Bolting is a key process in the growth and development of lettuce (Lactuca sativa L.). A high temperature can induce early bolting, which decreases both the quality and production of lettuce. However, knowledge of underlying lettuce bolting is still lacking. To better understand the molecular basis of bolting, a comparative proteomics analysis was conducted on lettuce stems, during the bolting period induced by a high temperature (33 °C) and a control temperature (20 °C) using iTRAQ-based proteomics, phenotypic measures, and biological verifications using qRT-PCR and Western blot. The high temperature induced lettuce bolting, while the control temperature did not. Of the 5454 identified proteins, 619 proteins presented differential abundance induced by high-temperature relative to the control group, of which 345 had an increased abundance and 274 had a decreased abundance. Proteins with an abundance level change were mainly enriched in pathways associated with photosynthesis and tryptophan metabolism involved in auxin (IAA) biosynthesis. Moreover, among the proteins with differential abundance, proteins associated with photosynthesis and tryptophan metabolism were increased. These findings indicate that a high temperature enhances the function of photosynthesis and IAA biosynthesis to promote the process of bolting, which is in line with the physiology and transcription level of IAA metabolism. Our data provide a first comprehensive dataset for gaining novel understanding of the molecular basis underlying lettuce bolting induced by high temperature. It is potentially important for further functional analysis and genetic manipulation for molecular breeding to breed new cultivars of lettuce to restrain early bolting, which is vital for improving vegetable quality.

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.

High temperature is a huge threat to lettuce production in the world, and spermidine (Spd) has been shown to improve heat tolerance in lettuce, but the action mechanism of Spd and the role of polyamine metabolism are still unclear. The effects of Spd and D-arginine (D-arg) on hydroponic lettuce seedlings under high-temperature stress by foliar spraying of Spd and D-arg were investigated. The results showed that high-temperature stress significantly inhibited the growth of lettuce seedlings, with a 33% decrease in total fresh weight and total dry weight; photosynthesis of lettuce seedlings was inhibited by high-temperature stress, and the inhibition was greater in the D-arg treatment, while the Spd recovery treatment increased net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), stomatal limit value (Ls), and intercellular CO₂ concentration (Ci). High-temperature stress significantly reduced the maximum photochemical efficiency (Fv/Fm), photochemical quenching coefficient (qP), electron transport rate (ETR), and photochemical efficiency of PSII (ΦPSII), increased the non-photochemical burst coefficient (NPQ) and reduced the use of light energy, which was alleviated by exogenous Spd. The increase in polyamine content may be due to an increase in polyamine synthase activity and a decrease in polyamine oxidase activity, as evidenced by changes in the expression levels of genes related to polyamine synthesis and metabolism enzymes. This evidence suggested that D-arg suppressed endogenous polyamine levels in lettuce and reduced its tolerance, whereas exogenous Spd promoted the synthesis and accumulation of polyamines in lettuce and increased its photosynthetic and oxidative stress levels, which had an impact on the tolerance of lettuce seedlings.

The present study aimed to compare polyphenols among red lettuce cultivars and identify suitable cultivars for the development and utilization of healthy vegetables. Polyphenols, mineral elements, and antioxidant activity were analyzed in the leaves of six red pigmented lettuce (Lactuca sativa L.) cultivars; thereafter, we assessed the anti-tumor effects of cultivar B-2, which displayed the highest antioxidant activity. Quadrupole–Orbitrap mass spectrometry analysis revealed four classes of polyphenols in these cultivars. The composition and contents of these metabolites varied significantly among cultivars and primarily depended on leaf color. The B-2 cultivar had the highest antioxidant potential than others because it contained the highest levels of polyphenols, especially anthocyanin, flavone, and phenolic acid; furthermore, this cultivar displayed anti-tumor effects against the human lung adenocarcinoma cell line A549, human hepatoma cell line Bel7402, human cancer colorectal adenoma cell line HCT-8, and HT-29 human colon cancer cell line. Hence, the new red-leaf lettuce cultivar B-2 has a distinct metabolite profile, with high potential for development and utilization of natural phytochemical and mineral resources in lettuces and can be used as a nutrient-dense food product.

High temperatures have significant impacts on heat-tolerant bolting in lettuce. In this study, it was found that high temperatures could facilitate the accumulation of GA in lettuce to induce bolting, with higher expression levels of two heat shock protein genes LsHsp70-3701 and LsHsp70-2711. By applying VIGS technology, these two Hsp70 genes were incompletely silenced and plant morphological changes under heat treatment of silenced plants were observed. The results showed that lower expression levels of these two genes could enhance bolting stem length of lettuce under high temperatures, which means these two proteins may play a significant role in heat-induced bolting tolerance. By using the yeast two-hybrid technique, it was found that a calmodulin protein could interact with LsHsp70 proteins in a high-temperature stress cDNA library, which was constructed for lettuce. Also, the Hsp70-calmodulin combination can be obtained at high temperatures. According to these results, it can be speculated that the interaction between Hsp70 and calmodulin could be induced under high temperatures and higher GA contents can be obtained at the same time. This study analyses the regulation of heat tolerance in lettuce and lays a foundation for additional studies of heat resistance in lettuce.