[Spatial transcriptomics techniques and its applications in plant research].

The heterogeneity of gene expression in plant cells plays a crucial role in determining the functional differences among tissues. Recent advancements in spatial transcriptome (ST) technology have significantly contributed to the study of specific biological questions in plants. This technology has been successfully applied to examine cell development, identification, and stress resistance. This review aims to explore the application of ST technology in plants by reviewing three aspects: the development of ST technology, its current application in plants, and future research directions. The review provides a systematic description of the development process of ST technology, with a focus on analyzing its progress in studying plant cell growth and differentiation, plant cell identification, and stress resistance. In addition, the challenges faced by ST technology in plant applications are summarized, along with proposed future directions for plant research, including the advantages of combining other omics technologies with ST technology to tackle scientific challenges in the field of plants.

The miR166 targets CsHDZ3 genes to negatively regulate drought tolerance in tea plant (Camellia sinensis).

Drought is the stressor with a significant adverse impact on the yield stability of tea plants. HD-ZIP III transcription factors (TFs) play important regulatory roles in plant growth, development, and stress responses. However, whether and how HD-ZIP III TFs are involved in drought response and tolerance in tea plants remains unclear. Here, we identified seven HD-ZIP III genes (CsHDZ3-1 to CsHDZ3-7) in tea plant genome. The evolutionary analysis demonstrated that CsHDZ3 members were subjected to purify selection. Subcellular localization analysis revealed that all seven CsHDZ3s located in the nucleus. Yeast self-activation and dual-luciferase reporter assays demonstrated that CsHDZ3-1 to CsHDZ3-4 have trans-activation ability whereas CsHDZ3-5 to CsHDZ3-7 served as transcriptional inhibitors. The qRT-PCR assay showed that all seven CsHDZ3 genes could respond to simulated natural drought stress and polyethylene glycol treatment. Further assays verified that all CsHDZ3 genes can be cleaved by csn-miR166. Overexpression of csn-miR166 inhibited the expression of seven CsHDZ3 genes and weakened drought tolerance of tea leaves. In contrast, suppression of csn-miR166 promoted the expression of seven CsHDZ3 genes and enhanced drought tolerance of tea leaves. These findings established the foundation for further understanding the mechanism of CsHDZ3-miR166 modules' participation in drought responses and tolerance.

Identification and Validation of the miR156 Family Involved in Drought Responses and Tolerance in Tea Plants (Camellia sinensis (L.) O. Kuntze).

The microRNA156 (miR156) family, one of the first miRNA families discovered in plants, plays various important roles in plant growth and resistance to various abiotic stresses. Previously, miR156s were shown to respond to drought stress, but miR156s in tea plants (Camellia sinensis (L.) O. Kuntze) have not been comprehensively identified and analyzed. Herein, we identify 47 mature sequences and 28 precursor sequences in tea plants. Our evolutionary analysis and multiple sequence alignment revealed that csn-miR156s were highly conserved during evolution and that the rates of the csn-miR156 members' evolution were different. The precursor sequences formed typical and stable stem-loop structures. The prediction of cis-acting elements in the CsMIR156s promoter region showed that the CsMIR156s had diverse cis-acting elements; of these, 12 CsMIR156s were found to be drought-responsive elements. The results of reverse transcription quantitative PCR (RT-qPCR) testing showed that csn-miR156 family members respond to drought and demonstrate different expression patterns under the conditions of drought stress. This suggests that csn-miR156 family members may be significantly involved in the response of tea plants to drought stress. Csn-miR156f-2-5p knockdown significantly reduced the Fv/Fm value and chlorophyll content and led to the accumulation of more-reactive oxygen species and proline compared with the control. The results of target gene prediction showed that csn-miR156f-2-5p targeted SQUAMOSA promoter binding protein-like (SPL) genes. Further analyses showed that CsSPL14 was targeted by csn-miR156f-2-5p, as confirmed through RT-qPCR, 5' RLM-RACE, and antisense oligonucleotide validation. Our results demonstrate that csn-miR156f-2-5p and CsSPL14 are involved in drought response and represent a new strategy for increasing drought tolerance via the breeding of tea plants.

Multiomics Analysis Reveals the Involvement of JsLHY in Controlling Aroma Production in Jasmine Flowers.

