The mitogen-activated protein kinase (MAPK) pathway is a widely distributed signaling cascade in eukaryotes and is involved in regulating plant growth, development, and stress responses. High temperature, a frequently occurring environmental stressor, causes premature bolting in lettuce with quality decline and yield loss. However, whether MAPKs play roles in thermally induced bolting remains poorly understood. In this study, 17 LsMAPK family members were identified from the lettuce genome. The physical and chemical properties, subcellular localization, chromosome localization, phylogeny, gene structure, family evolution, cis-acting elements, and phosphorylation sites of the LsMAPK gene family were evaluated via in silico analysis. According to phylogenetic relationships, LsMAPKs can be divided into four groups, A, B, C, and D, which is supported by analyses of gene structure and conserved domains. The collinearity analysis showed that there were 5 collinearity pairs among LsMAPKs, 8 with AtMAPKs, and 13 with SlMAPKs. The predicted cis-acting elements and potential phosphorylation sites were closely associated with hormones, stress resistance, growth, and development. Expression analysis showed that most LsMAPKs respond to high temperatures, among which LsMAPK4 is significantly and continuously upregulated upon heat treatments. Under heat stress, the stem length of the LsMAPK4-knockdown lines was significantly shorter than that of the control plants, and the microscope observations demonstrated that the differentiation time of flower buds at the stem apex was delayed accordingly. Therefore, silencing of LsMAPK4 significantly inhibited the high- temperature-accelerated bolting in lettuce, indicating that LsMPAK4 might be a potential regulator of lettuce bolting. This study provides a theoretical basis for a better understanding of the molecular mechanisms underlying the MAPK genes in high-temperature-induced bolting.

Lettuce (Lactuca sativa L.) being a cool‐season crop can bolt if the temperatures increase above 20 °C. When lettuce bolts, the plants start the reproductive growth cycle. The improvement in resistance to high temperature bolting has been an important goal of lettuce breeding. This paper verified that Lactuca sativa mitogen‐activated protein kinases 6 (LsMAPK6) played an important role in high temperature bolting. The full‐length CDS of LsMAPK6 gene was amplified by real‐time polymerase chain reaction (RT‐PCR) and analyzed by bioinformatics software. The expression of LsMAPK6 was determined by quantitative real‐time polymerase chain reaction (qRT‐PCR). The results showed that the length of LsMAPK6 was 1,182 bp, encoding 393 amino acids. The protein domain was STKc_TEY_MAPK and closely relevant to sunflower (Helianthus annuus L.), mouse‐ear cress [Arabidopsis thaliana (L.) Heynh.], and chrysanthemum (Chrysanthemum morifolium Ramat.). The qRT‐PCR showed that Lsmapk6 was mainly expressed in stem, stem apex, and taproot, and high temperature significantly promoted its expression. Virus‐induced gene silencing (VIGS) technology successfully silenced LsMAPK6 in lettuce, and the bolting of silenced lines was delayed under the action of high temperature and exogenous gibberellin. These suggest that LsMAPK6 plays an important role in promoting bolting. This study laid a foundation for further study on the function and regulation mechanism of MAPK6.