TY  - JOUR
T1  - Determination of Cold Tolerance in Local Alfalfa (M. sativa L.) Cultivars Grown in Türkiye under In Vitro Conditions through MsPYL3-3 and MsPYL5-1 Genes
TT  - Determination of Cold Tolerance in Local Alfalfa (Medicago sativa L.) Varieties Grown in Turkey under In Vitro Conditions through MsPYL3-3 and MsPYL5-1 Genes
AU  - İlhan, Doğan
AU  - Akçay, Mustafa
AU  - Yazıcılar, Büşra
AU  - Koç, Ümmü Gülsüm
AU  - Bezirganoglu, İsmail
PY  - 2025
DA  - June
Y2  - 2025
DO  - 10.46876/ja.1709167
JF  - Journal of Agriculture
JO  - JOAG
PB  - Barış EREN
WT  - DergiPark
SN  - 2636-8757
SP  - 65
EP  - 75
VL  - 8
IS  - 1
LA  - en
AB  - Alfalfa is an important legume cultivated worldwide. Cold stress adversely affects alfalfa’s growth and development. Recently, the use of phytohormone salicylic acid (SA) and nanoparticles are attracting attention as effective approaches for gaining tolerance in both plant development and stress defense mechanisms. This study investigated how the SA and magnesium oxide nanoparticles (MgO NPs) influence the expression of MsPYL3-3 and MsPYL5-1 genes in two alfalfa varieties, Denizli and Van, under cold stresses of 10°C and 4°C. Initially, plants were grown under controlled conditions (25°C, 16-hours light/8-hours dark photoperiod, 60% humidity) for one month before being exposed to cold treatments. The treatments included foliar applications of 1 mM and 2 mM SA, as well as 5 ppm and 20 ppm MgO nanoparticles. Gene expression was measured using RT-qPCR analysis. Results showed that lowering temperatures induced upregulation of MsPYL3-3 and MsPYL5-1 in both varieties. Moreover, treatment with SA and MgO nanoparticles further increased the expression levels of these genes. Notably, the gene expression responses varied between the Denizli and Van varieties, indicating genotype-specific differences.
KW  - Medicago sativa L.
KW  - Salicylic acid
KW  - MgO nanoparticle
KW  - MsPYL3-3
KW  - MsPYL5-1
N2  - Alfalfa is an important legume cultivated worldwide. Cold stress adversely affects alfalfa’s growth and development. Recently, the use of phytohormone salicylic acid (SA) and nanoparticles are attracting attention as effective approaches for gaining tolerance in both plant development and stress defense mechanisms. This study investigated how salicylic acid (SA) and magnesium oxide nanoparticles (MgO NPs) influence the expression of MsPYL3-3 and MsPYL5-1 genes in two alfalfa varieties, Denizli and Van, under cold stresses of 10°C and 4°C. Initially, plants were grown under controlled conditions (25°C, 16-hours light/8-hours dark photoperiod, 60% humidity) for one month before being exposed to cold treatments. The treatments included foliar applications of 1 mM and 2 mM SA, as well as 5 ppm and 20 ppm MgO nanoparticles. Gene expression was measured using RT-qPCR analysis. Results showed that lowering temperatures induced upregulation of MsPYL3-3 and MsPYL5-1 in both varieties. Moreover, treatment with SA and MgO nanoparticles further increased the expression levels of these genes. Notably, the gene expression responses varied between the Denizli and Van varieties, indicating genotype-specific differences.
CR  - Adhikari, L., Baral, R., Paudel, D., Min, D., Makaju, S. O., Poudel, H. P., Acharya, J. P., & Missaoui, A. M. (2022). Cold stress in plants: Strategies to improve cold tolerance in forage species. Plant Stress, 4, 100081.
CR  - Ahmed, S., Chaudhry, S. A., & Ikram, S. (2017). A review on biogenic synthesis of ZnO nanoparticles using plant extracts and microbes: a prospect towards green chemistry. Journal of Photochemistry and Photobiology B: Biology, 166, 272-284.