The Jasminum sambac flower is famous for its rich fragrance. However, our knowledge of the regulatory network for its aroma formation remains largely unknown and therefore needs further study. To this end, an integrated analysis of the volatilomics and transcriptomics of jasmine flowers at different flowering stages was performed. The results revealed many candidate transcription factors (TFs) may be involved in regulating the aroma formation of jasmine, among which the MYB-related TF LATE ELONGATED HYPOCOTYL (JsLHY) was identified as a hub gene. Using the DNA affinity purification sequencing method, dual-luciferase reporter, and yeast one-hybrid assays, we demonstrate that JsLHY can bind the gene promoter regions of six aroma-related structural genes (JsBEAT1, JsTPS34, JsCNL6, JsBPBT, JsAAAT5, and Js4CL7) and directly promote their expression. In addition, suppressing JsLHY expression decreased both the expression of JsLHY-bound genes and the content of related VOCs. The present study reveals how JsLHY participates in jasmine aroma formation.

Volatilomics Analysis of Jasmine Tea during Multiple Rounds of Scenting Processes.

Jasmine tea is reprocessed from finished tea by absorbing the floral aroma of jasmine (Jasminum sambac (L.) Aiton); this process is commonly known as "scenting". Making high-quality jasmine tea with a refreshing aroma requires repeated scenting. To date, the detailed volatile organic compounds (VOCs) and the formation of a refreshing aroma as the number of scenting processes increases are largely unknown and therefore need further study. To this end, integrated sensory evaluation, widely targeted volatilomics analysis, multivariate statistical analyses, and odor activity value (OAV) analysis were performed. The results showed that the aroma freshness, concentration, purity, and persistence of jasmine tea gradually intensifies as the number of scenting processes increases, and the last round of scenting process without drying plays a significant role in improving the refreshing aroma. A total of 887 VOCs was detected in jasmine tea samples, and their types and contents increased with the number of scenting processes. In addition, eight VOCs, including ethyl (methylthio)acetate, (Z)-3-hexen-1-ol acetate, (E)-2-hexenal, 2-nonenal, (Z)-3-hexen-1-ol, (6Z)-nonen-1-ol, ß-ionone, and benzyl acetate, were identified as key odorants responsible for the refreshing aroma of jasmine tea. This detailed information can expand our understanding of the formation of a refreshing aroma of jasmine tea.

RNA Methylome Reveals the m6A-mediated Regulation of Flavor Metabolites in Tea Leaves under Solar-withering.

The epitranscriptomic mark N6-methyladenosine (m6A), which is the predominant internal modification in RNA, is important for plant responses to diverse stresses. Multiple environmental stresses caused by the tea-withering process can greatly influence the accumulation of specialized metabolites and the formation of tea flavor. However, the effects of the m6A-mediated regulatory mechanism on flavor-related metabolic pathways in tea leaves remain relatively uncharacterized. We performed an integrated RNA methylome and transcriptome analysis to explore the m6A-mediated regulatory mechanism and its effects on flavonoid and terpenoid metabolism in tea (Camellia sinensis) leaves under solar-withering conditions. Dynamic changes in global m6A level in tea leaves were mainly controlled by two m6A erasers (CsALKBH4A and CsALKBH4B) during solar-withering treatments. Differentially methylated peak-associated genes following solar-withering treatments with different shading rates were assigned to terpenoid biosynthesis and spliceosome pathways. Further analyses indicated that CsALKBH4-driven RNA demethylation can directly affect the accumulation of volatile terpenoids by mediating the stability and abundance of terpenoid biosynthesis-related transcripts and also indirectly influence the flavonoid, catechin, and theaflavin contents by triggering alternative splicing-mediated regulation. Our findings revealed a novel layer of epitranscriptomic gene regulation in tea flavor-related metabolic pathways and established a link between the m6A-mediated regulatory mechanism and the formation of tea flavor under solar-withering conditions.

Analysis of the Genetic Diversity in Tea Plant Germplasm in Fujian Province Based on Restriction Site-Associated DNA Sequencing.

