Document Type : Original Article

Authors

1 Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Zanjan, Zanjan, Iran

2 Associate Professor, Department of Plant Production and Genetics, Faculty of Agriculture, University of Zanjan, Zanjan, Iran

Abstract

Introduction
The yield of wheat (Triticum aestivum L.), is greatly reduced under environmental stress and decreased temperatures. Therefore, adaptation mechanisms and cold resistance are crucial in this plant. Vernalization is known as one of the essential mechanisms for grain adaptation to environmental conditions, whereby the plant can accelerate the flowering process or flower after a long cold period. Vernalization also plays a significant role in the acquisition of cold resistance in wheat plants. When exposed to low temperatures, certain genes related to vernalization are activated, leading to changes in the plant's physiology and allowing it to a better cold stress tolerance. This process involves complex regulatory mechanisms depending on the cultivar and environmental conditions. Understanding the molecular basis of vernalization and the genes involved in cold resistance could assist in developing new strategies to improve wheat productivity in adverse environments.
Materials and methods
In this research, we investigated the expression of three genes, NAC, ERF, and TCP, related to vernalization in two wheat cultivars named Baz and NorthStar, which known as spring and winter cultivars, respectively. The plant samples were preserved in growth racks and applied for vernalization treatment after the tillering stage. RNA extraction was performed at this stage. Real-Time PCR technique was then utilized to analyze the gene expression. To better understanding the function of these genes in response to cold stress, the promoter of the three studied genes was analyzed by screening 500 nucleotides upstream of the wheat TSS. The vernalization treatment was applied at two levels of 14 and 21 days and compared to the control plants under 4°C.
Results and discussion
The results showed that the expression of all three genes (TCP, NAC, and ERF) decreased under the vernalization treatment. However, the expression of the TCP and NAC genes increased after 14 and 21 days of treatment in the NorthStar and Baz varieties, respectively. In general, the decreased level of expression was shown by increasing in the number of vernalization days. Notably, the expression of the ERF gene reduced in both tested varieties with the increased number of vernalization days. This trend was also observed in the expression of the NAC gene. However, the vice versa was observed for NAC gene in the Baz variety by an increased expression. In the Baz variety, the expression rate of the TCP gene decreased with an increase in the number of vernalization days, whereas in the NorthStar variety, the gene expression increased and then decreased after 14 and 21 days of treatment. Based on the abundance and diversity of the identified elements resulting from the analysis of the promoters of the studied genes, 28 types of regulatory elements were identified, many of which are binding sites for transcription factors responding to biotic and abiotic stresses Top of Form.
Conclusion
Despite the similarity of the pattern of expression changes of all three genes in the two investigated cultivars, the intensity of the changes in the two cultivars was not same, which could be due to different reactions to cold stress. The results show the complexity of gene expression regulation in wheat vernalization. Additionally, the multiplicity of stress-responsive transcription factor binding sites in the promoter region of these genes could be a justification for the complexity of regulating their expression during vernalization and response to cold stress.

