Dynamics in the activity of peroxidase and its isoforms in leaves of different apple cultivars

Background. Various approaches are used for identification of the most resistant fruit crop cultivars, including the analysis of different physiological and biochemical indicators. In Krasnodar Territory, Russia, one of the major stressors in summer is the hydrothermal stress. Drought and heat lead to an oxidative stress, as reactive oxygen species are produced in plant cells. Plants respond to oxidative damage by activating antioxidant enzymes, such as superoxide dismutase, catalase, and various peroxidases. Peroxidase is able to decompose hydrogen peroxide. Peroxidase activity was calculated under natural summertime changes in the hydrothermal pattern (control) and in simulated high-temperature conditions.

Materials and methods. Three apple cultivars of Russian breeding, ‘Fortuna’, ‘Soyuz’ and ‘Prikubanskoe’, and cv. ‘Ligol’ of Polish origin were studied. In the summers of 2018–2019, their leaf samples were analyzed to assess peroxidase activity and its isozyme composition under control and stress conditions. Native electrophoresis in polyacrylamide gel was used for separation of peroxidase isoforms. Malondialdehyde content was measured to identify oxidative stress levels in apple leaves.

Results. The tested indicators demonstrated a high degree of heterogeneity induced by both cultivar specificity and seasonal weather dynamics. Peroxidase isoforms with a molecular weight of 70 to 60 kDa, characterized by the maximum level of variability (1–4 isoforms), were isolated. Two other groups included 1–3 isoforms with a molecular weight of ~130–100 kDa, and one with a molecular weight of ~55 kDa. The highest enzyme activity was found in cvs. ‘Fortuna’ and ‘Soyuz’ in July 2018, the hottest month during the period of research. Under simulated conditions, the triploid cultivar ‘Soyuz’ was least susceptible to the stress impact.

1. Andreeva V.A. Peroxidase enzyme: participation in the protective mechanism of plants (Ferment peroksidaza: uchastiye v zashchitnom mekhanizme rasteniy). Moscow; 1988. [in Russian]

2. Araujo L., Stadnik M.J. Cultivar-specific and ulvan-induced resistance of apple plants to Glomerella leaf spot are associated with enhanced activity of peroxidases. Acta Scientiarum: Agronomy. 2013;35(3):287-293. DOI: 10.4025/actasciagron.v35i3.16174

3. Barnes M.F. Leaf peroxidase and catechol oxidase polymorphism and the identification of commercial apple varieties. New Zealand Journal of Crop and Horticultural Science. 1993;21(2):207-210. DOI: 10.1080/01140671.1993.9513769

4. Blokhina O., Virolainen E., Ferstedt K.V. Antioxidants, oxidative damage and oxygen deprivation stress: a review. Annals of Botany. 2003;91(2):179-194. DOI: 10.1093/aob/mcf118

5. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 1976;72:248-254. DOI: 10.1016/0003-2697(76)90527-3

6. Cosio C., Dunand C. Specific functions of individual class III peroxidase genes. Journal of Experimental Botany. 2009;60(2):391-408. DOI: 10.1093/jxb/ern318

7. Ermakov A.I. (ed.). Methods of biochemical research on plants (Metody biokhimicheskogo issledovaniya rasteniy). Leningrad; 1987. [in Russian]

8. Gao T., Zhang Z., Liu X., Wu Q., Chen Q., Liu Q. et al. Physiological and transcriptome analyses of the effects of exogenous dopamine on drought tolerance in apple. Plant Physiology and Biochemistry. 2020;148:260-272. DOI: 10.1016/j.plaphy.2020.01.022

9. Golyshkina L.V. Electrophoresis in polyacrylamide gel of protein systems of fruit crops (Elektroforez v poliakrilamidom gele belkovykh sistem plodovykh kultur). In: Breeding and variety investigation of horticultural crops (Selektsiya i sortorazvedeniye sadovykh kultur). Orel: VNIISPK; 2007. p.56-63. [in Russian]

10. Heath R.L., Packer L. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics. 1968;125(1):189-198. DOI: 10.1016/0003-9861(68)90654-1

11. Jia D., Jiang Q., van Nocker S., Gong X., Ma F. An apple (Malus domestica) NAC transcription factor enhances drought tolerance in transgenic apple plants. Plant Physiology and Biochemistry. 2019;139:504-512. DOI: 10.1016/j.plaphy.2019.04.011

12. Krivobochek V.G., Statsenko A.P., Gorehnik I.D., Kapustin D.A., Yurova Y.A. Use of variation peroxidase in evaluation of drought resistant spring wheat. Innovative Machinery and Technology. 2015;4:34-40. [in Russian]

13. Liu S., Chen S., Chen Ya, Guan Z., Yin D., Chen F. In vitro induced tetraploid of Dendranthema nankingense (Nakai) Tzvel. shows an improved level of abiotic stress tolerance. Scientia Horticulturae. 2011;127(3):411-419. DOI: 10.1016/j.scienta.2010.10.012

