A serine carboxypeptidase-like acyltransferase catalyzes consecutive four-step reactions of hydrolyzable tannin biosynthesis in Camellia oleifera.

Hydrolyzable tannins (HTs), a class of polyphenolic compounds found in dicotyledonous plants, are widely used in food and pharmaceutical industries because of their beneficial effects on human health. Although the biosynthesis of simple HTs has been verified at the enzymatic level, relevant genes have not yet been identified. Here, based on the parent ion-fragment ion pairs in the feature fragment data obtained using UPLC-Q-TOF-/MS/MS, galloyl phenolic compounds in the leaves of Camellia sinensis and C. oleifera were analyzed qualitatively and quantitatively. Correlation analysis between the transcript abundance of serine carboxypeptidase-like acyltransferases (SCPL-ATs) and the peak area of galloyl products in Camellia species showed that SCPL3 expression was highly correlated with HT biosynthesis. Enzymatic verification of the recombinant protein showed that CoSCPL3 from C. oleifera catalyzed the four consecutive steps involved in the conversion of digalloylglucose to pentagalloylglucose. We also identified the residues affecting the enzymatic activity of CoSCPL3 and determined that SCPL-AT catalyzes the synthesis of galloyl glycosides. The findings of this study provide a target gene for germplasm innovation of important cash crops that are rich in HTs, such as C. oleifera, strawberry, and walnut.

A flavonoid metabolon: cytochrome b5 enhances B-ring trihydroxylated flavan-3-ols synthesis in tea plants.

Flavan-3-ols are prominent phenolic compounds found abundantly in the young leaves of tea plants. The enzymes involved in flavan-3-ol biosynthesis in tea plants have been extensively investigated. However, the localization and associations of these numerous functional enzymes within cells have been largely neglected. In this study, we aimed to investigate the synthesis of flavan-3-ols in tea plants, particularly focusing on epigallocatechin gallate. Our analysis involving the DESI-MSI method to reveal a distinct distribution pattern of B-ring trihydroxylated flavonoids, primarily concentrated in the outer layer of buds. Subcellular localization showed that CsC4H, CsF3'H, and CsF3'5'H localizes endoplasmic reticulum. Protein-protein interaction studies demonstrated direct associations between CsC4H, CsF3'H, and cytoplasmic enzymes (CHS, CHI, F3H, DFR, FLS, and ANR), highlighting their interactions within the biosynthetic pathway. Notably, CsF3'5'H, the enzyme for B-ring trihydroxylation, did not directly interact with other enzymes. We identified cytochrome b5 isoform C serving as an essential redox partner, ensuring the proper functioning of CsF3'5'H. Our findings suggest the existence of distinct modules governing the synthesis of different B-ring hydroxylation compounds. This study provides valuable insights into the mechanisms underlying flavonoid diversity and efficient synthesis and enhances our understanding of the substantial accumulation of B-ring trihydroxylated flavan-3-ols in tea plants.

New insights into the function of plant tannase with promiscuous acyltransferase activity.

Plant tannases (TAs) or tannin acyl hydrolases, a class of recently reported carboxylesterases in tannin-rich plants, are involved in the degalloylation of two important groups of secondary metabolites: flavan-3-ol gallates and hydrolyzable tannins. In this paper, we have made new progress in studying the function of tea (Camellia sinensis) (Cs) TA-it is a hydrolase with promiscuous acyltransferase activity in vitro and in vivo and promotes the synthesis of simple galloyl glucoses and flavan-3-ol gallates in plants. We studied the functions of CsTA through enzyme analysis, protein mass spectrometry, and metabolic analysis of genetically modified plants. Firstly, CsTA was found to be not only a hydrolase but also an acyltransferase. In the two-step catalytic reaction where CsTA hydrolyzes the galloylated compounds epigallocatechin-3-gallate or 1,2,3,4,6-penta-O-galloyl-ß-d-glucose into their degalloylated forms, a long-lived covalently bound Ser159-linked galloyl-enzyme intermediate is also formed. Under nucleophilic attack, the galloyl group on the intermediate is transferred to the nucleophilic acyl acceptor (such as water, methanol, flavan-3-ols, and simple galloyl glucoses). Then, metabolic analysis suggested that transient overexpression of TAs in young strawberry (Fragaria × ananassa) fruits, young leaves of tea plants, and young leaves of Chinese bayberry (Myrica rubra) actually increased the total contents of simple galloyl glucoses and flavan-3-ol gallates. Overall, these findings provide new insights into the promiscuous acyltransferase activity of plant TA.

