Gene mining and genomics-assisted breeding empowered by the pangenome of tea plant Camellia sinensis.

Tea is one of the world's oldest crops and is cultivated to produce beverages with various flavours. Despite advances in sequencing technologies, the genetic mechanisms underlying key agronomic traits of tea remain unclear. In this study, we present a high-quality pangenome of 22 elite cultivars, representing broad genetic diversity in the species. Our analysis reveals that a recent long terminal repeat burst contributed nearly 20% of gene copies, introducing functional genetic variants that affect phenotypes such as leaf colour. Our graphical pangenome improves the efficiency of genome-wide association studies and allows the identification of key genes controlling bud flush timing. We also identified strong correlations between allelic variants and flavour-related chemistries. These findings deepen our understanding of the genetic basis of tea quality and provide valuable genomic resources to facilitate its genomics-assisted breeding.

Identification of key volatile and odor-active compounds in 10 main fragrance types of Fenghuang Dancong tea using HS-SPME/GC-MS combined with multivariate analysis.

Fenghuang Dancong tea (FHDC), a famous oolong tea originating from Guangdong Province in China, is known for its rich and unique fragrance. Nevertheless, the identification of the key aroma compounds with the difference fragrance types of FHDC remains uncertain. In order to characteristic the volatile components in different fragrance types of FHDC, 10 well-known fragrance types of FHDC and Tieguanyin (TGY) as a control were analyzed by headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography mass spectrometry (GC-MS). Results indicated that 172 volatile compounds were identified as common volatile compounds among all the tea samples. A total of 16 compounds were identified as key compounds that could be used to distinguish between FHDC and TGY. Among the 10 FHDC fragrance types, indole, hotrienol, benzyl nitrile, and jasmine lactone were found to be the most abundant compounds. Despite the presence of certain similarities in aroma components, each type exhibits unique fragrance characteristics as a result of variation in compound composition content and proportion. Furthermore, using statistical and odor activity value analysis, 20 aroma-active compounds were recognized as potential characteristic markers accountable for the diverse fragrance types of FHDC. This research enhances our comprehension of the various fragrance types of FHDC and provides reference values for their rapid identification in the market.

Characterization of the Aroma Profiles of Guangdong Black Teas Using Non-Targeted Metabolomics.

Guangdong black teas have diverse flavors and aromas. To explore the molecular basis of these aromas, we extracted and analyzed the volatile flavor compounds of 31 black tea samples from 7 districts (Yingde, Luokeng, Renhua, Meizhou, Chaozhou, Lianshan, and Heyuan) in Guangdong Province with headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). Then, 135 volatile flavor compounds (VFCs) were identified and grouped into 12 classes according to their chemical structure. Notably, alcohols accounted for 31.40-44.43% of total VFCs. The score plot of supervised partial least squares-discriminant analysis (PLS-DA) revealed good discrimination for most black tea samples. Additionally, 64 compounds with variable importance in projection > 1.0 were identified as differential odorants. Through an odor activity value analysis, eight volatile compounds were identified as the key active differential VFCs: linalool, methyl salicylate, phenylethyl alcohol, p-cresol, 3-methyl-butanoic acid, geraniol, benzaldehyde, and benzeneacetaldehyde. Thus, benzeneacetaldehyde and linalool in YJ-Yingde samples, benzaldehyde in Luokeng samples with an almond-like aroma, phenylethyl alcohol in the Heyuan samples, and p-cresol and 3-methyl-butanoic acid in the Chaozhou samples were the key volatile flavor compounds that could differentiate local black teas from other black teas. These findings will enrich the research in tea aroma chemistry and provide a method for identifying the origins of Guangdong black teas.

Identification and characterization of the key volatile flavor compounds in black teas from distinct regions worldwide.

