Walnuts are among the globally significant woody food and oil tree species. At high latitudes, they frequently experience late-frost damage, inducing low-temperature stress, which significantly affects walnut seedlings. The aim of this study was to investigate the physiological and biochemical alterations in walnut seedlings under low-temperature (LT) stress along with its underlying molecular mechanisms. Physiological indices were determined, and the transcriptome and miRNA were sequenced by sampling leaves (0 h, 6 h, 12 h, 24 h, and 48 h) of two-month-old live seedlings of walnuts treated with a low temperature of 4 °C. The results indicated that LT stress induced an increase in electrical conductivity and malondialdehyde content while simultaneously causing a reduction in Fv/Fm. From the transcriptome comparison between the control and treated groups, a total of 12,566 differentially expressed genes (DEGs) were identified, consisting of 6829 up-regulated and 5737 down-regulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the DEGs were primarily enriched in polysaccharide metabolic processes, responses to abscisic acid and phenylpropanoid biosynthesis pathways. Furthermore, the miRNA database identified 1052 miRNAs in response to low-temperature stress in walnuts; these miRNAs were found to target 7043 predicted genes. Through the integration and analysis of transcriptome and miRNA data, 244 differential DEGs were identified. Following GO and KEGG enrichment analyses of the differential target genes, we identified that these genes primarily regulate pathways involved in starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, and glycerophospholipid biosynthesis, as well as phenylalanine, tyrosine, and tryptophan biosynthesis, in walnut leaves under LT stress. Additionally, we conducted an in-depth analysis of the associations between differentially expressed genes (DEGs) and differentially expressed microRNAs (DEMs) within the starch and sucrose metabolism pathway. Real-time fluorescent quantitative PCR (qRT-PCR) validation of the expression patterns of a subset of differential genes confirmed the accuracy of the transcriptome data. This study unveils the potential molecular mechanisms underlying walnut’s response to low-temperature stress, providing valuable genetic resources for future research on the cold tolerance mechanisms of walnut in response to late-frost damage.

Walnut (Juglans regia L.) is a monoecious species with far more male than female flowers, yet the molecular mechanisms regulating sex differentiation remain largely unexplored. In this study, we investigated the effects of exogenous brassinolide (BR) on the female-to-male flower bud ratio and its regulatory mechanisms. We identified 23 April to 13 May as the critical physiological differentiation period for the ‘Xin xin 2’ variety. Exogenous application of 0.05 mg/L BR during this period significantly increased the female-to-male flower bud ratio across three consecutive years (2023–2025). Hormone metabolomics revealed that BR treatment induced sex-specific changes in ethylene and auxin contents. Transcriptomic analysis showed that BR differentially regulated MADS-MIKC genes in female and male buds. Integrated miRNA-mRNA analysis identified sex-specific regulatory modules targeting MADS-MIKC genes, including four in female buds and two in male buds. RT-qPCR and dual-luciferase assays confirmed that jre-miR156g directly targets the 3’-untranslated region of JrSPL5, with reciprocal expression patterns. Collectively, our findings demonstrate that exogenous BR increases the female-to-male flower bud ratio in walnut through multi-layered regulation involving hormone signaling, sex-specific expression of MADS-MIKC genes, and miRNA-mRNA regulatory cascades. This study provides a theoretical basis for breeding high-yielding walnut varieties by manipulating sex expression.