Plant Genetic Research
Scientific Journal

Identification, Functional Prediction, and Expression Analysis of Long Non-Coding RNAs in Lentil (Lens culinaris L.) Under Salt Stress

Document Type : Original Article

Authors

Saba Saeidi
Ahmad Ismaili
Seyed Sajad Sohrabi

Department of Plant Production and Genetic Engineering, Faculty of Agriculture, Lorestan University, Khorramabad, Iran

10.22034/pgr.2025.2081840.1027
Abstract
Throughout evolution, plants have developed complex molecular mechanisms to cope with biotic and abiotic stresses, many of which are driven by the reprogramming of gene expression. Multiple genetic factors regulate the molecular responses of plants to unfavorable environmental conditions, among which long non-coding RNAs (lncRNAs) play a significant role. LncRNAs play a pivotal role in regulating gene expression, epigenetic modifications, and signaling pathways, significantly contributing to responses against both biotic and abiotic stresses. In this study, for the first time, the identification, functional determination, and expression analysis of LncRNAs in lentil (Lens culinaris L.) under salt stress conditions were investigated. LncRNAs were identified using the lentil transcriptome reference, which was generated by assembling RNA sequencing data from the leaf and root tissues of lentil under both stress and control conditions. After filtering protein-coding sequences, several lncRNA sequences were identified using PLncPRO software. The relative expression levels of the identified sequences were measured, and the co-expression network with differentially expressed genes (DEGs) was constructed. To validate the RNA-seq data, the expression of selected lncRNAs was analyzed by qRT-PCR. The processing and refinement of the expression profile of lentil under salt stress resulted in the identification of 7677 lncRNA sequences, with 722 sequences being identified for the first time in lentil. Gene expression analysis revealed that 90 sequences in leaf tissue and 48 in root tissue showed increased expression under salt stress. Functional analysis of the co-expressed genes indicated that these genes were involved in biological processes such as translation, ion transport, and stress response. Metabolic pathway enrichment analysis identified 18 significant pathways, including S-adenosyl-L-methionine cycle, abscisic acid biosynthesis, and cytosolic glycolysis. The qRT-PCR validation confirmed the correlation of RNA-seq and qRT-PCR results with a 95% correlation coefficient. In conclusion, the results of this study revealed that the identified lncRNAs play a significant role in regulating key genes involved in essential metabolic pathways in lentil's molecular response to salt stress. Identifying and analyzing these lncRNAs in lentil can provide a better understanding of the molecular mechanisms underlying these stress responses. Investigating the interactions of these identified RNAs with other regulatory molecules, such as ABA and protein-coding genes, could contribute to the development of breeding strategies aimed at improving lentil’s tolerance to both biotic and abiotic stresses.

