Transcriptomic analysis reveals the molecular basis of photoperiod-regulated sex differentiation in tropical pumpkins (Cucurbita moschata Duch.).

BACKGROUND: Photoperiod, or the length of the day, has a significant impact on the flowering and sex differentiation of photoperiod-sensitive crops. The "miben" pumpkin (the main type of Cucurbita moschata Duch.) is well-known for its high yield and strong disease resistance. However, its cultivation has been limited due to its sensitivity to photoperiod. This sensitivity imposes challenges on its widespread cultivation and may result in suboptimal yields in regions with specific daylength conditions. As a consequence, efforts are being made to explore potential strategies or breeding techniques to enhance its adaptability to a broader range of photoperiods, thus unlocking its full cultivation potential and further promoting its valuable traits in agriculture. RESULTS: This study aimed to identify photoperiod-insensitive germplasm exhibiting no difference in sex differentiation under different day-length conditions. The investigation involved a phenotypic analysis of photoperiod-sensitive (PPS) and photoperiod-insensitive (PPIS) pumpkin materials exposed to different day lengths, including long days (LDs) and short days (SDs). The results revealed that female flower differentiation was significantly inhibited in PPS_LD, while no differences were observed in the other three groups (PPS_SD, PPIS_LD, and PPIS_SD). Transcriptome analysis was carried out for these four groups to explore the main-effect genes of sex differentiation responsive to photoperiod. The main-effect gene subclusters were identified based on the principal component and hierarchical cluster analyses. Further, functional annotations and enrichment analysis revealed significant upregulation of photoreceptors (CmCRY1, F-box/kelch-repeat protein), circadian rhythm-related genes (CmGI, CmPRR9, etc.), and CONSTANS (CO) in PPS_LD. Conversely, a significant downregulation was observed in most Nuclear Factor Y (NF-Y) transcription factors. Regarding the gibberellic acid (GA) signal transduction pathway, positive regulators of GA signaling (CmSCL3, CmSCL13, and so forth) displayed higher expression levels, while the negative regulators of GA signaling, CmGAI, exhibited lower expression levels in PPS_LD. Notably, this effect was not observed in the synthetic pathway genes. Furthermore, genes associated with ethylene synthesis and signal transduction (CmACO3, CmACO1, CmERF118, CmERF118-like1,2, CmWIN1-like, and CmRAP2-7-like) showed significant downregulation. CONCLUSIONS: This study offered a crucial theoretical and genetic basis for understanding how photoperiod influences the mechanism of female flower differentiation in pumpkins.

Decoding comparative taste and nutrition regulation in Chinese cabbage via integrated metabolome and transcriptome analysis.

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a widely consumed leafy vegetable known for its various health-beneficial nutrients. Caixin (ET and JY) represent distinct cultivars of Chinese cabbage that exhibit differential consumer preference attributed to variations in taste and nutritional content, with ET being characterized as sweeter and more nutritionally superior compared to JY. However, limited research has been conducted to explore regulation of flavor and nutrition-related quality traits in Chinese cabbage. In this pioneer study, comprehensive trans-meta-analysis was used to compare the metabolic and molecular underpinnings behind unique taste and nutritional profiles of ET and JY. 8-Methylsulfonyloctyl glucosinolates and Uridine 5'-diphospho-D-glucose exhibited the highest correlation coefficient in Pearson meta-meta-association, which modulate flavor and nutrition processes. While DAMs primarily featured L-Homomethionine, saccharic acid, 1,6-Di-O-caffeoyl-ß-D-glucose, and Rutin, with notable variations in expression between ET and JY. Conspicuously, DEGs encoding structural enzymes i.e. Glucosinolates (MAM, CYP, UGT), flavonoids (CHS, CHI, F3H) and sucrose (SPS, SPP, SUS) synthases were identified as key players in nutrient and flavor production. Multi-omics conjoint analysis revealed that saccharides, amino acids, ascorbates, flavonoids, organic acids and vitamins were positively correlated with taste and nutrition, and were found to be overexpressed in ET. While aliphatic glucosinolates were abundant in JY compared to ET, they might play a critical role in regulating quality traits. Besides, HPLC and RT-qPCR corroborated multi-omics data reliability. These findings offer novel insights into the mechanisms governing the regulation of taste and nutritional levels in Chinese cabbage.

An fusaric acid-based CRISPR library screen identifies MDH2 as a broad-spectrum regulator of Fusarium toxin-induced cell death.

