The effect of cold stress on the expression level of individual CBF genes in winter wheat varieties under simulated conditions

The expression levels of the CBF3, CBF15.2, CBF19, and CBF21 genes were studied using real-time PCR under simulated cold stress conditions in winter wheat varieties Irkutskaya, Girlyanda, and Cubus, which exhibit different levels of frost resistance. It was shown that the CBF3 gene is not expressed at +4 °C. Its expression level increases as the temperature decreases to –4 °C and reaches a maximum in the Girlyanda variety, exceeding the control values by 407.24 and 363.13 times after 2 and 24 hours, respectively. Two main expression peaks were revealed for the CBF15.2 gene in all studied varieties: at 2 hours after temperature decrease to –2 °C and –4 °C, respectively. The CBF19 gene expression profile was characterized by the most pronounced response to cold stress in the studied varieties when the temperature decreased to +4 °C, and 24 hours later when it decreased to –2 °C in the Irkutskaya and Cubus varieties. The greatest increase in the CBF21 gene expression level (44.61 times) was revealed for the Girlyanda variety 24 hours after the temperature decreased to –2 °C. The results show that the gene expression level increased at both the initial (CBF19 and CBF21 genes), intermediate (CBF3, CBF15.2, CBF19 and CBF21 genes) and final stages (CBF3 and CBF15.2 genes) of cold stress modeling. It depends on the genotype and is generally higher in the winter-hardy Girlyanda variety. The results can be used to assess the potential of wheat accessions to tolerate cold stress.

1. Medvedeva I. V., Zhigarev T. P., Vasilevskaya Zh. N., Tarasyuk N. V., Yarkovets A. I., Letko O. V., Kondratenko E. G., Palkovskaya E. M., Zharikova A. V. (eds.). Agriculture of the Republic of Belarus: Statistical booklet. Minsk, Information and Computing Center of the National Statistical Committee of the Republic of Belarus, 2025. 36 p. (in Russian).

2. Hyles J., Bloomfield M. T., Hunt J. R., Trethowan R. M. Trevaskis B. Phenology and related traits for wheat adaptation. Heredity, 2020, vol. 125, no. 6, pp. 417–430. https://doi.org/10.1038/s41437-020-0320-1

3. Schreiber M., Himmelbach A., Börner A., Mascher M. Genetic diversity and relationship between domesticated rye and its wild relatives as revealed through genotyping-by-sequencing. Evolutionary Applications, 2019, vol. 12, no. 1, pp. 66–77. https://doi.org/10.1111/eva.12624

4. Bond D. M., Dennis E. S., Finnegan E. J. The low temperature response pathways for cold acclimation and vernalization are in-dependent. Plant, Cell and Environment, 2011, vol. 34, no. 10, pp. 1737–1748. https://doi.org/10.1111/j.1365-3040.2011.02370.x

5. Trevaskis B., Hemming M. N., Peacock W. J., Dennis E. S. HvVRN2 Responds to Daylength, whereas HvVRN1 Is Regulated by Vernalization and Developmental Status. Plant Physiology, 2006, vol. 140, no. 4, pp. 1397–1405. https://doi.org/10.1104/pp.105.073486

6. Thomashow M. F. Molecular Basis of Plant Cold Acclimation: Insights Gained from Studying the CBF Cold Response Pathway. Plant Physiology, 2010, vol. 154, no. 2, pp. 571–577. https://doi.org/10.1104/pp.110.161794

7. Kosová K., Klíma M., Prášil I. T., Vítámvás P. COR/LEA Proteins as Indicators of Frost Tolerance in Triticeae: A Comparison of Controlled versus Field Conditions. Plants, 2021, vol. 10, no. 4, pp. 1–13. https://doi.org/10.3390/plants10040789

8. Motomura Y., Kobayashi F., Iehisa J. C. M., Takumi S. A major quantitative trait locus for cold-responsive gene expression is linked to frost-resistance gene Fr-A2 in common wheat. Breeding Science, 2013, vol. 63, no. 1, pp. 58–67. https://doi.org/10.1270/jsbbs.63.58