The mechanism of RGS5 regulating gastric cancer mismatch repair protein.

The incidence and mortality rates of gastric cancer (GC) remain alarmingly high worldwide, imposing a substantial healthcare burden. In this study, we utilized data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. A 4-gene prognostic model was developed to predict patient prognosis, and its accuracy was validated across multiple datasets. Patients with a low-risk score exhibited improved prognosis, elevated tumor mutation burden, heightened sensitivity to both immunotherapy and conventional chemotherapy. Notably, our investigation revealed that the key gene RGS5 positively modulates the expression of mismatch repair proteins via c-Myc. Furthermore, co-immunoprecipitation (COIP) assays demonstrated the interaction between RGS5 and c-Myc. Additionally, we confirmed that RGS5 regulates c-Myc through the ubiquitin-proteasome pathway. Moreover, RGS5 was identified as a positive regulator of PD-L1 expression and exhibited a negative correlation with the majority of immune cells. These findings underscore the potential of RGS5 as a novel biomarker and therapeutic target in the context of GC.

Deep learning and targeted metabolomics-based monitoring of chewing insects in tea plants and screening defense compounds.

Tea is an important cash crop that is often consumed by chewing pests, resulting in reduced yields and economic losses. It is important to establish a method to quickly identify the degree of damage to tea plants caused by leaf-eating insects and screen green control compounds. This study was performed through the combination of deep learning and targeted metabolomics, in vitro feeding experiment, enzymic analysis and transient genetic transformation. A small target damage detection model based on YOLOv5 with Transformer Prediction Head (TPH-YOLOv5) algorithm for the tea canopy level was established. Orthogonal partial least squares (OPLS) was used to analyze the correlation between the degree of damage and the phenolic metabolites. A potential defensive compound, (-)-epicatechin-3-O-caffeoate (EC-CA), was screened. In vitro feeding experiments showed that compared with EC and epicatechin gallate, Ectropis grisescens exhibited more significant antifeeding against EC-CA. In vitro enzymatic experiments showed that the hydroxycinnamoyl transferase (CsHCTs) recombinant protein has substrate promiscuity and can catalyze the synthesis of EC-CA. Transient overexpression of CsHCTs in tea leaves effectively reduced the degree of damage to tea leaves. This study provides important reference values and application prospects for the effective monitoring of pests in tea gardens and screening of green chemical control substances.

Aberration of social behavior and gut microbiota induced by cross-fostering implicating the gut-brain axis.

The gut microbiota and neurological development of neonatal mice are susceptible to environmental factors that may lead to altered behavior into adulthood. However, the role that changed gut microbiota and neurodevelopment early in life play in this needs to be clarified. In this study, by modeling early-life environmental changes by cross-fostering BALB/c mice, we revealed the effects of the environment during the critical period of postnatal development on adult social behavior and their relationship with the gut microbiota and the nervous system. The neural projections exist between the ascending colon and oxytocin neurons in the paraventricular nuclei (PVN), peripheral oxytocin levels and PVN neuron numbers decreased after cross-fostering, and sex-specific alteration in gut microbiota and its metabolites may be involved in social impairments and immune imbalances brought by cross-fostering via the gut-brain axis. Our findings also suggest that social cognitive impairment may result from a combination of PVN oxytocinergic neurons, gut microbiota, and metabolites.

Oxytocin neurons in the paraventricular nucleus and fear empathy among male mice.

