2,4-Dihydroxybenzoic Acid, a Novel SA Derivative, Controls Plant Immunity via UGT95B17-Mediated Glucosylation: A Case Study in Camellia Sinensis.

The plant hormone salicylic acid (SA) plays critical roles in plant innate immunity. Several SA derivatives and associated modification are identified, whereas the range and modes of action of SA-related metabolites remain elusive. Here, the study discovered 2,4-dihydroxybenzoic acid (2,4-DHBA) and its glycosylated form as native SA derivatives in plants whose accumulation is largely induced by SA application and Ps. camelliae-sinensis (Pcs) infection. CsSH1, a 4/5-hydroxylase, catalyzes the hydroxylation of SA to 2,4-DHBA, and UDP-glucosyltransferase UGT95B17 catalyzes the formation of 2,4-DHBA glucoside. Down-regulation reduced the accumulation of 2,4-DHBA glucosides and enhanced the sensitivity of tea plants to Pcs. Conversely, overexpression of UGT95B17 increased plant disease resistance. The exogenous application of 2,4-DHBA and 2,5-DHBA, as well as the accumulation of DHBA and plant resistance comparison, indicate that 2,4-DHBA functions as a potentially bioactive molecule and is stored mainly as a glucose conjugate in tea plants, differs from the mechanism described in Arabidopsis. When 2,4-DHBA is applied exogenously, UGT95B17-silenced tea plants accumulated more 2,4-DHBA than SA and showed induced resistance to Pcs infection. These results indicate that 2,4-DHBA glucosylation positively regulates disease resistance and highlight the role of 2,4-DHBA as potentially bioactive molecule in the establishment of basal resistance in tea plants.

Distribution of and Temporal Variation in Volatiles in Tea (Camellia sinensis) Flowers during the Opening Stages.

Tea (Camellia sinensis) flowers emit a large amount of volatiles that attract pollinators. However, few studies have characterized temporal and spatial variation in tea floral volatiles. To investigate the distribution of volatiles within tea flowers and their variation among opening stages, volatile components from different parts of tea flowers and different opening stages were collected by headspace solid-phase microextraction and analyzed by gas chromatography-mass spectrometry. A total of 51 volatile compounds of eight chemical classes were identified in the tea flowers. Volatile compounds were most abundant in tea flowers of the Shuchazao cultivar. Acetophenone, 1-phenylethanol, 2-phenylethanol, and benzyl alcohol were the most abundant volatiles. Terpenes were common in the sepals, and benzoids were common in the stamens. The fatty acid derivatives were mainly distributed in the pistils and receptacles and were less abundant in the petals, sepals, and stamens. During the opening phase of tea flowers, the volatile content increased 12-fold, which mainly stemmed from the increase in benzoids. These results enhance our understanding of the formation of volatiles in tea flowers.

Volatile compound-mediated plant-plant interactions under stress with the tea plant as a model.

Plants respond to environmental stimuli via the release of volatile organic compounds (VOCs), and neighboring plants constantly monitor and respond to these VOCs with great sensitivity and discrimination. This sensing can trigger increased plant fitness and reduce future plant damage through the priming of their own defenses. The defense mechanism in neighboring plants can either be induced by activation of the regulatory or transcriptional machinery, or it can be delayed by the absorption and storage of VOCs for the generation of an appropriate response later. Despite much research, many key questions remain on the role of VOCs in interplant communication and plant fitness. Here we review recent research on the VOCs induced by biotic (i.e. insects and pathogens) and abiotic (i.e. cold, drought, and salt) stresses, and elucidate the biosynthesis of stress-induced VOCs in tea plants. Our focus is on the role of stress-induced VOCs in complex ecological environments. Particularly, the roles of VOCs under abiotic stress are highlighted. Finally, we discuss pertinent questions and future research directions for advancing our understanding of plant interactions via VOCs.

(Z)-3-Hexenol integrates drought and cold stress signaling by activating abscisic acid glucosylation in tea plants.

