Genome-wide analysis of the SPL gene family in lettuce and its role in miR156/SPL module-mediated plant development and regeneration

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.