Association between statin use and dry eye disease in patients with hyperlipidemia: A population-based retrospective cohort study.

Higher serum cholesterol levels have been associated with an increased risk of dry eye disease (DED). The relationship between statin (HMG-CoA reductase inhibitor) use and DED in patients with hyperlipidemia remains unclear. To investigate the association between statin use and the risk of DED in patients with hyperlipidemia, we conducted a population-based retrospective cohort study utilizing data from Taiwan's Longitudinal Generation Tracking Database. Patients were categorized into statin users and nonusers, with a 5-year follow-up period. The study identified patients with newly diagnosed hyperlipidemia, excluding those with prior DED diagnoses. Matching and adjustments for covariates resulted in 41,931 individuals in each group. Patients receiving statin therapy were compared with those unexposed. Cumulative exposure doses were also evaluated to assess dose-response relationships. The primary outcome was the incidence of DED diagnosed during the follow-up period. Cox proportional hazards regression models estimated the risk of DED, and conditional logistic regression analyzed the dose-response effect of statin exposure. Among 41,931 matched pairs, statin users exhibited a slightly increased risk of developing DED compared with nonusers (adjusted hazard ratio, 1.06; 95% CI, 1.02-1.11; p < 0.01). However, no dose-response relationship was observed between statin exposure and DED risk. Statin use among patients with hyperlipidemia is associated with a marginally higher risk of DED. These findings underscore the importance of regular eye examinations in this patient population to facilitate early detection and management of DED.

Tree Longevity: Multifaceted Genetic Strategies and Beyond.

Old trees are remarkable for their ability to endure for centuries or even millennia, acting as recordkeepers of historical climate and custodians of genetic diversity. The secret to their longevity has long been a subject of fascination. Despite the challenges associated with studying old trees, such as massive size, slow growth rate, long lifespan and often remote habitat, accumulating studies have investigated the mechanisms underlying tree aging and longevity over the past decade. The recent publication of high-quality genomes of long-lived tree species, coupled with research on stem cell function and secondary metabolites in longevity, has brought us closer to unlocking the secrets of arboreal longevity. This review provides an overview of the global distribution of old trees and examines the environmental and anthropogenic factors that shape their presence. We summarize the contributions of physiological characteristics, stem cell activity, and immune system responses to their extraordinary longevity. We also explore the genetic and epigenetic 'longevity code', which consists of resistance and defense genes, DNA repair genes and patterns of DNA methylation modification. Further, we highlight key areas for future research that could enhance our understanding of the mechanisms underlying tree longevity.

Ammonia exposure impairs bone mineralization in zebrafish (Danio rerio) larvae.

Ammonia is a major pollutant of freshwater environments. Previous studies have indicated that ammonia exposure adversely affects the physiology of freshwater fish. However, its effect on bone mineralization in freshwater fish larvae remains unclear. In this study, zebrafish larvae were used as a model to investigate the effects of different ammonia levels (0, 2.5, 5, and 10 mM NH4Cl) on the survival rate, body length, and bone mineralization of fish. The survival rate of zebrafish embryos exposed to different NH4Cl concentrations for 8 days was not affected. In contrast, the body length and bone mineralization of zebrafish larvae at 8 days post fertilization (dpf) were significantly reduced at 5 and 10 mM NH4Cl exposure. Further investigations revealed that ammonia exposure decreased the mRNA expression of osteoblast-related genes and increased that of osteoclast-related genes. Additionally, exposure to 5 mM and 10 mM NH4Cl induced the production of reactive oxygen species (ROS). 10 mM-but not 5 mM-NH4Cl exposure reduced the calcium and phosphorus content in 8 dpf zebrafish larvae. In conclusion, ammonia exposure induces bone resorption, while decreasing the calcium and phosphorus content of the whole body and bone formation, resulting in impaired bone mineralization in fish larvae.

Conserved expression of the zebrafish syt4 gene in GABAergic neurons in the cerebellum of adult fishes revealed by mammalian SYT4 immunoreactive-like signals.