Keywords

Main Subjects

Abdullah, S.N.A., Azzeme, A.M., Yousefi, K., 2022. Fine-Tuning cold stress response through regulated cellular abundance and mechanistic actions of transcription factors. Frontiers in Plant Science. 13. https://doi.org/10.3389/fpls.2022.850216
Agrawal, P., Jha, B., 2010. Transcription factors in plants and ABA dependent and independent abiotic stress signalling. Biologia Plantarum. 54, 201–212. https://doi.org/10.1007/s10535-010-0038-7
Ait Barka, E., Audran, J., 1997. Response of champenoise grapevine to low temperatures: changes of shoot and bud proline concentrations in response to low temperatures and correlations with freezing tolerance. Journal of Horticultural Science. 72, 577-582. https://doi.org/10.1080/14620316.1997.11515546
Amasino, R.M., 2005. Vernalization and flowering time. Current opinion in biotechnology. 16, 154-158. https://doi.org/10.1016/j.copbio.2005.02.004
An, J.-P., Li, R., Qu, F.-J., You, C.-X., Wang, X.-F., Hao, Y.-J., 2018. An apple NAC transcription factor negatively regulates cold tolerance via CBF-dependent pathway. Journal of Plant Physiology. 221, 74-80. https://doi.org/10.1016/j.jplph.2017.12.009
Biłas, R., Szafran, K., Hnatuszko-Konka, K., Kononowicz, A.K., 2016. Cis-regulatory elements used to control gene expression in plants. Plant Cell, Tissue and Organ Culture (PCTOC). 127, 269-287. https://doi.org/10.1007/s11240-016-1057-7
Danisman, S. 2016. TCP transcription factors at the interface between environmental challenges and the plant’s growth responses. Frontiers in Plant Science. 7, 1930. https://doi.org/10.3389/fpls.2016.01930
Dong, W., Ai, X., Xu, F., Quan, T., Liu, S., Xia, G., 2012. Isolation and characterization of a bread wheat salinity responsive ERF transcription factor. Gene. 511, 38-45. https://doi.org/10.1016/j.gene.2012.09.039
Dowla, M. N. U., Edwards, I., O'Hara, G., Islam, S., Ma, W., 2018. Developing wheat for improved yield and adaptation under a changing climate: optimization of a few key genes. Engineering. 4, 514-522. https://doi.org/10.1016/j.eng.2018.06.005
Fang, Y., Zheng, Y., Lu, W., Li, J., Duan, Y., Zhang, S., Wang, Y., 2021. Roles of miR319-regulated TCPs in plant development and response to abiotic stress. The Crop Journal. 9, 17-28. https://doi.org/10.1016/j.cj.2020.07.007
Farokhpour, B., Ismaili, A., Eisvand, H.R., Sohrabi, S.M., 2019. Analysis of gene expression pattern of some members of NAC gene family in lentil (Lens culinaris M.) under cold stress. Agricultural Biotechnology Journal. 10, 111-131. [In Persian with English Summary]
Feng, Z.-J., Xu, S.-C., Liu, N., Zhang, G.-W., Hu, Q.-Z., Gong, Y.-M., 2018. Soybean TCP transcription factors: Evolution, classification, protein interaction and stress and hormone responsiveness. Plant Physiology and Biochemistry. 127, 129-142. https://doi.org/10.1016/j.plaphy.2018.03.020
Gahlaut, V., Jaiswal, V., Kumar, A., Gupta, P. K., 2016. Transcription factors involved in drought tolerance and their possible role in developing drought tolerant cultivars with emphasis on wheat (Triticum aestivum L.). Theoretical and Applied Genetics. 129, 2019-2042. https://doi.org/10.1007/s00122-016-2794-z
Giraud, E., Ng, S., Carrie, C., Duncan, O., Low, J., Lee, C. P., Van Aken, O., Millar, A. H., Murcha, M., Whelan, J., 2010. TCP transcription factors link the regulation of genes encoding mitochondrial proteins with the circadian clock in Arabidopsis thaliana. The Plant Cell. 22, 3921-3934. https://doi.org/10.1105/tpc.110.074518
Griffith, M., Yaish, M. W., 2004. Antifreeze proteins in overwintering plants: a tale of two activities. Trends in Plant Science. 9, 399-405. https://doi.org/10.1016/j.tplants.2004.06.007
Guo, J., Sun, B., He, H., Zhang, Y., Tian, H., Wang, B., 2021. Current understanding of bHLH transcription factors in plant abiotic stress tolerance. International Journal of Molecular Sciences. 22, 4921. https://doi.org/10.3390/ijms22094921
Hao, Y.J., Wei, W., Song, Q.X., Chen, H.W., Zhang, Y.Q., Wang, F., Zou, H.F., Lei, G., Tian, A.G., Zhang, W.K., 2011. Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants. The Plant Journal. 68, 302-313. https://doi.org/10.1111/j.1365-313X.2011.04687.x
He, F., Zhang, L., Zhao, G., Kang, J., Long, R., Li, M., Yang, Q., Chen, L., 2022. Genome-wide identification and expression analysis of the NAC gene family in Alfalfa revealed its potential roles in response to multiple abiotic stresses. International Journal of Molecular Sciences. 23, 10015. https://doi.org/10.3390/ijms231710015
