Abstract
<jats:title>ABSTRACT</jats:title> <jats:p> Tomato ( <jats:italic>Solanum lycopersicum</jats:italic> ) is classified as a day-neutral plant, whereas its wild relatives exhibit delayed flowering under long day (LD) conditions due to higher activity of the <jats:italic>SELF-PRUNING 5G</jats:italic> ( <jats:italic>SP5G</jats:italic> ). In <jats:italic>Arabidopsis thaliana</jats:italic> , CONSTANS (CO) activates <jats:italic>FLOWERING LOCUS T</jats:italic> ( <jats:italic>FT</jats:italic> ), a homolog of <jats:italic>SP5G</jats:italic> , but whether and how CO integrates with <jats:italic>SP5G</jats:italic> in tomato flowering was unclear. Here, we demonstrate that <jats:italic>SlCOL1</jats:italic> (the tomato CO homolog) delays flowering by directly activating <jats:italic>SP5G</jats:italic> in a <jats:italic>PHYTOCHROME B1</jats:italic> ( <jats:italic>PHYB1</jats:italic> )-dependent manner. Importantly, genetic and molecular analyses combining a photoperiod-responsive tomato line carrying the wild <jats:italic>SP5G</jats:italic> allele from <jats:italic>S. pennellii,</jats:italic> together with <jats:italic>SlCOL1</jats:italic> and flowering-pathway mutants, revealed synergistic crosstalk among the photoperiodic <jats:italic>SlCOL1–SP5G</jats:italic> module, age-dependent pathway (mediated mainly by the microRNA156– <jats:italic>SlSBP</jats:italic> module), gibberellin (GA) pathway, and <jats:italic>SINGLE FLOWER TRUSS</jats:italic> ( <jats:italic>SFT</jats:italic> ) pathway. Mechanistically, we show that SP5G forms a complex with miR156-targeted SlSBP13 to directly regulate <jats:italic>SFT</jats:italic> expression, and that GA may interfere with SP5G activity. Together, these findings revealed a coordinated network that integrates multiple flowering signals to modulate both shared and pathway-specific targets. Our findings provide a significant advance in understanding the molecular regulation of tomato flowering and offer promising avenues for breeding strategies optimized for diverse environmental conditions and latitudes. </jats:p>