ABSTRACT 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.
Qi et al. (2026) studied this question.