BACKGROUND: Recent studies have identified empathy deficit as a core impairment and diagnostic criterion for people with autism spectrum disorders; however, the improvement of empathy focuses primarily on behavioural interventions without the target regulation. We sought to compare brain regions associated with empathy-like behaviours of fear and pain, and to explore the role of the oxytocin-oxytocin receptor system in fear empathy. METHODS: We used C57BL mice to establish 2 models of fear empathy and pain empathy. We employed immunofluorescence histochemical techniques to observe the expression of c-Fos throughout the entire brain and subsequently quantified the number of c-Fos-positive cells in different brain regions. Furthermore, we employed chemogenetic technology to selectively manipulate these neurons in Oxt-Cre-/+ mice to identify the role of oxytocin in this process. RESULTS: The regions activated by fear empathy were the anterior cingulate cortex, basolateral amygdala, nucleus accumbens, paraventricular nucleus (PVN), lateral habenula, and ventral and dorsal hippocampus. The regions activated by pain empathy were the anterior cingulate cortex, basolateral amygdala, nucleus accumbens, and lateral habenula. We found that increasing the activity of oxytocin neurons in the PVN region enhanced the response to fear empathy. This enhancement may be mediated through oxytocin receptors. LIMITATIONS: This study included only male animals, which restricts the broader interpretation of the findings. Further investigations on circuit function need to be conducted. CONCLUSION: The brain regions implicated in the regulation of fear and pain empathy exhibit distinctions; the activity of PVN neurons was positively correlated with empathic behaviour in mice. These findings highlight the role of the PVN oxytocin pathway in regulating fear empathy and suggest the importance of oxytocin signalling in mediating empathetic responses.

An adaptive spacing of root-zone hole fertilization to improve production and fertilizer utilization of rapeseed.

BACKGROUND: Root-zone hole fertilization has a positive impact on enhancing crop production and fertilization efficiency. However, a suitable spacing for hole fertilization in rapeseed cultivation is unclear. To explore an adaptive hole spacing for improving rapeseed yield and fertilization efficiency, field experiments were conducted. Four spacings of hole fertilization were designed: 10 (FD10), 20 (FD20), 30 (FD30) and 40 cm (FD40), using no fertilization (F0) and deep-banded placement of fertilizer (DBP) as controls. The burial depth was 10 cm for FD and DBP treatments. RESULTS: Compared to DBP, hole fertilization impacted soil microenvironment, crop growth and yield components, resulting in a significant increase of 28.4% in seed yield and 25.6% in oil yield. Seed yield in FD20 (4345.43 kg ha-1) increased by 4.3%, 9.4% and 15.1% compared to FD10, FD30 and FD40, respectively. Fertilizer partial factor productivity under FD20 was 4.2%, 8.6% and 13.9% greater than FD10, FD30 and FD40, respectively; whereas the increase for agronomic efficiency was 6.0%, 12.7% and 21.0%, and the increase for N recovery efficiency was 39.5%, 52.5% and 62.9%, respectively. CONCLUSION: Fertilization with a hole spacing of 17 cm is a promising practice to maintain high production and fertilization efficiency when cultivating rapeseed. These results provide a theoretical foundation and scientific basis for improving rapeseed productivity and fertilizer utilization. © 2024 Society of Chemical Industry.

Effects of an inoculation dose of Issatchenkia terricola WJL-G4 on physicochemical properties, active substances, and antioxidant capacity of black, red, and white currant juice.

BACKGROUND: Due to the high level of organic acids - primarily citric acid - black, red, and white currants have an excessively sour taste, making taste adjustment during processing challenging. This study investigated and evaluated the effects of an inoculation dose of the acid-reducing yeast Issatchenkia terricola WJL-G4 on several aspect such as physicochemical properties, chromaticity, active substances, and antioxidant capacity. A sensory evaluation was also conducted. RESULTS: The results indicated that, when the inoculation dose increased from 2% to 12%, the total phenol, total flavonoid, and total anthocyanin content, and antioxidant capacity in currant juice decreased. A low inoculation dose (2-4%) was beneficial for preserving the total phenol and total flavonoid content. Although the levels of most phenolic compounds decreased, the concentrations of caffeic acid, p-coumaric acid, ferulic acid, rutin, and epicatechin were significantly higher than the control after fermentation. Overall acceptability and taste scores of fermented currants improved compared with those of the control group. CONCLUSION: This experiment provided an effective solution, with a theoretical basis, to the problems of the sour taste and harsh flavor of currant juice. © 2024 Society of Chemical Industry.

Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs.

Serine carboxypeptidase-like acyltransferases (SCPL-ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan-3-ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL-AT paralogs are involved in galloylation of flavan-3-ols: CsSCPL4, which contains the conserved catalytic triad S-D-H, and CsSCPL5, which has the alternative triad T-D-Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non-catalytic companion paralog (NCCP). Co-expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull-down and co-immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post-translational processing. Moreover, phylogenetic analyses revealed that their homologs co-exist in galloylated flavan-3-ol- or hydrolyzable tannin-rich plant species. Enzymatic assays further revealed the necessity of co-expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co-expression of SCPL-ATs and their NCCPs contributes to the acylation of flavan-3-ols in the plant kingdom.

AHSG, a Gene Promoting Tumour Proliferation, Migration and Invasion, is an Independent Prognostic Factor for Poor Overall Survival in Lung Adenocarcinoma.

OBJECTIVE: Recent studies have shown that the metabolic process-related gene AHSG is involved in multiple pathological processes of tumours. This study will explore the relationship between AHSG and lung adenocarcinoma. MATERIALS AND METHODS: Expression analysis, survival analysis and co-expression analysis of AHSG were performed using a public database, and cytological and molecular biology assays were performed to explore the role of AHSG in lung adenocarcinoma. RESULT: Compared with normal tissues, AHSG expression was significantly higher in cancer tissues in the TCGA-LUAD database, and pan-cancer analysis revealed abnormal AHSG expression in different kinds of tumours. Survival analysis revealed that compared with the low expression group, the patients in the high expression group had a significantly worse overall survival duration in the TCGA-LUAD database, and a subsequent study confirmed that AHSG expression could be an independent prognostic factor of overall survival in lung adenocarcinoma. AHSG-related genes are involved in multiple physiological and pathophysiological pathways. In subsequent cytological and molecular biology experiments, inhibition of AHSG expression suppressed proliferation, migration and invasion in lung adenocarcinoma cell lines, and the EMT process was blocked after knockdown of AHSG. CONCLUSION: AHSG could be used as a prognostic factor for OS in patients with lung adenocarcinoma. It can promote the biological behaviour of lung adenocarcinoma and may become a potential target for treatment, which is worthy of further study.

Clinical value of serum soluble receptor for advanced glycation end products in evaluating the condition of patients with bronchial asthma.

Objective: This study focused on the clinical value of serum soluble receptor for advanced glycation end products (sRAGE) levels in evaluating the severity of bronchial asthma (BA). Methods: Serum sRAGE expression was measured by using enzyme-linked immunosorbent assay, eosinophils (EOS) count was measured by using an automatic blood cell counter, and forced expiratory volume in 1 second (FEV1) was measured by pulmonary function analyzer in 120 patients with BA, 40 patients with non-BA pulmonary disease, and 40 healthy controls. Receiver operating characteristic curves were used to analyze the clinical value of sRAGE expression levels, EOS counts, and FEV1 level to assess the severity of illness in the patients with BA. Results: Compared with the healthy controls and the patients without BA, the patients with BA had the lowest serum sRAGE expression level (47.36 ± 6.3 ng/L versus 75.3 ± 6.3 ng/L versus 67.5 ± 5.06 ng/L; p < 0.05), the highest EOS count (231.2 ± 18.3 106/L versus 175.9 ± 15.6 106/L versus 197.8 ± 19.6 106/L; p < 0.05), and the lowest FEV1 level (1.19 ± 0.15 L versus 1.57 ± 0.2 L versus 1.3 ± 0.17 L; p < 0.05). Correlation analysis revealed that the serum sRAGE expression levels were notably negatively correlated with the EOS counts (r value of -0.471, p < 0.05) but significantly positively linked to FEV1 levels (r value of 0.362, p < 0.05). Serum sRAGE expression levels could help in accurately diagnosing patients with severe BA (area under the receiver operating characteristic curve (AUC) = 0.904), whereas prediction in the patients with mild BA was achieved by EOS counts (AUC = 0.857). Conclusion: The serum sRAGE level has potential value in diagnosing the severity of BA, which is conducive to identifying patients with severe BA and guiding in development of new therapeutic strategies.

