Adaptive responses of pear cultivars to low-temperature stress in the spring period

The pear is one of the most famous pome crops. It occupies about 7 % of the total area of perennial fruit crops in Russia. Orchard plantings are predominantly composed of foreign European cultivars. Spring frosts, which are typical for the southern regions of the country, lead to significant crop losses. This study determined the response characteristics of pear flower buds to low-temperature stress. The Crimean cultivar Dzhankoyskaya Pozdnyaya, two cultivars – Leven and Flamenco – of Krasnodar selection and the interspecific hybrid Kieffer were investigated. Flower buds at different developmental stages were exposed to a climatic chamber for 12 hours at temperatures –1.5…–2 °C. After stress exposure, the activity of certain antioxidant enzymes was determined, along with the content of phenolic compounds, malondialdehyde, and the gene expression level of its enzymes and proteins involved in cold adaptation. It was revealed that the autumn-ripening cultivar Kieffer, under conditions of the Krasnodar region, begins to bloom earlier than other studied cultivars, making it more susceptible to recurrent frosts. This is evidenced by high values of malondialdehyde and the activity level of superoxide dismutase. The Russian cultivars, Leven (winter cultivar) and Flamenco (summer cultivar), showed the highest activity of peroxidase and gene expression of PcDREB2, PcCAP160, PcCOR413, PcPOX1, with a reduced level of malondialdehyde. These cultivars typically emerged from dormancy later compared to Kieffer. The Crimean winter-ripening cultivar was closer to the interspecific hybrid in terms of the studied parameters but showed lower enzyme activity and gene expression levels. The obtained results suggest that under pear cultivation conditions in the southern region of the country, where spring frosts are possible, cultivars with flowering starting in the secondto-third decade of April and high indicators of antioxidant enzyme activity (primarily peroxidase) and gene expression levels of PcDREB2, PcCAP160, and PcCOR413 demonstrate greater resistance.

1. Ainsworth E.A., Gillespie K.M. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nat Protoc. 2007;2:875-877. doi 10.1038/nprot.2007.102

2. Asayesh Z.M., Arzani K., Mokhtassi-Bidgoli A., Abdollahi H. Enzymatic and non-enzymatic response of grafted and ungrafted young European pear (Pyrus communis L.) trees to drought stress. Sci Hortic. 2023;310:111745. doi 10.1016/j.scienta.2022.111745

3. Azarabadi S., Abdollahi H., Torabi M., Salehi Z., Nasiri J. ROS generation, oxidative burst and dynamic expression profiles of ROSscavenging enzymes of superoxide dismutase (SOD), catalase (CAT ) and ascorbate peroxidase (APX ) in response to Erwinia amylovora in pear (Pyrus communis L.). Eur J Plant Pathol. 2017;147:279-294. doi 10.1007/s10658-016-1000-0

4. Bandurko I.A. Assessment of the gene pool of the Pear by resistance to adverse factors of the winter period in the foothill zone of the NorthWest Caucasus. Science and Innovation. 2024;21:88-92 (in Russian)

5. Bonyanpour A.R., Jamali B. Seasonal enzymatic and non-enzymatic antioxidant responses in seven Iranian pomegranate cultivars. Adv Hortic Sci. 2020;34(3):265-276

6. Boyarkin A.N. A quick method for determining the activity of peroxidase. Biochemistry. 1951;16(4):352-355 (in Russian)

7. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem. 1976;72(1-2):248-254. doi 10.1016/0003-2697(76)90527-3

8. Busatto N., Giné-Bordonaba J., Larrigaudière C., Lindo-Garcia V., Farneti B., Biasioli F., Vrhovsek U., Costa F. Molecular and biochemical differences underlying the efficacy of lovastatin in preventing the onset of superficial scald in a susceptible and resistant Pyrus communis L. cultivar. Postharvest Biol Technol. 2021;173:111435. doi 10.1016/j.postharvbio.2020.111435

