Genome-wide identification of tea plant (Camellia sinensis) BAHD acyltransferases reveals their role in response to herbivorous pests.

BACKGROUND: BAHD acyltransferases are among the largest metabolic protein domain families in the genomes of terrestrial plants and play important roles in plant growth and development, aroma formation, and biotic and abiotic stress responses. Little is known about the BAHDs in the tea plant, a cash crop rich in secondary metabolites. RESULTS: In this study, 112 BAHD genes (CsBAHD01-CsBAHD112) were identified from the tea plant genome, with 85% (98/112) unevenly distributed across the 15 chromosomes. The number of BAHD gene family members has significantly expanded from wild tea plants to the assamica type to the sinensis type. Phylogenetic analysis showed that they could be classified into seven subgroups. Promoter cis-acting element analysis revealed that they contain a large number of light, phytohormones, and stress-responsive elements. Many members displayed tissue-specific expression patterns. CsBAHD05 was expressed at more than 500-fold higher levels in purple tea leaves than in green tea leaves. The genes exhibiting the most significant response to MeJA treatment and feeding by herbivorous pests were primarily concentrated in subgroups 5 and 6. The expression of 23 members of these two subgroups at different time points after feeding by tea green leafhoppers and tea geometrids was examined via qPCR, and the results revealed that the expression of CsBAHD93, CsBAHD94 and CsBAHD95 was significantly induced after the tea plants were subjected to feeding by both pricking and chewing pests. Moreover, based on the transcriptome data for tea plants being fed on by these two pests, a transcriptional regulatory network of different transcription factor genes coexpressed with these 23 members was constructed. CONCLUSIONS: Our study provides new insights into the role of BAHDs in the defense response of tea plants, and will facilitate in-depth studies of the molecular function of BAHDs in resistance to herbivorous pests.

Alternative splicing of CsWRKY21 positively regulates cold response in tea plant.

Alternative splicing (AS) was an important post-transcriptional mechanism that involved in plant resistance to adversity stress. WRKY transcription factors function as transcriptional activators or repressors to modulate plant growth, development and stress response. However, the role of alternate splicing of WRKY in cold tolerance is poorly understood in tea plants. In this study, we found that the CsWRKY21 transcription factor, a member of the WRKY IId subfamily, was induced by low temperature. Subcellular localization and transcriptional activity assays showed that CsWRKY21 localized to the nucleus and had no transcriptional activation activity. Y1H and dual-luciferase reporter assays showed that CsWRKY21 suppressed expression of CsABA8H and CsUGT by binding with their promoters. Transient overexpression of CsABA8H and CsUGT reduced abscisic acid (ABA) content in tobacco leaves. Furthermore, we discovered that CsWRKY21 undergoes AS in the 5'UTR region. The AS transcript CsWRKY21-b was induced at low temperature, up to 6 folds compared to the control, while the full-length CsWRKY21-a transcript did not significantly change. Western blot analysis showed that the retention of introns in the 5'UTR region of CsWRKY21-b led to higher CsWRKY21 protein content. These results revealed that alternative splicing of CsWRKY21 involved in cold tolerance of tea plant by regulating the protein expression level and then regulating the content of ABA, and provide insights into molecular mechanisms of low temperature defense mediated by AS in tea plant.

Usefulness of Self-Assessment of Gastrointestinal Symptoms: Web-Based Study in Anhui, China.

