A long non-coding RNA lncRNA18313 regulates resistance against cadmium stress in wheat

Abstract

Long non-coding RNAs (lncRNAs) have been demonstrated to play key roles in plant response and adaptation to heavy metal stresses. However, the exact biological functions and potential regulatory mechanism, especially in wheat's response to cadmium (Cd) stress, are still poorly understood. We have previously discovered a Cd stress-related lncRNA in wheat, namely TalncRNA18313. In this study, qRT-PCR analysis revealed that TalncRNA18313 was expressed extensively in wheat leaves, and its accumulation was highly induced by Cd stress. To further fully explore the function of lncRNA18313 in response to Cd stress, lncRNA18313 was cloned from wheat (Triticum aestivum L.), and was transformed into Arabidopsis. When TalncRNA18313 was heterologous expressed in Arabidopsis, the transgenic plants exhibited enhanced Cd tolerance characterized by lower malondialdehyde (MDA) levels and higher activities of key antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD). Subsequently, RNA-sequencing (RNA-seq) analysis demonstrated that 370 genes were differentially expressed in lncRNA18313 overexpressing transgenic lines under Cd stress comparing to wild type plants. Among the genes regulated by lncRNA18313, the most significantly enriched were those involved in transcriptional regulation and antioxidative defense responses. These results suggest that TalncRNA18313 plays a crucial role in improving Cd tolerance in wheat by modulating key stress-related pathways, particularly those critical for coping with oxidative damage and regulating gene expression under Cd stress. This discovery contributes to the expanding understanding of knowledge about the involvement of lncRNAs in plant stress responses and offers promising potential for improving crop resilience to environmental stresses.

Keywords: Cd stress; RNA-Seq; heavy metal; long non-coding RNA; wheat.

Figure 1

Characteristics and expression analysis of TalncRNA18313. (A) Analysis of the coding potential of lncRNA18313. (B) The expression levels of lncRNA18313 in wheat leaves treated with 100 μM CdCl₂ at different time points. (C) The expression levels of lncRNA18313 in different tissues of wheat. Gene expression was measured by qRT-PCR, with the expression of lncRNA18313 normalized against the TaEF-1α gene. Each bar represents the mean ± standard error of six replicates. Columns labeled with different letters indicate a significant difference (p <0.05), as determined by Duncan’s multiple range test.

Figure 2

Overexpression of TalncRNA18313 confers Cd tolerance in Arabidopsis. (A) Expression levels of TalncRNA18313 in wild-type (WT) and lncRNA18313- overexpressing (OE) Arabidopsis lines. The expression of TalncRNA18313 was normalized to the AtActin gene. (B) Phenotypes of rosette leaves in 6-week-old WT and TalncRNA18313-OE Arabidopsis seedlings exposed to 0 μM and 100 μM CdCl₂ for 4 days. (C) Determination of malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in WT and TalncRNA18313-OE lines under 100 μM CdCl₂ treatment for 4 days. Each bar represents the mean ± SD for three biological replicates. Columns labeled with different letters indicate significant differences (p < 0.05), as determined by Duncan’s multiple range test.

Figure 3

RNA-seq analysis of differentially expressed genes (DEGs) in lncRNA18313-OE and WT plants under 100 μM CdCl₂ conditions for 4 days. (A) Hierarchical clustering of all DEGs between WT and lncRNA18313-OE under Cd stress, based on log₁₀ RPKM (number of fragments per kilobase of transcript per million fragments mapped) values. The color scale (from blue to red) represents gene expression level from low to high. (B) Changes in DEGs between lncRNA18313-OE and WT plants under Cd stress. The number of up- and down-regulated genes between lncRNA18313-OE and WT is summarized. (C) Correlation between RNA-seq (y-axis) and qRT-PCR (x-axis) data, with the assay conducted for 10 randomly selected DEGs.

Figure 4

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes between lncRNA18313-OE and WT plants under 100 μM Cd stress. (A) GO enrichment analysis. The X-axis represents the GO term, while the Y-axis indicates the number of genes within each category. (B) KEGG enrichment analysis. The Y-axis indicates the KEGG pathway, the X-axis indicates rich factor, which is the ratio of differentially expressed gene numbers annotated in this pathway term to all gene numbers annotated in this pathway term. The color of the dot represents p-value, while the size of the dot represents the number of DEGs involved in the respective pathway.

Figure 5

Effects of overexpressing lncRNA18313 on the expression of differentially expressed genes (DEGs) related to antioxidant enzymes in Arabidopsis under Cd stress. (A) Heatmap of DEG expressions. The bar represents the scale of the expression level for each gene as indicated by blue (low expression) and red rectangles (high expression). (B) RT-qPCR analysis of the expression profiles of four selected transcripts. 0 μM, no Cd treatment; Cd stress, 100 μM CdCl₂. The bars (means ± SD, n = 3) labeled with different letters indicate significant differences (p < 0.05) between treatments as determined by one-way ANOVA with Duncan’s multiple range test.

Figure 6

Effects of overexpressing lncRNA18313 on the expression of DEGs related to transcription factors in Arabidopsis under Cd stress. (A) Heatmap of DEG expression associated with transcription factors. The color scale represents gene expression levels, with blue indicating low expression and red indicating high expression. (B) RT-qPCR analysis of expression profiles in four selected transcripts under Cd stress. 0 μM, no Cd treatment; Cd stress, 100 μM CdCl₂. Bars represent means ± SD (n = 3), with different letters indicating significant differences (p < 0.05) between treatments as determined by one-way ANOVA with Duncan’s multiple range test.