Synaptotagmin 4 (syt4) belongs to the synaptotagmin protein family, which has 17 and 28 family members in human and zebrafish, respectively. In zebrafish and rodents, syt4 is known to express abundantly in the entire central nervous system in the early developmental stages. In adult rodents, the gene expression shifts to be predominant in the cerebellum, mostly in Purkinje cells, a type of GABAergic neurons. However, there is no report of the expression pattern of syt4 in the adult zebrafish brain. Therefore, we hypothesize that the expression of syt4 is conserved in adult zebrafish and is specific to the GABAergic neurons, likely Purkinje cells, in the cerebellum. To examine the hypothesis, we first show that only one copy of syt4 gene remains in the zebrafish genome, and it is orthologous to the gene in other vertebrates. We further observe mammalian SYT4 antibody immunoreactive-like (mSYT4-ir) signals in several structures in the hindbrain including the medial divisions of the valvula cerebelli and the corpus cerebelli. In addition, our observations indicate the presence of mSYT4-ir signals in GABAergic neurons, most notably in the Purkinje cell layer of the molecular layer in the aforementioned structures. Conversely, mSYT4-ir signals are not observed in glutamatergic or cholinergic neurons. Therefore, we deduce that the syt4 gene in zebrafish exhibits a homologous expression pattern to those of previously studied vertebrate species, which is revealed by the positive immunoreactive-like signals of mammalian SYT4 antibodies.

Microbiota associated with urban forests.

Urban forests are essential for maintaining urban ecological stability. As decomposers, soil microorganisms play an indispensable role in the stability of urban forest ecosystems, promoting the material cycle of the ecosystems. This study used high-throughput sequencing technology to explore the bacteria in six forest stands, including Phyllostachys edulis (ZL), Metasequoia glyptostroboides (SSL), Cornus officinalis (SZY), mixed broad-leaved shrub forest (ZKG), mixed pine and cypress forest (SBL), and mixed broad-leaved tree forest (ZKQ). Meanwhile, the differences in fungal communities were investigated. The results show that ZL has the highest alpha diversity of bacterial communities, while its fungal community is the lowest; Proteobacteria is the most abundant bacterial phylum in the six forest stands; ZKQ has the highest fungal diversity. In addition, soil microbial communities are affected by environmental factors. Soil pH, organic matter (SOM), and available phosphorus (AP) significantly influence the compositions of urban forest soil microbial communities. This study revealed the differences in bulk soil (BS) microbial community structures among six forest stands and the relationship between environmental factors and soil microbial communities, which has important guiding significance for creating healthy and stable urban forests with profound ecological benefits.

Multi-omics profiling reveal responses of three major Dendrobium species from different growth years to medicinal components.

Dendrobium is a perennial herb found in Asia that is known for its medicinal and ornamental properties. Studies have shown that the stem is the primary medicinal component of Dendrobium spp. To investigate the effect of the species and age of Dendrobium (in years) on the content of its medicinal components, we collected the stems of 1-to-4-year-old D. officinale, D. moniliforme, and D. huoshanense, sequenced the transcriptome, metabolome, and microbiome, and analyzed the data in a comprehensive multi-omics study. We identified 10,426 differentially expressed genes (DEGs) with 644 differentially accumulated metabolites (DAMs) from 12 comparative groups and mapped the flavonoid pathway based on DEGs and DAMs. Transcriptomic and metabolomic data indicated a general trend of the accumulation of flavonoids exhibiting pharmacological effects in the three Dendrobium species. In addition, joint metabolome and microbiome analyses showed that actinobacteria was closely associated with flavonoid synthesis with increasing age. Our findings provide novel insights into the interactions of flavonoids of Dendrobium with the transcriptome and microbiome.

Perioperative Dexmedetomidine Infusion Improves Perioperative Care of Bariatric-Metabolic Surgery: A Single Center Experience with Meta-Analysis.