BACKGROUND: Gastrointestinal symptoms (GISs) are caused by a combination of biopsychosocial factors and are highly prevalent worldwide. Given their complex nature, coupled with ineffective communication of diagnoses by physicians, patients with intimate GISs often feel stigmatized. This, in turn, can inhibit their ability to express their thoughts and feelings adequately, leading them to over- or underreport their symptoms. Moreover, selective service-seeking for and reporting of GISs have a direct bearing on the stage of disease at presentation and, consequently, on the overall prognosis. OBJECTIVE: This study aimed to investigate the usefulness of a web-based self-assessment of GISs as a supplementary means to cope with potential over- or underreporting during routine consultations. METHODS: GIS data were collected using a novel web-based self-assessment tool (n=475) and from nonparticipative observation of doctor-patient consultations (n=447) and household surveys (n=10,552) in Anhui, China. Data analysis focused primarily on the description of the composition of respondents and the occurrence rates of GISs by sociodemographics, and by symptom solicitation methods and settings. Chi-square power tests were used when necessary to compare differences in the occurrence rates between relevant groups. The level of significance for the 2-sided test was set at α<.05. RESULTS: The average occurrence rates of both upper and lower GISs derived from the web-based self-assessment were higher than those from the observation (upper GISs: n=661, 20.9% vs n=382, 14.2%; P<.001; lower GISs: n=342, 12.9% vs n=250, 10.8%; P=.02). The differences in 6 of the 9 upper GISs and 3 of the 11 lower GISs studied were tested with statistical significance (P<.05); moreover, a higher frequency rate was recorded for symptoms with statistical significance via self-assessment than via observation. For upper GISs, the self-assessed versus observed differences ranged from 17.1% for bloating to 100% for bad mood after a meal, while for lower GISs, the differences ranged from -50.5% for hematochezia or melena to 100% for uncontrollable stool. Stomachache, regurgitation, and dysphagia had higher occurrence rates among participants of the self-assessment group than those of the household survey group (20% vs 12.7%, 14% vs 11%, and 3% vs 2.3%, respectively), while the opposite was observed for constipation (5% vs 10.9%), hematochezia or melena (4% vs 5%), and anorexia (4% vs 5.2%). All differences noted in the self-assessed occurrence rates of specific, persistent GISs between sociodemographic groups were tested for nonsignificance (P>.05), while the occurrence rates of any of the 6 persistent GISs among respondents aged 51-60 years was statistically higher than that among other age groups (P=.03). CONCLUSIONS: The web-based self-assessment tool piloted in this study is useful and acceptable for soliciting more comprehensive GISs, especially symptoms with concerns about stigmatization, privacy, and shame. Further studies are needed to integrate the web-based self-assessment with routine consultations and to evaluate its efficacy.

De novo full length transcriptome analysis of a naturally caffeine-free tea plant reveals specificity in secondary metabolic regulation.

Tea plants are crops with economic, health and cultural value. Catechin, caffeine and theanine are the main secondary metabolites of taste. In the process of germplasm collection, we found a resource in the Sandu Aquatic Autonomous County of Guizhou (SDT) that possessed significantly different characteristic metabolites compared with the cultivar 'Qiancha 1'. SDT is rich in theobromine and theophylline, possesses low levels of (-)-epicatechin-3-gallate, (-)-epigallocatechin-3-gallate, and theanine content, and is almost free of caffeine. However, research on this tea resource is limited. Full-length transcriptome analysis was performed to investigate the transcriptome and gene expression of these metabolites. In total, 78,809 unique transcripts were obtained, of which 65,263 were complete coding sequences. RNA-seq revealed 3415 differentially expressed transcripts in the tender leaves of 'Qiancha 1' and 'SDT'. Furthermore, 2665, 6231, and 2687 differentially expressed transcripts were found in different SDT tissues. These differentially expressed transcripts were enriched in flavonoid and amino acid metabolism processes. Co-expression network analysis identified five modules associated with metabolites and found that genes of caffeine synthase (TCS) may be responsible for the low caffeine content in SDT. Phenylalanine ammonia lyase (PAL), glutamine synthetase (GS), glutamate synthase (GOGAT), and arginine decarboxylase (ADC) play important roles in the synthesis of catechin and theanine. In addition, we identified that ethylene resposive factor (ERF) and WRKY transcription factors may be involved in theanine biosynthesis. Overall, our study provides candidate genes to improve understanding of the synthesis mechanisms of these metabolites and provides a basis for molecular breeding of tea plant.

Differences in service and antibiotics use following symptomatic respiratory tract infections between 2016 and 2021 in rural Anhui, China.

In the past 10-15 years, the government of China has made various efforts in tackling excessive antibiotics use. Yet, little is known about their effects at rural primary care settings. This study aimed to determine the impact of government policies and the COVID-19 pandemic on antibiotic prescribing practices at such settings utilizing data from separate studies carried out pre- and during the pandemic, in 2016 and 2021 in Anhui province, China, using identical sampling and survey approaches. Data on antibiotics prescribed, diagnosis, socio-demographic, etc., were obtained through non-participative observation and a structured exit survey. Data analysis comprised mainly descriptive comparisons of 1153 and 762 patients with respiratory infections recruited in 2016 and 2021, respectively. The overall antibiotics prescription rate decreased from 89.6% in 2016 to 69.1% in 2021, and the proportion of prescriptions for two or more classes of antibiotics was estimated as 35.9% in 2016 and 11.0% in 2021. There was a statistically significant decrease in the number of days from symptom onset to clinic visits between the year groups. In conclusion, measures to constrain excessive prescription of antibiotics have led to some improvements at the rural primary care level, and the COVID-19 pandemic has had varying effects on antibiotic use.

A monoterpene synthase gene cluster of tea plant (Camellia sinensis) potentially involved in constitutive and herbivore-induced terpene formation.