Leaf spot disease on Juglans regia caused by Ragnhildiana diffusa in China.

Juglans regia L. is commercially important for its edible nuts, which is a major species of walnut trees in Sichuan Province (Luo et al. 2020). In September 2021, brown leaf spot symptoms were observed on roughly 75% of 60 J. regia trees surveyed in an orchard of Chongzhou city (30°40'6''N, 103°40'18''E). Initially, the lesions measuring 2-10 mm were reddish to brown with a yellowish halo, then increased in size and coalesced to cover the whole leaf, eventually resulting in severe defoliation. Six symptomatic leaves from different trees were collected, and a single fungal isolate was obtained from each of the sampled leaves using single-spore isolation (Chomnunti et al. 2014). The isolates were incubated on potato dextrose agar (PDA) with a 12h photoperiod at 25 ℃, and deposited at the Culture Collection of Sichuan Agricultural University. Colonies were identical with black center and reddish-brown periphery, and the diameter reached 2 cm after 7 days. On the host, conidiophores were mostly reduced to conidiogenous cells, with prominent and thickened conidiogenous loci. Conidia were light green to light brown, and curved with a thickened and darked hilum at the base, 0-17 septate, tapering toward the distal end, and measuring 20-120 × 3-5 µm ((x ) ̅= 56 × 4, n = 30). Morphological characteristics fit the description of Ragnhildiana diffusa (Heald & F.A. Wolf) Videira & Crous (Synonym: Sirosporium diffusum (Heald & F. A. Wolf) Deighton) (Poletto et al. 2017). The internal transcribed spacer (ITS) region, the large subunit of the nrDNA (LSU), and RNA polymerase II second largest subunit (rpb2) were amplified by polymerase chain reaction and sequenced with primers ITS5/ITS4 (White et al. 1990), LR0R/LR5 (Vilgalys & Hester 1990), fRPB2-5F/Rpb2-R3 (Liu et al. 1999, Videira et al. 2017), respectively. The nucleotide blast of the two isolates (SICAUCC 22-0077, SICAUCC 22-0078) showed 99.7% and 99.5% (ITS, 472/473 bp, 471/473 bp), 100% (LSU, 725/725 bp, 725/725 bp), 99.8% (rpb2, 866/867 bp, 866/867 bp) identities with the ex-type strain of Ragnhildiana diffusa (CBS 106.14). The phylogenetic tree combined with ITS, LSU, and rpb2 genes and morphological characteristics confirmed the identification as R. diffusa. These sequences of the three gene regions of two isolates were deposited in GenBank with accession numbers ON409525 and ON409526 (ITS), ON409559 and ON409560 (LSU), ON417473 and ON417474 (rpb2), respectively. The isolate SICAUCC 22-0077 was used for pathogenicity test to fulfill Koch's postulates. Three leaves of each walnut seedlings (2-year-old seedlings) were inoculated by placing a mycelium plug onto fresh wounds on the upper leaf surface punctured via a fine needle (0.7 mm in diameter), and three replicate seedlings were inoculated. For the control, a sterile PDA plug was placed on the same number of replicate leaves on the plants. The inoculated and control plants were placed in a growth chamber at 25°C with relative humidity >80% and a 12-h photoperiod. Irregular light to dark brown spots developed on inoculated leaves after twenty days, and no symptoms were observed on controls. The re-isolation and examination of the fungus showed it to be morphologically and phylogenetically identical to the originally isolated pathogen. R. diffusa has been described on J. regia in Mexico (Farr & Rossman 2022). To our knowledge, this is the first report of R. diffusa causing brown leaf spot on J. regia in China. The identification of the pathogen will provide a basis for disease management in walnut planting areas.

