Inoculation of Cenococcum geophilum enhances heat tolerance in Pinus massoniana through integrated physiological, biochemical, and transcriptional reprogramming

Abstract

ABSTRACT Global warming is increasing the frequency of extreme heat events, threatening forest resilience. However, the physiological and molecular mechanisms underlying heat tolerance of ectomycorrhizal (ECM) inoculation in conifers remain poorly understood. This study investigates how the ECM fungus Cenococcum geophilum enhances thermotolerance in Pinus massoniana seedlings. We found that ECM inoculation significantly improved plant biomass and photosynthesis under both normal and high temperatures. Under heat stress, ECM symbiosis reduced oxidative damage by elevating the activities of antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], and peroxidase [POD]) and promoting nitric oxide (NO) accumulation via enhanced nitrate reductase (NR)- and nitric oxide synthase (NOS)-dependent pathways. Furthermore, ECM colonization reprogrammed proline metabolism, stimulating its biosynthesis through Δ1-pyrroline-5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT) while tissue-specifically regulating proline dehydrogenase (ProDH), thereby supporting osmotic adjustment in roots and energy maintenance in shoots. Transcriptomic analyses revealed that ECM primed the host at 25°C by activating defense signaling, reinforcing epidermal structures, and enhancing starch and sucrose metabolism. Under heat stress, ECM induced extensive transcriptional reorganization, upregulating pathways related to membrane lipid remodeling, cell wall modification, and carbon reallocation, while downregulating energy-costly processes, such as oxidative phosphorylation and RNA polymerase activity. This shift reflects an ECM-driven resource reallocation strategy that suppresses ROS production and prioritizes cellular integrity. Collectively, our study demonstrates that C. geophilum establishes an integrated mechanism involving physiological, biochemical, and transcriptional adjustments to enhance heat tolerance in P. massoniana, providing mechanistic insight into ECM-mediated climate resilience and underscoring the potential of using ECM fungi as an ecological tool to promote forest adaptation in a warming world. IMPORTANCE This study elucidates how the ectomycorrhizal fungus Cenococcum geophilum systemically enhances heat tolerance in Pinus massoniana by acting as a natural “heat shield,” revealing a symbiotic mechanism where the fungus primes the plant's antioxidant defenses, reprograms proline metabolism in a tissue-specific manner, and boosts nitric oxide signaling. Crucially, transcriptomic analysis shows the fungus drives a strategic resource reallocation under heat stress by upregulating pathways for cellular integrity while downregulating energy-intensive processes to minimize oxidative damage. These findings establish a detailed mechanistic framework for fungal-mediated climate resilience, highlighting that enhancing natural partnerships with soil fungi can fortify existing forests against increasing heatwaves by optimizing internal stress management for forest adaptation, offering a practical tool for ecosystem management in a warming world. This study elucidates how the ectomycorrhizal fungus Cenococcum geophilum systemically enhances heat tolerance in Pinus massoniana by acting as a natural “heat shield,” revealing a symbiotic mechanism where the fungus primes the plant's antioxidant defenses, reprograms proline metabolism in a tissue-specific manner, and boosts nitric oxide signaling. Crucially, transcriptomic analysis shows the fungus drives a strategic resource reallocation under heat stress by upregulating pathways for cellular integrity while downregulating energy-intensive processes to minimize oxidative damage. These findings establish a detailed mechanistic framework for fungal-mediated climate resilience, highlighting that enhancing natural partnerships with soil fungi can fortify existing forests against increasing heatwaves by optimizing internal stress management for forest adaptation, offering a practical tool for ecosystem management in a warming world.