CR  - Akçay, M., Yazıcılar, B., Kassa, S. B., Ilhan, D., Shadıdızajı, A., & Bezırganoglu, I. (2025). Synergistic effects of salicylic acid and magnesium oxide nanoparticles on cold stress and miRNA expression in alfalfa (Medicago sativa L.) genotypes. Plant Cell, Tissue and Organ Culture (PCTOC), 161(3), 1-18.
CR  - Arif, Y., Sami, F., Siddiqui, H., Bajguz, A., & Hayat, S. (2020). Salicylic acid in relation to other phytohormones in plant: A study towards physiology and signal transduction under challenging environment. Environmental and Experimental Botany, 175, 104040.
CR  - Bhardwaj, B., Singh, P., Kumar, A., Kumar, S., & Budhwar, V. (2020). Eco-friendly greener synthesis of nanoparticles. Advanced pharmaceutical bulletin, 10(4), 566.
CR  - Cofer, T., Engelberth, M., & Engelberth, J. (2018). Green leaf volatiles protect maize (zea mays) seedlings against damage from cold stress. Plant Cell & Environment, 41(7), 1673-1682. https://doi.org/10.1111/pce.13204
CR  - Deng, X., Wang, J., Li, Y., Wu, S., Yang, S., Chao, J., Chen, Y., Zhang, S., Shi, M., & Tian, W. (2018). Comparative transcriptome analysis reveals phytohormone signalings, heat shock module and ros scavenger mediate the cold-tolerance of rubber tree. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-23094-y
CR  - Gondor, O. K., Pál, M., Janda, T., & Szalai, G. (2022). The role of methyl salicylate in plant growth under stress conditions. Journal of Plant Physiology, 277, 153809.
CR  - Hall, J., Cobb, J., Iqbal, M., Abidali, M., Liu, Z., & Mount, D. (2008). Uvh6, a plant homolog of the human/yeast tfiih transcription factor subunit xpd/rad3, regulates cold-stress genes in arabidopsis thaliana. Plant Molecular Biology Reporter, 27(2), 217-228. https://doi.org/10.1007/s11105-008-0076-x
CR  - Hong, J. and Ryu, H. (2023). Identification and functional analysis of cold-signaling-related genes in panax ginseng. Journal of Plant Biotechnology, 50. https://doi.org/10.5010/jpb.2023.50.028.225
CR  - Hernández, J. A., Diaz-Vivancos, P., Barba-Espín, G., & Clemente-Moreno, M. J. (2017). On the role of salicylic acid in plant responses to environmental stresses. Salicylic acid: a multifaceted hormone, 17-34.
Iravani, S. (2011). Green synthesis of metal nanoparticles using plants. Green chemistry, 13(10), 2638-2650.
CR  - Karlidag, H., Yildirim, E., & Turan, M. (2009). Exogenous applications of salicylic acid affect quality and yield of strawberry grown under antifrost heated greenhouse conditions. Journal of Plant Nutrition and Soil Science, 172(2), 270-276.
CR  - Khan, M. I. R., Fatma, M., Per, T. S., Anjum, N. A., & Khan, N. A. (2015). Salicylic acid-induced abiotic stress tolerance and underlying mechanisms in plants. Frontiers in plant science, 6, 462.
CR  - Kidokoro, S., Watanabe, K., Ohori, T., Moriwaki, T., Maruyama, K., Mizoi, J., Htwe, N. M. P. S., Fujita, Y., Sekita, S., Shinozaki, K., Shinozaki, K. Y., & Yamaguchi‐Shinozaki, K. (2015). Soybean dreb1/cbf‐type transcription factors function in heat and drought as well as cold stress‐responsive gene expression. The Plant Journal, 81(3), 505-518. https://doi.org/10.1111/tpj.12746
CR  - Klessig, D. F., Choi, H. W., & Dempsey, D. M. A. (2018). Systemic acquired resistance and salicylic acid: past, present, and future. Molecular plant-microbe interactions, 31(9), 871-888.