Fujian province, an important tea-producing area in China, has abundant tea cultivars. To investigate the genetic relationships of tea plant cultivars in Fujian province and the characteristics of the tea plant varieties, a total of 70 tea cultivars from Fujian and other 12 provinces in China were subjected to restriction site-associated DNA sequencing (RAD-seq). A total of 60,258,975 single nucleotide polymorphism (SNP) sites were obtained. These 70 tea plant cultivars were divided into three groups based on analyzing the phylogenetic tree, principal component, and population structure. Selection pressure analysis indicated that nucleotide diversity was high in Southern China and genetically distinct from cultivars of Fujian tea plant cultivars, according to selection pressure analysis. The selected genes have significant enrichment in pathways associated with metabolism, photosynthesis, and respiration. There were ten characteristic volatiles screened by gas chromatography-mass spectrometry (GC-MS) coupled with multivariate statistical methods, among which the differences in the contents of methyl salicylate, 3-carene, cis-3-hexen-1-ol, (E)-4-hexen-1-ol, and 3-methylbutyraldehyde can be used as reference indicators of the geographical distribution of tea plants. Furthermore, a metabolome genome-wide association study (mGWAS) revealed that 438 candidate genes were related to the aroma metabolic pathway. Further analysis showed that 31 genes of all the selected genes were screened and revealed the reasons for the genetic differences in aroma among tea plant cultivars in Fujian and Southern China. These results reveal the genetic diversity in the Fujian tea plants as well as a theoretical basis for the conservation, development, and utilization of the Fujian highly aromatic tea plant cultivars.

A Comprehensive Investigation of Macro-Composition and Volatile Compounds in Spring-Picked and Autumn-Picked White Tea.

The flavour of white tea can be influenced by the season in which the fresh leaves are picked. In this study, the sensory evaluation results indicated that spring-picked white tea (SPWT) was stronger than autumn-picked white tea (APWT) in terms of the taste of umami, smoothness, astringency, and thickness as well as the aromas of flower and fresh. To explore key factors of sensory differences, a combination of biochemical composition determination, widely targeted volatilomics (WTV) analysis, multivariate statistical analysis, and odour activity value (OAV) analysis was employed. The phytochemical analysis showed that the free amino acid, tea polyphenol, and caffeine contents of SPWTs were significantly higher than those of APWTs, which may explain the higher umami, smoothness, thickness, and astringency scores of SPWTs than those of APWTs. The sabinene, (2E, 4E)-2, 4-octadienal, (-)-cis-rose oxide, caramel furanone, trans-rose oxide, and rose oxide contents were significantly higher in SPWTs than in APWTs, which may result in stronger flowery, fresh, and sweet aromas in SPWTs than in APWTs. Among these, (2E,4E)-2,4-octadienal and (-)-cis-rose oxide can be identified as key volatiles. This study provides an objective and accurate basis for classifying SPWTs and APWTs at the metabolite level.

Non-Targeted Metabolomics Analysis Revealed the Characteristic Non-Volatile and Volatile Metabolites in the Rougui Wuyi Rock Tea (Camellia sinensis) from Different Culturing Regions.

Rougui Wuyi Rock tea (WRT) with special flavor can be affected by multiple factors that are closely related to the culturing regions of tea plants. The present research adopted non-targeted metabolomics based on liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), aroma activity value method (OAV), and chemometrics to analyze the characteristic metabolites of three Rougui WRTs from different culturing regions. The results of sensory evaluation showed that the three Rougui Wuyi Rock teas had significantly different flavor qualities, especially in taste and aroma. Rougui (RG) had a heavy and mellow taste, while cinnamon-like odor Rougui (GPRG) and floral and fruity odor Rougui (HGRG) had a thick, sweet, and fresh taste. The cinnamon-like odor was more obvious and persistent in GPRG than in RG and HGRG. HGRG had floral and fruity characteristics such as clean and lasting, gentle, and heavy, which was more obvious than in RG and GPRG. The results of principal component analysis (PCA) showed that there were significant metabolic differences among the three Rougui WRTs. According to the projection value of variable importance (VIP) of the partial least squares discriminant analysis (PLS-DA), 24 differential non-volatile metabolites were identified. The PLSR analysis results showed that rutin, silibinin, arginine, lysine, dihydrocapsaicin, etc. may be the characteristic non-volatiles that form the different taste outlines of Rougui WRT. A total of 90 volatiles, including aldehydes, alcohols, esters, and hydrocarbons, were identified from the three flavors of Rougui WRT by using GC-MS. Based on OAV values and PLS-DA analysis, a total of 16 characteristic volatiles were identified. The PLSR analysis results showed that 1-penten-3-ol, α-pinene, 2-carene, ß-Pinene, dehydrolinalool, adipaldehyde, D-limonene, saffron aldehyde, and 6-methyl-5-hepten-2-one may be the characteristic volatiles that form the different aroma profile of Rougui WRT. These results provide the theoretical basis for understanding the characteristic metabolites that contribute to the distinctive flavors of Rougui WRT.