PURPOSE: This study aims to determine the effects of perioperative dexmedetomidine infusion (PDI) on Asian patients undergoing bariatric-metabolic surgery (BMS), focusing on the need for pain medications and management of postoperative nausea and vomiting (PONV), and to investigate the association with these variables, including patients' characteristics and BMS data. MATERIALS AND METHODS: A retrospective review of prospectively collected data was conducted in an Asian weight management center from August 2016 to October 2021. A total of 147 native patients with severe obesity were enrolled. All patients were informed of the full support of perioperative pain medications for BMS. The pain numeric rating scale scores, events of PONV, needs for pain medications, and the associated patients' characteristics were analyzed. A p-value of < 0.05 was considered statistically significant. Furthermore, to verify the effects of perioperative usage of dexmedetomidine for BMS, a systematic review with meta-analysis of currently available randomized control trials was performed. RESULTS: Among the 147 enrolled patients, 107 underwent laparoscopic sleeve gastrectomy and 40 underwent laparoscopic Roux-en-Y gastric bypass. PDI has been used as an adjunct multimodal analgesia for BMS in our institution since June 2017 (group D; n = 114). In comparison with those not administered with perioperative dexmedetomidine (group C; n = 33), lower pain numeric rating scale scores (2.52 ± 2.46 vs. 4.27 ± 2.95, p = 0.007) in the postanesthesia care unit, fewer PONV (32.46% vs. 51.52%; p = 0.046), and infrequent needs of additional pain medications (19.47% vs. 45.45%; p = 0.003) were observed in group D. Multivariable analysis demonstrated that type II diabetes mellitus was correlated with the decreased need of pain medications other than PDI (p = 0.035). Moreover, dexmedetomidine seemed to have a better analgesic effect for patients with longer surgical time based on our meta-analysis. CONCLUSION: Based on our limited experience, PDI could be a practical solution to alleviate pain and PONV in Asian patients undergoing BMS. Moreover, it might reduce the need for rescue painkillers with better postoperative pain management for patients with type II diabetes mellitus or longer surgical time.

Seasonal Changes in the Soil Microbial Community Structure in Urban Forests.

Urban forests play a crucial role in the overall health and stability of urban ecosystems. Soil microorganisms are vital to the functioning of urban forest ecosystems as they facilitate material cycling and contribute to environmental stability. This study utilized high-throughput sequencing technology to examine the structural characteristics of bacterial and fungal communities in the bulk soil of six different forest stands: Phyllostachys pubescens (ZL), Metasequoia glyptostroboides (SSL), Cornus officinalis (SZY), mixed broad-leaved shrub forest (ZKG), mixed pine and cypress forest (SBL), and mixed broad-leaved tree forest (ZKQ). Soil samples were collected from each forest stand, including the corners, center, and edges of each plot, and a combined sample was created from the first five samples. The results revealed that among the bacterial communities, ZKG exhibited the highest alpha diversity in spring, while ZL demonstrated the highest alpha diversity in both summer and autumn. Proteobacteria was the most abundant bacterial phylum in all six forest stand soils. The dominant fungal phylum across the six forest stands was identified as Ascomycota. Notably, the microbial community diversity of SBL bulk soil exhibited significant seasonal changes. Although ZL exhibited lower bacterial community diversity in spring, its fungal community diversity was the highest. The bulk soil microbial diversity of ZL and SSL surpassed that of the other forest stands, suggesting their importance in maintaining the stability of the urban forest ecosystem in the Zhuyu Bay Scenic Area. Furthermore, the diversity of the bulk soil microbial communities was higher in all six stands during spring compared to summer and autumn. Overall, this study provides valuable insights into the seasonal variations of bulk soil microbial communities in urban forests and identifies dominant tree species, offering guidance for tree species' selection and preservation in urban forest management.

Alternation of social behaviors for zebrafish (Danio rerio) in response to acute cold stress.