Volatile flavor compounds in 112 black teas from seven countries were analyzed by untargeted metabolomics using headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME/GC-MS). Multivariate statistical analysis and odor activity values (OAVs) were used to classify these samples and identify key odorants. A total of 140 volatile flavor compounds (VFCs), including 12 different groups, were identified, and alcohols were prevalent in China and India samples, accounting for 40.83% and 34.96% of the total VFCs, respectively. Eight volatile compounds with OAVs > 1 were identified as key active differential odorants in Chinese, Indian, and Sri Lankan samples, including linalool, pentanoic acid, methyl salicylate, hexanoic acid, 1-methyl-naphthalene, phenylethyl alcohol, geraniol, and ß-ionone. Linalool, pentanoic acid, and hexanoic acid in Indian black teas, phenylethyl alcohol in Chinese black teas, and 1-methyl-naphthalene, ß-ionone in Sri Lankan black teas could be used to discriminate different black tea groups. A total of 12-14 VFCs with OAVs > 1 were identified as key active aromatics in Chinese black tea sample. Linalool and benzeneacetaldehyde in Yingde (Guangdong) black tea, methyl salicylate in Taiwanese samples, and benzeneacetic acid in Anhui black tea could be used as biomarkers to distinguish them from other Chinese samples. Sensory evaluation results showed that most black teas presented the common sweet, floral odors, which were consistent with GC-MS analysis. These results will contribute to characterize the odor metabolome of black teas and provide biochemical basis for identifying the authenticity of different black teas. PRACTICAL APPLICATION: Linalool, pentanoic acid, and hexanoic acid in Indian black teas, phenylethyl alcohol in Chinese black teas, 1-methyl-naphthalene, ß-ionone, and methyl salicylate in Sri Lankan black teas could be used to discriminate black teas from the three countries. Linalool and benzeneacetaldehyde in Yingde black teas, methyl salicylate in Taiwanese black teas, and benzeneacetic acid in Anhui black tea are the potential biomarkers to distinguish these teas from other Chinese black teas.

Pre-clinical study of human umbilical cord mesenchymal stem cell transplantation for the treatment of traumatic brain injury: safety evaluation from immunogenic and oncogenic perspectives.

Stem cell therapy is a promising strategy for the treatment of traumatic brain injury (TBI). However, animal experiments are needed to evaluate safety; in particular, to examine the immunogenicity and tumorigenicity of human umbilical cord mesenchymal stem cells (huMSCs) before clinical application. In this study, huMSCs were harvested from human amniotic membrane and umbilical cord vascular tissue. A rat model of TBI was established using the controlled cortical impact method. Starting from the third day after injury, the rats were injected with 10 µL of 5 × 106/mL huMSCs by cerebral stereotaxis or with 500 µL of 1 × 106/mL huMSCs via the tail vein for 3 successive days. huMSC transplantation decreased the serum levels of proinflammatory cytokines in rats with TBI and increased the serum levels of anti-inflammatory cytokines, thereby exhibiting good immunoregulatory function. The transplanted huMSCs were distributed in the liver, lung and brain injury sites. No abnormal proliferation or tumorigenesis was found in these organs up to 12 months after transplantation. The transplanted huMSCs negligibly proliferated in vivo, and apoptosis was gradually observed at later stages. These findings suggest that huMSC transplantation for the treatment of traumatic brain injury displays good safety. In addition, huMSCs exhibit good immunoregulatory function, which can help prevent and reduce secondary brain injury caused by the rapid release of inflammatory factors after TBI. This study was approved by the Ethics Committee of Wuhan General Hospital of PLA (approval No. 20160054) on November 1, 2016.

A Comparative Metabolomic Analysis Reveals Difference Manufacture Suitability in "Yinghong 9" and "Huangyu" Teas (Camellia sinensis).