Monoterpenes and sesquiterpenes are the most abundant volatiles in tea plants and have dual functions in aroma quality formation and defense responses in tea plants. Terpene synthases (TPS) are the key enzymes for the synthesis of terpenes in plants; however, the functions of most of them in tea plants are still unknown. In this study, six putative terpene biosynthesis gene clusters were identified from the tea plant genome. Then we cloned three new TPS-b subfamily genes, CsTPS08, CsTPS10 and CsTPS58. In vitro enzyme assays showed that CsTPS08 and CsTPS58 are two multiple-product terpene synthases, with the former synthesizing linalool as the main product, and ß-myrcene, α-phellandrene, α-terpinolene, D-limonene, cis-ß-ocimene, trans-ß-ocimene and (4E,6Z)-allo-ocimene as minor products are also detected, while the latter catalyzing the formation of α-pinene and D-limonene using GPP as the substrate. No product of CsTPS10 was detected in the prokaryotic expression system, but geraniol production was detected when transiently expressed in tobacco leaves. CsTPS08 and CsTPS10 are two functional members of a monoterpene synthase gene cluster, which were significantly induced during both Ectropis oblique feeding and fresh leaf spreading treatments, suggesting that they have dual functions involved in tea plant pest defense and tea aroma quality regulation. In addition, the differences in their expression levels in different tea plant cultivars provide a possibility for the subsequent screening of tea plant resources with a specific aroma flavor. Our results deepen the understanding of terpenoid synthesis in tea plants.

TeaAS: a comprehensive database for alternative splicing in tea plants (Camellia sinensis).

Alternative splicing (AS) increases the diversity of transcripts and proteins through the selection of different splice sites and plays an important role in the growth, development and stress tolerance of plants. With the release of the reference genome of the tea plant (Camellia sinensis) and the development of transcriptome sequencing, researchers have reported the existence of AS in tea plants. However, there is a lack of a platform, centered on different RNA-seq datasets, that provides comprehensive information on AS.To facilitate access to information on AS and reveal the molecular function of AS in tea plants, we established the first comprehensive AS database for tea plants (TeaAS, http://www.teaas.cn/index.php ). In this study, 3.96 Tb reads from 66 different RNA-seq datasets were collected to identify AS events. TeaAS supports four methods of retrieval of AS information based on gene ID, gene name, annotation (non-redundant/Kyoto encyclopedia of genes and genomes/gene ontology annotation or chromosomal location) and RNA-seq data. It integrates data pertaining to genome annotation, type of AS event, transcript sequence, and isoforms expression levels from 66 RNA-seq datasets. The AS events resulting from different environmental conditions and that occurring in varied tissue types, and the expression levels of specific transcripts can be clearly identified through this online database. Moreover, it also provides two useful tools, Basic Local Alignment Search Tool and Generic Genome Browser, for sequence alignment and visualization of gene structure.The features of the TeaAS database make it a comprehensive AS bioinformatics platform for researchers, as well as a reference for studying AS events in woody crops. It could also be helpful for revealing the novel biological functions of AS in gene regulation in tea plants.

Dose-Effect Relationship Between Gastric Cancer and Common Digestive Tract Symptoms and Diagnoses in Anhui, China.

BACKGROUND: Early prevention and diagnosis are key to reducing the huge burden of gastric cancer (GC). Nearly half of the population worldwide are suffering from some form of digestive tract conditions (symptoms/diagnoses, DTCs) but their relations with GC are not well understood. We aim to explore the relationships (especially dose-effect relationships) between GC and DTCs. METHODS: This study used data from a community-based case-control study in Anhui, China during 2016-2019 and performed multivariate conditional logistic regression modeling of the associations between GC and DTCs. RESULTS: A total of 2255 participants (451 cases and 1804 controls) completed the study. Statistically significant relations (P<0.05) were found between GC and the presence of gastroesophageal reflux [odds ratio (OR)=1.41], odynophagia (OR=1.87), stomach discomfort (OR=1.86), poor appetite (OR=2.01) and Helicobacter pylori (H. pylori) infection (OR=4.39). When the DTCs were divided into duration grades, all these ORs presented an increasing trend (P<0.05), being 1.89 to 2.45 for gastroesophageal reflux, 1.63 to 3.78 for stomach discomfort, 2.36 to 5.29 for poor appetite, and 3.95 to 10.03 for H. pylori infection. When the DTCs were divided into severity grades, the ORs also witnessed an increasing trend (P<0.05), being 1.69 to 2.52 for gastroesophageal reflux, 2.44 to 3.56 for stomach discomfort, and 2.22 to 2.75 for poor appetite. When the DTCs were divided into duration-severity grades, the ORs displayed a much steeper increasing trend, being 0.49 to 4.96 for gastroesophageal reflux, 1.50 to 6.33 for odynophagia, 0.47 to 3.32 for stomach discomfort, and 0.40 to 10.47 for poor appetite. In contrast, the ORs for the lower DTCs were generally tested without statistical significance. CONCLUSION: The study revealed consistent dose-effect associations between GC and duration of gastroesophageal reflux, stomach discomfort, poor appetite, and H. pylori infection; severity of gastroesophageal reflux, stomach discomfort and poor appetite; and duration-severity of gastroesophageal reflux, odynophagia, stomach discomfort and poor appetite. These should inform future prevention, diagnosis and further research in patients with GC.

