Nitrate (NO3−) serves as a critical nitrogen source and signaling molecule essential for its growth and quality formation. Although substantial genetic variation in nitrogen use efficiency (NUE) has been documented among tea cultivars, a systematic characterization of nitrate (NO3−) absorption kinetics and the associated genome-wide transcriptional regulatory networks across varying nitrate concentrations remains lacking. This study employed 15N isotope labeling and transcriptome sequencing to systematically analyze the absorption characteristics and molecular response mechanisms of the cultivars ‘Longjing 43’ and ‘Zhongming 6 hao’ under varying NO3− concentrations. Results revealed significant differentiation in absorption strategies: ‘Zhongming 6 hao’ exhibited a significantly higher absorption rate at low concentrations, whereas ‘Longjing 43’ demonstrated enhanced performance at high concentrations. Transcriptome analysis indicated that both cultivars shared coordinated regulation of ‘photosynthesis’ and ‘nitrogen metabolism’ pathways. Furthermore, 14 nitrogen metabolism genes and 64 differentially expressed transcription factors (including MYB, NAC, and LBD families) were identified. Specifically, the CsNiR gene (encoding nitrite reductase) was functionally validated; silencing of CsNiR significantly reduced nitrite reductase activity, confirming its positive regulatory role. This study provided a theoretical framework and key candidate genes for breeding nitrogen-use-efficient varieties, which is essential for sustainable tea production.

Aluminum is toxic to most plants, but low concentrations of aluminum are conducive to the growth of tea plants. However, it is not clear whether aluminum treatment alters the rhizosphere microbiome of tea plants, especially growth-promoting rhizosphere bacteria. In this study, we used 16S rRNA sequencing to demonstrate that after aluminum treatment, the relative abundance of Proteobacteria in the rhizosphere bacterial community was the highest, with Burkholderia being enriched and the dominant strain. In addition, 53 strains of culturable rhizosphere bacteria, including 17 strains of Firmicutes, 23 strains of Proteobacteria, and 10 strains of Actinobacteria, were isolated and identified from the rhizosphere soil of tea plants. Further analysis of the 53 rhizosphere bacterial strains revealed that 21 strains exhibited four growth-promoting abilities. Among them, Bacillus NVLP_s (FNVLP) exhibited the maximum indole-3-acetic acid production capacity. Additionally, 51 strains could tolerate an aluminum concentration of at least 1 mol L-1, and Sinomonas gamaensis (ASG) exhibited the maximum aluminum tolerance ability, up to an aluminum concentration of 6 mmol L-1. Plant-bacteria interactions showed that ASG, FNVLP, Paraburkholderia hospita (PPH), and their synthetic community exhibited growth-promoting effects on rice roots. Furthermore, ASG, FNVLP, and PPH significantly alleviated aluminum stress in rice. Moreover, PPH and ASG promoted the growth of tea plants, especially the growth of lateral roots, irrespective of the presence of aluminum; and PPH inoculation enriched the Burkholderia community and improved carbohydrate metabolism and hormone biosynthesis and metabolism. Overall, a few bacterial strains with aluminum-tolerant and growth-promoting abilities were enriched in the rhizosphere and promoted the growth of tea plants after aluminum treatment. Thus, this study laid the foundation for further development and utilization of aluminum-tolerant, growth-promoting bacteria for the cultivation and management of tea plants.

Two types of bioactive products, catechins and caffeine, contribute to the flavor and health benefits associated with beverages made from plants. MYB184 has been characterized as a primary regulator of catechin and caffeine biosynthesis in tea plants. However, the coregulation mechanism of catechins and caffeine in beverage plants remains unknown. Here, we found that MYB184 is a major regulatory factor in the concurrence and co-evolution of caffeine and galloylated cis-catechins with different mechanisms in Camellia species. MYB184 orthologues showed conserved regulation of caffeine and catechin biosynthesis in coffee plants. Additionally, the conserved catechin-related repressor MYB206 negatively regulated caffeine biosynthesis via both interacting with MYB184 and directly targeting Caffeine Synthase1 (TCS1), playing crucial roles in the dynamic distribution of caffeine and catechins in developing leaves and metabolic homeostasis under stress conditions. Our findings provide insight into the convergent evolution of flavor-related compounds in beverage plants.