9. Du F., Xu J.-N., Li D., Wang X.-Y. The identification of novel and differentially expressed apple-tree genes under low-temperature stress using high-throughput Illumina sequencing. Mol Biol Rep. 2015;42: 569-580. doi 10.1007/s11033-014-3802-5

10. Dumanovic J., Nepovimova E., Natic M., Kuca K., Jacevic V. The significance of reactive oxygen species and antioxidant defense system in plants: a concise overview. Front Plant Sci. 2021;11:552969. doi 10.3389/fpls.2020.552969

11. Efimova M.V., Kolomeichuk L.V., Boyko E.V., Malofii M.K., Vidershpan A.N., Plyusnin I.N. Golovatskaya I.F., Murgan O.K., Kuznetsov Vl.V. Physiological mechanisms of Solanum tuberosum L. plants’ tolerance to chloride salinity. Russ J Plant Physiol. 2018; 65(3):394-403. doi 10.1134/S1021443718030020

12. Evers S.M., Knight T.M., Inouye D.W., Miller T.X., Salguero-Gómez R., Iler A.M., Compagnoni A. Lagged and dormant season climate better predict plant vital rates than climate during the growing season. Glob Chang Biol. 2021;27(9):1927-1941. doi 10.1111/gcb.15519

13. Gabay G., Flaishman M.A. Genetic and molecular regulation of chilling requirements in pear: breeding for climate change resilience. Front Plant Sci. 2024;15:1347527. doi 10.3389/fpls.2024.1347527

14. Hikmawanti N., Fatmawati S., Asri A.W. The effect of ethanol concentrations as the extraction solvent on antioxidant activity of katuk (Sauropus androgynus (L.) Merr.) leaves extracts. IOP Conf Ser Earth Environ Sci. 2021;755:012060. doi 10.1088/1755-1315/755/1/012060

15. Hussain S., Liu G., Liu D., Ahmed M., Hussain N., Teng Y. Study on the expression of dehydrin genes and activities of antioxidative enzymes in floral buds of two sand pear (Pyrus pyrifolia Nakai) cultivars requiring different chilling hours for bud break. Turk J Agric For. 2015;39(6):930-939. doi 10.3906/tar-1407-164

16. Klyukina A.V., Dragavtseva I.A., Oplachko R.A. Study of fruit crops varieties’ needs in temperature regime of their development phases (on the example of pear varieties). Fruit Growing and Viticulture of South Russia. 2024;89(5):49-58. doi 10.30679/2219-5335-2024-5-89-49-58 (in Russian)

17. Kolomiyts I.A. Biological analysis of the development of flower buds in an apple tree. Doklady Akademii Nauk SSSR. 1952;84(4):821-824 (in Russian)

18. Lee J.C., Park Y.S., Jeong H.N., Kim J.H., Heo J.Y. Temperature changes affected spring phenology and fruit quality of apples grown in high-latitude region of South Korea. Horticulturae. 2023;9(7): 794. doi 10.3390/horticulturae9070794

19. Li Y., Zhang J., Wang S., Zhang H., Liu Y., Yang M. Integrative transcriptomic and metabolomic analyses reveal the flavonoid biosynthesis of Pyrus hopeiensis flowers under cold stress. Hortic Plant J. 2023;9(3):395-413. doi 10.1016/j.hpj.2022.11.004

20. Lin S., Li Y., Zhao J., Guo W., Jiang M., Li X., Liu W., Zhang J., Yang M. Transcriptome analysis of biochemistry responses to lowtemperature stress in the flower organs of five pear varieties. Forests. 2023;14(3):490. doi 10.3390/f14030490

21. Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods. 2001;25(4):402-408. doi 10.1006/meth.2001.1262

22. New Methods of Radical Increase in Yields of Fruit Crops Based on the Theory of the Environmental and Genetic Organization of Quantitative Features in the Context of Climate Fluctuation. Krasnodar, 2023 (in Russian)

23. Nham N.T., Macnish A.J., Zakharov F., Mitcham E.J. ‘Bartlett’ pear fruit (Pyrus communis L.) ripening regulation by low temperatures involves genes associated with jasmonic acid, cold response, and transcription factors. Plant Sci. 2017;260:8-18. doi 10.1016/j.plantsci.2017.03.008