Jan, N., Andrabi, K. I., 2009. Cold resistance in plants: A mystery unresolved. Electronic Journal of Biotechnology. 12, 14-15. http://doi.org/10.2225/vol12-issue3-fulltext-3
Jin, H., Huang, F., Cheng, H., Song, H., Yu, D., 2013. Overexpression of the GmNAC2 gene, an NAC transcription factor, reduces abiotic stress tolerance in tobacco. Plant Molecular Biology Reporter. 31, 435-442. https://doi.org/10.1007/s11105-012-0514-7
Kakei, Y., Masuda, H., Nishizawa, N. K., Hattori, H., Aung, M. S., 2021. Elucidation of novel cis-regulatory elements and promoter structures involved in iron excess response mechanisms in rice using a bioinformatics approach. Frontiers in Plant Science. 12, 660303. https://doi.org/10.3389/fpls.2021.660303
Kidokoro, S., Shinozaki, K., Yamaguchi-Shinozaki, K., 2022. Transcriptional regulatory network of plant cold-stress responses. Trends in Plant Science. https://doi.org/10.1016/j.tplants.2022.01.008
Kosugi, S., Ohashi, Y., 2002. DNA binding and dimerization specificity and potential targets for the TCP protein family. The Plant Journal. 30, 337-348. https://doi.org/10.1046/j.1365-313X.2002.01294.x
Laudencia-Chingcuanco, D., Ganeshan, S., You, F., Fowler, B., Chibbar, R., Anderson, O., 2011. Genome-wide gene expression analysis supports a developmental model of low temperature tolerance gene regulation in wheat (Triticum aestivum L.). BMC genomics. 12, 1-20. https://doi.org/10.1186/1471-2164-12-299
Lawlor, D. W., Paul, M. J., 2014. Source/sink interactions underpin crop yield: the case for trehalose 6-phosphate/SnRK1 in improvement of wheat. Frontiers in Plant Science. 5, 418. https://doi.org/10.3389/fpls.2014.00418
Lee, D.-K., Jung, H., Jang, G., Jeong, J. S., Kim, Y. S., Ha, S.-H., Do Choi, Y., Kim, J.-K., 2016. Overexpression of the OsERF71 transcription factor alters rice root structure and drought resistance. Plant physiology. 172, 575-588. https://doi.org/10.1104/pp.16.00379
Lei, N., Yu, X., Li, S., Zeng, C., Zou, L., Liao, W., Peng, M., 2017. Phylogeny and expression pattern analysis of TCP transcription factors in cassava seedlings exposed to cold and/or drought stress. Scientific reports. 7, 1-13. http://dx.doi.org/10.1038/s41598-017-09398-5
Li, W.-y., Wang, C., Shi, H.-h., Wang, B., Wang, J.-x., Liu, Y.-s., Ma, J.-y., Tian, S.-y., Zhang, Y.-w., 2020. Genome-wide analysis of ethylene-response factor family in adzuki bean and functional determination of VaERF3 under saline-alkaline stress. Plant Physiology and Biochemistry. 147, 215-222. https://doi.org/10.1016/j.plaphy.2019.12.019
Li, X., Zhang, D., Li, H., Wang, Y., Zhang, Y., Wood, A.J., 2014. EsDREB2B, a novel truncated DREB2-type transcription factor in the desert legume Eremosparton songoricum, enhances tolerance to multiple abiotic stresses in yeast and transgenic tobacco. BMC Plant Biology. 14, 1-16. https://doi.org/10.1186/1471-2229-14-44
Liu, M.-M., Wang, M.-M., Yang, J., Wen, J., Guo, P.-C., Wu, Y.-W., Ke, Y.-Z., Li, P.-F., Li, J.-N., Du, H., 2019. Evolutionary and comparative expression analyses of TCP transcription factor gene family in land plants. International Journal of Molecular Sciences. 20, 3591. https://doi.org/10.3390/ijms20143591
Mao, H., Li, S., Chen, B., Jian, C., Mei, F., Zhang, Y., Li, F., Chen, N., Li, T., Du, L., 2022. Variation in cis-regulation of a NAC transcription factor contributes to drought tolerance in wheat. Molecular Plant. 15, 276-292. https://doi.org/10.1016/j.molp.2021.11.007
Marques, D. N., dos Reis, S. P., de Souza, C. R., 2017. Plant NAC transcription factors responsive to abiotic stresses. Plant Gene. 11, 170-179. https://doi.org/10.1016/j.plgene.2017.06.003
Marques, D. N., Reis, S. P. d., de Souza, C. R. B., 2017. Plant NAC transcription factors responsive to abiotic stresses. Plant Gene. 11, 170-179. https://doi.org/10.1016/j.plgene.2017.06.003
Mishra, P.K., Bisht, S.C., Ruwari, P., Selvakumar, G., Joshi, G.K., Bisht, J.K., Bhatt, J.C., Gupta, H.S., 2011. Alleviation of cold stress in inoculated wheat (Triticum aestivum L.) seedlings with psychrotolerant Pseudomonads from NW Himalayas. Archives of microbiology. 193, 497-513. https://doi.org/10.1007/s00203-011-0693-x
Mizoi, J., Shinozaki, K., Yamaguchi-Shinozaki, K., 2012. AP2/ERF family transcription factors in plant abiotic stress responses. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms., 1819, 86-96. https://doi.org/10.1016/j.bbagrm.2011.08.004