Fusarium mycotoxins are of great concern because they are the most common food-borne mycotoxins and environmental contaminants worldwide. Fusaric acid (FA), Deoxynivalenol (DON), Zearalenone (ZEA), T-2 toxin (T-2), and Fumonisin B1 (FB1) are important Fusarium toxins contaminating feeds and food and can cause serious health problems. FA can synergize with some other Fusarium toxins to enhance overall toxicity. However, the underlying molecular mechanism remains poorly understood. In this study, our CRISPR screening revealed Malate dehydrogenase 2 (MDH2) and Pyruvate dehydrogenase E1 subunit beta (PDHB) are the key genes for FA-induced cell death. Pathways associated with mitochondrial function, notably the TCA cycle, play a significant role in FA cytotoxicity. We found that MDH2 and PDHB depletion reduced FA-induced cell death, ROS accumulation, and the expression of caspase-3 and HIF-1α. The cell viability assays and flow cytometry demonstrated that MDH2 knockout but not PDHB decreased DON, ZEA, T-2, and FB1-induced cytotoxicity, apoptosis, and ROS accumulation. MDH2 inhibitor LW6 also decreased DON, ZEA, T-2, and FB1-induced toxicity. This suggested that MDH2, but not PDHB, is a common regulator of broad-spectrum Fusarium toxin (FA, DON, ZEA, T-2, and FB1)-induced cell death. Our work provides new avenues for the treatment of Fusarium toxin toxicity.

RPN1a, a 26S proteasome subunit, is required for innate immunity in Arabidopsis.

Accumulating evidence shows that proper degradation of proteins that affect defense responses in a positive or negative manner is critical in plant immunity. However, the role of plant degradation systems such as the 26S proteasome in plant immunity is not well understood. Loss-of-function mutations in EDR2 (ENHANCED DISEASE RESISTANCE 2) lead to increased resistance to the adapted biotrophic powdery mildew pathogen Golovinomyces cichoracearum. To study the molecular interactions between powdery mildew pathogen and Arabidopsis, we performed a screen for suppressors of edr2 and found that mutation in the gene that encodes RPN1a, a subunit of the 26S proteasome, suppressed edr2-associated disease resistance phenotypes. In addition, RPN1a is required for edr1- and pmr4-mediated powdery mildew resistance and mildew-induced cell death. Furthermore, we show that rpn1a displayed enhanced susceptibility to the fungal pathogen G. cichoracearum and to virulent and avirulent bacterial Pto DC3000 strains, which indicated that rpn1a has defects in basal defense and resistance (R) protein-mediated defense. RPN1a-GFP localizes to both the nucleus and cytoplasm. Accumulation of RPN1a is affected by salicylic acid (SA) and the rpn1a mutant has defects in SA accumulation upon Pto DC3000 infection. Further analysis revealed that two other subunits of the 26S proteasome, RPT2a and RPN8a are also involved in edr2-mediated disease resistance. Based on these results, we conclude that RPN1a is required for basal defense and R protein-mediated defense. Our data provide evidence that some subunits of the 26S proteasome are involved in innate immunity in Arabidopsis.

RNA-Seq analysis of gynoecious and weak female cucumber revealing the cell cycle pathway may regulate sex determination in cucumber.

This study aims to investigate key genes involved in molecular regulatory networks of cucumber sex determination. Genome-wide high-throughput RNA sequencing was performed for young apical buds of gynoecious and weak female cucumber at three growth stages (one-leaf one-bud, three-leaf one-bud, and five-leaf one-bud). Seven comparisons from the same cultivar at three different stages and at the same stage between the two cultivars were analyzed, and the results revealed that compared with differentially expressed genes (DEGs) in weak female cucumber, more genes were upregulated at the one-leaf one-bud stage and downregulated at the three-leaf one-bud stage in gynoecious cucumber. In addition, there were four kinds of gene expression trends (0, 1, 6, and 7), which were significantly enriched in gynoecious cucumber, while only two kinds of gene expression trends (5 and 6) were significantly enriched in weak female cucumber. Together with the data of the Gene Ontology (GO), pathway, gene expression trends and qRT-PCR, nine genes were identified and considered as candidate genes that may be involved in sex differentiation regulation in cucumber. These genes included Cs-MCM6, Cs-ACT3, Cs-XRCC4, Cs-MCM2, Cs-CDC45, Cs-Dpri, Cs-H2B, Cs-CDC20 and Cs-CNGC1. Among these genes, five genes (Cs-MCM6, Cs-MCM2, Cs-CDC45, Cs-Dpri, and Cs-CDC20) were involved in the cell cycle pathway, suggesting that the cell cycle pathway may play an important role in sex determination in cucumber.

