Comparative transcriptome analysis reveals key long noncoding RNAs for cadmium tolerance in Tibetan hull-less barley

Abstract

Cadmium (Cd) is one of the most hazardous and persistent heavy metal pollutants globally. Long noncoding RNAs (lncRNAs) play a crucial role in regulating plant gene expression under various abiotic stress conditions. This study investigated the response of the lncRNA transcriptome in the roots of two contrasting Tibetan hull-less barley genotypes, X178 (Cd-tolerant) and X38 (Cd-sensitive), to Cd stress using RNA sequencing. A total of 8299 novel lncRNAs were identified, with 5166 unique target genes associated with 2571 unique lncRNAs. Among these, 1884 target genes were regulated by cis-acting lncRNAs, while 3428 were regulated by trans-acting lncRNAs. By analyzing differential expression profiles in the two genotypes under Cd stress, 26 lncRNAs and 150 mRNAs were identified as potentially linked to Cd tolerance. Functional enrichment analysis revealed that the target genes were significantly enriched in detoxification and stress response functions, including pathways related to phenylalanine, tyrosine, tryptophan, ABC transporters, and secondary metabolites. Additionally, 12 lncRNAs forming 18 lncRNA-mRNA pairs were identified as key regulators of Cd tolerance. The functional roles of these lncRNA-mRNA interactions suggest that they modulate proteins such as DJ-1, EDR, PHT, and ABC transporters, which may contribute to the Cd tolerance observed in genotype X178. High-throughput sequencing results were validated by qRT-PCR. These findings deepen our understanding of lncRNAs as critical regulators of Cd tolerance in plants, offering valuable insights into the molecular mechanisms underlying heavy metal stress responses in crops.

Keywords: Cd toxicity; Hordeum vulgare var. nudum; high-throughput sequencing; lncRNA; mRNA; target genes.

Figure 1

Coding potential prediction of lncRNAs and mRNAs by CNCI, CPC2, LGC, and SwissProt/Pfam. Venn diagrams show number of predicted lncRNAs (left) and mRNAs (right).

Figure 2

Characterization of all lncRNAs and mRNAs. (A) Length distribution of lncRNAs and mRNAs. (B) The exon features of lncRNAs and mRNAs. (C) The transcripts number of genes and lncRNA loci.

Figure 3

Expression levels and densities of mRNA and lncRNA in Cd-tolerant (X178) and Cd-sensitive (X38) barley genotypes under 20 µmol L^-1 Cd stress. X-axis: RNA density; Y-axis: log₁₀(FPKM). Colors denote RNA type. Box plot: median (black line), quartiles (box), and data range (whiskers).

Figure 4

Root transcriptome profiles of Cd stress-responsive mRNAs and lncRNA in Cd-tolerant (X178) and Cd-sensitive (X38) barley genotypes under 20 µmol L^-1 Cd stress. log₂N, log₂N ≥ 1 are up-regulated, between 0 < |log₂N| < 1 are unchanged and log₂N ≤ − 1 are down-regulated.

Figure 5

Correlation between lncRNA/mRNA sequencing and qRT-PCR data. (a) Fold changes from sequencing (Y-axis) versus qRT-PCR (X-axis) for X178 and X38 under Cd stress. R² indicates linear regression fit quality. (b-k) Validation of six lncRNAs and four mRNAs by qRT-PCR in X178 (orange) and X38 (blue) under Cd stress. “Seq” denotes high-throughput sequencing data.

Figure 6

Annotation and function prediction of all mRNAs identified in four libraries. (A) Annotation of mRNAs by NR, NT, KEGG, KOG, Uniprot. Venn diagrams show number of mRNAs. (B) The species distribution of annotation results.

Figure 7

GO analysis of target genes of differentially expressed lncRNAs. GO classification of lncRNA target genes under Cd stress. The Y and X axes correspond to GO terms and the number of target genes (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Figure 8

KEGG enrichment analysis of target genes of differentially expressed lncRNAs. KEGG pathway classification of target genes of lncRNAs under Cd stress in X178 (A) and X38 (B), respectively. The Y axis corresponds to KEGG pathway, the X axis shows the enrichment ratio between the number of DEGs and all unigenes enriched in a particular pathway. The color of the dot represents p-value, and the size of the dot represents the number of DEGs mapped to the referent pathway.

Figure 9

A hypothetical schematic illustration of the mechanism that underlies Cd tolerance and adaptation at the lncRNAs-mRNA level in X178. Within each genotype, the fold change (Cd vs control) is log₂N, where changes in log₂N ≥ 1 are up-regulated, 0 < |log₂N| < 1 are unchanged and log₂N ≤ –1 are down-regulated. For the box representing the expression, red, blue, and gray indicate up-regulated, down-regulated, and unchanged, respectively; the left and right represent X178 and X38. DJ-1, Protein DJ-1 homolog B; EDR, Protein enhanced disease resistance 2; ABC T, ABC transporter C family member 13; PHT, Putrescine hydroxycinnamoyltransferase 1.