Fertilization depth effect on mechanized direct-seeded winter rapeseed yield and fertilizer use efficiency.

BACKGROUND: Deep fertilization is effective for improving crop yield and fertilizer use efficiency. However, its impact on mechanized direct-seeded rapeseed and the optimal fertilization depth are poorly understood. A field experiment was conducted to evaluate the fertilization depth effect on mechanized direct-seeded rapeseed growth. Five treatments were designed: surface broadcast fertilizer, no fertilization, and fertilizer banded placement at soil depths of 5 (D5), 10 (D10), and 15 cm (D15). RESULTS: Compared with surface broadcast fertilizer, deep fertilization generally increased seed yield and partial factor productivity by 11.0%, agronomic efficiency (AE) by 22.7%, and recovery efficiency (RE) by 79.2% due to the increase of root mass density (16.8%), plant height (8.6%), height of the first branch (10.6%), stem diameter (22.4%), shoot biomass (16.1%), and shoot nitrogen (35.7%), phosphorus (29.7%), and potassium (26.2%) uptake. D10 had the highest seed yield, oil yield, fertilizer use efficiency, and economic benefits at different fertilization depth treatments. Compared with D5 and D15 respectively, D10 increased seed yield by 5.4% and 46.0%, oil yield by 7.7% and 50.5%, partial factor productivity by 5.4% and 46.0%, AE by 9.0% and 99.5%, RE of nitrogen by 48.9% and 34.9%, RE of phosphorus by 83.1% and 38.0%, and RE of potassium by 57.5% and 32.5%. The economic benefits of D10 were CNY 867.31 ha-1 and CNY 4864.23 ha-1 higher than D5 and D15 respectively. CONCLUSION: Considering rapeseed growth and its economic benefits, this study shows that 10 cm is an appropriate placement depth with regard to mechanized direct-seeded winter rapeseed production. © 2022 Society of Chemical Industry.

Ba-modified peanut shell biochar (PSB): preparation and adsorption of Pb(II) from water.

The impact of Ba-modified peanut shell biochar (Ba-PSB) on Pb(II) removal was studied and BaCl2 was used as a modifier. It was shown that the PSB obtained at 750 °C had the best adsorption effect, and the Ba-PSB had a larger specific surface area and a good adsorption effect on Pb(II). At pH = 5, concentration was 400 mg/L, time was 14 h, and temperature was 55 °C, the loading amount of black peanut shell biochar (BPSB), red peanut shell biochar (RPSB), Ba-BPSB, and Ba-RPSB reached 128.050, 98.217, 379.330, and 364.910 mg/g, respectively. In addition, based on the non-linear fitting, it was found that the quasi-second-order kinetic model, and isothermal model could be applied to describe Pb(II) adsorption on PSB and Ba-PSB. The adsorption behavior of PSB unmodified and modified was a spontaneous process. Moreover, chemical modification of BPSB, RPSB, Ba-BPSB, and Ba-RPSB for hindering of -COOH and -OH groups revealed 81.81, 77.08, 86.90, and 83.65% removal of Pb(II), respectively, which was due to the participation of -COOH, while 17.61, 21.70, 12.77, and 15.06% was from -OH group, respectively. The increase of cation strength (Na+, K+, Ca2+, and Mg2+) will reduce the adsorption capacity of PSB for Pb(II).

The chromosome-scale reference genome of Rubus chingii Hu provides insight into the biosynthetic pathway of hydrolyzable tannins.