Association between cytokine gene polymorphisms and ankylosing spondylitis susceptibility: a systematic review and meta-analysis.

PURPOSE OF THE STUDY: The aim of this study was to perform a meta-analysis to derive precise estimation of the association of interleukin-23 receptor (IL-23R), IL-1 receptor 2 (IL-1R2), IL-12 beta (IL-12B), IL-10 and tumour necrosis factor (TNF)-α polymorphisms with ankylosing spondylitis (AS) susceptibility. STUDY DESIGN: A systematic literature search was conducted to identify the relevant studies. Pooled OR with 95% CI was calculated to assess the strength of the association in a fixed or random-effects model. RESULTS: A total of 13 917 cases and 19 849 controls in 43 eligible studies were included in the meta-analysis. Seventeen single-nucleotide polymorphisms (SNPs) in the abovementioned five cytokine genes were evaluated. The results indicate that the nine SNPs (rs11209026, rs1004819, rs10489629, rs11465804, rs1343151, rs11209032, rs1495965, rs7517847, rs2201841) of IL-23R are associated with AS susceptibility in all study subjects in the allelic model. Moreover, stratification by ethnicity identified a significant association between seven SNPs of IL-23R and AS susceptibility in Europeans and Americans, but not in Asians. In addition, the IL-10-819 C/T and TNF-α-857 C/T polymorphisms also confer susceptibility to AS, especially in Asian population. CONCLUSION: The results suggested that the genetic susceptibility for AS is associated with the nine SNPs of IL-23R in overall population. In the subgroup analysis, significant associations were shown in European and American population, but not in Asian population. Our results also suggest that IL-10-819 C/T and TNF-α-857 C/T polymorphism might be associated with AS risk, especially in Asian population.

Regional differences in smoking, drinking, and physical activities of Chinese residents.

AIMS: This article aims at analyzing regional differences in smoking, drinking, and physical activities in China and informing cross-regional initiatives addressing these behaviors. METHODS: Data were extracted from available large-scale surveys. Index of dissimilarity (ID), linear correlation, and geographical mapping were used to analyze regional differences in the prevalence of smoking (PS), smoking cessation (PSC), regular drinking (PRD), and physical activities (PPA). RESULTS: Significant differences existed across regions in all the behaviors (P < .01). IDs ranged from 0.04 to 0.42. IDs of PRD and PPA were much higher than IDs of PS and PSC. Linear correlation coefficients of the 4 indicators by regions varied substantially ranging (in absolute value) from 0.031 to 0.982. Characteristic formats of geographical distribution were found with PS, PSC, and PRD and PPA being in "high plateau," "low plateau," and "shifting" patterns, respectively. CONCLUSIONS: Distinct regional disparities in health behavior exist, and socioculturally and contextually competitive policies and interventions are needed.

Regional differences of maternal health care utilization in China.

OBJECTIVE: To describe regional differences in maternal health care (MHC) utilization in China. STUDY DESIGN: Cross-sectional comparisons of 4 MHC utilization indicators, namely, early (13 weeks within pregnancy) examinations rate (EER), prenatal examination (>4 times) rate (PER), hospital delivery rate (HDR), and postnatal visit (>1 time) rate (PVR), using index of dissimilarity (ID), linear correlation analysis, and geographical mapping. RESULTS: Significant differences existed across regions in all the indicators (P < .01). All the IDs for rural areas were higher than that for urban areas. The IDs for major regions ranged from 0.01 to 0.27. Linear correlation coefficients between MHC utilization indicators by regions varied from 0.007 to 0.889 (in absolute value, P < .05). Characteristic formats of geographical distribution were found with PER, EER, HDR, and PVR being in "high-plateau," "low-plateau," and "shifting" patterns, respectively. CONCLUSIONS: There exist substantial regional discrepancies in MHC utilization in China and future MHC-related policies should take account regional context.