[This corrects the article on p. 507 in vol. 9, PMID: 28337279.].

This study employed headspace solid-phase microextraction/solvent-assisted flavor evaporation combined with gas chromatography-mass spectrometry (HS-SPME/SAFE-GC-MS) and liquid chromatograph-mass spectrometer (LC-MS) to analyze volatile and non-volatile compounds of Congou black tea under different drying temperatures. The results demonstrated that 90RF (rough-fired) maximised floral/green aroma and umami taste. In contrast, 130RF enhanced fruity/roasted aromas with minimal astringency. Regarding over-fired drying, 60OF (over-fired) produced the highest umami taste, 75OF significantly enhanced floral aroma, whereas 90OF intensified fruity and sweet aromas and minimized astringency/bitterness. Analysis of key volatile compounds revealed that the pronounced floral and green aroma could be attributed to the high concentrations and the relative aroma characteristic impact (rACI) values of (E,E)-2,4-heptadienal and (Z)-3-hexen-1-ol. Simultaneously, the intensified fruity and sweet aroma was associated with an increased abundance of beta-damascenone. Furthermore, analysis of non-volatile metabolites in conjunction with a correlation analysis of taste attributes, revealed that amino acids, flavonoids (along with their glycosides), and phenolic acids were the primary constituents responsible for the observed differentiation in taste among the samples. This study showed 75 degrees C and 90 degrees C over-fired drying as the optimal condition for floral, fruity and sweet aromas intensity respectively, which offers a targeted approach for Congou black tea process control.

Abstract The albino leaves of cold-sensitive tea plants exhibited a marked reduction in chlorophyll content. However, the underlying regulatory mechanism remains elusive. In this study, we determined lower chlorophyll levels in tender leaves of cold-sensitive ‘Baiye 1’ compared to green leaves during the spring. Comparative transcriptome analyses identified the chlorophyll biosynthesis gene CsPORA, which was significantly downregulated in albino leaves. Functional assays confirmed that CsPORA positively regulates chlorophyll content. Electrophoretic mobility shift assay, yeast one-hybrid, dual-luciferase, and GUS staining results demonstrated that CsNAC87 binds to the promoter of CsPORA and suppresses its expression. Additionally, CsZAT12 physically interacts with CsNAC87 to enhance the repression. Overexpression of either CsZAT12 or CsNAC87 negatively regulated chlorophyll accumulation in tea plants and tobacco leaves, while co-transformation of CsZAT12 and CsNAC87 intensified the albino phenotype in tobacco leaves. Low temperature treatment of ‘Baiye 1’ triggered the marked upregulation of CsNAC87 and CsZAT12, leading to dramatical downregulation of CsPORA and leaf albinism. Collectively, our study reveals a novel cold-responsive CsZAT12-CsNAC87 module that negatively regulates chlorophyll synthesis by repressing CsPORA, providing new insights into chlorophyll metabolism in albino leaves of cold-sensitive tea cultivars.

Tumor tertiary lymphoid structures (TLS), especially mature TLS (mTLS), have been associated with better prognosis and improved responses to immune checkpoint blockade (ICB), but the underlying mechanisms remain incompletely understood. Here, by performing single-cell RNA, antigen receptor sequencing and spatial transcriptomics on tumor tissue from head and neck squamous cell carcinoma (HNSCC) patients with different statuses of TLS, we observe that mTLS are enriched with stem-like T cells, and B cells at various maturation stages. Notably, progenitor exhausted CD4+ T cells, with features resembling follicular helper T cells, support these responses, by activating B cells to produce plasma cells in the germinal center, and interacting with DC-LAMP+ dendritic cells to support CD8+ T cell activation. Conversely, non-mTLS tumors do not promote local anti-tumor immunity which is abundant of immunosuppressive cells or a lack of stem-like B and T cells. Furthermore, patients with mTLS manifest improved overall survival and response to ICB compared to those with non-mTLS. Overall, our study provides insights into mechanisms underlying mTLS-mediated intra-tumoral immunity events against cancer.

