Characterization of the SPL gene family and ethylenemediated regulation of the FT/TFL network in hops (Humulus lupulus)

Abstract

The transition from the vegetative to the reproductive phase is a critical determinant of yield in perennial crops. However, the molecular networks governing this process in hops (Humulus lupulus L.) remain largely unknown, particularly in non-traditional cultivation latitudes. In this study, we characterized the SQUAMOSA PROMOTERBINDING PROTEIN-LIKE (SPL) transcription factor family, which are considered central regulators of maturation and floral transition. We elucidated their integration with ethylene signaling and the phase-change genes HlFT3 (florigen) and HlTFL1 (antiflorigen), revealing how this network coordinates the reproductive development of the crop. We identified 17 HlSPL genes, categorized into nine distinct clades with high orthology to Cannabis sativa. Molecular characterization confirmed the presence of the highly conserved SBP domain and revealed a dense presence of hormone-responsive cis-regulatory elements in their promoter regions, specifically ethylene-responsive elements (EREs), suggesting a fundamental crosstalk between ontogenetic signals and ethylene signaling. To functionally contextualize this regulatory network, we integrated RNA-seq expression profiling across various tissues and cone development stages, revealing tissue-specific patterns and coordinated regulation among HlSPL members during inflorescence maturation. Furthermore, by modulating ethylene perception with silver thiosulfate (STS), we demonstrated a photoperiod-dependent control over the florigen/antiflorigen balance (HlFT3/HlTFL1). Our results indicate that ethylene acts by restricting HlFT3 under non-inductive conditions, while becoming indispensable for sustaining the florigenic pathway under short days. This work provides a functional characterization of the mechanisms controlling floral induction in hops, identifying essential genetic determinants for crop adaptation to low latitudes. These findings offer promising targets for CRISPR/Cas9-mediated breeding and biotechnological strategies aimed at optimizing yield and environmental resilience in new cultivation frontiers.