Yong-Xin Wang

Transcriptome-based discovery of AP2/ERF transcription factors related to temperature stress in tea plant (Camellia sinensis)

Tea plant (Camellia sinensis) is an important natural resource for the global supply of non-alcoholic beverage production. The extension of tea plant cultivation is challenged by biotic and abiotic stresses. Transcription factors (TFs) of the APETALA 2 (AP2)/ethylene-responsive factor (ERF) family are involved in growth and anti-stresses through multifaceted transcriptional regulation in plants. This study comprehensively analyzed AP2/ERF family TFs from C. sinensis on the basis of the transcriptome sequencing data of four tea plant cultivars, namely, ‘Yunnanshilixiang’, ‘Chawansanhao’, ‘Ruchengmaoyecha’, and ‘Anjibaicha’. A total of 89 putative AP2/ERF transcription factors with full-length AP2 domain were identified from C. sinensis and classified into five subfamilies, namely, AP2, dehydration-responsive-element-binding (DREB), ERF, related to ABI3/VP (RAV), and Soloist. All identified CsAP2/ERF genes presented relatively stable expression levels in the four tea plant cultivars. Many groups also showed cultivar specificity. Five CsAP2/ERF genes from each AP2/ERF subfamily (DREB, ERF, AP2, and RAV) were related to temperature stresses; these results indicated that AP2/ERF TFs may play important roles in abnormal temperature stress response in C. sinensis.

Transcriptome-wide identification of Camellia sinensis WRKY transcription factors in response to temperature stress

Tea plant [Camellia sinensis (L.) O. Kuntze] is a leaf-type healthy non-alcoholic beverage crop, which has been widely introduced worldwide. Tea is rich in various secondary metabolites, which are important for human health. However, varied climate and complex geography have posed challenges for tea plant survival. The WRKY gene family in plants is a large transcription factor family that is involved in biological processes related to stress defenses, development, and metabolite synthesis. Therefore, identification and analysis of WRKY family transcription factors in tea plant have a profound significance. In the present study, 50 putative C. sinensis WRKY proteins (CsWRKYs) with complete WRKY domain were identified and divided into three Groups (Group I-III) on the basis of phylogenetic analysis results. The distribution of WRKY family transcription factors among plantae, fungi, and protozoa showed that the number of WRKY genes increased in higher plant, whereas the number of these genes did not correspond to the evolutionary relationships of different species. Structural feature and annotation analysis results showed that CsWRKY proteins contained WRKYGQK/WRKYGKK domains and C2H2/C2HC-type zinc-finger structure: D-X18-R-X1-Y-X2-C-X4-7-C-X23-H motif; CsWRKY proteins may be associated with the biological processes of abiotic and biotic stresses, tissue development, and hormone and secondary metabolite biosynthesis. Temperature stresses suggested that the candidate CsWRKY genes were involved in responses to extreme temperatures. The current study established an extensive overview of the WRKY family transcription factors in tea plant. This study also provided a global survey of CsWRKY transcription factors and a foundation of future functional identification and molecular breeding.

Transcriptome-Wide Identification and Expression Analysis of the NAC Gene Family in Tea Plant [Camellia sinensis (L.) O. Kuntze]

In plants, the NAC (NAM-ATAF1/2-CUC) family of proteins constitutes several transcription factors and plays vital roles in diverse biological processes, such as growth, development, and adaption to adverse factors. Tea, as a non-alcoholic drink, is known for its bioactive ingredients and health efficacy. Currently, knowledge about NAC gene family in tea plant remains very limited. In this study, a total of 45 CsNAC genes encoding NAC proteins including three membrane-bound members were identified in tea plant through transcriptome analysis. CsNAC factors and Arabidopsis counterparts were clustered into 17 subgroups after phylogenetic analysis. Conserved motif analysis revealed that CsNAC proteins with a close evolutionary relationship possessed uniform or similar motif compositions. The distribution of NAC family MTFs (membrane-associated transcription factors) among higher plants of whose genome-wide has been completed revealed that the existence of doubled TMs (transmembrane motifs) may be specific to fabids. Transcriptome analysis exhibited the expression profiles of CsNAC genes in different tea plant cultivars under non-stress conditions. Nine CsNAC genes, including the predicted stress-related and membrane-bound genes, were examined through qRT-PCR (quantitative real time polymerase chain reaction) in two tea plant cultivars, namely, ‘Huangjinya’ and ‘Yingshuang’. The expression patterns of these genes were investigated in different tissues (root, stem, mature leaf, young leaf and bud) and under diverse environmental stresses (drought, salt, heat, cold and abscisic acid). Several CsNAC genes, including CsNAC17 and CsNAC30 that are highly orthologous to known stress-responsive ANAC072/RD26 were identified as highly responsive to abiotic stress. This study provides a global survey of tea plant NAC proteins, and would be helpful for the improvement of stress resistance in tea plant via genetic engineering.