"Yinghong 9" is a widely cultivated large-leaf variety in South China, and the black tea made from it has a high aroma and strong sweet flavor. "Huangyu" is a light-sensitive tea variety with yellow leaves. It was cultivated from the bud-mutation of "Yinghong 9" and has a very low level of chlorophyll during young shoot development. Due to chlorophyll being involved in carbon fixation and assimilation, the changes in photosynthesis might potentially affect the accumulation of flavor metabolites, as well as the quality of "Huangyu" tea. Although "Huangyu" has a golden yellow color and high amino acid content, the mechanism underlying the formation of leaf color and drinking value remains unclear. The widely targeted metabolomics and GC-MS analysis were performed to reveal the differences of key metabolites in fresh and fermented leaves between "Yinghong 9" and "Huangyu." The results showed that tea polyphenols, total chlorophyll, and carotenoids were more abundant in "Yinghong 9." Targeted metabolomics analysis indicated that kaempferol-3-glycoside was more abundant in "Yinghong 9," while "Huangyu" had a higher ratio of kaempferol-3-glucoside to kaempferol-3-galactoside. Compared with "Yinghong 9" fresh leaves, the contents of zeaxanthin and zeaxanthin palmitate were significantly higher in "Huangyu." The contents of α-farnesene, ß-cyclocitral, nerolidol, and trans-geranylacetone, which were from carotenoid degradation and involved in flowery-fruity-like flavor in "Huangyu" fermented leaves, were higher than those of "Yinghong 9." Our results indicated that "Huangyu" was suitable for manufacturing non-fermented tea because of its yellow leaf and flowery-fruity-like compounds from carotenoid degradation.

Genome-wide association analysis identified molecular markers associated with important tea flavor-related metabolites.

The characteristic secondary metabolites in tea (theanine, caffeine, and catechins) are important factors contributing to unique tea flavors. However, there has been relatively little research on molecular markers related to these metabolites. Thus, we conducted a genome-wide association analysis of the levels of these tea flavor-related metabolites in three seasons. The theanine, caffeine, and catechin levels in Population 1 comprising 191 tea plant germplasms were examined, which revealed that their heritability exceeded 0.5 in the analyzed seasons, with the following rank order (highest to lowest heritabilities): (+)-catechin > (-)-gallocatechin gallate > caffeine = (-)-epicatechin > (-)-epigallocatechin-3-gallate > theanine > (-)-epigallocatechin > (-)-epicatechin-3-gallate > catechin gallate > (+)-gallocatechin. The SNPs detected by amplified-fragment SNP and methylation sequencing divided Population 1 into three groups and seven subgroups. An association analysis yielded 307 SNP markers related to theanine, caffeine, and catechins that were common to all three seasons. Some of the markers were pleiotropic. The functional annotation of 180 key genes at the SNP loci revealed that FLS, UGT, MYB, and WD40 domain-containing proteins, as well as ATP-binding cassette transporters, may be important for catechin synthesis. KEGG and GO analyses indicated that these genes are associated with metabolic pathways and secondary metabolite biosynthesis. Moreover, in Population 2 (98 tea plant germplasm resources), 30 candidate SNPs were verified, including 17 SNPs that were significantly or extremely significantly associated with specific metabolite levels. These results will provide a foundation for future research on important flavor-related metabolites and may help accelerate the breeding of new tea varieties.

Identification of key metabolites based on non-targeted metabolomics and chemometrics analyses provides insights into bitterness in Kucha [Camellia kucha (Chang et Wang) Chang].

Camellia kucha (Chang et Wang) Chang is a special tea in China, which is extremely bitter but beneficial for human health. However, there are no systematic studies on Kucha metabolites, especially those associated with bitterness. In this study, a non-targeted metabolomics approach based on UHPLC-LTQ-Orbitrap-MS was applied to comprehensively profile the characteristic metabolites of two Kucha cultivars by comparison with three common tea cultivars. A total of 90 differential metabolites were identified. Among them, eight key metabolites (theacrine, 2,4-dimethyl-1H-indole, EGCG, dihydrokaempferol, panasenoside, 3-cresotinic acid, 3-methylglutaconic acid, and L-histidine) were more abundant in Kucha than in the controls, most of which were positively correlated with bitterness. Furthermore, quantitative analysis of some important catechins and alkaloids by HPLC implied absolutely higher concentrations of EGCG and theacrine in Kucha, which was similar to the metabolomics results. These results will be contribute to future research on the bitter and nutritional properties of Kucha.

Histone tales: lysine methylation, a protagonist in Arabidopsis development.

Histone methylation plays a fundamental role in the epigenetic regulation of gene expression driven by developmental and environmental cues in plants, including Arabidopsis. Histone methyltransferases and demethylases act as 'writers' and 'erasers' of methylation at lysine and/or arginine residues of core histones, respectively. A third group of proteins, the 'readers', recognize and interpret the methylation marks. Emerging evidence confirms the crucial roles of histone methylation in multiple biological processes throughout the plant life cycle. In this review, we summarize the regulatory mechanisms of lysine methylation, especially at histone H3 tails, and focus on the recent advances regarding the roles of lysine methylation in Arabidopsis development, from seed performance to reproductive development, and in callus formation.