Low temperature is one of the most common abiotic stresses for aquatic ectotherms. Ambient low temperatures reduce the metabolic rate of teleosts, therefore, teleosts have developed strategies to modulate their physiological status for energy saving in response to cold stress, including behaviors, circulatory system, respiratory function, and metabolic adjustments. Many teleosts are social animals and they can live in large schools to serve a variety of functions, including predator avoidance, foraging efficiency, and reproduction. However, the impacts of acute cold stress on social behaviors of fish remain unclear. In the present study, we test the hypothesis that zebrafish alter their social behaviors for energy saving as a strategy in response to acute cold stress. We found that acute cold stress increased shoaling behavior that reflected a save-energy strategy for fish to forage and escape from the predators under cold stress. The aggressive levels measured by fighting behavior tests and mirror fighting tests were reduced by cold treatment. In addition, we also found that acute cold stress impaired the learning ability but did not affect memory. Our findings provided evidence that acute cold stress alters the social behaviors of aquatic ectotherms for energy saving; knowledge of their responses to cold is essential for their conservation and management.

Investigation of ammonia-induced lethal toxicity toward ion regulation in zebrafish embryos.

Ammonia is an environmental pollutant that is toxic to all aquatic animals. However, the mechanism of ammonia toxicity toward the ion regulatory function of early-stage fish has not been fully documented. We addressed this issue using zebrafish embryos as a model. We hypothesized that ammonia might impair ion regulation by inducing oxidative stress, mitochondrial dysfunction, and cell death of epidermal ionocytes and keratinocytes in zebrafish embryos. After exposure to various concentrations (10- 30 mM) of NH4Cl for 96 h, mortality increased up to 50 % and 100 % at 25 and 30 mM, respectively. Whole-embryo sodium, potassium, and calcium contents decreased at ≥10 mM, suggesting dysfunction of ion regulation. Numbers of H+-ATPase-rich (HR) cells and Na+/K+-ATPase-rich (NaR) cells (two ionocyte subtypes) were not significantly altered at 15 or 20 mM, while the mitochondrial abundance significantly decreased and reactive oxygen species (ROS) levels significantly increased in ionocytes. Moreover, caspase-3-dependent apoptosis was found in epidermal keratinocytes. Whole-embryo transcript levels of several genes involved in ion regulation, antioxidation, and apoptosis were upregulated after ammonia exposure. In conclusion, ammonia exposure was shown to induce oxidative stress and mitochondrial dysfunction in ionocytes and apoptosis in keratinocytes, thereby impairing ion regulation and ultimately leading to the death of zebrafish embryos.

Fenpropathrin causes alterations in locomotion and social behaviors in zebrafish (Danio rerio).

Fenpropathrin is one of the widely used pyrethroid pesticides in agriculture and is frequently detected in the environment, groundwater, and food. While fenpropathrin was found to have neurotoxic effects in mammals, it remains unclear whether it has similar effects on fish. Here, we used adult zebrafish to investigate the impacts of fenpropathrin on fish social behaviors and neural activity. Exposure of adult zebrafish to 500 ppb of fenpropathrin for 72 h increased anxiety levels but decreased physical fitness, as measured by a novel tank diving test and swimming tunnel test. Fish exposed to fenpropathrin appeared to spend more time in the conspecific zone of the tank, possibly seeking greater comfort from their companions. Although learning, memory, and aggressive behavior did not change, fish exposed to fenpropathrin appeared to have shorter fighting durations. The immunocytochemical results showed the tyrosine hydroxylase antibody-labeled dopaminergic neurons in the teleost posterior tuberculum decreased in the zebrafish brain. According to a quantitative polymerase chain reaction (qPCR) analysis of the brain, exposure to fenpropathrin resulted in a decrease in the messenger (m)RNA expression of monoamine oxidase (mao), an enzyme that facilitates the deamination of dopamine. In contrast, the mRNA expression of the sncga gene, which may trigger Parkinson's disease, was found to have increased. There were no changes observed in expressions of genes related to antioxidants and apoptosis between the control and fenpropathrin-exposed groups. We provide evidence to demonstrate the defect of the neurotoxicity of fenpropathrin toward dopaminergic neurons in adult zebrafish.

UV-B promotes flavonoid biosynthesis in Ginkgo biloba by inducing the GbHY5-GbMYB1-GbFLS module.

