Basic helix-loop-helix (bHLH) transcription factors regulate diverse plant processes, particularly anthocyanin biosynthesis through the MYB-bHLH-WD40 complex. Despite snapdragon (Antirrhinum majus) serving as a classical model for studying flower pigmentation genetics, its bHLH gene family has rarely been comprehensively characterized. Here, we performed a genome-wide identification and systematic characterization of the bHLH gene family in A. majus, with a focus on candidates involved in anthocyanin biosynthesis. A total of 150 AmbHLH genes were identified and subjected to in-silico analyses, including phylogenetic classification, structural analysis, and promoter cis-element characterization. Comparative transcriptomic profiling between anthocyanin-poor (“SIPPE50”, Green) and anthocyanin-rich (“JI2R”, Red) snapdragon lines highlighted eight differentially expressed AmbHLHs. AmbHLH001, AmbHLH002, and AmbHLH042 showed significant upregulation in the anthocyanin-rich line and showed positive correlations with the expression of key anthocyanin biosynthetic genes. Among these, AmbHLH002 was prioritized as a candidate and was assessed via heterologous overexpression in tomatoes. Notably, AmbHLH002 is a newly identified regulator whose overexpression in tomato resulted in visible purple pigmentation and increased anthocyanin accumulation. These findings support the view that AmbHLH002 acts as a positive regulator, with phylogenetic evidence for conservation of anthocyanin biosynthesis, presenting valuable potential for engineering pigmentation traits in ornamental plants and serving as a candidate visible marker for plant genetic transformation.

Lettuce (Lactuca sativa) is a globally important leafy vegetable. Understanding the genetic factors underlying its growth and regeneration is critical for advancing agricultural productivity and biotechnological applications. To address this, the study aimed to comprehensively identify and characterize the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family in lettuce and investigate their potential roles in plant development and regeneration. As a result, 22 SPL genes were identified within the lettuce genome. Fourteen of these genes contain recognition sites for microRNA156, suggesting post-transcriptional regulation. Each LsSPL protein has the highly conserved SBP domain and is predicted to localize in the nucleus. Analysis of public RNA-seq datasets revealed tissue-specific expression patterns of the 22 LsSPL genes, with five highly expressed in leaves, four in roots, and three in stems, indicating their distinct roles in plant development. Overexpression of lettuce miRNA156c (miR156-OX) led to reduced leaf size and delayed flowering time, whereas suppression of miR156 (miR156-STTM) resulted in increased leaf size. Surprisingly, cotyledon explants from miR156-OX lettuce lines exhibited a 1.9-fold increase in shoot regeneration compared to wild-type, whereas miR156-STTM lines exhibited a 54.3% decrease. This enhanced in vitro shoot regeneration was also observed in ectopic miR156-overexpression tomato lines, suggesting a conserved mechanism. Quantitative RT-PCR analysis confirmed the downregulation of LsSPL13A.1, LsSPL13A.2, and LsSPL12.2 in miR156-OX lines and their upregulation in miR156-STTM lines after 5 days of callus induction, implicating their specific roles in in vitro organogenesis and plant regeneration. This comprehensive analysis provides valuable insights into the SPL gene family and the miR156-SPLs regulatory network, specifically highlighting its role in regeneration. These findings hold the potential for improving plant growth, development, and biotechnological applications.