Intra-tumoral bacteria are pivotal in the initiation and progression of head and neck squamous cell carcinoma (HNSCC), exerting a significant influence on tumor cell biology, immune responses, and the tumor microenvironment (TME). Different types and distribution of bacteria threaten the balance of metabolism and the immune environment of tumor cells. Taking advantage of this disrupted homeostasis, intra-tumoral bacteria stimulate the secretion of metabolites or influence specific immune cell types to produce inflammatory or chemokines, thereby influencing the anti-tumor immune response while regulating the level of inflammation and immunosuppression within the TME. Some intra-tumoral bacteria are used as diagnostic and prognostic markers in clinical practice. Based on the unique characteristics of bacteria, the use of engineered bacteria and outer membrane vesicles for drug delivery and biological intervention is a promising new therapeutic strategy. The presence of intra-tumoral bacteria also makes chemoradiotherapy tolerable, resulting in a poor treatment effect. However, due to the immune-related complexity of intra-tumoral bacteria, there may be unexpected effects in immunotherapy. In this review the patterns of intra-tumoral bacteria involvement in HNSCC are discussed, elucidating the dual roles, while exploring the relevance to anti-tumor immune responses in the clinical context and the prospects and limitations of the use of bacteria in targeted therapy.

Background: Signaling pathways play crucial roles in tumor cells. However, functional heterogeneity of signaling pathways in skin cutaneous melanoma (SKCM) has not been established. Methods: Based on a recent computational pipeline, pathway activities between SKCM and normal samples were identified. Results: The results showed that high activities in 12 pathways were associated with poor prognoses, while high activities in 17 pathways were associated with favorable prognoses. Interestingly, elevated metabolic pathway activity was unfavorable, whereas elevated immune activity was favorable for SKCM. Unfavorably elevated metabolic pathways strongly correlated with Wnt/betacatenin signaling. Conversely, favorable pathways, such as glycosaminoglycan biosynthesis and keratan sulfate, were strongly correlated with anti-tumor pathways. Moreover, the activities of favorable pathways were strongly positively correlated with infiltrating CD8+ T cells, macrophages M1, immune score, and stromal score, all of which were favorable for SKCM. Conclusion: Taken together, our study provides insights into the characteristics of several pathways in SKCM.

The Fragile X Mental Retardation 1 (FMR1) gene is well-known for its role in Fragile X syndrome, a neurodevelopmental disorder, but emerging evidence suggests its involvement in regulating cellular metabolism, with implications for cancer biology. FMR1 encodes the Fragile X mental retardation protein (FMRP), an RNA-binding protein that controls various cellular processes, including translation, synaptic plasticity, and RNA metabolism. Recent studies have uncovered novel links between FMR1, metabolic regulation, and tumorigenesis. This review discusses the role of FMR1 in cellular metabolism and its potential involvement in cancer, focusing on glycolysis, mitochondrial metabolism, lipid metabolism, immune cell metabolism, and tumor immune evasion, and as a potential target to enhance immunotherapy, and highlights future research directions to elucidate its mechanistic roles in cancer.