Ginkgo biloba (ginkgo) leaves have medicinal value due to their high levels of secondary metabolites, such as flavonoids. We found that the flavonoid content in ginkgo leaves increases significantly at high altitudes (Qinghai-Tibet Plateau). Considering that high UV-B radiation is among the key environmental characteristics of the Qinghai-Tibet Plateau, we carried out simulated UV-B treatments on ginkgo seedlings and found that the flavonoid content of the leaves increased significantly following the treatments. Combined with results from our previous studies, we determined that the transcription factor GbHY5 may play a key role in responses to UV-B radiation. Overexpression of GbHY5 significantly promoted the accumulation of flavonoids in both ginkgo callus and Arabidopsis thaliana. Furthermore, yeast two-hybrid and real-time quantitative PCR showed that GbHY5 promoted the expression of GbMYB1 by interacting with GbMYB1 protein. Overexpression of GbMYB1 in ginkgo callus and A. thaliana also significantly promoted flavonoid biosynthesis. GbFLS encodes a key enzyme in flavonoid biosynthesis, and its promoter has binding elements of GbHY5 and GbMYB1. A dual-luciferase reporter assay indicated that while GbHY5 and GbMYB1 activated the expression of GbFLS individually, their co-expression achieved greater activation. Our analyses reveal the molecular mechanisms by which the UV-B-induced GbHY5-GbMYB1-GbFLS module promotes flavonoid biosynthesis in ginkgo, and they provide insight into the use of UV-B radiation to enhance the flavonoid content of ginkgo leaves.

Metabolomics based on GC-MS revealed hub metabolites of pecan seeds germinating at different temperatures.

BACKGROUND: As an important plant source of food and edible oils, pecans are rich in metabolites. Few studies have focused on metabolites involved in pecan seed germination at different temperatures. RESULTS: In our study, we germinated pecan seeds at different temperatures and found that, the germination rate and water content were highest at 30°C. It was found that the radicle of pecan seeds could sense seed coat cracking by observing the microstructure and cell ultra-structure of the seeds at the early stage of germination. We compared the metabolomes of seeds at different temperatures with different germination processes. A total of 349 metabolites were identified, including 138 primary metabolites and 211 secondary metabolites. KEGG enrichment analysis indicated that the differential metabolites were mainly enriched in the metabolic pathways, amino acid synthesis pathways and ABC transporters. Using weighted gene co-expression network analysis (WGCNA), three modules of closely related metabolites were identified. In the brown module, most of hub metabolites were amino substances, whereas in the blue module, many hub metabolites were sugars. CONCLUSIONS: Amino acids and carbohydrates play an important role in pecan seed germination. Differential metaboliteanalysis showed that 30°C was the temperature at which metabolites differed most significantly. This study provides useful information for further research on the seedling establishment of pecan seeds.

Long Non-Coding RNA lncWOX11a Suppresses Adventitious Root Formation of Poplar by Regulating the Expression of PeWOX11a.

Long non-coding RNAs (lncRNAs), a class of poorly conserved transcripts without protein-encoding ability, are widely involved in plant organogenesis and stress responses by mediating the transmission and expression of genetic information at the transcriptional, posttranscriptional, and epigenetic levels. Here, we cloned and characterized a novel lncRNA molecule through sequence alignment, Sanger sequencing, transient expression in protoplasts, and genetic transformation in poplar. lncWOX11a is a 215 bp transcript located on poplar chromosome 13, ~50 kbp upstream of PeWOX11a on the reverse strand, and the lncRNA may fold into a series of complex stem-loop structures. Despite the small open reading frame (sORF) of 51 bp within lncWOX11a, bioinformatics analysis and protoplast transfection revealed that lncWOX11a has no protein-coding ability. The overexpression of lncWOX11a led to a decrease in the quantity of adventitious roots on the cuttings of transgenic poplars. Further, cis-regulatory module prediction and CRISPR/Cas9 knockout experiments with poplar protoplasts demonstrated that lncWOX11a acts as a negative regulator of adventitious rooting by downregulating the WUSCHEL-related homeobox gene WOX11, which is supposed to activate adventitious root development in plants. Collectively, our findings imply that lncWOX11a is essential for modulating the formation and development of adventitious roots.