Cold and drought stresses severely limit crop production and can occur simultaneously. Although some transcription factors and hormones have been characterized in plants subjected each stress, the role of metabolites, especially volatiles, in response to cold and drought stress exposure is rarely studied due to lack of suitable models. Here, we established a model for studying the role of volatiles in tea (Camellia sinensis) plants experiencing cold and drought stresses simultaneously. Using this model, we showed that volatiles induced by cold stress promote drought tolerance in tea plants by mediating reactive oxygen species and stomatal conductance. Needle trap microextraction combined with GC-MS identified the volatiles involved in the crosstalk and showed that cold-induced (Z)-3-hexenol improved the drought tolerance of tea plants. In addition, silencing C. sinensis alcohol dehydrogenase 2 (CsADH2) led to reduced (Z)-3-hexenol production and significantly reduced drought tolerance in response to simultaneous cold and drought stress. Transcriptome and metabolite analyses, together with plant hormone comparison and abscisic acid (ABA) biosynthesis pathway inhibition experiments, further confirmed the roles of ABA in (Z)-3-hexenol-induced drought tolerance of tea plants. (Z)-3-Hexenol application and gene silencing results supported the hypothesis that (Z)-3-hexenol plays a role in the integration of cold and drought tolerance by stimulating the dual-function glucosyltransferase UGT85A53, thereby altering ABA homeostasis in tea plants. Overall, we present a model for studying the roles of metabolites in plants under multiple stresses and reveal the roles of volatiles in integrating cold and drought stresses in plants.

Interleukin-17 Promotes the Infiltration of CD8+ T Cells into the Brain in a Mouse Model for Alzheimer's Disease.

BACKGROUND: Interleukin-17 (IL-17) family cytokines play critical roles in inflammation and pathogen resistance. Inflammation in the central nervous system, denoted as neuroinflammation, promotes the onset and progression of Alzheimer's disease (AD). Previous studies showed that IL-17A neutralizing antibody treatment alleviated Amyloid ß (Aß) burden in rodent models of AD, while overexpression of IL-17A in mouse lateral ventricles rescued part of the AD pathology. However, the involvement of IL-17 in AD and its mechanism of action remain largely unknown. METHODS: To investigate the role of IL-17 in AD, we crossed mice lacking the common receptor of IL-17 signaling (IL-17RA knockout mice) to the APP/PS1 mouse model of AD. We then analyzed the composition of immune cells and cytokines/chemokines during different phases of AD pathology, and interrogated the underlying mechanism by which IL-17 may regulate immune cell infiltration into AD brains. RESULTS: Ablation of IL-17RA in APP/PS1 mice decreased infiltration of CD8+ T cells and myeloid cells to mouse brain. IL-17 was able to promote the production of myeloid- and T cell-attracting chemokines CXCL1 and CXCL9/10 in primary glial cells. We also observed that IL-17 is upregulated in the late stage of AD development, and ectopic expression of IL-17 via adenoviral infection to the cortex trended towards worsened cognition in APP/PS1 mice, suggesting a pathogenic role of excessive IL-17 in AD. CONCLUSION: Our data show that IL-17 signaling promotes neuroinflammation in AD by accelerating the infiltration of CD8+ T lymphocytes and Gr1+ CD11b+ myeloid cells.

The effect of tuina on ulcerative colitis model mice analyzed by gut microbiota and proteomics.