Rubus chingii Hu (Fu-Pen-Zi), a perennial woody plant in the Rosaceae family, is a characteristic traditional Chinese medicinal plant because of its unique pharmacological effects. There are abundant hydrolyzable tannin (HT) components in R. chingii that provide health benefits. Here, an R. chingii chromosome-scale genome and related functional analysis provide insights into the biosynthetic pathway of HTs. In total, sequence data of 231.21 Mb (155 scaffolds with an N50 of 8.2 Mb) were assembled into seven chromosomes with an average length of 31.4 Mb, and 33 130 protein-coding genes were predicted, 89.28% of which were functionally annotated. Evolutionary analysis showed that R. chingii was most closely related to Rubus occidentalis, from which it was predicted to have diverged 22.46 million years ago (Table S8). Comparative genomic analysis showed that there was a tandem gene cluster of UGT, carboxylesterase (CXE) and SCPL genes on chromosome 02 of R. chingii, including 11 CXE, eight UGT, and six SCPL genes, which may be critical for the synthesis of HTs. In vitro enzyme assays indicated that the proteins encoded by the CXE (LG02.4273) and UGT (LG02.4102) genes have tannin hydrolase and gallic acid glycosyltransferase functions, respectively. The genomic sequence of R. chingii will be a valuable resource for comparative genomic analysis within the Rosaceae family and will be useful for understanding the biosynthesis of HTs.

Molecular and biochemical characterization of two 4-coumarate: CoA ligase genes in tea plant (Camellia sinensis).

KEY MESSAGE: Two 4-coumarate: CoA ligase genes in tea plant involved in phenylpropanoids biosynthesis and response to environmental stresses. Tea plant is rich in flavonoids benefiting human health. Lignin is essential for tea plant growth. Both flavonoids and lignin defend plants from stresses. The biosynthesis of lignin and flavonoids shares a key intermediate, 4-coumaroyl-CoA, which is formed from 4-coumaric acid catalyzed by 4-coumaric acid: CoA ligase (4CL). Herein, we report two 4CL paralogs from tea plant, Cs4CL1 and Cs4CL2, which are a member of class I and II of this gene family, respectively. Cs4CL1 was mainly expressed in roots and stems, while Cs4CL2 was mainly expressed in leaves. The promoter of Cs4CL1 had AC, nine types of light sensitive (LSE), four types of stress-inducible (SIE), and two types of meristem-specific elements (MSE). The promoter of Cs4CL2 also had AC and nine types of LSEs, but only had two types of SIEs and did not have MSEs. In addition, the LSEs varied in the two promoters. Based on the different features of regulatory elements, three stress treatments were tested to understand their expression responses to different conditions. The resulting data indicated that the expression of Cs4CL1 was sensitive to mechanical wounding, while the expression of Cs4CL2 was UV-B-inducible. Enzymatic assays showed that both recombinant Cs4CL1 and Cs4CL2 transformed 4-coumaric acid (CM), ferulic acid (FR), and caffeic acid (CF) to their corresponding CoA ethers. Kinetic analysis indicated that the recombinant Cs4CL1 preferred to catalyze CF, while the recombinant Cs4CL2 favored to catalyze CM. The overexpression of both Cs4CL1 and Cs4CL2 increased the levels of chlorogenic acid and total lignin in transgenic tobacco seedlings. In addition, the overexpression of Cs4CL2 consistently increased the levels of three flavonoid compounds. These findings indicate the differences of Cs4CL1 and Cs4CL2 in the phenylpropanoid metabolism.

The Functional Characterization of Carboxylesterases Involved in the Degradation of Volatile Esters Produced in Strawberry Fruits.

Volatile ester compounds are important contributors to the flavor of strawberry, which affect consumer preference. Here, the GC-MS results showed that volatile esters are the basic aroma components of strawberry, banana, apple, pear, and peach, and the volatile esters were significantly accumulated with the maturation of strawberry fruits. The main purpose of this study is to discuss the relationship between carboxylesterases (CXEs) and the accumulation of volatile ester components in strawberries. FaCXE2 and FaCXE3 were found to have the activity of hydrolyzing hexyl acetate, Z-3-hexenyl acetate, and E-2-hexenyl acetate to the corresponding alcohols. The enzyme kinetics results showed that FaCXE3 had the higher affinity for hexyl acetate, E-2-hexenyl acetate, and Z-3-hexenyl acetate compared with FaCXE2. The volatile esters were mainly accumulated at the maturity stages in strawberry fruits, less at the early stages, and the least during the following maturation stages. The expression of FaCXE2 gradually increased with fruit ripening and the expression level of FaCXE3 showed a decreasing trend, which suggested the complexity of the true function of CXEs. The transient expression of FaCXE2 and FaCXE3 genes in strawberry fruits resulted in a significantly decreased content of volatile esters, such as Z-3-hexenyl acetate, methyl hexanoate, methyl butyrate, and other volatile esters. Taken together, FaCXE2 and FaCXE3 are indeed involved in the regulation of the synthesis and degradation of strawberry volatile esters.

