JrSPL13 integrates miR156, WRKY21, and hormonal signaling to modulate circadian-regulated flowering in walnut (Juglans regia)

The SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) gene family is integral to plant growth and development, notably influencing flowering and, consequently, yield in walnut trees. Nevertheless, existing research concerning the regulatory functions of SPL genes in the flowering of walnut ( Juglans regia L.) is predominantly confined to molecular characterization, with scant insights into their functional mechanisms. In this study, we successfully cloned JrSPL13 encoding a protein with a conserved SBP domain that is localized within the nucleus. qRT-PCR analysis revealed that the expression of JrSPL13 was significantly upregulated by exogenous hormones such as IAA and 6-BA, which in turn facilitated floral induction. Moreover, JrSPL13 expression exhibited an inverse correlation with miR156 levels, implying potential post-transcriptional regulation by miR156. Yeast one-hybrid and dual-luciferase transient expression assays provided evidence that JrWRKY21 directly binds to and activates the promoter of JrSPL13 . Overexpression of JrSPL13 in Arabidopsis thaliana resulted in flowering across all transgenic lines, with the OE-1 line flowering on average 8-days earlier than the wild type. qRT-PCR analysis further revealed that flowering-related genes, including FT , SOC1 , AP1 , and CO , were significantly upregulated in transgenic Arabidopsis , with FT expression showing a 15-fold increase. Transcriptome sequencing has additionally demonstrated an upregulation of genes associated with circadian rhythm, including FKF1 , TOC1 , PRR5 , and ELF3 . These findings suggest that JrSPL13 is modulated by multiple factors, such as miR156, WRKY transcription factors, and hormones, and plays a role in influencing plant circadian rhythms to regulate flowering through various genetic pathways. This study provides novel insights into the molecular mechanisms governing flowering in industrial walnut production and practical guidance for optimizing walnut cultivation techniques. Furthermore, it establishes a theoretical framework for the genetic enhancement of flowering traits in woody plants. • Nuclear-localized JrSPL13 cloned in walnut, induced by IAA/6-BA to promote flowering. • Dual regulation: miR156 suppresses JrSPL13 and JrWRKY21 activates its promoter. • 8-day early flowering in JrSPL13 -OE Arabidopsis via 15-fold FT upregulation. • Circadian genes ( FKF1 / TOC1 / PRR5 / ELF3 ) upregulated by JrSPL13 during floral transition. • Multi-pathway control: Hormones, miRNAs, and TFs converge on JrSPL13 for flowering.