High Albumin Level Is Associated With Regression of Glucose Metabolism Disorders Upon Resolution of Acute Liver Inflammation in Hepatitis B-Related Cirrhosis.

Background and Aim: To investigate the short-term dynamic changes and the factors associated with regression of glucose metabolism disorders in patients with hepatitis flare of chronic hepatitis B virus (HBV) infection. Methods: In this study, 118 patients with severe hepatitis flare of chronic HBV infection were prospectively studied. Oral glucose tolerance test was performed on admission and during follow-up to evaluate dynamic changes in glucose metabolism disorders. The factors associated with regression of glucose metabolism disorders were identified using univariate and multivariate logistic regression analyses. Results: The prevalence of diabetes was significantly higher in 70 (47.1%) patients with liver cirrhosis than that in 48 (16.8%) patients without liver cirrhosis. The prevalence of impaired glucose tolerance in patients with liver cirrhosis (35.7%) was significantly lower than that in patients without liver cirrhosis (47.8%). After a follow-up of 20.0 ± 18.7 days, 28 of 31 (90.3%) patients without liver cirrhosis experienced regression of glucose metabolism disorders. Additionally, 30 (54.5%) patients with liver cirrhosis experienced regression of glucose metabolism disorders after 42.0 ± 36.2 days. In patients with liver cirrhosis, those with regression of glucose metabolism disorders had significantly higher levels of homeostasis model assessment-ß-cell function, albumin (ALB), and a significantly lower level of fibrosis-4 score. ALB was identified as an independent factor associated with the regression of glucose metabolism disorders in patients with liver cirrhosis. Conclusion: Severe acute liver inflammation aggravates glucose metabolism disorders in patients with hepatitis B-related liver cirrhosis and high ALB level is associated with regression of glucose metabolism disorders upon resolution of acute liver inflammation.

Diabetes is associated with poor short-term prognosis in patients with hepatitis B virus-related acute-on-chronic liver failure.

BACKGROUND/PURPOSE: Associations between the disturbances in glucose homeostasis and prognosis in patients with hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) remain unclear. This study was conducted to investigate the clinical characteristics of disturbances in glucose homeostasis and their associations with 90-day mortality in patients with HBV-related ACLF. METHODS: Ninety-six patients with HBV-related ACLF without pre-existing diabetes were prospectively included. Glucose abnormalities were diagnosed based on fasting plasma glucose and oral glucose tolerance test results on admission and during follow-up. Homeostasis model assessment was used to establish insulin resistance (HOMA2-IR), insulin sensitivity (HOMA2-IS) and HOMA2-ß-cell function (HOMA2-ß). Multivariate Cox proportional hazards analysis was used to identify independent risk factors for death within 90 days after admission. RESULTS: Among 96 patients with ACLF, 51 (53.1%) had diabetes, 29 (30.2%) had impaired glucose tolerance (IGT), and 17 (17.7%) had hypoglycemia. Patients with diabetes had significantly lower levels of HOMA2-ß than did patients with normal glucose tolerance. Of 22 patients with diabetes or IGT and without anti-hyperglycemic treatment, 8 (36.4%) exhibited regression of their glucose metabolism disorders after a follow-up of 32.8 ± 28.8 days, and higher platelet levels were associated with regression. Twenty-five patients (25.0%) with ACLF died of liver failure within 90 days. Diabetes [odds ratio (OR) 3.601, 95% confidence interval (CI) 1.342-9.661] and age (OR 1.045, 95% CI 1.010-1.082) were the independent risk factors associated with mortality. CONCLUSION: Impaired pancreatic ß-cell function is related to diabetes development, and diabetes is associated with high mortality in patients with chronic HBV-related ACLF.

