Elevated temperature affects the expression of signaling molecules in quail testes meiosis I prophase, but spermatogenesis remains normal.

Spermatogenesis in eukaryotes is a process that occurs within a very narrow temperature threshold, typically not exceeding 36 °C. SPO11 was isolated from the temperature-sensitive mutant receptor of Saccharomyces cerevisiae and is thought to be the only protein that functions during meiosis. This suggested that SPO11 may be the key protein that influenced the temperature of spermatogenesis not exceeding 36 °C. Elevated temperatures typically damage the spermatogenic cells. Birds have a core body temperature of 41-42 °C, and their testis are located inside their bodies, providing an alternative perspective to investigate the potential impact of temperature threshold on spermatogenesis. The objective of this study was to ascertain whether elevated ambient temperatures affect spermatogenesis in birds and whether SPO11 is the key gene affecting the temperature threshold for spermatogenesis. STRA8, SCP3, SPO11, γ-H2AX, and RAD51 were all crucial components in the process of meiotic initiation, synapsis, DNA double-strand break (DSB) induction, homologous chromosome crossover recombination, and repair of DSB. In this study, 39-day-old Japanese quail were subjected to heat stress (HS) at 38 °C for 8 h per day for 3 (3d HS) and 13 (13d HS) consecutive days and analyzed the expression of meiotic signaling molecules (STRA8, SCP3, SPO11, γ-H2AX, and RAD51) using molecular biology techniques, including Immunohistochemistry (IHC), Western Blot (WB), and Real-time Quantitative Polymerase Chain Reaction (qRT-PCR). We found that spermatogenesis was normal in both groups exposed to HS. Meiotic signaling molecules were expressed normally in the 3d HS group. All detected signaling molecules were normally expressed in the 13d HS group, except for SPO11, which showed a significant increase in expression, indicating that SPO11 was temperature-sensitive. We examined the localized expression of each meiotic signaling molecule in quail testis, explored the temperature sensitivity of SPO11, and determined that quail testis can undergo normal spermatogenesis at ambient temperatures exceeding 36 °C. This study concluded that SPO11 is not the key protein influencing spermatogenesis in birds. These findings enhance our understanding of avian spermatogenesis.

Potassium single-atoms anchoring on three-dimensional porous N-doped carbon material as sensing material for boosting electrochemical sensing of hydrogen peroxide.

For the first time potassium single-atoms (K SA) are explored as the sensing material to boost electrochemical sensing of hydrogen peroxide (H2O2). The N-doped carbon material with a three-dimensional porous structure (3D NG) was prepared using NaCl as the template, and K SA were anchored to the surface of 3D NG through high-temperature pyrolysis. The structure of K SA/3D NG was characterized by TEM, HAADF-STEM, XPS, and XRD. The results of electrochemical studies indicate that K SA play a crucial role in promoting the electrocatalytic reduction of H2O2, which not only optimized the adsorption strength for H2O2 but also improved the electron transfer rate, therefore improving the sensitivity for detecting H2O2. This study demonstrates the excellent electrocatalytic activity of K SA, which provides a promising sensing material for the detection of H2O2 and lays the foundation for the application of alkali metal single-atoms in the field of electrochemical sensing.

Screening and characterization of a novel linear B-cell epitope on orf virus F1L protein.

Background: Orf, also known as contagious ecthyma (CE), is an acute, contagious zoonotic disease caused by the orf virus (ORFV). The F1L protein is a major immunodominant protein on the surface of ORFV and can induce the production of neutralizing antibodies. Methods: The prokaryotic expression system was used to produce the recombinant F1L protein of ORFV, which was subsequently purified and used to immunize mice. Positive hybridoma clones were screened using an indirect enzyme-linked immunosorbent assay (ELISA). The reactivity and specificity of the monoclonal antibody (mAb) were verified through Western blot and indirect immunofluorescence (IFA). The linear antigenic epitope specific to the mAb was identified through Western blot, using truncated F1L proteins expressed in eukaryotic cells. A multiple sequence alignment of the ORFV reference strains was performed to evaluate the degree of conservation of the identified epitope. Results: After three rounds of subcloning, a mAb named Ba-F1L was produced. Ba-F1L was found to react with both the exogenously expressed F1L protein and the native F1L protein from ORFV-infected cells, as confirmed by Western blot and IFA. The mAb recognized the core epitope 103CKSTCPKEM111, which is highly conserved among various ORFV strains, as shown by homologous sequence alignment. Conclusion: The mAb produced in the present study can be used as a diagnostic reagent for detecting ORFV and as a basic tool for exploring the mechanisms of orf pathogenesis. In addition, the identified linear epitope may be valuable for the development of epitope-based vaccines.