24. Program and Methodology of Varietalization of Fruit, Berry and Walnut Crops. Orel, 1999 (in Russian)

25. Queiroz C., da Silva A.J.R., Lopes M.L.M., Fialho E., Valente-Mesquita V.L. Polyphenol oxidase activity, phenolic acid composition and browning in cashew apple (Anacardium occidentale L.) after processing. Food Chem. 2011;125(1):128-132. doi 10.1016/j.foodchem.2010.08.048

26. Şahin Ö., Dumanoğlu H., Sarikamiş G., Javadisaber J., Ergül A., Aydemir B.Ç. Tolerance of Pyrus spp. and Cydonia oblonga as pear rootstocks to iron chlorosis determined by in vitro growth, antioxidant and molecular responses. Sci Hortic. 2022;296:110911. doi 10.1016/j.scienta.2022.110911

27. Shitt P.G. The Study of the Growth and Development of Fruit and Berry Plants. Moscow, 1958 (in Russian)

28. Sotnik A.I., Babina R.D., Khoruzhy P.G. Comparative assessment of resistance of the generative organs of pear varieties (Pirus communis L.) to low-temperature stresses under the conditions of Crimea. Izvestia Orenburg State Agrarian University. 2017;3(65):72-74 (in Russian)

29. Sundyreva M.A., Stepanov I.V., Suprun I.I., Ushakova Y.V. A modified protocol of RNA isolation from mature leaves of grapes for RT-PCR. Scientific Journal of Kuban State Agrarian University. 2018;143:16- 30. doi 10.21515/1990-4665-143-012 (in Russian)

30. Suzuki N., Koussevitzky S., Mittler R., Miller G. ROS and redox signalling in the response of plants to abiotic stress. Plant Cell Environ. 2012;35(2):259-270. doi 10.1111/j.1365-3040.2011.02336.x

31. Treml J., Mejkal K. Flavonoids as potent scavengers of hydroxyl radicals. Compr Rev Food Sci Food Saf. 2016;15(4):720-738. doi 10.1111/1541-4337.12204

32. 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. Int J Mol Sci. 2018;19(1):316. doi 10.3390/ijms19010316

33. Xiao L.J., Asseng S., Wang X.T., Xia J.X., Zhang P., Liu L.L., Tang L., Cao W., Zhu Y., Liu B. Simulating the effects of low-temperature stress on wheat biomass growth and yield. Agric For Meteorol. 2022;326:109191. doi 10.1016/j.agrformet.2022.109191

34. Yang T., Huang X.-S. Deep sequencing-based characterization of transcriptome of Pyrus ussuriensis in response to cold stress. Gene. 2018;661:109-118. doi 10.1016/j.gene.2018.03.067

35. Zhai R., Liu J., Liu F., Zhao Y., Liu L., Fang C., Wang H., Li X., Wang Z., Ma F., Xu L. Melatonin limited ethylene production, softening and reduced physiology disorder in pear (Pyrus communis L.) fruit during senescence. Postharvest Biol Technol. 2018;139:38-46. doi 10.1016/j.postharvbio.2018.01.017

36. Zhang S. Recent advances of polyphenol oxidases in plants. Molecules. 2023;28(5):2158. doi 10.3390/molecules28052158

37. Zhang Y., Wu L., Liu L., Jia B., Ye Z., Tang X., Heng W., Liu L. Functional analysis of PbbZIP11 transcription factor in response to cold stress in Arabidopsis and pear. Plants. 2023;13(1):24. doi 10.3390/plants13010024

38. Zhao Y., Hu M.Y., Wang Q., Yan X.K., Lu M.Y., Wu C.H., Li H.Y., Zhang M.J. Climate variation and its influence on the cold tolerance and phenology periods of pear cultivars in Jilin, China. Int J Fruit Sci. 2023;23(1):165-180. doi 10.1080/15538362.2023.2249995