L-Theanine Content and Related Gene Expression: Novel Insights into Theanine Biosynthesis and Hydrolysis among Different Tea Plant (Camellia sinensis L.) Tissues and Cultivars

L-Theanine content has tissues and cultivars specificity in tea plant (Camellia sinensis L.), the correlations of theanine metabolic related genes expression profiles with theanine contents were explored in this study. L-theanine contents in the bud and 1st leaf, 2nd leaf, 3rd leaf, old leaf, stem, and lateral root were determined by HPLC from three C. sinensis cultivars, namely ‘Huangjinya’, ‘Anjibaicha’, and ‘Yingshuang’, respectively. The theanine contents in leaves and root of ‘Huangjinya’ were the highest, followed by ‘Anjibaicha’, and ‘Yingshuang’. The theanine contents in the leaves reduced as the leaf mature gradually, and in stem were the least. Seventeen genes encoding enzymes involved in theanine metabolism were identified from GenBank and our tea transcriptome database, including CsTS1, CsTS2, CsGS1, CsGS2, CsGOGAT-Fe, CsGOGAT-NAD(P)H, CsGDH1, CsGDH2, CsALT, CsSAMDC, CsADC, CsCuAO, CsPAO, CsNiR, CsNR, CsGGT1, and CsGGT3. The transcript profiles of those seventeen genes in the different tissues of three tea plant cultivars were analyzed comparatively. Among the different cultivars, the transcript levels of most selected genes in ‘Huangjinya’ were significantly higher than that in the ‘Anjibaicha’ and ‘Yingshuang’. Among the different tissues, the transcript levels of CsTS2, CsGS1, and CsGDH2 almost showed positive correlation with the theanine contents, while the other genes showed negative correlation with the theanine contents in most cases. The theanine contents showed correlations with related genes expression levels among cultivars and tissues of tea plant, and were determined by the integrated effect of the metabolic related genes.

Transcriptome-Based Analysis of Dof Family Transcription Factors and Their Responses to Abiotic Stress in Tea Plant (Camellia sinensis)

Tea plant (Camellia sinensis (L.) O. Kuntze) is affected by abiotic stress during its growth and development. DNA-binding with one finger (Dof) transcription factors (TFs) play important roles in abiotic stress tolerance of plants. In this study, a total of 29 putative Dof TFs were identified based on transcriptome of tea plant, and the conserved domains and common motifs of these CsDof TFs were predicted and analyzed. The 29 CsDof proteins were divided into 7 groups (A, B1, B2, C1, C2.1, C2.2, and D2), and the interaction networks of Dof proteins in C. sinensis were established according to the data in Arabidopsis. Gene expression was analyzed in “Yingshuang” and “Huangjinya” under four experimental stresses by qRT-PCR. CsDof genes were expressed differentially and related to different abiotic stress conditions. In total, our results might suggest that there is a potential relationship between CsDof factors and tea plant stress resistance.

Genome-wide identification and expression analysis of GRAS family transcription factors in tea plant ( Camellia sinensis )

GRAS proteins are important transcription factors that play multifarious roles in regulating the growth and development as well as stress responses of plants. Tea plant is an economically important leaf -type beverage crop. Information concerning GRAS family transcription factors in tea plant is insufficient. In this study, 52 CsGRAS genes encoding GRAS proteins were identified from tea plant genome database. Phylogenetic analysis of the identified GRAS proteins from tea plant, Arabidopsis, and rice divided these proteins into at least 13 subgroups. Conserved motif analysis revealed that the gene structure and motif compositions of the proteins were considerably conserved among the same subgroup. Functional divergence analysis indicated that the shifted evolutionary rate might act as a major evolutionary force driving subfamily-specific functional diversification. Transcriptome analysis showed that the transcriptional levels of CsGRAS genes under non-stress conditions varied among different tea plant cultivars. qRT-PCR analysis revealed tissue and development stage-specific expression patterns of CsGRAS genes in tea plant. The expression patterns of CsGRAS genes in response to abiotic stresses and gibberellin treatment suggested the possible multiple functions of these genes. This study provides insights into the potential functions of GRAS genes.