Selecting effective eletrocatalyst from Cu single-atoms and nanoparticles for realizing highly sensitive electrochemical sensing of glucose and H2O2.

Which is more suitable as a sensing material between metal single-atoms and nanoparticles? Herein, electrocatalytic behaviors of copper single-atoms (Cu SAs) and copper nanoparticles (CuNPs) toward H2O2 reduction and glucose oxidation were studied. Surprisingly, the electrocatalytic activity of Cu SAs and CuNPs showed significant differences in H2O2 reduction and glucose oxidation. Compared with CuNPs, Cu SAs exhibit outstanding activity in the electrocatalytic reduction of H2O2 but exhibit weak activity in the electrocatalytic oxidation of glucose. On the contrary, CuNPs exhibit excellent activity in the electrochemical oxidation of glucose but have very weak electrocatalytic activity towards H2O2 reduction. DFT results show that H2O2 reduction is more favourable with Cu SAs; however, the electrochemical oxidation of glucose with CuNPs requires overcoming much lower energy barriers than that with Cu SAs. This study proves that both metal single-atoms and nanoparticles are not omnipotent, which provides ideas for constructing highly active sensing materials.

Molecular detection and phylogenetic analysis of Orf viruses from goats in Jiangxi province, China.

Orf is a zoonosis caused by the Orf virus (ORFV), which is endemic in goats, sheep, and wild ruminants worldwide. Orf infection is prevalent in China, with outbreaks reported in several provinces. Currently, there is limited information available regarding the characterization of ORFV strains in Jiangxi province. This study investigated an acute outbreak of Orf that occurred in 2021 in a goat herd in the Jiangxi province of China. Clinical signs in this case included lesions on the lips, nose, and inside the mouth. The presence of ORFV was confirmed from tissue samples by polymerase chain reaction (PCR). The nucleotide sequences of the B2L and F1L genes were fully sequenced and used to construct phylogenetic trees. The results of this investigation identified the ORFV JXxy2021 as the cause of the outbreak. The phylogenetic analysis revealed that the ORFV strain JXxy2021 had the highest similarity to the ORFV strains GO and FJ-SL from the neighboring province of Fujian. This suggests that JXxy2021 was likely transmitted from Fujian province. The results have provided valuable information on the genetic characteristics of JXxy2021 and the endemic situations of Orf in China.

Differential alterations of CXCR3, CXCR5 and CX3CR1 in patients with immune thrombocytopenia.

As a versatile element for maintaining homeostasis, the chemokine system has been reported to be implicated in the pathogenesis of immune thrombocytopenia (ITP). However, research pertaining to chemokine receptors and related ligands in adult ITP is still limited. The states of several typical chemokine receptors and cognate ligands in the circulation were comparatively assessed through various methodologies. Multiple variable analyses of correlation matrixes were conducted to characterize the correlation signatures of various chemokine receptors or candidate ligands with platelet counts. Our data illustrated a significant decrease in relative CXCR3 expression and elevated plasma levels of CXCL4, 9-11, 13, and CCL3 chemokines in ITP patients with varied platelet counts. Flow cytometry assays revealed eminently diminished CXCR3 levels on T and B lymphocytes and increased CXCR5 on cytotoxic T cell (Tc) subsets in ITP patients with certain platelet counts. Meanwhile, circulating CX3CR1 levels were markedly higher on T cells with a concomitant increase in plasma CX3CL1 level in ITP patients, highlighting the importance of aberrant alterations of the CX3CR1-CX3CL1 axis in ITP pathogenesis. Spearman's correlation analyses revealed a strong positive association of peripheral CXCL4 mRNA level, and negative correlations of plasma CXCL4 concentration and certain chemokine receptors with platelet counts, which might serve as a potential biomarker of platelet destruction in ITP development. Overall, these results indicate that the differential expression patterns and distinct activation states of peripheral chemokine network, and the subsequent expansion of circulating CXCR5+ Tc cells and CX3CR1+ T cells, may be a hallmark during ITP progression, which ultimately contributes to thrombocytopenia in ITP patients.

