Mechanism of Engineered Rhizobia Overexpressing Cytokinin Improve Heat Stress Response in Alfalfa

Heat stress is a major abiotic stress that limits alfalfa (Medicago sativa L.) productivity. To test whether cytokinin-overexpressing engineered rhizobia could enhance heat tolerance, we inoculated alfalfa with modified rhizobia and evaluated their physiological and molecular responses. We found that inoculation with engineered rhizobia significantly increased trans-Zeatin content in alfalfa. Compared with control rhizobia-inoculated plants, engineered rhizobia-inoculated plants exhibited increased plant height, fresh/dry weight, relative water content, and photosynthetic efficiency (total chlorophyll and carotenoids), alongside reduced hydrogen peroxide (H₂O₂) and superoxide (O₂.^-) levels under heat stress. RNA-seq analysis revealed that engineered rhizobia upregulated heat stress-responsive genes in alfalfa, which was further verified by qRT-PCR. Metabolomics analyses showed significant alterations in phenylpropanoid, flavonoid, phenolic acid, and salicylic acid metabolic pathways in engineered rhizobia-inoculated plants under heat stress. Contrary to conventional approaches, our results demonstrate that cytokinin-overexpressing rhizobia not only enhance alfalfa heat tolerance but also activate multi-pathway stress responses. Collectively, these findings propose a novel strategy for developing heat-tolerant alfalfa through engineered rhizobia-mediated cytokinin biosynthesis, which helps to promote the development of sustainable alfalfa breeding for heat-tolerant varieties.