Integrative 16S rRNA and transcriptome analysis reveals the molecular mechanisms underlying salt- tolerant germination in highland barley (Hordeum vulgare var. coeleste Linnaeus) seeds

TL;DR

This study identifies molecular and microbial mechanisms that help highland barley seeds tolerate high salt levels during germination.

Contribution

The study integrates 16S rRNA and transcriptome data to reveal novel genetic and microbial factors involved in salt tolerance in highland barley.

Findings

Salt-tolerant variety 37 showed 98% germination under 500 mmol/L NaCl with lower Na+ accumulation and Na+/K+ ratio.

Endophytic bacterium Brevundimonas was more abundant in salt-tolerant seeds and correlated with higher K+ content.

Transcriptome analysis identified 1,467 DEGs in salt-tolerant seeds, enriched in potassium homeostasis and oxidative stress response pathways.

Abstract

Salt stress is a critical abiotic factor that impairs crop seed germination and limits agricultural productivity. Elucidating the mechanisms governing salt tolerance is essential for development of salt-tolerant crop varieties. In this investigation, 217 accessions of highland barley (Hordeum vulgare var. coeleste Linnaeus) were evaluated. Germination assays conducted under 200 mmol/L and 500 mmol/L NaCl conditions identified a salt-tolerant variety 37 and a salt-sensitive variety 44. By integrating transcriptome sequencing, 16S rRNA sequencing, and Na+/K+ content analysis, we systematically investigated the molecular mechanisms underlying salt-tolerant germination in highland barley seeds. Our findings revealed that the salt-tolerant variety 37 maintained a high germination rate of 98% under 500 mmol/L NaCl stress, with lower Na+ accumulation (4.24 g/kg) and a lower Na+/K+ ratio (2.59) compared to the salt-sensitive variety 44 (Na+ accumulation: 4.89 g/kg, Na+/K+ ratio: 3.62). Analysis of 16S rRNA sequencing data showed a significant increase in the abundance of the endophytic bacterium Brevundimonas in salt-tolerant variety 37 under high-salt conditions, which was positively correlated with K+ content. In contrast, the dominant bacterium Rhodococcus in salt-sensitive variety 44 exhibited a positive correlation with Na+ content and the Na+/K+ ratio. Transcriptome sequencing identified 1,467 and 1,644 differentially expressed genes (DEGs) in salt-tolerant variety 37 and salt-sensitive variety 44, respectively. Pathway enrichment analysis indicated that DEGs in salt-tolerant variety 37 were primarily associated with “potassium ion homeostasis” and “response to oxidative stress”. Weighted gene co-expression network analysis (WGCNA) identified 5 co-expression modules, among which the MEyellow module was correlated with Na+ content (r = 0.59). Ten core genes were identified, including WRKY transcription factor (HORVU.MOREX.r3.3HG0268090) and receptor protein kinase (RPK; HORVU.MOREX.r3.4HG0331910). A total of 174 HvRPK genes were identified, distributed across 7 chromosomes with a predominant localization on chromosome 2. These genes exhibited functional conservation and were involved in salt stress signaling pathways. Phylogenetic, collinearity, and cis-element analyses further supported their regulatory role in salt stress responses. This study clarifies the key mechanisms underlying salt-tolerant germination in highland barley seeds, providing valuable insights and genetic resources for the molecular breeding of salt-tolerant crops.

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