LsFUL-LsSMU2 module positively controls bolting time in leaf lettuce (Lactuca sativa L.) under high temperature.

High temperature (HT) is an environmental factor that considerably affects plant physiology, development, crop yield, and economic value. HT can cause diseases and early bolting of leaf lettuce, thereby reducing the yield and quality of leaf lettuce. Herein, we used two leaf lettuce (Lactuca sativa L.) cultivars (bolting-resistant 'S24' and bolting-sensitive 'S39') to investigate the key factors and molecular mechanism impacting bolting. We found that 14 MADS-box genes implicated in bolting and flowering, LsMADS54 (also referred to as L. sativa FRUITFULL, LsFUL), was significantly up-regulated 1,000 times after 5-d HT treatment and that HT-induced up-regulation of LsFUL was higher in bolting-sensitive than in resistant cultivars. The overexpression lines of LsFUL exhibited an earlier bolting time than that in the non-transformed 'S39'(CK). However, the RNA interference, and CRISPR-Cas9-mediated knockout lines of LsFUL exhibited a later bolting time than that in CK. In addition, we found that L. sativa SUPPRESSORS OF MEC-8 AND UNC-52 PROTEIN 2 (LsSMU2) and L. sativa CONSTANS-LIKE PROTEIN 5 (LsCOL5) interact with LsFUL, and these interactions could stimulate or prevent bolting. We observed that elevated temperature stimulated the abundance of LsSMU2 in the stem, which collaborated with LsFUL to accelerate bolting. Conversely, room temperature (RT) condition led to relatively more stable LsCOL5, which worked with LsFUL to postpone bolting. In summary, our findings demonstrate a molecular regulatory module of LsSMU2-LsFUL associated with HT-induced premature bolting, which serves as a reference for understanding HT-induced premature bolting phenomenon in leaf lettuce.

Reversal of the immunosuppressive tumor microenvironment via platinum-based neoadjuvant chemotherapy in cervical cancer.

Background: Immunotherapy favors patients with tumors; however, only 3-26.3% of patients with cervical cancer benefit from single-agent immune checkpoint inhibitors. Combined immunotherapy and chemotherapy has been explored against tumor; however, the combination remains controversial. This study aimed to investigate the tumor immune microenvironment (TIME) and the effects of platinum-based neoadjuvant chemotherapy (NACT) in cervical cancer to identify the clinical value of combining chemotherapy with immunotherapy. Methods: Multiplex immunohistochemistry (IHC) with 11 markers (cluster of differentiation [CD]3, CD8, CD4, CD11c, CD68, forkhead box P3 [Foxp3], programmed cell death 1 [PD-1], programmed cell death 1 ligand 1 [PD-L1], indoleamine 2,3-dioxygenase [IDO], cyclin-dependent kinase inhibitor 2A [p16], and cytokeratin [CK]) was performed to evaluate TIME from 108 matched pre- and post-NACT cervical cancer samples. The mechanism of antitumor immunity triggered by NACT was explored using RNA sequencing (RNA-seq) from four paired samples and subsequently verified in 41 samples using IHC. Results: The infiltration rate of the CD8+ T cells in treatment-naive cervical cancer was 0.73%, and those of Foxp3+ regulatory T cells (Tregs) and IDO+ cells were 0.87% and 17.15%, respectively. Moreover, immunoreactive T cells, dendritic cells, and macrophages were more in the stromal than the intratumor region. NACT increased dendritic, CD3+ T, CD8+ T, and CD4+ T cells and decreased Tregs. The aforementioned alterations occurred predominantly in the stromal region and were primarily in responders. Non-responders primarily showed decreased Tregs and no increase in CD8+ T or dendritic cell infiltration. Furthermore, dendritic cells interacted more closely with CD3+ T cells after NACT, an effect primarily observed in responders. RNA-seq data revealed activation of the antigen receptor-mediated signaling pathway and upregulation of major histocompatibility complex (MHC) I and MHC II after chemotherapy, validated using IHC. Conclusions: NACT can reduce Tregs, and when tumor cells are effectively killed, antigen presentation is enhanced, subsequently activating antitumor immunity finitely. Our study provides the molecular characteristics and theoretical basis for the simultaneous or sequential combination of platinum-based NACT and immunotherapy for cervical cancer.

