Cultivated lettuce (Lactuca sativa L.) is a major leafy crop and an emerging model for functional genomics within the Asteraceae family, supported by high-quality reference genomes and efficient transformation systems. Although CRISPR/Cas technology offers powerful opportunities for crop improvement, editing efficiency depends on optimized construct architecture and reliable guide RNA (gRNA) validation. However, a rapid platform for evaluating CRISPR reagents in lettuce is still lacking. Here, we developed an efficient hairyroot-based system to accelerate CRISPR/Cas genome editing optimization in L. sativa. Four Agrobacterium rhizogenes strains were compared for hairy root induction in two cultivars, ‘Saladin’ and ‘Osiride’, identifying strain ATCC15834 as the most effective based on transformation frequency and root production. Using this platform, we evaluated multiple CRISPR construct configurations, including alternative promoters for nuclease and gRNA expression. A plant-derived promoter combined with At-pU6-26 variant significantly improved editing efficiency. As a proof of concept, we targeted LsHB2, the putative ortholog of Arabidopsis thaliana ATHB2, a key regulator of the shade avoidance response using SpCas9, SaCas9, and LbCas12a nucleases. The system enabled rapid genotyping and quantitative indel profiling. Overall, this workflow provides a robust framework for efficient guide selection and construct optimization in lettuce genome editing.

ATHB1, an Arabidopsis thaliana homeodomain-leucine zipper (HD-Zip) transcription factor, is involved in the control of leaf development and hypocotyl elongation under short-day conditions. As growth adaptation to environmental conditions is essential for plant resilience, we investigated the role of ATHB1 in the interaction between transcriptional regulatory networks and hormone signaling pathways. We found that wounding, flooding and ethylene induce ATHB1 expression. In addition, we found that the ethylene signal transduction pathway is also involved in an age-dependent ATHB1 expression increase in leaves. Conversely, methyl jasmonate (MeJA) application decreases the ATHB1 transcript level. By exploiting mutant and over-expressing (OE) lines, we also found that the ATHB1 level influences plant sensitivity to the inhibitory effect of MeJA treatment on growth. To gain deeper insights into the regulatory pathways affected by ATHB1, we performed a microarray analysis comparing the transcriptome of wild-type and athb1 mutant plants following exposure to MeJA. Remarkably, although the response to the MeJA treatment was not impaired in athb1, several genes involved in jasmonate and salycilic acid signaling were already downregulated in athb1 seedlings under normal conditions compared to the wild type. Thus, our study suggests that ATHB1 may integrate different hormone signaling pathways to influence plant growth under various stress conditions.