Transcriptome-wide identification and expression profile analysis of the bHLH family genes in Camellia sinensis

The tea plant is an important commercial horticulture crop cultivated worldwide. Yield and quality of this plant are influenced by abiotic stress. The bHLH family transcription factors play a pivotal role in the growth and development, including abiotic stress response, of plants. A growing number of bHLH proteins have been functionally characterized in plants. However, few studies have focused on the bHLH proteins in tea plants. In this study, 120 CsbHLH TFs were identified from tea plants using computational prediction method. Structural analysis detected 23 conservative residues, with over 50% identities in the bHLH domain. Moreover, 103 CsbHLH proteins were assumed to bind DNA and encompassed 98 E-Box binders and 85 G-Box binders. The CsbHLH proteins were grouped into 20 subfamilies based on phylogenetic analysis and a previous classification system. A survey of transcriptome profiling screened 22 and 39 CsbHLH genes that were upregulated under heat and drought stress. Nine CsbHLH genes were validated using qRT-PCR. Results were approximately in accordance with transcriptome data. These genes could be induced by one or more abiotic stresses.

Identification of genes revealed differential expression profiles and lignin accumulation during leaf and stem development in tea plant (Camellia sinensis (L.) O. Kuntze)

Lignin is a complex aromatic heteropolymer that plays essential roles in mechanical support, water transport, and response to biotic and abiotic stresses. The tea plant is a leaf-type beverage crop, which serves as a resource for non-alcoholic beverage tea. The content and distribution of lignin in tea plant leaves seriously affect the quality of tea. However, the biosynthetic pathways of lignin remain to be characterized in the tea plant. In the present study, lignin accumulation was investigated in tea plant leaves and stems at three developmental stages. The lignin content continuously increased during leaf and stem development in both tea plant cultivars ‘Fudingdabai’ and ‘Suchazao.’ The lignin distribution and anatomical characteristics of the tea plant leaves coincided with lignin accumulation and showed that lignin is mainly distributed in the epidermis, xylem, and vascular bundle sheath. ‘Suchazao’ exhibits a low lignin content and lacks a vascular bundle sheath. Twelve genes encoding the enzymes involved in the lignin biosynthesis of tea plant were identified and included CsPAL, CsC4H, Cs4CL, CsHCT, CsC3H, CsCCoAOMT, CsCCR, CsCAD, CsF5H, CsCOMT, CsPER, and CsLAC. The expression profiling of lignin biosynthesis-related genes and analysis of lignin accumulation may help elaborate the regulatory mechanisms of lignin biosynthesis in tea plant.

Differentially expressed protein and gene analysis revealed the effects of temperature on changes in ascorbic acid metabolism in harvested tea leaves

Tea is an important non-alcoholic beverage worldwide. Tea quality is determined by numerous secondary metabolites in harvested tea leaves, including tea polyphenols, theanine, caffeine, and ascorbic acid (AsA). AsA metabolism in harvested tea leaves is affected by storage and transportation temperature. However, the molecular mechanisms underlying AsA metabolism in harvested tea leaves exposed to different storage and transportation temperature conditions remain unclear. Here we performed RP-HPLC to detect dynamic changes in AsA content in tea leaves subjected to high- (38 °C), low- (4 °C), or room-temperature (25 °C) treatments. The AsA distribution and levels in the treated tea leaves were analyzed using cytological–anatomical characterization methods. The differentially expressed CsAPX1 and CsDHAR2 proteins, which are involved in the AsA recycling pathway, were identified from the corresponding proteomic data using iTRAQ. We also analyzed the expression profiles of 18 genes involved in AsA metabolism, including CsAPX1 and CsDHAR2. AsA was mainly distributed in tea leaf mesophyll cells. High- and low-temperature treatments upregulated the CsAPX1 and CsDHAR2 proteins and induced CsAPX and CsDHAR2 gene expression. These results indicated that the CsAPX1 and CsDHAR2 proteins might have critical roles in AsA recycling in tea leaves. Our results provide a foundation for the in-depth investigation of AsA metabolism in tea leaves during storage and transportation, and they will promote better tea flavor in tea production.Tea: storage temperature impacts vitamin C metabolismHigh and low temperatures induce the expression of enzymes involved in vitamin C metabolism in tea leaves. A research team from Nanjing Agricultural University, China, led by Jing Zhuang collected fresh leaves from 1-year-old tea plants of the Longjing #43 varietal and then stored the leaves for 4 h at 4 °C (cold), 38 °C (hot), or 25 °C (room temperature). They found that absolute levels of vitamin C—an antioxidant molecule also known as ascorbic acid—were highest after storage at the low temperature and lowest after storage at the high temperature. However, the expression levels of genes involved in vitamin C synthesis and breakdown were elevated at both high and low temperatures. The findings could aid the tea industry in developing storage and transportation strategies that optimize leaf quality and flavor.