Neoadjuvant chemotherapy plus camrelizumab for locally advanced cervical cancer (NACI study): a multicentre, single-arm, phase 2 trial.

BACKGROUND: Locally advanced cervical cancer constitutes around 37% of cervical cancer cases globally and has a poor prognosis due to limited therapeutic options. Immune checkpoint inhibitors in the neoadjuvant setting could address these challenges. We aimed to investigate the efficacy and safety of neoadjuvant chemo-immunotherapy for locally advanced cervical cancer. METHODS: In this single-arm, phase 2 trial, which was done across eight tertiary hospitals in China, we enrolled patients aged 18-70 years with untreated cervical cancer (IB3, IIA2, or IIB/IIIC1r with a tumour diameter ≥4 cm [International Federation of Gynecology and Obstetrics, 2018]) and an Eastern Cooperative Oncology Group performance status of 0 or 1. Eligible patients underwent one cycle of priming doublet chemotherapy (75-80 mg/m2 cisplatin, intravenously, plus 260 mg/m2 nab-paclitaxel, intravenously), followed by two cycles of a combination of chemotherapy (cisplatin plus nab-paclitaxel) on day 1 with camrelizumab (200 mg, intravenously) on day 2, with a 3-week interval between treatment cycles. Patients with stable disease or progressive disease received concurrent chemoradiotherapy, and patients with a complete response or partial response proceeded to radical surgery. The primary endpoint was the objective response rate, by independent central reviewer according to Response Evaluation Criteria in Solid Tumours, version 1.1. Activity and safety were analysed in patients who received at least one dose of camrelizumab. This study is registered with ClinicalTrials.gov, NCT04516616, and is ongoing. FINDINGS: Between Dec 1, 2020, and Feb 10, 2023, 85 patients were enrolled and all received at least one dose of camrelizumab. Median age was 51 years (IQR 46-57) and no data on race or ethnicity were collected. At data cutoff (April 30, 2023), median follow-up was 11·0 months (IQR 6·0-14·5). An objective response was noted in 83 (98% [95% CI 92-100]) patients, including 16 (19%) patients who had a complete response and 67 (79%) who had a partial response. The most common grade 3-4 treatment-related adverse events during neoadjuvant chemo-immunotherapy were lymphopenia (21 [25%] of 85), neutropenia (ten [12%]), and leukopenia (seven [8%]). No serious adverse events or treatment-related deaths occurred. INTERPRETATION: Neoadjuvant chemo-immunotherapy showed promising antitumour activity and a manageable adverse event profile in patients with locally advanced cervical cancer. The combination of neoadjuvant chemo-immunotherapy with radical surgery holds potential as a novel therapeutic approach for locally advanced cervical cancer. FUNDING: National Key Technology Research and Development Program of China and the National Clinical Research Center of Obstetrics and Gynecology.

HDACs alters negatively to the tumor immune microenvironment in gynecologic cancers.

The role of histone deacetylases (HDACs) in the tumor immune microenvironment of gynecologic tumors remains unexplored. We integrated data from The Cancer Genome Atlas and Human Protein Atlas to examine HDAC expression in breast, cervical, ovarian, and endometrial cancers. Elevated HDAC expression correlated with poor prognosis and highly malignant cancer subtypes. Gene Set Enrichment Analysis revealed positive associations between HDAC expression and tumor proliferation signature, while negative associations were found with tumor inflammation signature. Increased HDAC expression was linked to reduced infiltration of natural killer (NK), NKT, and CD8+ T cells, along with negative associations with the expression of PSMB10, NKG7, CCL5, CD27, HLA-DQA1, and HLA-DQB1. In a murine 4T1 breast cancer model, treatment with suberoylanilide hydroxamic acid (SAHA; HDAC inhibitor) and PD-1 antibody significantly inhibited tumor growth and infiltration of CD3+ and CD8+ T cells. Real-time polymerase chain reaction revealed upregulated expressions of Psmb10, Nkg7, Ccl5, Cd8a, Cxcr6, and Cxcl9 genes, while Ctnnb1 and Myc genes were inhibited, indicating tumor suppression and immune microenvironment activation. Our study revealed that HDACs play tumor-promoting and immunosuppressive roles in gynecologic cancers, suggesting HDAC inhibitors as potential therapeutic agents for these cancers.

