Quantitative proteomic analyses reveal that energy metabolism and protein biosynthesis reinitiation are responsible for the initiation of bolting induced by high temperature in lettuce (Lactuca sativa L.).

BACKGROUND: Lettuce (Lactuca sativa L.), one of the most economically important leaf vegetables, exhibits early bolting under high-temperature conditions. Early bolting leads to loss of commodity value and edibility, leading to considerable loss and waste of resources. However, the initiation and molecular mechanism underlying early bolting induced by high temperature remain largely elusive. RESULTS: In order to better understand this phenomenon, we defined the lettuce bolting starting period, and the high temperature (33 °C) and controlled temperature (20 °C) induced bolting starting phase of proteomics is analyzed, based on the iTRAQ-based proteomics, phenotypic measurement, and biological validation by RT-qPCR. Morphological and microscopic observation showed that the initiation of bolting occurred 8 days after high-temperature treatment. Fructose accumulated rapidly after high-temperature treatment. During initiation of bolting, of the 3305 identified proteins, a total of 93 proteins exhibited differential abundances, 38 of which were upregulated and 55 downregulated. Approximately 38% of the proteins were involved in metabolic pathways and were clustered mainly in energy metabolism and protein synthesis. Furthermore, some proteins involved in sugar synthesis were differentially expressed and were also associated with energy production. CONCLUSIONS: This report is the first to report on the metabolic changes involved in the initiation of bolting in lettuce. Our study suggested that energy metabolism and ribosomal proteins are pivotal components during initiation of bolting. This study could provide a potential regulatory mechanism for the initiation of early bolting by high temperature, which could have applications in the manipulation of lettuce for breeding.

Quantitative Proteomics Analysis of Lettuce (Lactuca sativa L.) Reveals Molecular Basis-Associated Auxin and Photosynthesis with Bolting Induced by High Temperature.

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 zinc-finger transcription factor BcMF20 and its orthologs in Cruciferae which are required for pollen development.

Brassica campestris Male Fertility 20 (BcMF20) is a typical zinc-finger transcription factor that was previously isolated from flower buds of Chinese cabbage (Brassica campestris ssp. chinensis). By applying expression pattern analysis, it can be known that BcMF20 was specifically and strongly expressed in tapetum and pollen, beginning from the uninucleate stage, and was maintained during the mature-pollen stage. As BcMF20 was highly conserved in Cruciferae, it can be indicated that this zinc-finger transcription factor is important during the growth of Cruciferae. In this study, 12 C2H2-type zinc-finger TFs which shared high homology with BcMF20 were found from NCBI via BLAST. A new molecular phylogenetic tree was constructed by the comparison between BcMF20 and these 12 C2H2-type zinc-finger TFs with NJ method. By analyzing this phylogenetic tree, the evolution of BcMF20 was discussed. Then, antisense RNA technology was applied in the transgenesis of Arabidopsis thaliana to get the deletion mutants of BcMF20, so that its function during the pollen development can be identified. The results showed: BcMF20 are in the same clade with three genes from Arabidopsis. The inhibition of BcMF20 expression led to smaller amounts of and lower rate in germination of pollen and lower rate in fruit setting in certain transgenetic plants. This also led to the complete collapse of pollen grains. By SEM and TEM, pollen morphology and anther development processes were observed. In the middle uninucleate microspore stage, a relatively thin or even no primexine was formed in microspores. This may result in the malformation of the pollen wall and finally cause the deformity of pollens. Above all, it can be indicated that BcMF20 may act as a part of regulation mechanisms of TAZ1 and MS1. Together they play a role in a genetic pathway in the tapetum to act on proliferation of tapetal cells and keep the normal development of pollens.

Beneficial Phytochemicals with Anti-Tumor Potential Revealed through Metabolic Profiling of New Red Pigmented Lettuces (Lactuca sativa L.).

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.

[Effects of temperature on leaf lettuce vernalization.] / 温度对叶用莴苣春化的影响.

To investigate the effects of different temperatures on the vernalization of leaf lettuce, and declare their type, two easy bolting leaf lettuce varieties of GB-30 and GB-31 were selected as material, which were treated by 4 ℃, 20 ℃ and 25 ℃ for 20 d respectively and afterwards treated by high temperature stress. The process of flower bud differentiation was observed by using paraffin section technology, and combined the condition of bolting and flowering to estimate whether or not it underwent vernalization, and defined its vernalization type. The results showed that, two varieties of GB-30 and GB-31 appeared bolting to different degrees at the 8th day under high temperature stress after temperature treatments in the early stage. Different temperatures in the early stage all made flower bud differentiated of two varieties. 4 ℃ treatment did not advance the flower bud differentiation, while the high temperature in later time accelerated this progress. Furthermore, the days required for the two varieties to complete development stages differed under different temperature treatments. The effective accumulated temperature whether from pregermination to flowering or from high temperature stress to flowering of two varieties were also different. The leaf lettuce without low temperature treatment in early stage could enter into the flower bud differentiation, bolting, budding and flowering stages, and it could be considered as non-low temperature vernalization plant. The high temperature treatment in later stage could obviously promote its bolting and flowering. In addition, the effective accumulated temperature had to reach about 2500 ℃·d from germination to blossom.