Genome-Wide Investigation of N6-Methyladenosine Regulatory Genes and Their Roles in Tea (Camellia sinensis) Leaves During Withering Process.

N6-methyladenosine (m6A), one of the internal modifications of RNA molecules, can directly influence RNA abundance and function without altering the nucleotide sequence, and plays a pivotal role in response to diverse environmental stresses. The precise m6A regulatory mechanism comprises three types of components, namely, m6A writers, erasers, and readers. To date, the research focusing on m6A regulatory genes in plant kingdom is still in its infancy. Here, a total of 34 m6A regulatory genes were identified from the chromosome-scale genome of tea plants. The expansion of m6A regulatory genes was driven mainly by whole-genome duplication (WGD) and segmental duplication, and the duplicated gene pairs evolved through purifying selection. Gene structure analysis revealed that the sequence variation contributed to the functional diversification of m6A regulatory genes. Expression pattern analysis showed that most m6A regulatory genes were differentially expressed under environmental stresses and tea-withering stage. These observations indicated that m6A regulatory genes play essential roles in response to environmental stresses and tea-withering stage. We also found that RNA methylation and DNA methylation formed a negative feedback by interacting with each other's methylation regulatory genes. This study provided a foundation for understanding the m6A-mediated regulatory mechanism in tea plants under environmental stresses and tea-withering stage.

Noninvasive assessment of liver fibrosis for predicting acute-on-chronic liver failure in patients with chronic hepatitis B.

BACKGROUND AND AIM: The aim of this study was to evaluate the accuracy of serum markers of liver fibrosis for predicting progression to acute-on-chronic liver failure (ACLF) in patients with acute exacerbation (AE) and severe AE (SAE) of chronic hepatitis B virus (HBV) infection. METHODS: The predictive accuracy of aminotransferase-to-platelet ratio index (APRI), Fibrosis-4, Lok index, and Forns index for progression to ACLF was evaluated via receiver operating characteristic (ROC) curve and area under the ROC (AUROC) in 441 and 130 patients with AE and SAE. RESULTS: After admission, 24 (5.8%) and 25 (19.2%) patients with AE and SAE, respectively, progressed to ACLF. The Lok index was one of the independent risk factors associated with progression to ACLF in patients with AE and SAE. The AUROC of Lok index for diagnosing liver cirrhosis was 0.815 (0.774-0.851) in patients with AE and 0.715 (0.629-0.791) in patients with SAE. The AUROC of Lok index for predicting progression to ACLF in patients with AE and SAE was 0.756 (0.711-0.797) and 0.866 (0.795-0.919), respectively. In patients with AE and SAE, the cut-off values of the Lok index for predicting ACLF were higher and lower, respectively, than those for diagnosing liver cirrhosis. CONCLUSION: The Lok index has predictive accuracy regarding progression to ACLF in patients with AE and SAE. Different thresholds of liver fibrosis are needed for determining progression to ACLF in patients with different severity of liver injury during acute exacerbation of chronic HBV infection.

Validation and Refinement of the Baveno VI Criteria for Ruling Out High-Risk Varices.

In the past decade, numerous studies have evaluated the roles of noninvasive methods for diagnosing or excluding varices and high-risk varices in patients with liver cirrhosis. The Baveno VI criteria recommend the use of a simple algorithm based on a liver stiffness measurement < 20 kPa through transient elastography and a platelet count > 150 × 109/L for ruling out high-risk varices in patients with compensated advanced chronic liver disease. A large number of studies have validated the clinical usefulness of Baveno VI criteria for excluding high-risk varices. Several strategies have been proposed to refine the Baveno VI criteria; however, currently there is no review to summarize the diagnostic accuracy and limitations of the Baveno VI criteria after extensive validation. In this review, we summarize the diagnostic accuracy and limitations of the Baveno VI criteria after extensive validation. We also discuss methods to refine these criteria.

Hepatocyte Endoplasmic Reticulum Stress Inhibits Hepatitis B Virus Secretion and Delays Intracellular Hepatitis B Virus Clearance After Entecavir Treatment.