Tea green leafhopper infestations affect tea plant growth by altering the synthesis of brassinolide.

Tea green leafhoppers are insects widely distributed in major tea-growing areas. At present, less attention has been paid to the study on effect of tea green leafhopper infestation on tea growth phenotype. In this study, tea green leafhoppers were used to treat tea branches in laboratory and co-treated with brassinolide (BL), the highest bioactivity of brassinosteroids (BRs), in tea garden. The results showed that the expression of genes related to BRs synthesis was inhibited and BL content was reduced in tea shoots after infestation by tea green leafhoppers. In addition, area of each leaf position, length and diameter of internodes, and the biomass of the tender shoots of tea plant were decreased after infestation by tea green leafhoppers. The number of trichomes, leaf thickness, palisade tissue thickness and cuticle thickness of tea shoots were increased after tea green leafhoppers infestation. BL spraying could partially recover the phenotypic changes of tea branches caused by tea green leafhoppers infestation. Further studies showed that tea green leafhoppers infestation may regulate the expression of CsDWF4 (a key gene for BL synthesis) through transcription factors CsFP1 and CsTCP1a, which finally affect the BL content. Moreover, BL was applied to inhibit the tea green leafhoppers infestation on tea shoots. In conclusion, our study revealed the effect of plant hormone BL-mediated tea green leafhoppers infestation on the growth phenotype of tea plants.

Dual single atomic Ni sites constructing Janus hollow graphene for boosting electrochemical sensing of glucose.

Single atom catalysts (SACs) have attracted attention due to their excellent catalysis activity under specific reactions and conditions. However, the low density of SACs greatly limits catalytic performance. The three-dimensional graphene hollow nanospheres (GHSs) with very thin shell structure can be used as excellent carrier materials. Not only can its outer surface be used to anchor metal single atoms, but its inner surface can also provide rich sites. Here, a novel step-by-step assembly strategy is reported to anchor nickel single atoms (Ni SAs) on the inner and outer surfaces of GHSs (Ni SAs/GHSs/Ni SAs), which significantly increases the loading capacity of Ni SAs (4.8 wt%). Compared to conventional materials that only anchor Ni SAs to the outer surface of the carrier (Ni SAs/GHSs), Ni SAs/GHSs/Ni SAs exhibits significantly higher electrocatalytic activity toward glucose oxidation in alkaline media. The sensitivity of Ni SAs/GHSs/Ni SAs/GCE is nearly five times higher than that of Ni SAs/GHSs/GCE. Moreover, the sensor based on Ni SAs/GHSs/Ni SAs can detect glucose in a wide concentration range of 0.8 µM-1.1244 mM with a low detection limit of 0.19 µM (S/N = 3). This study not only provides an effective sensing material for glucose detection, but also opens a new avenue to construct high-density metal SACs.

The Circadian Clock Gene PHYTOCLOCK1 Mediates the Diurnal Emission of the Anti-Insect Volatile Benzyl Nitrile from Damaged Tea (Camellia sinensis) Plants.

Benzyl nitrile from tea plants attacked by various pests displays a diurnal pattern, which may be closely regulated by the endogenous circadian clock. However, the molecular mechanism by the circadian clock of tea plants that regulates the biosynthesis and release of volatiles remains unclear. In this study, the circadian clock gene CsPCL1 can activate both the expression of the benzyl nitrile biosynthesis-related gene CsCYP79 and the jasmonic acid signaling-related transcription factor CsMYC2 involved in upregulating CsCYP79 gene, thereby resulting in the accumulation and release of benzyl nitrile. Therefore, the anti-insect function of benzyl nitrile was explored in the laboratory. The application of slow-release beads of benzyl nitrile in tea plantations significantly reduced the number of tea geometrids and had positive effects on the yield of fresh tea leaves. These findings reveal the potential utility of herbivore-induced plant volatiles for the green control of pests in tea plantations.

Intrinsic tensile ductility in strain hardening multiprincipal element metallic glass.

Traditional metallic glasses (MGs), based on one or two principal elements, are notoriously known for their lack of tensile ductility at room temperature. Here, we developed a multiprincipal element MG (MPEMG), which exhibits a gigapascal yield strength, significant strain hardening that almost doubles its yield strength, and 2% uniform tensile ductility at room temperature. These remarkable properties stem from the heterogeneous amorphous structure of our MPEMG, which is composed of atoms with significant size mismatch but similar atomic fractions. In sharp contrast to traditional MGs, shear banding in our glass triggers local elemental segregation and subsequent ordering, which transforms shear softening to hardening, hence resulting in shear-band self-halting and extensive plastic flows. Our findings reveal a promising pathway to design stronger, more ductile glasses that can be applied in a wide range of technological fields.

