Regulation of heat tolerance by iron chlorine E6 in “84 K” poplar tissue culture seedlings: A study coupled with metabolomic and transcriptomic analysis

Populus spp, vital fast-growing timber and ecological restoration trees globally, have growth and survival directly linked to forest ecosystem stability. With global warming intensifying heat stress events, Populus often faces growth inhibition, membrane damage, and metabolic disorders, threatening forest resilience. Iron chloride E6, a novel plant growth regulator, shows potential in enhancing stress resistance. This study aimed to improve thermotolerance of “84 K” poplar tissue cultured seedlings by screening optimal ICE6 concentration and elucidating its mechanism. Phenotypic observation initially screened concentrations from 0.01 to 0.08 μg/mL, with 0.04 μg/mL selected. High temperature stress tests measured physiological indices (chlorophyll, root activity, malondialdehyde, osmotic substances, antioxidant enzymes). Integrated transcriptomic and metabolomic analyses, along with gene expression profiling, were conducted. Results showed 0.04 μg/mL ICE6 significantly promoted seedling growth. Under stress, it reduced MDA accumulation, slowed relative conductivity increase, and enhanced osmotic and antioxidant capacities. Transcriptomics identified 3789 differentially expressed genes, while metabolomics detected 819 differentially accumulated metabolites. Key genes correlated with dehydroascorbic acid and 2,3-diketo- L -gulonic acid. Mechanistically, 0.04 μg/mL ICE6 maintained membrane integrity, alleviated oxidative damage, boosted antioxidants, and upregulated sugars/proline. Regulation involved hormone signaling, phenylpropanoid biosynthesis, and flavonoid related antioxidant metabolism. This study represents the first application of ICE6 in the large-scale propagation system of poplar tissue-cultured plantlets, filling a research gap in the use of novel regulators for stress resistance in forest tree tissue-cultured plantlets. By analyzing key targets mediating heat tolerance via ICE6 at transcriptional and metabolic levels, it provides insights for genetic improvement of poplar stress resistance from these dual perspectives. Furthermore, this work lays a theoretical and technical foundation for directional regulation of stress resistance during industrial production of tissue-cultured plantlets, ultimately contributing to enhancing the resilience of forest ecosystems under climate change. • We first validated ICE6 function in the woody plant “84 K”. • We quantified ICE6-mediated physiological enhancement of “84 K” poplar under heat stress. • Elucidated the thermotolerance mechanism mediated by ICE6 under high temperature. • We revealed ICE6's regulation of key pathways, providing actionable targets for tree thermotolerance improvement.