Identification, genomic localization, and functional validation of salt-stress-related lncRNAs in Indian Mustard (Brassica juncea L.)

Abstract

Indian Mustard (Brassica juncea L.) is a globally cultivated winter oilseed crop of the rapeseed-mustard group. It is predominantly grown in the semi-arid northwest agroclimatic zone of India, characterized by high soil salinity. Enhancing tolerance to salt stress in B. juncea is therefore crucial for sustaining its production in this region. Long non-coding RNAs (lncRNAs) play critical roles in coordinating gene expression under various abiotic stresses, including salt stress, but their involvement in the salt stress response in B. juncea remains largely unknown. In this study, we conducted RNA-seq analysis on control, salt-stressed, and salt-shocked young leaves of the salt-tolerant B. juncea cv CS-52. We identified a total of 3,602 differentially expressed transcripts between stress versus control and shock versus control samples. Among these, 61 were identified as potential lncRNAs, with 21 specific to salt stress and 40 specific to salt shock. Of the 21 lncRNAs specific to salt stress, 15 were upregulated and six were downregulated, while all 40 lncRNAs unique to salt shock were downregulated. Chromosomal distribution analysis of the lncRNAs revealed their uneven placement across 18 chromosomes in B. juncea. RNA-RNA interaction analysis between salt stress-upregulated lncRNAs and salt stress-related miRNAs identified 26 interactions between 10 lncRNAs and 23 miRNAs and predicted 13 interactions between six miRNAs and 13 mRNAs. Finally, six lncRNA-miRNA-mRNA interaction networks were established, involving five lncRNAs, 13 miRNAs, and 23 mRNAs. RT-qPCR analysis revealed the upregulation of four out of five lncRNAs, along with their target mRNAs, supporting their involvement in the salt stress response in B. juncea.

Keywords: Brassica juncea; Salt shock; Salt stress; eTMs; lncRNA.

Fig. 1

Workflow for analysing RNA-seq data

Fig. 2

VENN diagram showing the number of differentially expressed transcripts between salt stress versus control and salt shock versus control samples

Fig. 3

Chromosomal location of the salinity-related lncRNAs in B. juncea

Fig. 4

Functional classification of GO term assigned differentially expressed trancripts into (a) biological process (b), molecular functions, and (c), cellular components

Fig. 5

RT-qPCR expression analysis, (a) lncRNA (Bju_lncRNA006) and target mRNA (BjuVA01G16450), (b) lncRNA (Bju_lncRNA019) and target mRNA (BjuVB04G12640), (c) lncRNA (Bju_lncRNA025) and target mRNAs (BjuVA06G46870; BjuVB04G12370; BjuVA08G30140), and (d) lncRNA (Bju_lncRNA043) and target mRNAs (BjuVB02G28640; BjuVB02G56810; BjuVB06G37680; BjuVA07G33590; BjuVA07G14640) under salt-stress conditions in B. juncea. Variables with the same letter have no statistically significant difference in their means