Ultra-Efficient and Cost-Effective Platinum Nanomembrane Electrocatalyst for Sustainable Hydrogen Production.

Hydrogen production through hydrogen evolution reaction (HER) offers a promising solution to combat climate change by replacing fossil fuels with clean energy sources. However, the widespread adoption of efficient electrocatalysts, such as platinum (Pt), has been hindered by their high cost. In this study, we developed an easy-to-implement method to create ultrathin Pt nanomembranes, which catalyze HER at a cost significantly lower than commercial Pt/C and comparable to non-noble metal electrocatalysts. These Pt nanomembranes consist of highly distorted Pt nanocrystals and exhibit a heterogeneous elastic strain field, a characteristic rarely seen in conventional crystals. This unique feature results in significantly higher electrocatalytic efficiency than various forms of Pt electrocatalysts, including Pt/C, Pt foils, and numerous Pt single-atom or single-cluster catalysts. Our research offers a promising approach to develop highly efficient and cost-effective low-dimensional electrocatalysts for sustainable hydrogen production, potentially addressing the challenges posed by the climate crisis.

Biosynthetic Pathway and Bioactivity of Vanillin, a Highly Abundant Metabolite Distributed in the Root Cortex of Tea Plants (Camellia sinensis).

Volatiles are important for plant root stress resistance. The diseases in tea root are serious, causing major losses. The volatile composition in tea root and whether it can resist diseases remain unclear. In this study, the volatile composition in different tea tissues was revealed. The vanillin content was higher in the root (mainly in root cortex) than in aerial parts. The antifungal effects of vanillin on pathogenic fungi in tea root were equal to or greater than those of other metabolites. O-methyltransferase (CsOMT), a key enzyme in one of two biosynthetic pathways of vanillin, converted protocatechualdehyde to vanillin in vitro. Furthermore, its characteristics and kinetic parameters were studied. In Arabidopsis thaliana protoplasts, the transiently expressed CsOMT was localized in the cytoplasm and nucleus. These findings have clarified the formation and bioactivities of volatiles in tea roots and provided a theoretical basis for understanding how tea plants resist root diseases.

Effect of the biosynthesis of the volatile compound phenylacetaldehyde on chloroplast modifications in tea (Camellia sinensis) plants.

Plant volatile compounds have important physiological and ecological functions. Phenylacetaldehyde (PAld), a volatile phenylpropanoid/benzenoid, accumulates in the leaves of tea (Camellia sinensis) plants grown under continuous shading. This study was conducted to determine whether PAld production is correlated with light and to elucidate the physiological functions of PAld in tea plants. Specifically, the upstream mechanism modulating PAld biosynthesis in tea plants under different light conditions as well as the effects of PAld on chloroplast/chlorophyll were investigated. The biosynthesis of PAld was inhibited under light, whereas it was induced in darkness. The structural gene encoding aromatic amino acid aminotransferase 1 (CsAAAT1) was expressed at a high level in darkness, consistent with its importance for PAld accumulation. Additionally, the results of a transcriptional activation assay and an electrophoretic mobility shift assay indicated CsAAAT1 expression was slightly activated by phytochrome-interacting factor 3-2 (CsPIF3-2), which is a light-responsive transcription factor. Furthermore, PAld might promote the excitation of chlorophyll in dark-treated chloroplasts and mediate electron energy transfer in cells. However, the accumulated PAld can degrade chloroplasts and chlorophyll, with potentially detrimental effects on photosynthesis. Moreover, PAld biosynthesis is inhibited in tea leaves by red and blue light, thereby decreasing the adverse effects of PAld on chloroplasts during daytime. In conclusion, the regulated biosynthesis of PAld in tea plants under light and in darkness leads to chloroplast modifications. The results of this study have expanded our understanding of the biosynthesis and functions of volatile phenylpropanoids/benzenoids in tea leaves.

Ultrastrong yet Ductile 2D Titanium Nanomaterial for On-Skin Conformal Triboelectric Sensing.