Biochar immobilized plant growth-promoting rhizobacteria enhanced the physicochemical properties, agronomic characters and microbial communities during lettuce seedling.

Spent mushroom substrate (SMS) is the by-products of mushroom production, which is mainly composed of disintegrated lignocellulosic biomass, mushroom mycelia and some minerals. The huge output and the lack of effective utilization methods make SMS becoming a serious environmental problem. In order to improve the application of SMS and SMS derived biochar (SBC), composted SMS (CSMS), SBC, combined plant growth-promoting rhizobacteria (PGPR, Bacillus subtilis BUABN-01 and Arthrobacter pascens BUAYN-122) and SBC immobilized PGPR (BCP) were applied in the lettuce seedling. Seven substrate treatments were used, including (1) CK, commercial control; (2) T1, CSMS based blank control; (3) T2, T1 with combined PGPR (9:1, v/v); (4) T3, T1 with SBC (19:1, v/v); (5) T4, T1 with SBC (9:1, v/v); (6) T5, T1 with BCP (19:1, v/v); (7) T6, T1 with BCP (9:1, v/v). The physicochemical properties of substrate, agronomic and physicochemical properties of lettuce and rhizospheric bacterial and fungal communities were investigated. The addition of SBC and BCP significantly (p < 0.05) improved the total nitrogen and available potassium content. The 5% (v/v) BCP addiction treatment (T5) represented the highest fresh weight of aboveground and underground, leave number, chlorophyll content and leaf anthocyanin content, and the lowest root malondialdehyde content. Moreover, high throughput sequencing revealed that the biochar immobilization enhanced the adaptability of PGPR. The addition of PGPR, SBC and BCP significantly enriched the unique bacterial biomarkers. The co-occurrence network analysis revealed that 5% BCP greatly increased the network complexity of rhizospheric microorganisms and improved the correlations of the two PGPR with other microorganisms. Furthermore, microbial functional prediction indicated that BCP enhanced the nutrient transport of rhizospheric microorganisms. This study showed the BCP can increase the agronomic properties of lettuce and improve the rhizospheric microbial community.

Cancer-associated fibroblasts: Just on the opposite side of antitumour immunity?

The tumour microenvironment (TME) is critical for the initiation, progression, and metastasis of tumours, and cancer-associated fibroblasts (CAFs) are the most dominant cells and have attracted interest as targets for cancer therapy among the stromal components within the TME. Currently, most of the identified CAF subpopulations are believed to exhibit suppressive effects on antitumour immunity. However, accumulating evidence indicates the presence of immunostimulatory CAF subpopulations, which play an important role in the maintenance and amplification of antitumour immunity, in the TME. Undoubtedly, these findings provide novel insights into CAF heterogeneity. Herein, we focus on summarizing CAF subpopulations that promote antitumour immunity, the surface markers of these populations, and possible immunostimulatory mechanisms in the context of recent advances in research on CAF subpopulations. In addition, we discuss the possibility of new therapies targeting CAF subpopulations and conclude with a brief description of some prospective avenues for CAF research.

Neoadjuvant camrelizumab plus chemotherapy for locally advanced cervical cancer (NACI Study): a study protocol of a prospective, single-arm, phase II trial.