Volatile components and nutritional qualities of Viscum articulatum Burm.f. parasitic on ancient tea trees.

Volatile flavor compounds (VFCs) and nutrients in Viscum articulatum Burm.f. parasitic on ancient tea trees (named TM) were studied in this research by headspace solid-phase microextraction (HS-SPME)/gas chromatography-mass spectrometry (GC-MS) and conventional methods. Sixty-six volatile compounds belonging to different classes were identified by GC-MS. The ketones, alcohols, and aldehydes were the principal aroma groups in TM according to principle component analysis (PCA). The most abundant aroma components in TM included benzaldehyde (9.64%), geranylacetone (7.92%), epoxy-ß-ionone (7.71%), ß-linalool (7.35%), methyl salicylate (6.96%), and hotrienol (6.14%), significantly higher than CKs (p < .05). The positive PC1 and PC2 in TM were correlated with benzaldehyde, hotrienol, methyl salicylate, and geranylacetone. The mistletoes could be differentiated from CKs due to the difference in aroma compounds. Clean and fresh, woody and nutty odor with minor floral scent was the characteristics of TM. Analysis of the nutritional components showed that contents of polyphenols and catechins in TM were at trace levels, significantly lower than CKs (p < .05). The total contents of polyphenols, amino acids, carbohydrates, and caffeine in TM were significantly lower from the total soluble solids (p < .05), indicating that there were still lots of compounds undetected in TM. The sensory test showed that the taste and aroma in TM can be accepted, which indicates TM could be developed into alternative tea drinks in the future.

GC-MS-based metabolomic study reveals dynamic changes of chemical compositions during black tea processing.

The chemical composition of black tea during tea processing is in a state of flux. However, the dynamic changes of this sophisticated metabolic process are far from clear. GC-MS-based metabolomic analyses were performed to examine changes in volatile and non-volatile compounds throughout the five stages of tea processing. The results showed that the most striking differences were observed at the withering and rolling stages, during which 62 non-volatile and 47 volatile compounds were significantly changed. The levels of most monosaccharides decreased at the withering stage and increased in subsequent stages while di-saccharides decreased as the process progressed. Free amino acids increased sharply at the withering stage, and most kept increasing or remained stable afterwards. However, levels of catechin, epicatechin, epigallocatechin, and epigallocatechin gallate decreased after withering and remained at low levels afterwards. Among the 47 volatile compounds with altered levels, phenylpropanoids/benzenoids and carotenoid-derived volatiles, which contribute to the honey-like and rose-like fragrances and quality of Danxia2 tea, kept increasing during the processing, among them eight were newly produced. Furthermore, 19 volatiles with a grassy odor decreased during processing. This study provides a comprehensive profile of metabolic changes during black tea processing, which is potentially important for both quality control and improvement of the flavor of black teas.

Elevated expression of PTCD3 correlates with tumor progression and predicts poor prognosis in patients with prostate cancer.

Pentatricopeptide repeat domain protein 3 (PTCD3) is a mitochondrial RNA­binding protein that serves a role in mitochondrial translation. PTCD3 was originally reported as an oncogene that is involved in breast cancer and lymphoma. However, the expression and function of PTCD3 in prostate cancer (PCa) are unknown. Therefore, the aim of the present study was to investigate the expression of PTCD3 and its clinical significance in PCa. Immunohistochemistry and dataset analyses revealed that PTCD3 protein expression levels were enhanced in human PCa tissues and mouse PCa models. PTCD3 expression levels were positively correlated with advanced PCa pathological grade and clinical stage. Additionally, PTCD3 mRNA expression was positively correlated with tissue malignancy, high Gleason score and distant metastasis in The Cancer Genome Atlas dataset. Kaplan­Meier analysis revealed that high PTCD3 levels can predict the increased biochemical recurrence (BCR)­free survival in all patients with or without metastasis. The overexpression of PTCD3 could be used as an independent prognostic marker of poor BCR­free survival. Immunofluorescence and western blot analysis in human PCa cell lines further confirmed that PTCD3 levels were associated with the hormone independence of PCa. Therefore, the present study revealed that PTCD3 levels may serve as a novel biomarker for PCa prognosis.