Tuina can effectively alleviate ulcerative colitis-related symptoms, but the mechanism of action is unknown. The purpose of this research is to explore potential pathways for the treatment of tuina through gut microbiota and proteomics techniques. Thirty-two male BALB/c mice were divided into four groups, the control, model, mesalazine, and tuina groups. The ulcerative colitis model was established by freely drinking a 3% dextran sulphate sodium solution for 7 days. The mesalazine group and the tuina group, respectively, received 7 days of mesalazine and tuina treatment. Subsequently, their body weights, feces properties, colon length, histomorphological changes, gut microbiota, and colon proteomics were determined. Body weights, disease activity index score, colon histological scores, and microbiota diversity were restored in the tuina group. At the phylum level, Firmicutes was increased and Bacteroidota decreased. At the family level, Lachnospiraceae increased and Prevotellaceae decreased. At the genus level, the Lachnospiraceae_NK4A136_group was increased. Proteomics detected 370 differentially expressed proteins regulated by tuina, enriched to a total of 304 pathways, including biotin metabolism, Notch signaling pathway, linoleic acid metabolism, and autophagy. Tuina can effectively improve the symptoms of weight loss, fecal properties, and colon inflammation in ulcerative colitis mice and restore the gut microbiota diversity, adjusting the relative abundance of microbiota. The therapeutic effects of tuina may be achieved by modulating the signaling pathways of biotin metabolism, Notch signaling pathway, linoleic acid metabolism, and autophagy.

Predicting Dynamic Riverine Nitrogen Export in Unmonitored Watersheds: Leveraging Insights of AI from Data-Rich Regions.

Terrestrial export of nitrogen is a critical Earth system process, but its global dynamics remain difficult to predict at a high spatiotemporal resolution. Here, we use deep learning (DL) to model daily riverine nitrogen export in response to hydrometeorological and anthropogenic drivers. Long short-term memory (LSTM) models for the daily concentration and flux of dissolved inorganic nitrogen (DIN) were built in a coastal watershed in southeastern China with a typical subtropical monsoon climate. The DL models exhibited excellent accuracy for both DIN concentration and flux, with Nash-Sutcliffe efficiency coefficients (NSEs) up to 0.67 and 0.92, respectively, a performance unlikely to be achieved by generic process-based models with comparable data quality. The flux model ensemble, without retraining, performed well (mean NSE = 0.32-0.84) in seven distinct watersheds in Asia, Europe, and North America, and retraining with multi-watershed data further improved the lowest NSE from 0.32 to 0.68. DL interpretation confirmed that interbasin consistency of riverine nitrogen export exists across different continents, which stems from the similarities in rainfall-runoff relationships. The multi-watershed flux model projects 0.60-12.4% increases in the nitrogen export to oceans from the studied watersheds under a 20% increase in fertilizer consumption, which rises to 6.7-20.1% with a 10% increase in runoff, indicating the synergistic effect of human activities and climate change. The DL-based method represents a successful case of explainable artificial intelligence in environmental science, providing a potential shortcut to a consistent understanding of the global daily-resolution dynamics of riverine nitrogen export under the currently limited data conditions.

A Review on the Immunomodulatory Mechanism of Acupuncture in the Treatment of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease with a high prevalence and canceration rate. The immune disorder is one of the recognized mechanisms. Acupuncture is widely used to treat patients with IBD. In recent years, an increasing number of studies have proven the effectiveness of acupuncture in the treatment of IBD, and some progress has been made in the mechanism. In this paper, we reviewed the studies related to acupuncture for IBD and focused on the immunomodulatory mechanism. We found that acupuncture could regulate the innate and adaptive immunity of IBD patients in many ways. Acupuncture exerts innate immunomodulatory effects by regulating intestinal epithelial barrier, toll-like receptors, NLRP3 inflammasomes, oxidative stress, and endoplasmic reticulum stress and exerts adaptive immunomodulation by regulating the balance of Th17/Treg and Th1/Th2 cells. In addition, acupuncture can also regulate intestinal flora.

Salicylic acid carboxyl glucosyltransferase UGT87E7 regulates disease resistance in Camellia sinensis.

