Quantitative RT-PCR Analysis of Plant Growth-Promoting Rhizobacteria-Induced Transcriptional Responses in Barley (<i>Hordeum vulgare</i> L.) under High Salt and Drought Stress Conditions

Salinity and drought are two major environmental stresses that significantly impact global agricultural productivity. Plant growth-promoting rhizobacteria (PGPR) are essential bioinoculants that contribute to sustainable agriculture by enhancing plant resilience to such stresses. Given the importance of motility for effective rhizosphere colonization, this study initially screened various PGPR strains for their motility and chemotactic behaviors. Except for Pseudomonas fluorescens F113, all of the tested strains exhibited high motility. Based on these results, two strains, i.e., P. fluorescens SBW25 and Pseudomonas putida KT2440 were selected for further evaluation of their ability to promote barley growth under abiotic stress conditions. Barley plants inoculated with selected PGPR strains were subjected to salt (200 mM and 1000 mM) and drought stress conditions, and their biochemical and molecular responses were compared to noninoculated plants. Biochemical analysis revealed that inoculated plants have significantly higher levels of osmolytes such as soluble sugars (78 mg/g in SBW25), proline (12 mg/g in SBW25), free amino acids (5.8 mg/g in SBW25), and antioxidant enzymes (POD: 0.72, CAT: 0.78 U/mg in SBW25, and APx: 4.1 U/mg in KT2440) compared to controls under stress conditions. At the molecular level, qRT-PCR analysis revealed that PGPR strain P. putida KT2440 significantly upregulated key genes in the jasmonic acid defense pathway, including FAD3 (∼3900%), LOX1 (1600%), AOS (380%), and AOC (540%) under high salt (1000 mM) stress. Both PGPR strains also induced a marked increase in ethylene biosynthesis gene ACCO expression (3400% and 1500%) compared to control (∼200%), under high salt stress, while downregulated ACCS (50% and 95.2%) and ACCO (50% and 93.8%) under drought stress, as opposed to their controls (ACCS: 2600%, ACCO: 10,600%). Additionally, the MAPKK gene (3250%) indicated a significantly strong induction under high salt stress with P. putida KT2440. Other stress-related genes, such as NHX1 (8500% in P. putida KT2440), NRT2.2 (1800% in P. fluorescens SBW25), CAT2 (1200% in P. fluorescens), and GR (420% in P. putida), were also upregulated under different stress conditions. Overall, most of the phytohormone biosynthesis genes were upregulated under high salt (1000 mM) and downregulated under drought stress conditions. In summary, our results demonstrated that these PGPR strains can enhance salinity and drought tolerance in barley by modulating key defense pathways, ion and nitrate transporters, and antioxidant enzymes. These findings highlight the potential use of these Pseudomonas strains in improving stress tolerance in barley and related crops. Additionally, the candidate genes identified in this study could serve as valuable markers for breeding stress-tolerant plants.