9. Kobayashi F., Ishibashi M., Takumi S. Transcriptional activation of Cor/Lea genes and increase in abiotic stress tole rance through expression of a wheat DREB2 homolog in transgenic tobacco. Transgenic Research, 2008, vol. 17, no. 5, pp. 755–767. https://doi.org/10.1007/s11248-007-9158-z

10. Gilmour S. J., Fowler S. G., Thomashow M. F. Arabidopsis Transcriptional Activators CBF1, CBF2, and CBF3 have Matching Functional Activities. Plant Molecular Biology, 2004, vol. 54, no. 5, pp. 767–781. https://doi.org/10.1023/B:PLAN.0000040902.06881.d4

11. Morran S., Eini O., Pyvovarenko T., Parent B., Singh R., Ismagul A., Eliby S., Shirley N., Langridge P., Lopato S. Improve-ment of stress tolerance of wheat and barley by modulation of expression of DREB/CBF factors. Plant Biotechnology Journal, 2011, vol. 9, no. 2, pp. 230–249. https://doi.org/10.1111/j.1467-7652.2010.00547.x

12. Mohseni S., Che H., Djillali Z., Dumont E., Nankeu J., Danyluk J. Wheat CBF gene family: identification of polymorphisms in the CBF coding sequence. Genome, 2012, vol. 55, no. 12, pp. 865–881. https://doi.org/10.1139/gen-2012-0112

13. The International Wheat Genome Sequencing Consortium (IWGSC), Appels R., Eversole K., Stein N., Feuillet C., Keller B., Rogers J. [et al.]. Shifting the limits in wheat research and breeding using a fully annotated reference genome. Science , 2018, vol. 361, no. 6403, p. eaar7191. https://doi.org/10.1126/science.aar7191

14. Sutton F., Chen D.-G., Ge X., Kenefick D. Cbfgenes of the Fr-A2 allele are differentially regulated between long-term cold acclimated crown tissue of freeze-resistant and – susceptible, winter wheat mutant lines. BMC Plant Biology, 2009, vol. 9, art. 34. https://doi.org/10.1186/1471-2229-9-34

15. Todorovska E. G., Kolev S., Christov N. K., Balint A., Kocsy G., Vagujfalvi A., Galiba G. The expression of CBFgenes at Fr­2 locus is associated with the level of frost tolerance in Bulgarian winter wheat cultivars. Biotechnology and Biotechnological Equipment, 2014, vol. 28, no. 3, pp. 392–401. https://doi.org/10.1080/13102818.2014.944401

16. Vagujfalvi A., Aprile A., Miller A., Dubcovsky J., Delugu G., Galiba G., Cattivelli L. The expression of several Cbf genes at the Fr­A2 locus is linked to frost resistance in wheat. Molecular Genetics and Genomics, 2005, vol. 274, no. 5, pp. 506–514. https://doi.org/10.1007/s00438-005-0047-y

17. Knox A. K., Li C. X., Vagujfalvi A., Galiba G., Stockinger E. J., Dubcovsky J. Identification of candidate CBF genes for the frost tolerance locus Fr­Am2 in Triticum monococcum. Plant Molecular Biology, 2008, vol. 67, no. 3, pp. 257–270. https://doi.org/10.1007/s11103-008-9316-6

18. Jurczyk B., Pociecha E., Janeczko A., Paczyński R., Rapacz M. Assessment of candidate reference genes for the expression studies with brassinosteroids in Lolium perenne and Triticum aestivum. Journal of Plant Physiology, 2014, vol. 171, no. 16, pp. 1541–1544. https://doi.org/10.1016/j.jplph.2014.07.008

19. Livak K. J., Schmittgen T. D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods, 2001, vol. 25, no. 4. pp. 402–408. https://doi.org/10.1006/meth.2001.1262

20. Winfield M. O., Lu C., Wilson I. D., Coghill J. A., Edwards K. J. Plant responses to cold: transcriptome analysis of wheat. Plant Biotechnology Journal, 2010, vol. 8, no. 7, pp. 749–771. https://doi.org/10.1111/j.1467-7652.2010.00536.x