Conventional titanium (e.g., bulk or thin films) is well-known for its relatively high mechanical strength, excellent corrosion resistance, and superior biocompatibility, which are suitable for biomedical engineering and wearable devices. However, the strength of conventional titanium often trades off its ductility, and their use in wearable devices has not been explored yet. In this work, we fabricated a series of large-sized 2D titanium nanomaterials with the method of polymer surface buckling enabled exfoliation (PSBEE), which possess a unique heterogeneous nanostructure containing nanosized titanium, titanium oxide, and MXene-like phases. As a result, these 2D titaniums exhibit both superb mechanical strength (6-13 GPa) and remarkable ductility (25-35%) at room temperature, outperforming all other titanium-based materials reported so far. More interestingly, we demonstrate that the 2D titanium nanomaterials also showed good performance in triboelectric sensing and can be used to fabricate self-powered, on-skin conformal triboelectric sensors with good mechanical reliability.

Contrasting terpene emissions from canopy and understory vegetation in response to increases in nitrogen deposition and seasonal changes in precipitation.

Given global change and shifts in climate are expected to increase BVOC emissions, the quantification of links between environmental conditions, plant physiology, and terpene emission dynamics is required to improve model predictions of ecosystem responses to increasing nitrogen deposition and changes in precipitation regimes. Here, we conducted a two-factor field experiment in sub-tropical forest plots to determine effects of N addition (N), precipitation change (PC), and NP (N and PC combined treatment) on wet and dry season terpene emissions and leaf photosynthetic parameters from canopy and understory species. Changes of ß-ocimene and sabinene under PC and NP in the wet season (0.4-5.6-fold change) were the largest contributor to changes in total terpene emissions. In the dry season, the standardized total terpene emission rate was enhanced by 144.9% under N addition and 185.7% under PC for the understory species, while the total terpene emission rate was lower under NP than N addition and PC, indicating that N addition tended to moderate increases in PC-induced understory total terpene emissions. In the wet season, the total terpene emission rate under N and PC was close to ambient conditions for the canopy species, while the total terpene emission rate was enhanced by 54.6% under NP, indicating that N and PC combined treatment had an additive effect on canopy total terpene emissions. Total terpene emission rates increased with rates of net leaf photosynthesis (Pn) and transpiration (Tr) and there was a decoupling between terpene emission rates and Pn under NP, indicating that complex effects between PC and N decreased the regularity of single-factor effects. We recommend that N and PC interaction effects are included in models for the prediction of terpene emissions, particularly from canopy vegetation during the wet season as a major source of forest ecosystem terpene emissions.

Involvement of histone deacetylase CsHDA2 in regulating (E)-nerolidol formation in tea (Camellia sinensis) exposed to tea green leafhopper infestation.

Herbivore-induced plant volatiles (HIPVs) help the tea plant (Camellia sinensis) adapt to environmental stress, and they are also quality-related components of tea. However, the upstream mechanism regulating the herbivore-induced expression of volatile biosynthesis genes is unclear, especially at the level of epigenetic regulation. In this study, similar to the effects of a tea green leafhopper infestation, treatments with exogenous jasmonic acid (JA) and histone deacetylase inhibitors significantly increased the (E)-nerolidol content in tea and induced the expression of the associated biosynthesis gene CsNES. Furthermore, a key transcription factor related to JA signaling, myelocytomatosis 2 (CsMYC2), interacted with histone deacetylase 2 (CsHDA2) in vitro and in vivo. A tea green leafhopper infestation inhibited CsHDA2 expression and decreased CsHDA2 abundance. Moreover, the tea green leafhopper infestation increased H3 and H4 acetylation levels in the promoter region of CsNES, which in turn upregulated the expression of CsNES and increased the (E)-nerolidol content. In this study, we revealed the effects of histone acetylations on the accumulation of HIPVs, while also confirming that CsHDA2-CsMYC2 is an important transcriptional regulatory module for the accumulation of (E)-nerolidol induced by tea green leafhoppers. The results of this study may be useful for characterizing plant aromatic compounds and the main upstream stress-responsive signaling molecules. Furthermore, the study findings will assist researchers clarify the epigenetic regulation influencing plant secondary metabolism in response to external stress.

Natural allelic variation confers high resistance to sweet potato weevils in sweet potato.