TaWRKY51 promotes lateral root formation through negative regulation of ethylene biosynthesis in wheat (Triticum aestivum L.).

Common wheat (Triticum aestivum L.) is an important staple food crop worldwide. Lateral roots (LRs), as the major component of root architecture, affect water and nutrient uptake in wheat. The phytohormone ethylene is known to affect LR formation; however, the factor(s) modulating ethylene during this process have not yet been elucidated in wheat. Here we identified wheat TaWRKY51 as a key factor that functions in LR formation by modulating ethylene biosynthesis. Wheat TaWRKY51RNA interference lines (TaWRKY51-RNAi) and the homozygous mutants tawrky51-2a and tawrky51-2b all produced fewer LRs than the wild type and negative transgenic plants, whereas the TaWRKY51 overexpression lines (TaWRKY51-OE) had the opposite phenotype. Transcription analysis revealed that 1-aminocyclopropane-1-carboxylic acid synthase (ACS) genes (TaACS2, TaACS7 and TaACS8) involved in ethylene biosynthesis were downregulated in TaWRKY51-OE lines but upregulated in TaWRKY51-RNAi lines. The rate of ethylene production also decreased in TaWRKY51-OE lines but increased in TaWRKY51-RNAi lines compared with their respective negative transgenic controls. Electrophoretic mobility shift and transient expression assays revealed that TaWRKY51 inhibits the expression of ACS genes by binding to the W-box cis-element present in their promoter region. Moreover, overexpression of ACS2 or exogenous application of 1-aminocyclopropane-1-carboxylic acid reversed the phenotype of enhanced LR number in TaWRKY51-OE Arabidopsis lines, and overexpression of TaWRKY51 in the ethylene-overproducing mutant eto1-1 rescued its LR defect phenotype. In addition, genetic evidence demonstrates that TaWRKY51-regulated LR formation is also dependent on ethylene and auxin signaling pathways. Our findings reveal a molecular genetic mechanism by which a WRKY gene coordinates ethylene production and LR formation in wheat.

Comparative proteomics reveals the physiological differences between winter tender shoots and spring tender shoots of a novel tea (Camellia sinensis L.) cultivar evergrowing in winter.

BACKGROUND: A recently discovered tea [Camellia sinensis (L.) O. Kuntze] cultivar can generate tender shoots in winter. We performed comparative proteomics to analyze the differentially accumulated proteins between winter and spring tender shoots of this clonal cultivar to reveal the physiological basis of its evergrowing character during winter. RESULTS: We extracted proteins from the winter and spring tender shoots (newly formed two leaves and a bud) of the evergrowing tea cultivar "Dongcha11" respectively. Thirty-three differentially accumulated high-confidence proteins were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF / TOF MS). Among these, 24 proteins had increased abundance while nine showed were decreased abundance in winter tender shoots as compared with the spring tender shoots. We categorized the differentially accumulated proteins into eight critical biological processes based on protein function annotation including photosynthesis, cell structure, protein synthesis & destination, transporters, metabolism of sugars and polysaccharides, secondary metabolism, disease/defense and proteins with unknown functions. Proteins with increased abundance in winter tender shoots were mainly related to the processes of photosynthesis, cytoskeleton and protein synthesis, whereas those with decreased abundance were correlated to metabolism and the secondary metabolism of polyphenolic flavonoids. Biochemical analysis showed that the total contents of soluble sugar and amino acid were higher in winter tender shoots while tea polyphenols were lower as compared with spring tender shoots. CONCLUSIONS: Our study suggested that the simultaneous increase in the abundance of photosynthesis-related proteins rubisco, plastocyanin, and ATP synthase delta chain, metabolism-related proteins eIF4 and protease subunits, and the cytoskeleton-structure associated proteins phosphatidylinositol transfer protein and profilin may be because of the adaptation of the evergrowing tea cultivar "Dongcha11" to low temperature and light conditions. Histone H4, Histone H2A.1, putative In2.1 protein and protein lin-28 homologs may also regulate the development of winter shoots and their response to adverse conditions.