14. Ma Y.H., Ma F.W., Zhang J.K., Li M.J., Wang Y.H., Liang D. Effects of high temperature on activities and gene expression of enzymes involved in ascorbate-glutathione cycle in apple leaves. Plant Science. 2008;175(6):761- 766. DOI: 10.1016/j.plantsci.2008.07.010

15. Manganaris A.G., Alston F.H. Inheritance and linkage relationships of peroxidase isoenzymes in apple. Theoretical and Applied Genetics. 1992;83(3):392-399. DOI: 10.1007/BF00224288

16. Mei Y., Sun H., Du G., Wang X., Lyu D. Exogenous chlorogenic acid alleviates oxidative stress in apple leaves by enhancing antioxidant capacity. Scientia Horticulturae. 2020;274:109676. DOI: 10.1016/j.scienta.2020.109676

17. Møller I.M., Jensen P.E., Hansson A. Oxidative modifications to cellular components in plants. Annual Review of Plant Biology. 2007;58:459-481. DOI: 10.1146/annurev.arplant.58.032806.103946

18. Petrokas R., Stanys V. Leaf peroxidase isozyme polymorphism of wild apple. Agronomy Research. 2008;6(2):531-541.

19. Prisedsky Y., Kabar A., Lykholat Y., Martynova N., Shupranova L. Activity and isoenzyme composition of peroxidase in the vegetative organs of Japanese quince under steppe zone conditions. Biologija. 2017;63(2):185-192. DOI: 10.6001/biologija.v63i2.3530

20. Rachenko M.A., Rachenko Ye.I., Zhivetiev M.A., Ro mano va I.M., Graskova I.A. Activity and isoforms of peroxidase in leaves of apple tree varieties, distinguished by resistance to scab. Vestnik of the Russian Academy of Agricultural Sciences. 2014;5:20-25. [in Russian]

21. Rácz A., Hideg É., Czégény G. Selective responses of class III plant peroxidase isoforms to environmentally relevant UV-B doses. Journal of Plant Physiology. 2018;221:101-106. DOI: 10.1016/j.jplph.2017.12.010

22. Radyukina N.L., Ivanov Y.V., Shevyakova N.I. Methods for assessing the content of reactive oxygen species, low molecular weight antioxidants and the activities of the main antioxidant enzymes (Metody otsenki soderzhaniya aktivnykh form kisloroda, nizkomolekulyarnykh antioksidantov i aktivnostey osnovnykh antioksidantnykh fermentov). In: Vl.V. Kuznetsov, V.V. Kuznetsov, G.A. Romanov (eds). Molecular-genetic and biochemical methods in modern plant biology (Molekulyarnogene ticheskiye i biokhimicheskiye metody v sovremennoy biologii rasteniy). Moscow; 2012. p.355-356. [in Russian]

23. Ranieri A., Castagna A., Baldan A., Soldatini G.F. Iron deficiency differently affects peroxidase isoforms in sunflower. Journal of Experimental Botany. 2001;52(354):25-35. DOI: 10.1093/jexbot/52.354.25

24. Ulyanovskaya E.V. Summer apple varieties: Souz, Zolotoe letnee, Feua, Fortuna. Fruit Growing and Viticulture of South Russia. 2020;65(5):1-18. [in Russian] DOI: 10.30679/2219-5335-2020-5-65-1-18

25. Wei Z., Gao T., Liang B., Zhao Q., Ma F., Li C. Effects of exogenous melatonin on methyl viologen-mediated oxidative stress in apple leaf. International Journal of Molecular Sciences. 2018;19(1):316. DOI: 10.3390/ijms19010316

26. Yamada M., Hidaka T., Fukamachi H. Heat tolerance in leaves of tropical fruit crops as measured by chlorophyll fluorescence. Scientia Horticulturae. 1996;67(1-2):39-48. DOI: 10.1016/S0304-4238(96)00931-4

27. Yanchevskaya T.G., Grits A.N., Kolomiets E.I., Romanovskaya T.V., Yarullina L.G., Ibragimov R.I. et al. Stimulation of cellular mechanisms of anti–virus sta bility of potatoes by the action of the preparation based on Bacillus subtilis bacteria. Applied Biochemistry and Microbiology.·2018;54(3):324-330. DOI: 10.7868/S0555109918030108

28. Zhang F., Xue H., Lu X., Zhang B., Wang F., Ma Y. et al. Autotetraploidization enhances drought stress tolerance in two apple cultivars. Trees. 2015;29:1773-1780. DOI: 10.1007/s00468-015-1258-4

29. Zhu Y., Jia X.,·Wu Y., Hu Y., Cheng L., Zhao T. et al. Quantitative proteomic analysis of Malus halliana exposed to salt-alkali mixed stress reveals alterations in energy metabolism and stress regulation. Plant Growth Regulation. 2020;90(2):205-222. DOI: 10.1007/s10725-019-00563-6