyme of protein metabolism, the glutamate dehydrogenase (GDH), was carried out using embryos of germinating seeds of the spring barley ( Hordeum vulgare L.) cultivars. A totlal of 12 cultivars of spring barley contrasting in resistance to Pb2+ (6 tolerant cultivars and 6 sensitive cultivars) were studied. To analyze the composition of isoenzyme the germinating embryos of viable seeds were used with root length of 1 mm after 1 day germination. Homogenized embryos of seeds were subjected to electrophoresis using 7.5 % polyacrylamide gel (PAGE) units with an alkaline buffer system (pH 8.9). The analysis showed the presence of following isoenzymes from enzyme systems of superoxide dismutase, peroxidase and glutamate dehydrogenase: an obligate SODII, PERI and GDHI as well as rare SODI, SODIII + SODIV (always occurring together), SODV, PERII and GDHII. The frequencies of rare alleles were calculated and the highest frequencies were found for alleles SODI and SODIII + SODIV being the same in sensitive and resistant cultivars. A reliability of differences in the frequencies of rare allelic forms in the sensitive and resistant cultivars was assessed using φ-criterion of the angular Fisher transformation. The calculated value of Fisher’s φ-criterion was proved to exceed the critical level. Thus the studied rare enzyme isoforms could be regarded as biochemical markers of sensitivity or tolerance to lead in barley plants. As our research ascertained, SODV and PERII are more common in lead sensitive cultivars, while GDHII is mostly found in tolerant ones. Frequency of SODIII and SODIV also reliably differed in the resistant and tolerant cultivars. The paper discusses the molecular genetic mechanisms of plant HM-resistance. It is first experimentally shown that spring barley plant polymorphism on resistance to lead is associated with biochemical polymorphism and correlates with a specific complex of superoxide dismutase, peroxidase and glutamate dehydrogenase isoenzymes. The data obtained can be used in selection programs aimed to producing lead-resistant barley cultivars.