Plant immune response following pathogenic infection is regulated by plant hormones, and salicylic acid (SA) and its sugar conjugates play important roles in establishing basal resistance. Here, the important pathogen Pseudopestalotiopsis camelliae-sinensis (Pcs) was isolated from tea gray blight, one of the most destructive diseases in tea plantations. Transcriptomic analysis led to the discovery of the putative Camellia sinensis UDP-glucosyltransferase CsUGT87E7 whose expression was significantly induced by SA application and Pcs infection. Recombinant CsUGT87E7 glucosylates SA with a Km value of 12 µM to form SA glucose ester (SGE). Downregulation reduced the accumulation of SGE, and CsUGT87E7-silenced tea plants exhibited greater susceptibility to pathogen infection than control plants. Similarly, CsUGT87E7-silenced tea leaves accumulated significantly less SA after infection and showed reduced expression of pathogenesis-related genes. These results suggest that CsUGT87E7 is an SA carboxyl glucosyltransferase that plays a positive role in plant disease resistance by modulating SA homeostasis through a mechanism distinct from that described in Arabidopsis (Arabidopsis thaliana). This study provides insight into the mechanisms of SA metabolism and highlights the role of SGE in the modulation of plant disease resistance.

Eugenol functions as a signal mediating cold and drought tolerance via UGT71A59-mediated glucosylation in tea plants.

Cold and drought stress are the most critical stresses encountered by crops and occur simultaneously under field conditions. However, it is unclear whether volatiles contribute to both cold and drought tolerance, and if so, by what mechanisms they act. Here, we show that airborne eugenol can be taken up by the tea (Camellia sinensis) plant and metabolized into glycosides, thus enhancing cold and drought tolerance of tea plants. A uridine diphosphate (UDP)-glucosyltransferase, UGT71A59, was discovered, whose expression is strongly induced by multiple abiotic stresses. UGT71A59 specifically catalyzes glucosylation of eugenol glucoside in vitro and in vivo. Suppression of UGT71A59 expression in tea reduced the accumulation of eugenol glucoside, lowered reactive oxygen species (ROS) scavenging capacity, and ultimately impaired cold and drought stress tolerance. Exposure to airborne eugenol triggered a marked increase in UGT71A59 expression, eugenol glucoside accumulation, and cold tolerance by modulating ROS accumulation and CBF1 expression. It also promoted drought tolerance by altering abscisic acid homeostasis and stomatal closure. CBF1 and CBF3 play positive roles in eugenol-induced cold tolerance and CBF2 may be a negative regulator of eugenol-induced cold tolerance in tea plants. These results provide evidence that eugenol functions as a signal in cold and drought tolerance regulation and shed new light on the biological functions of volatiles in the response to multiple abiotic stresses in plants.

Safety of Five Tuina Manipulations in Rats with Deep Vein Thrombosis.

OBJECTIVE: To study the effects of five tuina manipulations in rats with deep vein thrombosis (DVT) and to explore how to safely perform tuina in the treatment of thrombotic diseases. METHODS: Seventy-two male Sprague-Dawley (SD) rats were randomly divided into the model, pointing manipulation, plucking manipulation, kneading manipulation, pushing manipulation, and pulling manipulation groups (n = 12). DVT model was established by incomplete ligation. The tuina intervention was started on the next day after modeling and applied once a day 10 times by the manipulation simulators. On the 3rd and 10th days after intervention, respectively, the effects of tuina on thrombosis were evaluated based on thrombus elasticity, blood coagulation, fibrinolytic function and blood rheology with the ultrasound elastography, four coagulation tests, enzyme linked immunosorbent assay (ELISA), and hemorheology tests. RESULTS: In the pointing manipulation group, the strain rate ratio, 6-ketoprostaglandin F1α (6-Keto-PGF1α), and high shear rate were decreased, and the thromboxane B2 (TXB2) content was increased (P < 0.05). In the plucking manipulation group, the D-dimer and 6-Keto-PGF1α contents were increased, prothrombin time (PT) was shortened, and activated partial thromboplastin time (APTT) was activated, and the high shear rate and plasma viscosity were decreased (P < 0.05). In the kneading manipulation group, APTT was shortened, and 6-Keto-PGF1α, high shear rate, and plasma viscosity were decreased (P < 0.05). In the pushing manipulation group, the strain rate ratio, low shear rate, and high shear rate were all decreased (P < 0.05). In the pulling manipulation group, both the strain rate ratio and the low shear rate were decreased (P < 0.05). The 6-Keto-PGF1α changes on the 3rd and 10th days after intervention were opposite in the pushing manipulation group and the pulling manipulation group (P < 0.05). CONCLUSION: The pointing, pushing, and pulling manipulations seem to be safe in the early period of thrombosis, but the risk is likely to be elevated as the treatment course of intervention increases. The plucking and kneading manipulations potentially have certain risks in the treatment of DVT in rats.