Keywords

Salt stress
Gene expression regulation
Lentil
LncRNA

Subjects

Anee, T.I., Sewelam, N.A., Bautista, N.S., Hirayama, T. and Suzuki, N. (2025). Roles of ROS and NO in Plant Responses to Individual and Combined Salt Stress and Waterlogging. Antioxidants, 14(12): 1455. https://doi.org/10.3390/antiox14121455   
Bae, N., Shim, S.H., Alavilli, H., Do, H., Park, M., Lee, D.W., Lee, J.H., Lee, H., Li, X., Lee, C.H., Jeon, J.S. and Lee, B.H. (2025). Enhanced salt stress tolerance in plants without growth penalty through increased photosynthesis activity by plastocyanin from Antarctic moss. Plant Journal, 121(1): e17168. https://doi.org/10.1111/tpj.17168  
Baum, M. and Kumar, J. (2024). Lentil genome sequencing: Establishing a comprehensive platform for molecular breeding. In The Lentil Genome: Genetics, Genomics and Breeding (pp. 217-236). https://doi.org/10.1016/B978-0-443-19409-2.00008-9  
Chekanova, J.A. (2015). Long non-coding RNAs and their functions in plants. Curr Opin Plant Biol, 27: 207-216. https://doi.org/10.1016/j.pbi.2015.08.003  
Dhakate, P., Ambreen, H., Chakraborty, A., Singh, B., Yadav, M., Tiwari, M., Singh, G., Pandey, V. and Bhatia, S. (2020). Comprehending lncRNA-mediated gene regulation during abiotic stresses and reproductive development in legumes. In Long Noncoding RNAs in Plants: Roles in Development and Stress (pp. 151-176). https://doi.org/10.1016/B978-0-12-821452-7.00010-6  
Fu, H. and Yang, Y. (2023). How Plants Tolerate Salt Stress. Current Issues in Molecular Biology, 45(7): 5914-5934. https://doi.org/10.3390/cimb45070374  
Goudarzi, M., Ismaili, A., Sohrabi, S.S., Nazarian-Firouzabadi, F. and Eisvand, H.R. (2024). Transcriptomic analysis of salt-stress-responsive genes in lentil roots and leaves. Plant Biotechnology Reports, 18(7): 907-925. https://doi.org/10.1007/s11816-024-00937-x  
Haghi Asl, N., Darvishzadeh, R., Alipour, H., Fatanatvash, S. and Arzhang, S. (2025). Genomic selection for grain yield in maize (Zea mays L.) under normal and salinity stress conditions. Plant Genetic Research, 12(1): 1-20. (In Persian). https://doi.org/10.22034/pgr.2025.2062236.1000
Hossain, M.S., Hasanuzzaman, M., Rahman, A., Nahar, K., Mahmud, J.A. and Fujita, M. (2019). Heat shock-induced salt stress tolerance in lentil (Lens culinaris Medik.). Russian Journal of Plant Physiology, 66(3): 450-460. https://doi.org/10.1134/S1021443719030075  
Ismaili, A., Sohrabi, S.S., Hosseini, S.Z., Namdarian, R. and Godarzi, D. (2016). Genotypic correlation and path analysis of some traits related to oil yield and grain yield in canola (Brassica napus L.) under non-stress and water deficit stress conditions.‏ Iranian Journal of Field Crops Research, 4(4): 646-664 (In Persian). https://doi.org/10.22067/gsc.v14i4.41019
Khadivar, R., Ismaili, A., Sohrabi, S.S. and Torabi Podeh, H. (2023). identification and functional prediction of long non-coding RNAs Responsive to drought stress in Lens culinaris L. Plant Genetic Research. 9(2): 55-70 (In Persian). http://doi.org/10.22034/pgr.9.2.5
Kumar, N., Bharadwaj, C., Sahu, S., Shiv, A., Shrivastava, A.K., Reddy, S.P.P., Soren, K.R., Patil, B.S., Pal, M., Soni, A., Roorkiwal, M. and Varshney, R.K. (2021). Genome-wide identification and functional prediction of salt- stress related long non-coding RNAs (lncRNAs) in chickpea (Cicer arietinum L.). Physiology and Molecular Biology of Plants, 27(11): 2605-2619. https://doi.org/10.1007/s12298-021-01093-0  
Li, J., He, H., Li, Y., Wang, A. and He, L.F. (2022). Role of long non-coding RNA in plant responses to abiotic stresses. Acta Physiologiae Plantarum, 44(5): 54. https://doi.org/10.1007/s11738-022-03387-6  
Li, X. and Zhang, N. (2025). Methionine as a key player in salt stress adaptation in plants. Plant Physiology, 198(1): kiaf169. https://doi.org/10.1093/plphys/kiaf169   
Li, Z., Zhou, H., Xu, G., Zhang, P., Zhai, N., Zheng, Q., Liu, P., Jin, L., Bai, G. and Zhang, H. (2023). Genome-wide analysis of long noncoding RNAs in response to salt stress in Nicotiana tabacum. BMC Plant Biology, 23(1): 646. https://doi.org/10.1186/s12870-023-04659-0  