Mukhopadhyay, P., Tyagi, A.K., 2015. OsTCP19 influences developmental and abiotic stress signaling by modulatingABI4-mediated pathways. Scientific reports. 5, 1-12. https://doi.org/10.1038/srep09998
Nakano, T., Suzuki, K., Fujimura, T., Shinshi, H., 2006. Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant physiology. 140, 411-432. https://doi.org/10.1104/pp.105.073783
Ohama, N., Sato, H., Shinozaki, K., Yamaguchi-Shinozaki, K., 2017. Transcriptional regulatory network of plant heat stress response. Trends in Plant Science. 22, 53-65. https://doi.org/10.1016/j.tplants.2016.08.015
Paolacci, A.R., Tanzarella, O.A., Porceddu, E., Ciaffi, M., 2009. Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC molecular biology. 10, 1-27. https://doi.org/10.1186/1471-2199-10-11
Rawson, H. M., Zajac, M., Penrose, L.D.J., 1998. Effect of seedling temperature and its duration on development of wheat cultivars differing in vernalization response. Field Crops Research. 57, 289-300. https://doi.org/10.1016/S0378-4290(98)00073-2
Rosenzweig, C., Tubiello, F. N., 1996. Effects of changes in minimum and maximum temperature on wheat yields in the central US A simulation study. Agricultural and Forest Meteorology. 80, 215-230. https://doi.org/10.1016/0168-1923(95)02299-6
Rushton, P.J., Somssich, I.E., Ringler, P., Shen, Q.J., 2010. WRKY transcription factors. Trends in Plant Science. 15, 247-258. https://doi.org/10.1016/j.tplants.2010.02.006
Sablowski, R.W., Meyerowitz, E.M., 1998. A homolog of NO APICAL MERISTEM is an immediate target of the floral homeotic genes APETALA3/PISTILLATA. Cell. 92, 93-103. https://doi.org/10.1016/S0092-8674(00)80902-2
Sarvepalli, K., Nath, U., 2011. Hyper‐activation of the TCP4 transcription factor in Arabidopsis thaliana accelerates multiple aspects of plant maturation. The Plant Journal. 67, 595-607. https://doi.org/10.1111/j.1365-313X.2011.04616.x
Seki, M., Kamei, A., Yamaguchi-Shinozaki, K., Shinozaki, K., 2003. Molecular responses to drought, salinity and frost: common and different paths for plant protection. Current opinion in biotechnology. 14, 194-199. https://doi.org/10.1016/S0958-1669(03)00030-2
Tran, L.-S.P., Nakashima, K., Sakuma, Y., Simpson, S.D., Fujita, Y., Maruyama, K., Fujita, M., Seki, M., Shinozaki, K., Yamaguchi-Shinozaki, K., 2004. Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. The Plant Cell. 16, 2481-2498. https://doi.org/10.1105/tpc.104.022699
Trupiano, D., Yordanov, Y., Regan, S., Meilan, R., Tschaplinski, T., Scippa, G. S., Busov, V. 2013. Identification, characterization of an AP2/ERF transcription factor that promotes adventitious, lateral root formation in Populus. Planta. 238, 271-282. https://doi.org/10.1007/s00425-013-1890-4
Verma, S., Bhatia, S., 2019. A comprehensive analysis of the B3 superfamily identifies tissue-specific and stress-responsive genes in chickpea (Cicer arietinum L.). 3 Biotechnology. 9, 1-17. https://doi.org/10.1007/s13205-019-1875-5
Wang, G., Zhang, S., Ma, X., Wang, Y., Kong, F., Meng, Q., 2016. A stress-associated NAC transcription factor (SlNAC35) from tomato plays a positive role in biotic and abiotic stresses. Physiologia Plantarum. 158, 45-64. https://doi.org/10.1111/ppl.12444
Wang, H., Wang, H., Shao, H., Tang, X., 2016. Recent advances in utilizing transcription factors to improve plant abiotic stress tolerance by transgenic technology. Frontiers in Plant Science. 7, 67. https://doi.org/10.3389/fpls.2016.00067
Wang, S.-t., Sun, X.-l., Hoshino, Y., Yu, Y., Jia, B., Sun, Z.-w., Sun, M.-z., Duan, X.-b., Zhu, Y.-m., 2014. MicroRNA319 positively regulates cold tolerance by targeting OsPCF6 and OsTCP21 in rice (Oryza sativa L.). PLoS One. 9, e91357. https://doi.org/10.1371/journal.pone.0091357
Winfield, M. O., Lu, C., Wilson, I. D., Coghill, J. A., Edwards, K. J., 2010. Plant responses to cold: transcriptome analysis of wheat. Plant biotechnology journal. 8, 749-771. https://doi.org/10.1111/j.1467-7652.2010.00536.x
Xu, Z.-S., Chen, M., Li, L.-C., Ma, Y.-Z. 2008. Functions of the ERF transcription factor family in plants. Botany. 86, 969-977.
Zhang, T., Zhao, Y., Wang, Y., Liu, Z., Gao, C., 2018. Comprehensive analysis of MYB gene family and their expressions under abiotic stresses and hormone treatments in Tamarix hispida. Frontiers in Plant Science. 9, 1303. https://doi.org/10.3389/fpls.2018.01303