INTRODUCTION: Neoadjuvant chemotherapy (NACT) is an emerging approach for locally advanced cervical cancer (LACC). However, the clinical response and postoperative adjuvant radiation or chemoradiation trimodality treatment resulted in controversy. PD-1 inhibitors have shown promising role in recurrent or metastatic cervical cancer, and there is preclinical evidence of the activation and synergistic effects of NACT on PD-1 inhibitors. This study aims to evaluate the efficacy and safety of the preoperative PD-1 inhibitor camrelizumab combined with NACT for LACC. METHODS AND ANALYSIS: The study is designed as a multicentre, open-label, single-arm, prospective phase II study. A total of 82 patients will receive neoadjuvant chemo-immunotherapy, defined as one cycle of cisplatin (75-80 mg/m2, intravenously) plus nab-paclitaxel (260 mg/m2, intravenously) NACT and subsequent two cycles of camrelizumab (200 mg, intravenously) combined with NACT. After neoadjuvant chemo-immunotherapy, patients exhibiting complete response and partial response will undergo radical surgery and subsequent adjuvant therapy. In contrast, patients with stable disease and progressive disease will transfer to concurrent chemoradiotherapy (CCRT). Following surgery, patients will receive adjuvant CCRT or radiotherapy. The primary endpoint is the objective response rate. The secondary endpoints are the pathological complete response, patients requiring postoperative adjuvant therapy, safety of neoadjuvant chemo-immunotherapy, surgical complication, event-free survival, and overall survival. An additional aim is to dynamically evaluate peripheral immune responses and local immunological microenvironments and their association with neoadjuvant immunotherapy. ETHICS AND DISSEMINATION: This trial was approved by the Medical Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (S2020-112). This study is among the first to evaluate the efficacy and safety of neoadjuvant chemo-immunotherapy in LACC. The findings of this research will promote neoadjuvant anti-PD-1 immunotherapy with radical surgery as a new therapeutic strategy. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT04516616).

GA signaling protein LsRGL1 interacts with the abscisic acid signaling-related gene LsWRKY70 to affect the bolting of leaf lettuce.

A variety of endogenous hormone signals, developmental cues, and environmental stressors can trigger and promote leaf lettuce bolting. One such factor is gibberellin (GA), which has been linked to bolting. However, the signaling pathways and the mechanisms that regulate the process have not been discussed in full detail. To clarify the potential role of GAs in leaf lettuce, significant enrichment of GA pathway genes was found by RNA-seq, among which the LsRGL1 gene was considered significant. Upon overexpression of LsRGL1, a noticeable inhibition of leaf lettuce bolting was observed, whereas its knockdown by RNA interference led to an increase in bolting. In situ hybridization analysis indicated a significant accumulation of LsRGL1 in the stem tip cells of overexpressing plants. Leaf lettuce plants stably expressing LsRGL1 were examined concerning differentially expressed genes through RNA-seq analysis, and the data indicated enhanced enrichment of these genes in the 'plant hormone signal transduction' and 'phenylpropanoid biosynthesis' pathways. Additionally, significant changes in LsWRKY70 gene expression were identified in COG (Clusters of Orthologous Groups) functional classification. The results of yeast one-hybrid, ß-glucuronidase (GUS), and biolayer interferometry (BLI) experiments showed that LsRGL1 proteins directly bind to the LsWRKY70 promoter. Silencing LsWRKY70 by virus-induced gene silencing (VIGS) can delay bolting, regulate the expression of endogenous hormones, abscisic acid (ABA)-linked genes, and flowering genes, and improve the nutritional quality of leaf lettuce. These results strongly associate the positive regulation of bolting with LsWRKY70 by identifying its vital functions in the GA-mediated signaling pathway. The data obtained in this research are invaluable for further experiments concerning the development and growth of leaf lettuce.

LettuceGDB: The community database for lettuce genetics and omics.

As a globally popular leafy vegetable and a representative plant of the Asteraceae family, lettuce has great economic and academic significance. In the last decade, high-throughput sequencing, phenotyping, and other multi-omics data in lettuce have accumulated on a large scale, thus increasing the demand for an integrative lettuce database. Here, we report the establishment of a comprehensive lettuce database, LettuceGDB (https://www.lettucegdb.com/). As an omics data hub, the current LettuceGDB includes two reference genomes with detailed annotations; re-sequencing data from over 1000 lettuce varieties; a collection of more than 1300 worldwide germplasms and millions of accompanying phenotypic records obtained with manual and cutting-edge phenomics technologies; re-analyses of 256 RNA sequencing datasets; a complete miRNAome; extensive metabolite information for representative varieties and wild relatives; epigenetic data on the genome-wide chromatin accessibility landscape; and various lettuce research papers published in the last decade. Five hierarchically accessible functions (Genome, Genotype, Germplasm, Phenotype, and O-Omics) have been developed with a user-friendly interface to enable convenient data access. Eight built-in tools (Assembly Converter, Search Gene, BLAST, JBrowse, Primer Design, Gene Annotation, Tissue Expression, Literature, and Data) are available for data downloading and browsing, functional gene exploration, and experimental practice. A community forum is also available for information sharing, and a summary of current research progress on different aspects of lettuce is included. We believe that LettuceGDB can be a comprehensive functional database amenable to data mining and database-driven exploration, useful for both scientific research and lettuce breeding.

