# Transcriptomic Analysis Reveals the Role of Long Non-Coding RNAs in Response to Drought Stress in Tibetan Hulless Barley

## TL;DR

This study identifies long non-coding RNAs (lncRNAs) involved in drought tolerance in Tibetan hulless barley, revealing their role in regulating stress responses through gene networks.

## Contribution

The study provides new insights into lncRNA-mediated drought stress responses in Tibetan hulless barley using RNA-seq and co-expression network analysis.

## Key findings

- Most lncRNAs in Tibetan hulless barley are short, single-exon, and intergenic, with nearby genes enriched in stress-response pathways.
- WGCNA identified 11 drought-related gene modules with co-expressed lncRNAs and protein-coding genes.
- 12 high-confidence lncRNA-PCG pairs were validated, some involving transcription factors, suggesting regulatory roles in drought adaptation.

## Abstract

The high-altitude ecosystems of the Qinghai-Xizang (Tibet) Plateau (QTP) endowed Tibetan hulless barley (Hordeum vulgare var. nudum) with remarkable resistance to abiotic stresses, making it an excellent crop model for exploring the drought-tolerance mechanism. Here, we characterized all long noncoding RNAs (lncRNAs) with the RNA sequencing (RNA-seq) data from drought-tolerant (Z772) and drought-sensitive (Z013) cultivars subjected to varying drought durations (0, 1, and 5 h). Then, we conducted serval analysis including structural features, differential expression, Weighted Gene Co-expression Network Analysis (WGCNA), Gene Ontology (GO) enrichment, and quantitative real-time polymerase chain reaction (qRT-PCR) analysis to identify the lncRNAs that may be involved in the drought stress response. We found that most lncRNAs were short, single-exon, and intergenic, and their nearby protein-coding genes (PCGs) were enriched in stress-response pathways. These results suggested that the specifically expressed lncRNAs appear to be directly related to the drought tolerance of the cultivars, and lncRNAs may regulate drought stress responses through both cis-regulatory and trans-regulatory mechanisms by controlling transcription factors (TFs) in Tibetan hulless barley.

LncRNAs, a type of RNAs exceeding 200 nucleotides (nt) and lacking representative open reading frames (ORFs), have emerged as crucial regulatory molecules that modulate numerous growth and development processes in plants. While substantial progress has been made in interpreting the functions and regulatory mechanisms of coding RNAs, the study of lncRNAs in Tibetan hulless barley remains incomplete. To elucidate the coordination of drought stress responses in Tibetan hulless barely by lncRNAs, we analyzed the previously published RNA-seq data from two cultivars of hulless barley, drought-tolerant (Z772) and drought-sensitive (Z013), subjected to varying durations of drought treatment (0, 1, and 5 h). Initially, we identified a total of 2877 lncRNAs through a strict pipeline, of which 2179 were co-expressed in both cultivars. Additionally, 331 and 367 lncRNAs showed cultivar-specific expression patterns in Z772 and Z013, respectively. Given the trans-regulatory functions of lncRNAs, we utilized WGCNA and uncovered 11 modules that were enriched in drought-responsive pathways. Within these modules, lncRNAs and neighboring PCGs were co-clustered in key control modules. The GO enrichment analysis of potential lncRNA-PCG pairs primarily involved processes related to the response to water deprivation, regulation of abiotic stress, and RNA metabolic processes. Notably, 12 high-confidence lncRNA-PCG pairs displayed concordant expression profiles, with some annotated as TFs. Two of these pairs were validated by qRT-PCR in the Tibetan hulless barley cultivar Kunlun 14. These findings suggested that lncRNAs may participate in regulatory networks involved in drought adaptation in Tibetan hulless barley, offering novel insights into the drought resistance mechanisms of Poaceae crops and potential targets for breeding drought-resistant varieties.