Determination of salt tolerance levels of walnut cultivars and identification of genes responsible for salt stress in walnut by rnaseq analysis

Walnut (Juglans regia L.) is highly sensitive to salinity stress, posing a significant challenge for sustainable cultivation under increasing soil and water salinity. This study aimed to evaluate the morphological, physiological, and molecular responses of walnut genotypes to salt stress over two consecutive years (2021–2022). In the first year, 21 genotypes were subjected to four NaCl concentrations (75, 150, 225, and 300 mM). Morphophysiological assessments revealed significant reductions in growth parameters with increasing salinity, though genotypic variations were evident. The genotype 'Serr' maintained the highest plant height, leaf number, relative water content (RWC), and root dry weight across all salt treatments, while 'Sölez-1' showed the highest SPAD value, leaf number, and 100% survival rate. Conversely, 'Fernor' exhibited the most severe symptoms under salt stress, including elevated leaf temperature, decreased RWC and SPAD values, and a survival rate reduced to 41% under 300 mM NaCl. Based on these findings, 'Serr', 'Sölez-1', and 'Maraş-18' were classified as salt-tolerant, while 'Fernor', 'Chandler', and 'Hartley' were categorized as sensitive. In the second year, these six genotypes were further evaluated under 150 mM NaCl. 'Serr' maintained favorable physiological traits, including high RWC, broader leaf area, less negative osmotic potential, increased proline accumulation, limited Na⁺ and Cl⁻ accumulation, and elevated K⁺ and Ca²⁺ levels, indicating effective ion homeostasis. In contrast, 'Fernor' displayed disrupted water relations and ionic imbalance under stress. RNA-Seq analyses were performed using Serr (salt-tolerant) and Fernor (salt-sensitive) walnut genotypes to elucidate transcriptional responses under 150 mM NaCl-induced salt stress. A total of 14,153 novel transcripts were identified, and differential gene expression (DGE) analysis revealed 161 candidate genes potentially associated with salt stress response. Functional categorization of these genes through Gene Ontology (GO) enrichment analysis indicated significant enrichment in biological processes such as ion transport, osmotic adjustment, oxidative stress mitigation, and signal transduction pathways. To validate RNA-Seq results, 12 candidate genes were selected based on their putative functional relevance, and 14 specific primer pairs were designed for quantitative real-time PCR (qPCR) analysis. The qPCR validation confirmed consistent expression trends with RNA-Seq data for seven genes. Notably, LOC108990701, encoding Δ¹-pyrroline-5-carboxylate synthetase (P5CS), was significantly upregulated under salt stress and implicated in proline biosynthesis and osmotic adjustment. Similarly, LOC108995962, associated with transmembrane potassium transport and stomatal regulation, exhibited increased expression, suggesting its role in maintaining ionic balance. LOC108990546, a gene involved in oxidative stress response, and LOC109012163, encoding a lipoxygenase enzyme linked to cellular redox homeostasis, were also differentially expressed and demonstrated strong correlation between qPCR and transcriptomic data. In conclusion, this study provides a comprehensive understanding of the multilevel responses of walnut to salinity and identifies promising candidate genes for molecular breeding aimed at improving salt tolerance in walnut. Keywords: Walnut, J. regia, Salt Stress, RNA-Seq