Functional demonstration of plant flavonoid carbocations proposed to be involved in the biosynthesis of proanthocyanidins.

The plant flavonoid dogma proposes that labile plant flavonoid carbocations (PFCs) play vital roles in the biosynthesis of proanthocyanidins (PAs). However, whether PFCs exist in plants and how PFCs function remain unclear. Here, we report the use of an integrative strategy including enzymatic assays, mutant analysis, metabolic engineering, isotope labeling and metabolic profiling to capture PFCs and demonstrate their functions. In anthocyanidin reductase (ANR) assays, an (-)-epicatechin conjugate was captured in protic polar nucleophilic methanol alone or methanol-HCl extracts. Tandem mass spectrum (MS/MS) analysis characterized this compound as an (-)-epicatechin-4-O-methyl (EOM) ether, which resulted from (-)-epicatechin carbocation and the methyl group of methanol. Acid-based catalysis of procyanidin B2 and B3 produced four compounds, which were annotated as two EOM and two (+)-catechin-4-O-methyl (COM) ethers. Metabolic profiling of seven PA pathway mutants showed an absence or reduction of two EOM ether isomers in seeds. Camellia sinensis ANRa (CsANRa), leucoanthocyanidin reductase c (CsLARc), and CsMYB5b (a transcription factor) were independently overexpressed for successful PA engineering in tobacco. The EOM ether was remarkably increased in CsANRa and CsMYB5b transgenic flowers. Further metabolic profiling for eight green tea tissues revealed two EOM and two COM ethers associated with PA biosynthesis. Moreover, an incubation of (-)-epicatechin or (+)-catechin with epicatechin carbocation in CsANRa transgenic flower extracts formed dimeric procyanidin B1 or B2, demonstrating the role of flavan-3-ol carbocation in the formation of PAs. Taken together, these findings indicated that flavan-3-ol carbocations exist in extracts and are involved in the biosynthesis of PAs of plants.

Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality.

Tea, one of the world's most important beverage crops, provides numerous secondary metabolites that account for its rich taste and health benefits. Here we present a high-quality sequence of the genome of tea, Camellia sinensis var. sinensis (CSS), using both Illumina and PacBio sequencing technologies. At least 64% of the 3.1-Gb genome assembly consists of repetitive sequences, and the rest yields 33,932 high-confidence predictions of encoded proteins. Divergence between two major lineages, CSS and Camellia sinensis var. assamica (CSA), is calculated to ∼0.38 to 1.54 million years ago (Mya). Analysis of genic collinearity reveals that the tea genome is the product of two rounds of whole-genome duplications (WGDs) that occurred ∼30 to 40 and ∼90 to 100 Mya. We provide evidence that these WGD events, and subsequent paralogous duplications, had major impacts on the copy numbers of secondary metabolite genes, particularly genes critical to producing three key quality compounds: catechins, theanine, and caffeine. Analyses of transcriptome and phytochemistry data show that amplification and transcriptional divergence of genes encoding a large acyltransferase family and leucoanthocyanidin reductases are associated with the characteristic young leaf accumulation of monomeric galloylated catechins in tea, while functional divergence of a single member of the glutamine synthetase gene family yielded theanine synthetase. This genome sequence will facilitate understanding of tea genome evolution and tea metabolite pathways, and will promote germplasm utilization for breeding improved tea varieties.