A Review on the Mechanism of Tuina Promoting the Recovery of Peripheral Nerve Injury.

Tuina, as one of the characteristic external therapies of Traditional Chinese Medicine (TCM), has been used to treat the disease caused by peripheral nerve injury (PNI) for thousands of years. An increasing number of clinical trials and animal experiments have demonstrated that tuina can improve the symptoms and promote the recovery of damaged nerves. This review focuses on the mechanistic studies of tuina in promoting the recovery of PNI, which might provide a neurobiological foundation for the effects of tuina. Although many mechanisms underlying the effects of tuina on nerve repair have been identified, there are still many unknown problems, such as the key substance or way for tuina to work, so further investigation is warranted.

An Investigation into the Rehabilitative Mechanism of Tuina in the Treatment of Sciatic Nerve Injury.

OBJECTIVE: To explore the effect of tuina on the gene expression at the point of nerve injury in rats with sciatic nerve injury (SNI) and to elucidate the repair mechanism of tuina promoting the functional recovery of peripheral nerve injury. METHODS: In the Sham group, the right sciatic nerve was exposed without clamping. The SNI model was established using the sciatic nerve clamp method on the right leg and then randomly divided into the SNI group and the Tuina group. Seven days after modeling, the Tuina group was treated daily with a "massage and tuina manipulation simulator" (Patent No. ZL 2007 0187403.1), which was used daily to stimulate Yinmen (BL37), Yanglingquan (GB34), and Chengshan (BL57) with point-pressing method, plucking method, and kneading method. The stimulating force was 4N, and the stimulating frequency was 60 times per minute; each method and each point were used for 1 minute, totaling 9 minutes (1 min/acupoint/method × 3 methods × 3 acupoints). Treatment was administered for 21 days, followed by a 1-day rest after the 10th treatment, for a total of 20 times of intervention. The sciatic function index (SFI) was used to evaluate the fine movements of the hind limbs of rats in each group. The ultrastructural changes at the point of nerve injury were observed by transmission electron microscopy, and the gene changes at the point of nerve injury were detected using RNA-sequencing (RNA-seq) technology. RESULTS: Compared with the baseline, the SFI of the SNI group and the Tuina group decreased significantly at the 0th intervention (7 days after molding); compared with the SNI group, the SFI of the Tuina group increased at the 10th intervention (P < 0.05) and increased significantly at the 15th and 20th intervention (P < 0.01). Compared with the Sham group, the myelin sheath integrity of the sciatic nerve in the SNI group was destroyed and the myelin sheath collapsed seriously, even forming myelin sheath ball, accompanied with severe axonal atrophy and mitochondrial degeneration. The tuina intervention could significantly improve the ultrastructure of the nerve injury point, and the nerve fiber myelin sheath in the Tuina group remained intact, without obvious axonal swelling or atrophy. Atrophic thread granules could be seen in the axon, but there were no vacuolated mitochondria. RNA-seq results showed that there were differences at 221 genes at the point of nerve injury between the Tuina group and the SNI group and the differentially expressed genes (DEGs) are enriched in the biological processes related to the regulation of myocyte. Regulations include the regulation of striated muscle cell differentiation, myoblast differentiation, and myotube differentiation. CONCLUSION: Tuina can improve the fine motor recovery and protect the myelin integrity in rats with peripheral nerve injury, and this is achieved by changing the gene sequence at the injured point.

