A variety of endogenous hormone signals, developmental cues, and environmental stressors can trigger and promote leaf lettuce bolting. One such factor is gibberellin (GA), which has been linked to bolting. However, the signaling pathways and the mechanisms that regulate the process have not been discussed in full detail. To clarify the potential role of GAs in leaf lettuce, significant enrichment of GA pathway genes was found by RNA-seq, among which the LsRGL1 gene was considered significant. Upon overexpression of LsRGL1, a noticeable inhibition of leaf lettuce bolting was observed, whereas its knockdown by RNA interference led to an increase in bolting. In situ hybridization analysis indicated a significant accumulation of LsRGL1 in the stem tip cells of overexpressing plants. Leaf lettuce plants stably expressing LsRGL1 were examined concerning differentially expressed genes through RNA-seq analysis, and the data indicated enhanced enrichment of these genes in the ‘plant hormone signal transduction’ and ‘phenylpropanoid biosynthesis’ pathways. Additionally, significant changes in LsWRKY70 gene expression were identified in COG (Clusters of Orthologous Groups) functional classification. The results of yeast one-hybrid, β-glucuronidase (GUS), and biolayer interferometry (BLI) experiments showed that LsRGL1 proteins directly bind to the LsWRKY70 promoter. Silencing LsWRKY70 by virus-induced gene silencing (VIGS) can delay bolting, regulate the expression of endogenous hormones, abscisic acid (ABA)-linked genes, and flowering genes, and improve the nutritional quality of leaf lettuce. These results strongly associate the positive regulation of bolting with LsWRKY70 by identifying its vital functions in the GA-mediated signaling pathway. The data obtained in this research are invaluable for further experiments concerning the development and growth of leaf lettuce.

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