Retention of ion channel genes expression increases Japanese medaka survival during seawater reacclimation.

Euryhaline teleosts exhibit varying acclimability to survive in environments that alternate between being hypotonic and hypertonic. Such ability is conferred by ion channels expressed by ionocytes, the ion-regulating cells in the gills or skin. However, switching between environments is physiologically challenging, because most channels can only perform unidirectional ion transportation. Coordination between acute responses, such as gene expression, and long-term responses, such as cell differentiation, is believed to strongly facilitate adaptability. Moreover, the pre-acclimation to half seawater salinity can improve the survivability of Japanese medaka (Oryzias latipes) during direct transfer to seawater; here, the ionocytes preserve hypertonic acclimability while performing hypotonic functions. Whether acclimability can be similarly induced in a closed species and their corresponding responses in terms of ion channel expression remain unclear. In the present study, Japanese medaka pre-acclimated in brackish water were noted to have higher survival rates while retaining higher expression of the three ion channel genes ATP1a1a.1, ATP1b1b, and SLC12a2a. This retention was maintained up to 2 weeks after the fish were transferred back into freshwater. Notably, this induced acclimability was not found in its close kin, Indian medaka (Oryzias dancena), the natural habitat of which is brackish water. In conclusion, Japanese medaka surpassed Indian medaka in seawater acclimability after experiencing exposure to brackish water, and this ability coincided with seawater-retention gene expression.

Associations of Warfarin Use with Risks of Ischemic Cerebrovascular Events and Major Bleeding in Patients with Hyperthyroidism-Related Atrial Fibrillation.

The use of oral anticoagulants for patients with new-onset hyperthyroidism-related atrial fibrillation (AF) is controversial. We aimed to evaluate the clinical benefits of warfarin therapy in this population. This retrospective cohort study used a data-cut of Taiwan Health and Welfare Database between 2000 and 2016. We compared warfarin users and nonusers among AF patients with hyperthyroidism. We used 1:2 propensity score matching to balance covariates and Cox regression model to calculate hazard ratios (HRs). The primary outcome was risk of ischemic stroke/transient ischemic attack (TIA), and the secondary outcome was major bleeding. After propensity score matching, we defined 90 and 168 hyperthyroidism-related AF patients with mean (SD) age of 59.9 ± 13.5 and 59.2 ± 14.6 in the warfarin-treated group and untreated group separately. The mean (SD) CHA2DS2-VASc scores for the two groups were 2.1 ± 1.6 and 1.8 ± 1.5, respectively. Patients with hyperthyroidism-related AF receiving warfarin had no significant risk of ischemic stroke/TIA (adjusted HR: 1.16, 95% confidence interval [CI]: 0.52-2.56, p = 0.717) compared to nonusers. There was a comparable risk of major bleeding between those receiving warfarin or not (adjusted HR: 0.91, 95% CI: 0.56-1.47, p = 0.702). The active-comparator design also demonstrated that warfarin use had no significant association with the risk of stroke/TIA versus aspirin use (adjusted HR: 2.43; 95% CI: 0.68-8.70). In conclusion, anticoagulation therapy did not have a statistically significant benefit on ischemic stroke/TIA nor risk of bleeding, among patients with new-onset hyperthyroidism-related AF under a low CHA2DS2-VASc score, by comparing those without use.

Investigation of children's habits of smartphone usage and parental awareness of myopia control in underdeveloped areas of China.