Suppression of edr2-mediated powdery mildew resistance, cell death and ethylene-induced senescence by mutations in ALD1 in Arabidopsis.

EDR2 is a negative regulator of the defense response and cell death in Arabidopsis. Loss-of-function of EDR2 leads to enhanced resistance to powdery mildew. To identify new components in the EDR2 signal transduction pathway, mutations that suppress edr2 resistant phenotypes were screened. Three mutants, edts5-1, edts5-2 and edts5-3 (edrtwo suppressor 5), were identified. The EDTS5 gene was identified by map-based cloning and previously was shown to encode an aminotransferase (ALD1). Therefore we renamed these three alleles ald1-10, ald1-11 and ald1-12, respectively. Mutations in ALD1 suppressed all edr2-mediated phenotypes, including powdery mildew resistance, programmed cell death and ethylene-induced senescence. Accumulation of hydrogen peroxide in edr2 was also suppressed by ald1 mutation. The expression of defense-related genes was up-regulated in the edr2 mutant, and the up-regulation of those genes in edr2 was suppressed in the edr2/ald1 double mutant. The ald1 single mutant displayed delayed ethylene-induced senescence. In addition, ald1 mutation suppressed edr1-mediated powdery mildew resistance, but could not suppress the edr1/edr2 double-mutant phenotype. These data demonstrate that ALD1 plays important roles in edr2-mediated defense responses, and senescence and revealed a crosstalk between ethylene and salicylic acid signaling mediated by ALD1 and EDR2.

[The relationships of Zhang Jing-yue's three theories of the Supreme Ultimate].

Under the influence of Supreme Ultimate of Neo-Confucianism, Zhang Jing-yue put forward the three theories of the Supreme Ultimate, viz., Supreme Ultimate of the Primordial Qi, Supreme Ultimate of the Heart, and Supreme Ultimate of the Vital Gate. These three theories were of trinity relationship, forming the basic frame of Jingyue's system of medical theories. Among them, the theory of Supreme Ultimate of the Primordial Qi was the foundation of natural concept in his medical theories, the theory of Supreme Ultimate of the Vital Gate was the teleology of his medical theories, and the theory of Supreme Ultimate of the Heart, as a cognitive method, was the methodology of his medical theories. Jing-yue' s medical theories were constructed on these three corner-stays, which was a tremendous leap forward in the development of traditional Chinese medicine and Neo-Confucianism.

Antiviral activity of Morus alba L. extract against pseudorabies virus.

ETHNOPHARMACOLOGICAL RELEVANCE: Morus alba L. are widely used as ethnomedicine and functional food in China, Japan, Korea and other Asian countries. Morus alba L. have a variety of pharmacological activity such as antiviral, antioxidation, anti-cholesterol, anticancer, hypoglycemia, and neuroprotection. Morus alba L. has demonstrated antiviral efficacy against influenza viruses, SARS-CoV-2 and so on, but its potential activity against pseudorabies virus (PRV) remains uncertain. AIM OF THE STUDY: This study endeavors to delve into the anti-pseudorabies virus (PRV) potential of the ethanol extract of Morus alba L. leaves (MLE), while simultaneously elucidating its underlying mechanism of action. MATERIALS AND METHODS: The anti-PRV activities of Morus alba L. extracts at different concentrations were evaluated by qPCR and immunoblotting. The inhibitory effects of MLE on PRV replication in three distinct treatment modes (pretreatment, co-treatment, and post-treatment) were detected by qPCR and indirect immunofluorescence assays. qPCR was used to investigate the effects of MLE on PRV attachment, entrance, and cytokine expression in PRV-infected cells. The chemical components in MLE were analyzed by UPLC-MS/MS. RESULTS: MLE significantly inhibits PRV replication and protein expression in a dose-dependent manner. MLE displays inhibitory effects against PRV at three different modes of treatment. The most significant inhibitory effect of MLE was observed when used in co-treatment mode, resulting in an inhibition rate of 99.42%. MLE inhibits PRV infection in the early stage. MLE inhibits PRV infection by affecting viral attachment and viral entry. Furthermore, MLE exerts its inhibition on PRV replication by mitigating the heightened expression of cytokines (TNF-α and IFN-α) triggered by PRV. Analysis of its chemical composition highlights phenolic acids and flavonoids as the principal constituents of MLE. CONCLUSION: The results illustrate that MLE effectively impedes PRV infection by suppressing viral adsorption and entry, while also curbing the expression of antiviral cytokines. Therefore, MLE may be a potential resource for creating new medications to treat human and animal PRV infections.