Using RNA-Seq to Explore the Repair Mechanism of the Three Methods and Three-Acupoint Technique on DRGs in Sciatic Nerve Injured Rats.

Objective: To study the effects of the three methods and three-acupoint technique on DRG gene expression in SNI model rats and to elucidate the molecular mechanism of the three methods and three-acupoint technique on promoting recovery in peripheral nerve injury. Methods: 27 male SD rats were randomly divided into three groups: a Sham group, the SNI group, and the Tuina group. The Tuina group was treated with a tuina manipulation simulator to simulate massage on points, controlling for both quality and quantity. Point-pressing, plucking, and kneading methods were administered quantitatively at Yinmen (BL37), Chengshan (BL57), and Yanglingquan (GB34) points on the affected side once a day, beginning 7 days after modeling. Intervention was applied once a day for 10 days, then 1 day of rest, followed by 10 more days of intervention, totally equaling 20 times of intervention. The effect of the three methods and three-point technique on the recovery of injured rats was evaluated using behavior analysis. RNA sequencing (RNA-Seq) analysis of differentially expressed genes in DRGs of the three groups of rats was also performed. GO and KEGG enrichment was analyzed and verified using real-time PCR. Results: RNA-Seq combined with database information showed that the number of differentially expressed genes in DRG was the largest in the Tuina group compared with the SNI group, totaling 226. GO function is enriched in the positive regulation of cell processes, ion binding, protein binding, neuron, response to pressure, response to metal ions, neuron projection, and other biological processes. GO function is also enriched in the Wnt, IL-17, and MAPK signaling pathways in the KEGG database. PCR results were consistent with those of RNA sequencing, suggesting that the results of transcriptome sequencing were reliable. Conclusion: The three methods and three-acupoint technique can promote the recovery of SNI model rats by altering the gene sequence in DRGs.

Continuous flow, large-scale, microbial fuel cell system for the sustained treatment of swine waste.

Microbial fuel cells (MFCs) have long held the promise of being a cost-effective technology for the energy-neutral treatment of wastewater. However, successful pilot-scale demonstrations for this technology are still limited to very few. Here, we present a large-scale MFC system, composed of 12 MFCs with a total volume of 110 L, successfully treating swine wastewater at a small educational farm. The system was operated for over 200 days in continuous mode with hydraulic residence time of 4 hr. Very stable electrochemical and waste treatment performance was observed with up to 65% of chemical oxygen demand (COD) removed and a maximum treatment rate of 5.0 kg COD/m3 .day. Robust microbial enrichment was performed and adapted to metabolize and transform a diversity of compounds present. The Net Energy Recovery (NER = 0.11 kWhr/kg COD) is not only competitive with conventional cogeneration processes, but is in fact sufficient to sustain the operational energy requirements of the system. PRACTITIONER POINTS: This study demonstrates the design and operation of a large-scale microbial fuel cells (MFC) system for continuous treatment of swine wastewater. The system achieved a high chemical oxygen demand removal rate within a short hydraulic residence time. This study moves one-step closer to applying MFC technology for real wastewater treatment.

IL-17 inhibits CXCL9/10-mediated recruitment of CD8+ cytotoxic T cells and regulatory T cells to colorectal tumors.