HMGA1 As a Potential Prognostic and Therapeutic Biomarker in Breast Cancer.

Background: High-mobility group AT-hook1 (HMGA1) protein plays an important role in various diseases. However, the contribution of HMGA1 in breast cancer remains to be tapped. Methods: The expression of HMGA1 was analyzed in The Cancer Genome Atlas (TCGA) and TIMER database, and immunohistochemistry was performed in 39 breast cancer (BC) patients. The correlation between HMGA1 expression and prognosis was evaluated using Kaplan-Meier plotter (KM plotter) in patients with breast cancer. Then, cBioPortal and bc-GenExMiner were requisitioned to analyze the contribution of HMGA1 expression to clinical features. In order to reveal the function of HMGA1 in breast cancer cells, enrichment analysis was performed using the clusterProfiler R software package. Moreover, CCK8 assay, EdU assay, and Cell Cycle Assay were performed to assess the proliferation, and transwell assay was used to evaluate cell migration and invasion. Flow cytometry was used to explore the role of HMGA1 on cell apoptosis. After that, the effect of HMGA1 on signaling pathways in BC cells was detected by western blot. Results: HMGA1 was highly expressed in a variety of tumors tissues, including BC. High HMGA1 expression was correlated with poor prognosis in BC patients. Meanwhile, HMGA1 expression was increased in molecular phenotypes with poor prognosis (ER-, PR-, and HER2+) and associated with high-grade group, lymph node metastasis, and NPI (Nottingham Prognostic Index). Further, function analysis revealed HMGA1 was enriched in DNA replication and cell cycle pathways in breast cancer. Moreover, knockdown of HMGA1 caused apoptosis, inhibited proliferation, migration, and invasion of MCF-7 and MDA-MB-231 cells, in which the oncogenic signaling pathway of PI3K/AKT/MMP9 played a critical role. Conclusions: HMGA1 was important for breast cancer progression and was a critical prognostic indicator, prompting a potential therapeutic target of breast cancer.

Neoadjuvant chemotherapy for patients with international federation of gynecology and obstetrics stages IB3 and IIA2 cervical cancer: a multicenter prospective trial.

BACKGROUND: Preoperative neoadjuvant chemotherapy (NACT) has been widely used in developing countries for the treatment of patients with International Federation of Gynecology and Obstetrics (FIGO) stages IB3 and IIA2 cervical cancer. However, the effectiveness of NACT and treatment options for NACT-insensitive patients have been concerning. This study will assess prognostic differences between NACT and primary surgery treatment (PST), determine factors associated with prognosis, and explore better adjuvant treatment modalities for NACT-insensitive patients. METHODS: This study analyzed clinical characteristics, pathological characteristics, treatment options, and follow-up information of 774 patients with FIGO stages IB3 and IIA2 cervical cancer from 28 centers from January 2016 to October 2019 who participated in a multicenter, prospective, randomized controlled trial. RESULTS: For patients undergoing NACT, the 5-year OS and PFS rate was 85.8 and 80.5% respectively. They were similar in the PST group. There was no significant difference in OS and PFS between clinical response (CR)/partial response (PR) groups and stable disease (SD)/progressive disease (PD) groups. Apart from deep cervical invasion (p = 0.046) affecting OS for patients undergoing NACT, no other clinical and pathological factors were associated with OS. 97.8% of NACT-insensitive patients opted for surgery. If these patients did not have intermediate- or high-risk factors, whether they had undergone postoperative adjuvant therapy was irrelevant to their prognosis, whereas for patients with intermediate- or high-risk factors, adjuvant chemotherapy resulted in better PFS (chemotherapy vs. no therapy, p < 0.001; chemotherapy vs. radiotherapy, p = 0.019) and OS (chemotherapy vs. no therapy, p < 0.001; chemotherapy vs. radiotherapy, p = 0.002). CONCLUSIONS: NACT could be a choice for patients with FIGO stages IB3 and IIA2 cervical cancer. The main risk factor influencing prognosis in the NACT group is deep cervical invasion. After systematic treatment, insensitivity to NACT does not indicate a poorer prognosis. For NACT-insensitive patients, Chinese prefer surgery. Postoperative adjuvant therapy in patients with no intermediate- or high-risk factors does not improve prognosis, and chemotherapy in patients with intermediate- and high-risk factors is more effective than radiation therapy and other treatments. TRIAL REGISTRATION: The study was prospectively registered on ClinicalTrials.gov (NCT03308591); date of registration: 12/10/2017.