AIM: To investigate the behaviors of smartphone usage and parental knowledge of vision health among primary students in the rural areas of China. METHODS: In this school-based, cross-sectional study, a total of 52 606 parents of students from 30 primary schools in the Xingguo County were investigated through an online questionnaire from July 2020 to August 2020. The self-designed questionnaire contained three parts: the demographic factors of both children and parents, parental knowledge and attitude toward myopia, and the preventive treatment of myopia. RESULTS: A total of 52 485 appropriately answered questionnaires were received, showing an effective response rate of 95.1%. The average age of the primary students was 10.1±0.98y and the prevalence of myopia among the primary students was 40.3%. The age of myopia occurrence in elementary students was significantly correlated with the parents' educational level (95%CI: 0.82-0.98, P=0.013), children's gender (95%CI: 1.08-1.20, P<0.001), school location (county or countryside) (95%CI: 0.59-0.66, P<0.001), children's smartphone ownership (95%CI: 1.09-1.26, P<0.001), and the average time spent on smartphone per day (95%CI: 0.78-0.88, P<0.001). School location in the county town, high family income, and high parents' educational level significantly affected both parents' myopia awareness and children's vision-threatening behaviors (P<0.01). Left-behind children showed a higher incidence of vision-threatening habits than those who lived with their parents (P<0.01). CONCLUSION: The results reveal the current situation of myopia development among rural primary school students and their parents. This survey will serve as a guidance for designing myopic prevention policies in the rural areas of China.

A Comparison of the Flavonoid Biosynthesis Mechanisms of Dendrobium Species by Analyzing the Transcriptome and Metabolome.

Dendrobium huoshanense, Dendrobium officinale, and Dendrobium moniliforme, as precious Chinese medicinal materials, have a variety of medicinal properties. Flavonoids are important medicinal components of Dendrobium, but their accumulation rules and biosynthesis mechanisms remain unclear. To explore the similarities and differences of flavonoid accumulation and biosynthesis in these three Dendrobium species, we performed flavonoid content determination, widely-targeted metabolomics and transcriptome sequencing on 1-4 years old Dendrobium species. The results showed that in different growth years, D. huoshanense stems had the highest flavonoid content in the second year of growth, while D. officinale and D. moniliforme stems had the highest flavonoid content in the third year of growth. A total of 644 differentially accumulated metabolites (DAMs) and 10,426 differentially expressed genes (DEGs) were identified by metabolomic and transcriptomic analysis. It was found that DAMs and DEGs were not only enriched in the general pathway of "flavonoid biosynthesis", but also in multiple sub-pathways such as "Flavone biosynthesis", and "Flavonol biosynthesis" and "Isoflavonoid biosynthesis". According to a combined transcriptome and metabolome analysis, the expression levels of the F3'H gene (LOC110096779) and two F3'5'H genes (LOC110101765 and LOC110103762) may be the main genes responsible for the differences in flavonoid accumulation. As a result of this study, we have not only determined the optimal harvesting period for three Dendrobium plants, but also identified the key genes involved in flavonoid biosynthesis and provided a basis for further study of the molecular mechanism of flavonoid synthesis.

Analysis of the different growth years accumulation of flavonoids in Dendrobium moniliforme (L.) Sw. by the integration of metabolomic and transcriptomic approaches.

Dendrobium moniliforme (L.) Sw. is a valuable herbal crop, and flavonoids are primarily distributed as active ingredients in the stem, but the composition and synthesis mechanisms of flavonoids in different growth years are not clear. The accumulation of flavonoids in D. moniliforme from four different years was investigated, using a combined metabolomics and transcriptomics approach in this study. The phenylpropanoid and flavonoid biosynthetic pathways were significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs). The widely targeted metabolomics technique revealed a total of 173 kinds of flavonoid metabolites. The metabolomics data confirmed the trend of total flavonoids (TF) content in stems of D. moniliforme, with chalcone, naringenin, eriodictyol, dihydroquercetin, and other flavonoids considerably up-accumulating in the third year. Twenty DEGs were detected that regulate flavonoid synthesis and the expression of these genes in different growth years was verified using real-time quantitative PCR (qRT-PCR). Furthermore, a comprehensive regulatory network was built for flavonoid biosynthesis and it was discovered that there is one FLS gene, one CCR gene and two MYB transcription factors (TFs) with a high connection with flavonoid biosynthesis by weighted gene co-expression network analysis (WGCNA). In this study, the correlation between genes involved in flavonoid biosynthesis and metabolites was revealed, and a new regulatory mechanism related to flavonoid biosynthesis in D. moniliforme was proposed. These results provide an important reference for the farmers involved in the cultivation of D. moniliforme.