Background: The aim of this study was to explore the effects of endoplasmic reticulum (ER) stress on hepatitis B virus (HBV) replication and the antiviral effect of entecavir (ETV). Methods: Thapsigargin (TG) and stearic acid (SA) were used to induce ER stress in HepG2.2.15 cells and HepAD38 cells that contained an integrated HBV genome, while ETV was used to inhibit HBV replication. The expression levels of glucose-regulated protein 78 (GRP78) and phosphorylated eukaryotic translation initiation factor 2 subunit alpha (p-eIF2α) were measured by western blotting. Intracellular HBV DNA was determined by qPCR; HBsAg by western blotting; HBV RNA by real-time RT-qPCR; HBsAg and HBeAg in supernatants by enzyme-linked immunosorbent assay (ELISA); and HBV DNA in supernatants by qPCR. Results: TG and SA induced ER stress in HepG2.2.15 cells and HepAD38 cells from 12 to 48 h post treatment. However, 4-phenylbutyric acid (PBA) partly alleviated the TG-induced ER stress. Moreover, TG inhibited HBsAg, HBeAg, and HBV DNA secretion from 12 to 48 h, while different concentrations of SA inhibited HBsAg and HBV DNA secretion at 48 h. TG promoted intracellular HBV DNA and HBsAg accumulation and the transcription of the HBV 3.5-kb mRNA and S mRNA. PBA treatment restored the secretion of HBsAg and HBV DNA. Finally, ER stress accelerated extracellular HBV DNA clearance but delayed intracellular HBV DNA clearance after ETV treatment. Conclusions: Hepatocyte ER stress promoted intracellular HBV DNA and HBsAg accumulation by inhibiting their secretion. Our study also suggested that hepatocyte ER stress delayed intracellular HBV DNA clearance after ETV treatment.

Liver stiffness and serum markers for excluding high-risk varices in patients who do not meet Baveno VI criteria.

BACKGROUND: The Baveno VI criteria for predicting esophageal varices, i.e., liver stiffness measurement (LSM) < 20 kPa and platelet (PLT) count > 150 × 109/L, identify patients who can safely avoid gastroscopy screening. However, they require further refinement. AIM: To evaluate the utility of LSM and serum markers of liver fibrosis in ruling out high-risk varices (HRV) in patients who do not meet Baveno VI criteria. METHODS: Data from 132 patients with hepatitis B virus (HBV)-related compensated liver cirrhosis who did not meet the Baveno VI criteria were retrospectively reviewed. MedCalc 15.8 was used to calculate receiver operating characteristic (ROC) curves, and the accuracy of LSM, PLT count, aspartate aminotransferase (AST)-to-PLT ratio index, Fibrosis-4, and the Lok index in predicting HRV were evaluated according to the area under each ROC curve (AUROC). The utility of LSM, PLT, and serum markers of liver fibrosis stratified by alanine transaminase (ALT) and total bilirubin (TBil) levels was evaluated for ruling out HRV. RESULTS: In all patients who did not meet the Baveno VI criteria, the independent risk factors for HRV were LSM and ALT. Only the AUROC of Lok index was above 0.7 for predicting HRV, and at a cutoff value of 0.4531 it could further spare 24.2% of gastroscopies without missing HRVs. The prevalence of HRV was significantly lower in patients with ALT or TBil ≥ 2 upper limit of normal (ULN) (14.3%) than in patients with both ALT and TBil < 2 ULN (34.1%) (P = 0.018). In the 41 patients with ALT and TBil < 2 ULN, LSM had an AUROC for predicting HRV of 0.821. LSM < 20.6 kPa spared 39.0% of gastroscopies without missing HRVs. In the 91 patients with ALT or TBiL ≥ 2 ULN, the Lok index and PLT had AUROCs of 0.814 and 0.741, respectively. Lok index ≤ 0.5596 or PLT > 100 × 109/L further spared 39.6% and 43.9% of gastroscopies, respectively, without missing HRVs. CONCLUSION: In HBV-related compensated cirrhosis patients who do not meet Baveno VI criteria, the LSM, PLT, or Lok index cutoff stratified by ALT and TBil accurately identifies more patients without HRV.