While mTOR plays a key role in the development of pulmonary arterial hypertension (PAH), its suppressor, AMPKα, acts as an inhibitor. Although mTOR-driven transcriptional upregulation of the plasma membrane exchanger and amino acid transporter xCT, encoded by the Slc7a11 gene, is critical for cell proliferation and tumorigenesis, the involvement of xCT in PAH remains unexplored. In this study, we found that xCT expression was elevated in hypoxia-treated human pulmonary arterial endothelial cells (HPAECs) and the lungs of hypoxia-exposed mice and Sugen5416/hypoxia (SuHx)-induced PAH mice. Knockout of xCT prevented the development of PAH and right heart failure in SuHx-conditioned mice. The xCT inhibitor sulfasalazine prevented and reversed SuHx-induced PAH in mice. Deleting and inhibiting xCT activated AMPKα and inactivated mTOR in mouse lungs with PAH and in HPAECs. Sulfasalazine suppressed mTOR through activation of AMPKα in HPAECs. The mTOR inhibitor rapamycin reduced xCT expression, activated AMPKα, and suppressed mTOR in HPAECs. These findings suggest that xCT promotes the development of PAH, likely through suppression of AMPKα and activation of mTOR. Blockage of xCT and mTOR or activation of AMPKα by existing drugs such as sulfasalazine, sirolimus, and metformin may offer readily therapeutic strategies for PAH.

Understanding both local and systemic immunity is essential to optimizing the effectiveness of immunotherapy. However, the dynamic alterations in systemic immunity during tumor development are yet to be clearly defined. Here, we identified a previously unrecognized connection that bridges the interaction between the spleen and tumor through erythroid progenitor cells (EPCs), which suppress tumor immunity and promote tumor progression. We performed the single- cell RNA-seq and RNA-seq to demonstrate the presence of EPCs and identify the characteristic and an immunomodulatory role of EPCs during tumor progression. These tumor- hijacked EPCs proliferate in situ in spleens and impaired systemic and local antitumor response through the interaction between tumor and spleen. Specifically, the splenic CD45- EPCs secreted heparin- binding growth factor to regulate PD- L1- mediated immunosuppression of splenic CD45+EPCs. Educated CD45+ EPCs from the spleen then migrated to the tumors via the CCL5/CCR5 axis, thereby weakening local antitumor immunity. Consequently, targeting EPCs not only revitalized antitumor immunity but also improved the anti- PD- L1 effect by promoting intratumoral T cell infiltration. Importantly, CD45+ EPCs are associated with immunosuppression and reduced survival in patients with head and neck squamous cell carcinoma. Collectively, these findings reveal the role of EPCs in orchestrating the interaction between the spleen and tumor, which could have significant implications for the development of more effective cancer immunotherapy.

Objectives This study evaluates Fasudil, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, for its potential to inhibit oral squamous cell carcinoma (OSCC) growth and explores phospho-cofilin as a potential biomarker for prediction treatment efficiency of Fasudil in OSCC. Design A cohort of 109 OSCC patients provided tissue samples for phospho-cofilin expression analysis and survival analysis. The study examined the effect of Fasudil on OSCC cell lines HSC-3, UM1, and CAL33, assessing tumor growth inhibition through various in vitro and in vivo experiments. ROCK inhibition response and downstream mechanisms were explored by RNA sequencing, q-PCR, and immunofluorescence. Results High phospho-cofilin expression in OSCC tissues correlated with poor patient outcomes and was a reliable biomarker for ROCK activity. Fasudil inhibited growth in OSCC cell lines, particularly those with high phospho-cofilin expression. ROCK inhibition led to downregulation of Yes-associated protein (YAP) activity, resulting in suppressed tumor proliferation and increased apoptosis both in vitro and in vivo. Conclusions Inhibition of ROCK/phospho-cofilin/YAP by Fasudil could suppress OSCC proliferation, while phospho-cofilin served as a potential biomarker of OSCC.