BACKGROUND: The IL-17 family cytokines are potent drivers of colorectal cancer (CRC) development. We and others have shown that IL-17 mainly signals to tumor cells to promote CRC, but the underlying mechanism remains unclear. IL-17 also dampens Th1-armed anti-tumor immunity, in part by attracting myeloid cells to tumor. Whether IL-17 controls the activity of adaptive immune cells in a more direct manner, however, is unknown. METHODS: Using mouse models of sporadic or inducible colorectal cancers, we ablated IL-17RA in the whole body or specifically in colorectal tumor cells. We also performed adoptive bone marrow reconstitution to knockout CXCR3 in hematopoietic cells. Histological and immunological experimental methods were used to reveal the link among IL-17, chemokine production, and CRC development. RESULTS: Loss of IL-17 signaling in mouse CRC resulted in marked increase in the recruitment of CD8+ cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs), starting from early stage CRC lesions. This is accompanied by the increased expression of anti-inflammatory cytokines IL-10 and TGF-ß. IL-17 signaling also inhibits the production of T cell attracting chemokines CXCL9 and CXCL10 by tumor cells. Conversely, the inability of hematopoietic cells to respond to CXCL9/10 resulted in decreased tumor infiltration by CTLs and Tregs, decreased levels of IL-10 and TGF-ß, and increased numbers of tumor lesions. Blockade of IL-17 signaling resulted in increased expression of immune checkpoint markers. On the other hand, treatment of mouse CRC with anti-CTLA-4 antibody led to increased expression of pro-tumor IL-17. CONCLUSION: IL-17 signals to colorectal tumor cells and inhibits their production of CXCL9/10 chemokines. By doing so, IL-17 inhibits the infiltration of CD8+ CTLs and Tregs to CRC, thus promoting CRC development. Cancer immunotherapy may be benefited by the use of anti-IL-17 agents as adjuvant therapies, which serve to block both IL-17-mediated tumor promotion and T cell exclusion.

Shower effect of a rainfall onset on the heat accumulated during a preceding dry spell.

Popular perception claims that rain following a hot day brings relief, indicating a bio-meteorological perspective of 'rainy' forecasts. However, the hypothesis has rarely been examined on India which experiences distinct pre- and post-monsoon seasons with continuous dry days, occasionally interrupted by thunderstorms or cyclones. The current study analyzes 54 years of observed daily meteorological records across India to assess the impact of shower effect, defined as the amount of change in the temperature on the first day of a wet spell that succeeds a dry spell. Nine combinations of low to high probability rainfall events on the first day of a wet spell and short to prolonged dry spell categories are evaluated. Results indicate that the north, the northeastern, and the eastern states of India witness a decrease in the maximum and minimum temperatures, up to 5 °C during the pre-monsoon season while mostly exhibiting a statistically insignificant long-term temporal trend. During the post-monsoon season, a rainfall event decreases the maximum temperature, providing significant relief by reducing the heat index (HI) warning from 'Caution' to 'Normal', but is unable to lower the HI warning from 'danger' during the pre-monsoon season.

Caffeine Content and Related Gene Expression: Novel Insight into Caffeine Metabolism in Camellia Plants Containing Low, Normal, and High Caffeine Concentrations.

Caffeine is a crucial secondary metabolic product in tea plants. Although the presence of caffeine in tea plants has been identified, the molecular mechanisms regulating relevant caffeine metabolism remain unclear. For the elucidation of the caffeine biosynthesis and catabolism in Camellia plants, fresh, germinated leaves from four Camellia plants with low (2), normal (1), and high (1) caffeine concentrations, namely, low-caffeine tea 1 (LCT1, Camellia crassicolumna), low-caffeine tea 2 (LCT2, C. crassicolumna), Shuchazao (SCZ, C. sinensis), and Yunkang 43 (YK43, C. sinensis) were used in this research. Transcriptome and purine alkaloids analyses of these Camellia leaves were performed using RNA-Seq and liquid chromatography-mass spectrometry (LC-MS). Moreover, 15N-caffeine tracing was performed to determine the metabolic fate of caffeine in leaves of these plants. Caffeine content was correlated with related gene expression levels, and a quantitative real-time (qRT) PCR analysis of specific genes showed a consistent tendency with the obtained transcriptomic analysis. On the basis of the results of stable isotope-labeled tracer experiments, we discovered a degradation pathway of caffeine to theobromine. These findings could assist researchers in understanding the caffeine-related mechanisms in Camellia plants containing low, normal, and high caffeine content and be applied to caffeine regulation and breeding improvement in future research.