Livak, K.J. and Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods, 25(4): 402-408.‏ https://doi.org/10.1006/meth.2001.1262
Mahajan, M., Poor, P., Kaur, H., Aher, R.R., Palakolanu, S.R. and Khan, M.I.R. (2025a). Salt stress tolerance and abscisic acid in plants: associating role of plant growth regulators and transcription factors. Plant Physiology and Biochemistry, 228: 110303. https://doi.org/10.1016/j.plaphy.2025.110303  
Mahdavian, K. (2023). Effect of calcium chloride and salicylic acid on antioxidative responses of lentils (Lens culinaris Medik.) under salt stress. Iranian Journal of Plant Physiology, 13(2): 4541-4554. https://doi.org/10.30495/ijpp.2023.704670  
Paul, A., Mondal, S., Pal, A., Biswas, S., Chakraborty, K., Mazumder, A., Biswas, A.K. and Kundu, R. (2023). Seed priming with NaCl helps to improve tissue tolerance, potassium retention ability of plants, and protects the photosynthetic ability in two different legumes, chickpea and lentil, under salt stress. Planta, 257(6): 111. https://doi.org/10.1007/s00425-023-04150-y   
Saha, C., Saha, S. and Bhattacharyya, N.P. (2025). LncRNAOmics: a comprehensive review of long non-coding RNAs in plants. Genes, 16(7): 765. https://doi.org/https://doi.org/10.3390/genes16070765  
Shi, B., Li, K., Xu, R., Zhang, F., Yu, Z., Ding, Z. and Tian, H. (2025). Methionine-mediated trade-off between plant growth and salt tolerance. Plant Physiology, 197(3): kiaf074. https://doi.org/10.1093/plphys/kiaf074  
Sun, X., Tian, R., Zhao, M. and Zhang, W.H. (2026). The lncRNA MtCIR2 suppresses seed germination by PRC1-dependent H2A ubiquitination in Medicago truncatula under salt stress. Plant Journal, 125(1): e70682. https://doi.org/10.1111/tpj.70682  
Tiwari, A., Joshi, S., Joshi, R. and Bohra, A. (2024). Omics advancements in plant abiotic stress. In Current Omics Advancement in Plant Abiotic Stress Biology: a volume in Developments in Applied Microbiology and Biotechnology (pp. 23-37). https://doi.org/10.1016/B978-0-443-21625-1.00002-6  
Tiwari, V., Kumar, A. and Singh, P. (2021). Effects of Salt Stress on Physiology of Crop Plants: At Cellular Level. In Physiology of Salt Stress in Plants: Perception, Signalling, Omics and Tolerance Mechanism (pp. 16-37). https://doi.org/10.1002/9781119700517.ch2  
Tripathi, A., Singh, D., Bhati, J., Singh, D., Taunk, J., Alkahtani, J., Al-Hashimi, A. and Singh, M.P. (2025). Genome wide identification of MATE and ALMT gene family in lentil (Lens culinaris Medikus) and expression profiling under Al stress condition. BMC Plant Biology, 25(1): 88. https://doi.org/10.1186/s12870-025-06086-9   
Wang, T.Z., Liu, M., Zhao, M.G., Chen, R. and Zhang, W.H. (2015). Identification and characterization of long non-coding RNAs involved in osmotic and salt stress in Medicago truncatula using genome-wide high-throughput sequencing. BMC Plant Biology, 15: 131. https://doi.org/10.1186/s12870-015-0530-5  
Xifang, Z., Wang, S., Han, Y., Zhao, Q., Xu, P., Yan, Q., Wu, F. and Zhang, J. (2021). Genome-wide profiling of the potential regulatory network of lncRNA and mRNA in Melilotus albus under salt stress. Environmental and Experimental Botany, 189: 104548. https://doi.org/https://doi.org/10.1016/j.envexpbot.2021.104548  
Zhao, S., Zhang, Q., Liu, M., Zhou, H., Ma, C. and Wang, P. (2021). Regulation of plant responses to salt stress. International Journal of Molecular Sciences, 22(9): 4609. https://doi.org/10.3390/ijms22094609   
Zong, X., Wang, S., Han, Y., Zhao, Q., Xu, P., Yan, Q., Wu, F. and Zhang, J. (2021). Genome-wide profiling of the potential regulatory network of lncRNA and mRNA in Melilotus albus under salt stress. Environmental and Experimental Botany, 189: 104548. https://doi.org/10.1016/j.envexpbot.2021.104548  
Zulfiqar, F. and Ashraf, M. (2023). Proline Alleviates Abiotic Stress Induced Oxidative Stress in Plants. Journal of Plant Growth Regulation, 42(8): 4629-4651. https://doi.org/10.1007/s00344-022-10839-3  

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