Effects of Pu-erh ripened tea on hyperuricemic mice studied by serum metabolomics.

To evaluate effects of Pu-erh ripened tea in hyperuricemic mice, a mouse hyperuricemia model was developed by oral administration of potassium oxonate for 7 d. Serum metabolomics, based on gas chromatography-mass spectrometry, was used to generate metabolic profiles from normal control, hyperuricemic and allopurinol-treated hyperuricemic mice, as well as hyperuricemic mice given Pu-erh ripened tea at three doses. Pu-erh ripened tea significantly lowered serum uric acid levels. Twelve potential biomarkers associated with hyperuricemia were identified. Pu-erh ripened tea and allopurinol differed in their metabolic effects in the hyperuricemic mice. Levels of glutamic acid, indolelactate, L-allothreonine, nicotinoylglycine, isoleucine, l-cysteine and glycocyamine, all involved in amino acid metabolism, were significantly changed in hyperuricemic mice treated Pu-erh ripened tea. Thus, modulating amino acid metabolism might be the primary mechanism of anti-hyperuricemia by Pu-erh ripened tea.

Optimization of beam transformation system for laser-diode bars.

An optimized beam transformation system (BTS) is proposed to improve the beam quality of laser-diode bars. Through this optimized design, the deterioration of beam quality after the BTS can be significantly reduced. Both the simulation and experimental results demonstrate that the optimized system enables the beam quality of a mini-bar (9 emitters) approximately equal to 5.0 mm × 3.6 mrad in the fast-axis and slow-axis. After beam shaping by the optimized BTS, the laser-diode beam can be coupled into a 100 µm core, 0.15 numerical aperture (NA) fiber with an output power of over 100 W and an electric-optical efficiency of 46.8%.

High brightness laser-diode device emitting 160 watts from a 100 µm/NA 0.22 fiber.

A practical method of achieving a high-brightness and high-power fiber-coupled laser-diode device is demonstrated both by experiment and ZEMAX software simulation, which is obtained by a beam transformation system, free-space beam combining, and polarization beam combining based on a mini-bar laser-diode chip. Using this method, fiber-coupled laser-diode module output power from the multimode fiber with 100 µm core diameter and 0.22 numerical aperture (NA) could reach 174 W, with equalizing brightness of 14.2 MW/(cm2·sr). By this method, much wider applications of fiber-coupled laser-diodes are anticipated.

Beam shaping design for compact and high-brightness fiber-coupled laser-diode system.

Fiber-coupled laser diodes have become essential sources for fiber laser pumping and direct energy applications. A compact and high-brightness fiber-coupled system has been designed based on a significant beam shaping method. The laser-diode stack consists of eight mini-bars and is effectively coupled into a standard 100 µm core diameter and NA=0.22 fiber. The simulative result indicates that the module will have an output power over 440 W. Using this technique, compactness and high-brightness production of a fiber-coupled laser-diode module is possible.

High brightness beam shaping and fiber coupling of laser-diode bars.

The strong beam quality mismatch in the fast and slow axes of laser-diode bars requires a significant beam shaping method to reach the parameters needed for fiber coupling. An effective solution to this problem is proposed that is based on a right-angle prism array and a distributed cylinder-lens stack. Coupling 12 mini-bars into a standard 100 µm core diameter and 0.15 numerical aperture fiber is achieved, and the output power can reach 400 W. Using this technique, production of compact and high brightness fiber-coupled laser-diode modules is possible.

Laser differential reflection-confocal focal-length measurement.

A new laser differential reflection-confocal focal-length measurement (DRCFM) method is proposed for the high-accuracy measurement of the lens focal length. DRCFM uses weak light reflected from the lens last surface to determine the vertex position of this surface. Differential confocal technology is then used to identify precisely the lens focus and vertex of the lens last surface, thereby enabling the precise measurement of the lens focal length. Compared with existing measurement methods, DRCFM has high accuracy and strong anti-interference capability. Theoretical analyses and experimental results indicate that the DRCFM relative measurement error is less than 10 ppm.