Multi-omics analysis of leaf and root-nodule responses to combined heat and drought in R4-stage soybean

The R4 full-pod stage is a yield-sensitive transition from pod establishment toward seed filling, when developing reproductive sinks depend on sustained carbon and nitrogen supply. Combined heat and drought (HD) can severely limit soybean ( Glycine max L.) productivity at this stage, but the mechanisms coordinating tissue responses and post-stress recovery remain unclear. We compared two cultivars originating from contrasting latitudes at the R4 stage: lanceolate-leaved Heinong 65 (HN65) from northeastern China and ovate-leaved Qihuang 34 (QH34) from the Huang-Huai-Hai region. HN65 showed less wilting, lower acute reactive oxygen species (ROS) accumulation rate, and stronger gas-exchange recovery, while QH34 showed a significant HD-associated reduction in seed weight per plant. MicroRNA (miRNA) and mRNA sequencing of leaves and root nodules at HD 3 d and the seven-day recovery point (re 7 d), together with quantitative leaf proteomics, resolved four levels of tolerance-associated regulation. First, seven HN65-specific or cultivar-shared HD-responsive candidate miRNA-target modules showed stage-dependent patterns. Dual-luciferase assays supported direct regulation of RUN1 and ROQ1 by miR1510b-3p . Second, joint analysis of tissue transcriptomes and the leaf proteome revealed complementary molecular responses in leaves and nodules. Third, stage-restricted genes with significant cultivar × treatment interactions indicated a stronger acute stress response in HN65 than in QH34, but lower immune-related expression in HN65 at re 7 d. Fourth, 14 genes with concordant mRNA and protein changes in leaves were associated with protein quality control, chloroplast function, and photosynthetic adjustment; all were hubs or candidate hubs in both mRNA and protein WGCNA networks. Of these, chloroplast localization was experimentally confirmed for two proteins and predicted for six others. Hairy-root composite assays further showed that overexpression of the functionally uncharacterized LHDI1 , which displayed a diffuse nuclear-cytoplasmic distribution similar to the GFP control, improved survival and leaf water retention and reduced ROS accumulation under HD stress. Together, these results define a tissue-and stage-dependent response framework and identify candidate regulators for improving soybean performance under combined heat and drought.