CR  - Kumari, R., & Nath, M. (2018). Synthesis and characterization of novel trimethyltin (IV) and tributylltin (IV) complexes of anticoagulant, WARFARIN: Potential DNA binding and plasmid cleaving agents. Inorganic Chemistry Communications, 95, 40-46.
CR  - Li, Y., Liu, G. B., Gao, H. W., Wang, Z., Zhao, H. M., Xie, N., & Wang, Y. H. (2009). Study on comprehensive evaluation of drought resistance of Medicago sativa L. germplasm at seedling stage. Acta Agrestia Sinica, 17(6), 807.
CR  - Liu, Y., Cai, Y., Li, Y., Zhang, X., Shi, N., Zhao, J., & Yang, H. (2022). Dynamic changes in the transcriptome landscape of arabidopsis thaliana in response to cold stress. Frontiers in Plant Science, 13. https://doi.org/10.3389/fpls.2022.983460
CR  - Ma, Z., Huang, B., Xu, S., Chen, Y., Li, S. and Lin, S. 2015. Isolation of high-quality total RNA from chinese fir (Cunninghamia lanceolata (Lamb.) Hook). PLoS One, 10(6), e0130234.
CR  - Miura, K., Ohta, M., Nakazawa, M., Ono, M., & Hasegawa, P. (2011). Ice1 ser403 is necessary for protein stabilization and regulation of cold signaling and tolerance. The Plant Journal, 67(2), 269-279. https://doi.org/10.1111/j.1365-313x.2011.04589.x
CR  - Miura, K., & Tada, Y. (2014). Regulation of water, salinity, and cold stress responses by salicylic acid. Frontiers in plant science, 5, 4.
CR  - Nejati, M., Rostami, M., Mirzaei, H., Rahimi-Nasrabadi, M., Vosoughifar, M., Nasab, A. S., & Ganjali, M. R. (2022). Green methods for the preparation of MgO nanomaterials and their drug delivery, anti-cancer and anti-bacterial potentials: A review. Inorganic Chemistry Communications, 136, 109107.
CR  - Nian, L., Zhang, X., Yi, X., Liu, X., Ain, N. U., Yang, Y., Li, X., Haider, F. U., & Zhu, X. (2021). Genome-wide identification of ABA receptor PYL/RCAR gene family and their response to cold stress in Medicago sativa L. Physiology and Molecular Biology of Plants, 27, 1979-1995.
CR  - Pugazhendhi, A., Prabhu, R., Muruganantham, K., Shanmuganathan, R., & Natarajan, S. (2019). Anticancer, antimicrobial and photocatalytic activities of green synthesized magnesium oxide nanoparticles (MgONPs) using aqueous extract of Sargassum wightii. Journal of Photochemistry and Photobiology B: Biology, 190, 86-97.
CR  - Ren, C., Kuang, Y., Lin, Y., Guo, Y., Li, H., Fan, P., Li, S., & Liang, Z. (2022). Overexpression of grape ABA receptor gene VaPYL4 enhances tolerance to multiple abiotic stresses in Arabidopsis. BMC Plant Biology, 22(1), 271.
CR  - Samac, D. A., & Jung, H. J. G, Lamb JFS (2006): Development of alfalfa (Medicago sativa L.) as a feedstock for production of ethanol and other bio products.
CR  - Seki, M., Narusaka, M., Ishida, J., Nanjo, T., Fujita, M., Oono, Y., Kamiya, A., Nakajima, M., Enju, A., Sakurai, T., Satou, M., Akiyama, K., Taji, T., Shinozaki, K. Y., Carninci, P., Kawai, J., Hayashizaki, Y., & Shinozaki, K. (2002). Monitoring the expression profiles of 7000 arabidopsis genes under drought, cold and high‐salinity stresses using a full length cdna microarray. The Plant Journal, 31(3), 279-292. https://doi.org/10.1046/j.1365-313x.2002.01359.x
CR  - Shang, Y., Hasan, M. K., Ahammed, G. J., Li, M., Yin, H., & Zhou, J. (2019). Applications of nanotechnology in plant growth and crop protection: a review. Molecules, 24(14), 2558.