Transcriptome and Metabolome Analysis Revealed That Exogenous Spermidine-Modulated Flavone Enhances the Heat Tolerance of Lettuce.

Lettuce is sensitive to high temperature, and exogenous spermidine can improve heat tolerance in lettuce, but its intrinsic mechanism is still unclear. We analyzed the effects of exogenous spermidine on the leaf physiological metabolism, transcriptome and metabolome of lettuce seedlings under high-temperature stress using the heat-sensitive lettuce variety 'Beisansheng No. 3' as the material. The results showed that exogenous spermidine increased the total fresh weight, total dry weight, root length, chlorophyll content and total flavonoid content, increased the activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and decreased malondialdehyde (MDA) content in lettuce under high temperature stress. Transcriptome and metabolome analyses revealed 818 differentially expressed genes (DEGs) and 393 metabolites between water spray and spermidine spray treatments under high temperature stress, and 75 genes from 13 transcription factors (TF) families were included in the DEGs. The Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis of DEG contains pathways for plant-pathogen interactions, photosynthesis-antennal proteins, mitogen-activated protein kinase (MAPK) signaling pathway and flavonoid biosynthesis. A total of 19 genes related to flavonoid synthesis were detected. Most of these 19 DEGs were down-regulated under high temperature stress and up-regulated after spermidine application, which may be responsible for the increase in total flavonoid content. We provide a possible source and conjecture for exploring the mechanism of exogenous spermidine-mediated heat tolerance in lettuce.

Effects of selenate and selenite on selenium accumulation and speciation in lettuce.

Lettuce is a common vegetable in hydroponic production. In this paper, a selenium (Se)-biofortification method was provided. The Se content, speciation, and the effects of different concentrations of selenate and selenite on lettuce growth and amino acids were investigated. The results showed that lettuce had strong ability to accumulate exogenous selenium, and inorganic Se could be effectively converted into organic Se. The proportion of organic Se in the shoots under treatment with 4 µmol L-1 selenite was 100%. Selenomethionine was the main organic Se, accounting for 51% (selenate) and 90% (selenite) of the total Se. Adding Se improves photosynthesis of lettuce and promotes growth. The growth with 2 µmol L-1 selenate and 4 µmol L-1 selenite was better than CK, and the shoot fresh weight was increased by 143.22% and 166.98%, respectively. Furthermore, the optimum Se application is 2 µmol L-1, and some areas can apply 4 µmol L-1 selenite. But Se-excessive areas are not recommended to grow selenium-rich foods. Therefore, lettuce has strong biofortification potential.

Genome-Wide Identification and Expression Analysis of MAPK Gene Family in Lettuce (Lactuca sativa L.) and Functional Analysis of LsMAPK4 in High- Temperature-Induced Bolting.

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.

LsARF3 mediates thermally induced bolting through promoting the expression of LsCO in lettuce (Lactuca sativa L.).