Sweet potato (Ipomoea batatas L.) is a major root crop worldwide. Sweet potato weevils (SPWs) pose one of the most significant challenges to sweet potato production in tropical and subtropical regions, causing deleterious economic and environmental effects. Characterizing the mechanisms underlying natural resistance to SPWs is therefore crucial; however, the genetic basis of host SPW resistance (SPWR) remains unclear. Here we obtained two sweet potato germplasm with high SPWR and, by map-based cloning, revealed two major SPW-resistant genes-SPWR1 and SPWR2-that are important regulators of natural defence against SPWs. The SPW-induced WRKY transcriptional factor SPWR1 directly activates the expression of SPWR2, and SPWR2, the conserved dehydroquinate synthase, promotes the accumulation of quinate derivative metabolites that confer SPWR in sweet potato. Generally, our results provide new insights into the molecular mechanism underlying sweet potato-SPW interactions and will aid future efforts to achieve eco-friendly SPW management.

Heterologous Expression and Characterization of Tea (Camellia sinensis) Polyamine Oxidase Homologs and Their Involvement in Stresses.

Polyamine oxidase (PAO) is a key enzyme maintaining polyamine homeostasis, which affects plant physiological activities. Until now, the gene members and function of PAOs in tea (Camellia sinenesis) have not been fully identified. Here, through the expression in Escherichia coli and Nicotiana benthamiana, we identified six genes annotated as CsPAO in tea genome and transcriptome and determined their enzyme reaction modes and gene expression profiles in tea cultivar 'Yinghong 9'. We found that CsPAO1,2,3 could catalyze spermine, thermospermine, and norspermidine, and CsPAO2,3 could catalyze spermidine in the back-conversion mode, which indicated that the precursor of γ-aminobutyric acid might originate from the oxidation of putrescin but not spermidine. We further investigated the changes of CsPAO activity with temperature and pH and their stability. Kinetic parameters suggested that CsPAO2 was the major PAO modifying polyamine composition in tea, and it could be inactivated by ß-hydroxyethylhydrazine and aminoguanidine. Putrescine content and CsPAO2 expression were high in tea flowers. CsPAO2 responded to wound, drought, and salt stress; CsPAO1 might be the main member responding to cold stress; anoxia induced CsPAO3. We conclude that in terms of phylogenetic tree, enzyme characteristics, and expression profile, CsPAO2 might be the dominant CsPAO in the polyamine degradation pathway.

Light synergistically promotes the tea green leafhopper infestation-induced accumulation of linalool oxides and their glucosides in tea (Camellia sinensis).

Linalool, which is one of the most representative aroma substances in tea, is transformed into other aroma-related compounds, including linalool 3,6-oxides and linalool 3,7-oxides. The objective of this study was to elucidate the linalool oxide synthesis pathway and its response to stress in tea. By feeding experiment, chemical synthesis, and compound analysis, it was found that linalool can be transformed to linalool oxides via 6,7-epoxylinalool. The conversion rate from 6,7-epoxylinalool to linalool oxides was relatively high under acidic conditions. Four linalool oxide glucosides obtained from tea were structurally characterized. Additionally, tea green leafhopper infestation was observed to activate the whole metabolic flow from linalool into linalool oxides and their glucosides (p < 0.01). Moreover, light treatments further increased the accumulation of linalool oxides and their glucosides (p < 0.05). These results will be useful for elucidating the mechanism mediating linalool oxides content changes in response to stress in tea.

Mechanism underlying the carotenoid accumulation in shaded tea leaves.

Carotenoids contribute to tea leaf coloration and are the precursors of important aromatic compounds. Shading can promote the accumulation of carotenoids in tea leaves, but the underlying mechanism remains unknown. In the study, we analyzed the content and composition of carotenoids, and transcript levels and functions of related genes in carotenoid biosynthesis using HPLC, qRT-PCR, and heterologous expression system. It was found that long-term shading (14 days, 90% shading) significantly increased the total carotenoid content in tea leaves, and increased the expression of non-mevalonate pathway (MEP) genes (CsDXS1 and CsDXS3) and key genes in carotenoid synthesis pathway (CsPSY, CsLCYB, and CsLCYE). Long-term exposure to darkness (14 days, 0 lx) decreased the transcription of most carotenoid biosynthetic genes and adversely affected carotenoid accumulation. Furthermore, CsDXS1, CsDXS3, CsPSY, CsLCYB, and CsLCYE were functionally identified and contributed to the enhanced accumulation of carotenoids in tea leaves in response to long-term shading.