CR  - Shen, Y., He, L., Yang, Z., & Xiong, Y. (2020). Corrosion behavior of different coatings prepared on the surface of AZ80 magnesium alloy in simulated body fluid. Journal of Materials Engineering and Performance, 29, 1609-1621.
CR  - Sun, C., Zhu, L., Cao, L., Qi, H., Liu, H., Zhao, F., & Han, X. (2022). Pks5 confers cold tolerance by controlling stomatal movement and regulating cold-responsive genes in arabidopsis. Life, 12(10), 1633. https://doi.org/10.3390/life12101633
CR  - Szczerba, A., Płażek, A., Pastuszak, J., Kopeć, P., Hornyák, M., & Dubert, F. (2021). Effect of low temperature on germination, growth, and seed yield of four soybean (Glycine max L.) cultivars. Agronomy, 11(4), 800.
CR  - Vijayakumar, H., Thamilarasan, S., Shanmugam, A., Natarajan, S., Jung, H., Park, J., Kim, H., Chung, M. Y., & Nou, I. (2016). Glutathione transferases superfamily: cold-inducible expression of distinct gst genes in brassica oleracea. International Journal of Molecular Sciences, 17(8), 1211. https://doi.org/10.3390/ijms17081211
CR  - Wang, W., Wang, X., Zhang, J., Huang, M., Cai, J., Zhou, Q., Dai, T., & Jiang, D. (2020). Salicylic acid and cold priming induce late-spring freezing tolerance by maintaining cellular redox homeostasis and protecting photosynthetic apparatus in wheat. Plant Growth Regulation, 90, 109-121.
CR  - Wu, L., Zhang, Z., Zhang, H., Wang, X. C., & Huang, R. (2008). Transcriptional modulation of ethylene response factor protein JERF3 in the oxidative stress response enhances tolerance of tobacco seedlings to salt, drought, and freezing. Plant physiology, 148(4), 1953-1963.
CR  - Yang, S. S., Xu, W. W., Tesfaye, M., Lamb, J. F., Jung, H. J. G., VandenBosch, K. A., Vance, C. P., & Gronwald, J. W. (2010). Transcript profiling of two alfalfa genotypes with contrasting cell wall composition in stems using a cross-species platform: optimizing analysis by masking biased probes. BMC genomics, 11, 1-18.
CR  - Yu, G., Jiang, L., Ma, X., Xu, Z., Liu, M., Shan, S., & Cheng, X. (2014). A soybean c2h2-type zinc finger gene gmzf1 enhanced cold tolerance in transgenic arabidopsis. Plos One, 9(10), e109399. https://doi.org/10.1371/journal.pone.0109399
CR  - Zhang, F., Huang, L., Wang, W., Zhao, X., Zhu, L., Fu, B., & Li, Z. (2012). Genome-wide gene expression profiling of introgressed indica rice alleles associated with seedling cold tolerance improvement in a japonica rice background. BMC Genomics, 13(1). https://doi.org/10.1186/1471-2164-13-461
CR  - Zhao, C., Zhang, Z., Xie, S., Si, T., Li, Y., & Zhu, J. (2016). Mutational evidence for the critical role of cbf transcription factors in cold acclimation in arabidopsis. Plant Physiology, 171(4), 2744-2759. https://doi.org/10.1104/pp.16.00533
CR  - Zhao, H., Nie, K., Zhou, H., Yan, X., Zhan, Q., Zheng, Y., & Song, C. P. (2020). ABI5 modulates seed germination via feedback regulation of the expression of the PYR/PYL/RCAR ABA receptor genes. New Phytologist, 228(2), 596-608.
UR  - https://doi.org/10.46876/ja.1709167
L1  - https://dergipark.org.tr/tr/download/article-file/4914554
ER  -