Peroxidase activity and isoenzyme composition in Robinia pseudoacacia L. leaves under the influence of Parectopa robiniella Clemens, 1863

Іn recent times R. pseudoacacia has been suffering from populations of new pests, which cause serious economic damage to this strategic species. Therefore, studying the features of plant defense responses against harmful insects is of great importance for biodiversity conservation for preserving this species in natural and artificial plantings. The aim of the study was to analyze the peculiarities of the activity and isoenzyme composition of peroxidase in R. pseudoacacia leaves during attacks by the invading insect P. robiniella. In the green plantations of Dnipro city, as the object of research, six groups of Robinia pseudoacacia L. model trees of different ages (5 and 35–40 years of age) with the existing leaf damages by P. robiniella leaf blotch miner were identified. Differences in benzidine-peroxidase activity were found in the leaves of 5- and 40-year-old R. pseudoacacia trees. In 35‒40-year-old trees with the presence of leaf blotch mi­ners, the leaves showed a tendency to increase BPOD activity by 24.6% vs control. While in young plants, a significant increase in peroxidase activity was found by 3.8 times due to mechanical damage of the leaves by the miner compared to the control. The revealed significant increase (by 63.2%) in the activity of guaiacol-peroxidase of black locust indicates its active participation in enhancing the barrier functions of leaf cells against P. robiniella, and it indicates the promising use of the enzyme as a biomarker for pest influence. The composition of benzidine-peroxidase isoenzymes was analyzed by electrofocusing. The main pattern of changes in the expressiveness of benzidine-peroxidase due to damage of leaves by a pest was a quantitative redistribution of activity between different molecular forms of peroxidase. Several enzyme isoforms from the highly acidic pH zone showed a significant activity increase in R. pseudoacacia leaves damaged by P. robiniella; this especially applies to the peroxidase system of young black locust plants. The increase in bensidine- and guajacol-peroxidase activity under the influence of leaf miner may be explained by a change in the gene expression of individual isoforms of peroxidases, which is confirmed by the results of studying the relative share of peroxidase molecular forms with isoelectrofocusing. Calculation of the internal diversity index (KD) of IEF benzidine-peroxidase spectra showed that the studied peroxidase spectra of young and medieval trees of control and experimental samples of R. pseudoacacia leaves differ significantly in the level of complexity. Under the influence of the invader the level of complexity increased. The study of the reaction of black locust and the mechanisms of its protection against mechanical damage by phytophages with the involvement of a larger range of enzymes in further research can serve as a theoretical basis for species selection and management to restore it under biotic stress.

The Subcellular Localization and Oligomerization Preferences of NME1/NME2 upon Radiation-Induced DNA Damage

Nucleoside diphosphate kinases (NDPK/NME/Nm23) are enzymes composed of subunits NME1/NDPK A and NME2/NDPK B, responsible for the maintenance of the cellular (d)NTP pool and involved in other cellular processes, such as metastasis suppression and DNA damage repair. Although eukaryotic NDPKs are active only as hexamers, it is unclear whether other NME functions require the hexameric form, and how the isoenzyme composition varies in different cellular compartments. To examine the effect of DNA damage on intracellular localization of NME1 and NME2 and the composition of NME oligomers in the nucleus and the cytoplasm, we used live-cell imaging and the FRET/FLIM technique. We showed that exogenous NME1 and NME2 proteins co-localize in the cytoplasm of non-irradiated cells, and move simultaneously to the nucleus after gamma irradiation. The FRET/FLIM experiments imply that, after DNA damage, there is a slight shift in the homomer/heteromer balance between the nucleus and the cytoplasm. Collectively, our results indicate that, after irradiation, NME1 and NME2 engage in mutual functions in the nucleus, possibly performing specific functions in their homomeric states. Finally, we demonstrated that fluorophores fused to the N-termini of NME polypeptides produce the largest FRET effect and thus recommend this orientation for use in similar studies.

Dynamics of the isoenzyme composition of peroxidase and pigments in the needles of the introduced species of Picea (L.) Karst. in the taiga zone (Karelia)

The study was conducted at the Botanical Garden of Petrozavodsk State University (middle taiga sub-zone). The subjects of the study were an indigenous species (P. abies (L.) Karst.), and five introduced species (P. pungens Engelm. f. glauca Regel., P. pungens Engelm. f. viridis Regel., P. glauca (Mill.) Britt., P. omorica (Pane) Purk., P. mariana Britt., P. obovata Ledeb.). The study established high variability of the isoperoxidase spectrum in the Picea species needles during the circannual cycle. Molecular forms of peroxidase typical for growth and dormant periods were determined. Some Picea species were found to have isoenzymes appearing only during the deep dormant period. An increase in the heterogeneity of the needles isoperoxidase spectrum and appearance of molecular forms of the enzyme typical for the dormant period were observed in the indigenous and introduced Picea species in the course of adaptation to unfavorable winter conditions. The isoenzyme system rearrangement ensures plants tolerance to external factors and homeostasis regulation. The content of chlorophyll and carotenoids in the needles of the studied species undergoes significant seasonal changes and is largely determined by their biological characteristics. Pigments concentration naturally increases by the end of the vegetative period and decreases slightly in winter. The total number of pigments in the needles of the indigenous and introduced species is almost the same, indicating a similar rate of stock formation. By the dormant period, the ratio of chlorophylls to carotenoids increases and reaches approximately the same level in all Picea species. The Picea species introduced in Karelia adapt to low winter temperatures with the same physiological changes as the indigenous ones. These include changes in the isoenzyme composition of peroxidase, the dynamics of the pigments content in the needles, and the ratio of chlorophylls to carotenoids. Potential tolerance of the studied plant species to unfavorable environmental factors is affected by the extreme factor of tension that does not exceed the threshold value.

Activity and isoenzyme composition of peroxidase in the vegetative organs of Japanese quince under steppe zone conditions

Parameters of peroxidase complex are indicators of the adaptable processes which are taking place in plants under the environment influence. In this work the general activity dynamics and peroxidase isoenzyme composition in vegetative organs of Japanese quince (Chaenomeles Lindl.) were studied. The four species introduced in a steppe zone of Ukraine were researched: Ch. japonica (Thunb.) Lindl., Ch. speciosa (Sweet) Nak., Ch. cathayensis (Hemsl.) Schneid., Ch. × superba (Frahm) Red.). It was determined that the enzyme activity of these species possesses specific features subject to the phases of seasonal development and in response to the action of the hydrothermal stress. The highest enzyme activity in the period of intensive growth and flowering, with a subsequent sharp drop towards the middle of summer and further smoother decrease during the phase of physiological rest were observed in the leaves of Ch. speciosa and Ch. cathayensis. A higher peroxidase activity was a characteristic of Ch. japonica and Ch. × superba in the middle of the vegetative period, which can be considered a display of adaptation processes in the conditions of an unfavourable hydrothermal regime. The dynamics of changes in the quantitative composition of isoperoxidases was similar to the dynamics of enzyme activity. As a result of the generalization of the obtained data it is possible to consider that Ch. japonica and Ch. × superba are the steadiest in the conditions of the region.