Lettuce (Lactuca sativa L.) is a leafy vegetable whose edible organs usually are leaf or stems, and thus high-temperature induced bolting followed by flower initiation is an undesirable trait in lettuce production. However, the molecular mechanism that controls lettuce bolting and flowering upon thermal treatments is largely unknown. Here, we identified a Lettuce auxin response factor 3 (LsARF3), the expression of which was enhanced by heat and auxin treatments. Interestingly, LsARF3 is preferentially expressed in stem apex, suggesting it might be associated with lettuce bolting. Transgenic lettuce overexpressing LsARF3 displayed early bolting and flowering, whereas knockout of LsARF3 dramatically delayed bolting and flowering in lettuce under normal or high temperature conditions. Furthermore, Exogenous application of IAA failed to rescue the late-bolting and -flowering phenotype of lsarf3 mutants. Several floral integrator genes including LsCO, LsFT, and LsLFY were co-expressed with LsARF3 in the overexpression and knockout lettuce plants. Yeast one-hybrid (Y1H) experiments suggested that LsARF3 could physically interact with the LsCO promoter, which was further confirmed by a dual luciferase assay in tobacco leaves. The results indicated that LsARF3 might directly modulate the expression of LsCO in lettuce. Therefore, these results demonstrate that LsARF3 could promote lettuce bolting in response to the high temperature by directly or indirectly activating the expression of floral genes such as LsCO, which provides new insights into lettuce bolting in the context of ARFs signaling and heat response.

Molecular basis of high temperature-induced bolting in lettuce revealed by multi-omics analysis.

BACKGROUND: High temperature induces early bolting in lettuce (Lactuca sativa L.), which affects both quality and production. However, the molecular mechanism underlying high temperature-induced bolting is still limited. RESULTS: We performed systematical analysis of morphology, transcriptome, miRNAs and methylome in lettuce under high temperature treatment. Through a comparison of RNA-Seq data between the control and the high temperature treated lettuces at different time points totally identified 2944 up-regulated genes and 2203 down-regulated genes, which cover three floral pathways including photoperiod, age and gibberellin (GA) pathways. Genome wide analysis of miRNAs and methylome during high temperature treatment indicated miRNAs and DNA methylation might play a role controlling gene expression during high temperature-induced bolting. miRNA targets included some protein kinase family proteins, which potentially play crucial roles in this process. CONCLUSIONS: Together, our results propose a possible regulation network involved in high temperature-induced bolting.

LsMYB15 Regulates Bolting in Leaf Lettuce (Lactuca sativa L.) Under High-Temperature Stress.

High temperature is one of the primary environmental stress factors affecting the bolting of leaf lettuce. To determine the potential role of melatonin in regulating high-temperature induced bolting in leaf lettuce (Lactuca sativa L.), we conducted melatonin treatment of the bolting-sensitive cultivar "S39." The results showed that 100 µmol L-1 melatonin treatment significantly promoted growth, and melatonin treatment delayed high-temperature-induced bolting in lettuce. RNA-seq analysis revealed that the differentially expressed genes (DEGs) involved in "plant hormone signal transduction" and "phenylpropanoid biosynthesis" were significantly enriched during high-temperature and melatonin treatment. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis suggested that the expression patterns of abscisic acid (ABA)-related genes positively correlated with stem length during leaf lettuce development. Furthermore, weighted gene co-expression network analysis (WGCNA) demonstrated that MYB15 may play an important role in melatonin-induced resistance to high temperatures. Silencing the LsMYB15 gene in leaf lettuce resulted in early bolting, and exogenous melatonin delayed early bolting in leaf lettuce at high temperatures. Our study provides valuable data for future studies of leaf lettuce quality.

Application of exogenous auxin and gibberellin regulates the bolting of lettuce (Lactuca sativa L.).

Plant bolting is regulated and controlled by various internal and external factors. We aimed to provide an improved method for breeding to determine whether there is a synergism between hormones and to explore the regulatory effect of plant hormones on the bolting of leaf lettuce. Lettuce plants were sprayed with exogenous auxin and gibberellin separately or in combination. The specific bolting period was determined by the change in stem length and cytological observation. The dynamic changes in endogenous hormones and genes closely related to bolting were analyzed. Treatment with gibberellin alone and the combined application of auxin and gibberellin induced bolting on the fourth day, and treatment with auxin alone resulted in bolting on the eighth day. In the early bolting stage, the auxin contents in the stems of the treatment groups, especially the combined gibberellin and auxin group, were higher than those of the control group. After the application of exogenous auxin and gibberellin, we found that the expression of the ARF8 and GID1 genes was upregulated. Based on the results of our study, combined treatment with exogenous gibberellin and auxin was the best method to promote the bolting of leaf lettuce, and the ARF8 and GID1 genes are closely related to this process.

Role of Spermidine in Photosynthesis and Polyamine Metabolism in Lettuce Seedlings under High-Temperature Stress.

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.

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.