Despite significant progress in characterizing the omics landscape of head and neck squamous cell carcinoma (HNSCC), the development of precision therapies remains limited. One key factor contributing to this challenge is the marked molecular heterogeneity of HNSCC. Further investigation of molecular profiles within HNSCC may facilitate the improvement in more effective precision treatments. Here, we focus on the dysregulation of PDZ and LIM domain protein 3 (PDLIM3) in HNSCC. The expression levels of PDLIM3 were analyzed using public datasets to assess its potential role in tumor progression. We found that PDLIM3 was downregulated in pan-cancer and HNSCC. The prognostic significance of PDLIM3 was evaluated through tissue microarray, and the downregulation of PDLIM3 was correlated with poor HNSCC prognosis. Investigating the implications of PDLIM3 for tumor metastatic ability in vitro, we found that PDLIM3 suppressed the migration and invasion of HNSCC, accompanied by partially impeding the process of epithelial-mesenchymal transition (EMT). Furthermore, PDLIM3 inhibited the transcriptional activity of Yes-associated protein (YAP), suggesting that YAP may be involved in the PDLIM3-mediated suppression of HNSCC metastatic ability. Our findings identify a potential signaling axis wherein PDLIM3 regulates YAP-EMT, thereby influencing tumor metastatic ability, and suggest the potential role of PDLIM3 as a tumor suppressor and prognostic biomarker for HNSCC.

A novel variegated tea cultivar exhibiting a stable variegated phenotype was recently identified, demonstrating significantly elevated amino acid content concomitantly with reduced polyphenolic compound levels compared to conventional green-leaf varieties. Nevertheless, the underlying mechanism remains unclear. Here, variegated leaves and normal leaves of 'Huangshanzhong’ tea plant were used to perform pigment content analysis and comparative transcriptome analysis. The chlorophyll content in variegated leaves significantly decreased compared to normal leaves, while the ratio of Chl a to Chl b was enhanced. Multiple genes (CsrpiA, CsGAPDH, CsPGAM, CsPK and CsOGDH) involved in sugar metabolism exhibited downregulated expression in variegated leaves. Key genes involved in the photosynthetic pathway were down-regulated in variegated leaves, including those encoding light-harvesting protein complex chlorophyll a/b binding proteins (CsLhca1, CsLhca4, CsLhcb1 and CsLhcb3) and photosystem II complex proteins (CspsbP and CspsbW). Meanwhile, genes involved in chlorophyll degradation metabolism (CsSGR and CsCLH1) were upregulated in variegated leaves. Compared to the wild type, transgenic plants overexpressing CsCLH1 and CsCLH2 exhibited no significant changes in chlorophyll content. Enzyme activity assays showed that CsCLH1 could degrade chlorophyll in vitro. Subcellular localization results revealed that CsCLH1 and CsCLH2 were localized in the cytoplasm and nucleus. These findings suggest that impaired photosynthetic system function, suppressed carbohydrate synthesis, and accelerated degradation of photosynthetic pigments collectively contribute to the variegated phenotype in tea leaves. This study advances our understanding of mechanisms underlying leaf variegation in plants.

The application of microbial fertilizers in tea cultivation has emerged a promising strategy to enhance tea quality and promote sustainable agricultural practices. However, the underlying mechanism remains unclear. In this study, the impacts of endophytic bacteria V4 on tea fresh leaves and dried tea were investigated. The results demonstrated that astringency in V4 sample was significantly reduced, while the umami and sweetness tastes, along with flowery and roasted aromas were significantly enhanced compared to the SW sample. Analysis of key quality-related compounds revealed a significant decrease in tea polyphenols and catechins, accompanied by a marked increase in free amino acids and soluble sugars. Evaluation of aroma characteristic impact (ACI) values of key volatile compounds revealed that β-ionone, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-3,6-dimethylpyrazine and 2,3-diethyl-5-methylpyrazine in V4 sample were significantly higher than those in SW sample. Transcriptomic analysis indicated downregulation of key catechin biosynthesis genes (such as CsLAR, CsANS and CsSCPL) in tea leaves treated with V4, while theanine biosynthetic genes such as CsAlaDC and CsTS were upregulated. This study provides a sustainable and innovative cultivation strategy for significantly improving tea flavor and enhancing product quality.