Alternative splicing of CsbHLH133 regulates geraniol biosynthesis in tea plants.

Geraniol is one of the most abundant aromatic compounds in fresh tea leaves and contributes to the pleasant odor of tea products. Additionally, it functions as an airborne signal that interacts with other members of the ecosystem. To date, the regulation of the geraniol biosynthesis in tea plants remains to be investigated. In this study, a correlation test of the content of geraniol and its glycosides with gene expression data revealed that nudix hydrolase, CsNudix26, and its transcription factor, CsbHLH133 are involved in geraniol biosynthesis. In vitro enzyme assays and metabolic analyses of genetically modified tea plants confirmed that CsNudix26 is responsible for the formation of geraniol. Yeast one-hybrid, dual-luciferase reporter, and EMSA assays were used to verify the binding of CsbHLH133 to the CsNudix26 promoter. Overexpression of CsbHLH133 in tea leaves enhanced CsNudix26 expression and geraniol accumulation, whereas CsbHLH133 silencing reduced CsNudix26 transcript levels and geraniol content. Interestingly, CsbHLH133-AS, produced by alternative splicing, was discovered and proved to be the primary transcript expressed in response to various environmental stresses. Furthermore, geraniol release was found to be affected by various factors that alter the expression patterns of CsbHLH133 and CsbHLH133-AS. Our findings indicate that distinct transcript splicing patterns of CsbHLH133 regulate geraniol biosynthesis in tea plants in response to different regulatory factors.

The auxin-responsive CsSPL9-CsGH3.4 module finely regulates auxin levels to suppress the development of adventitious roots in tea (Camellia sinensis).

The cutting technique is extensively used in tea breeding, with key emphasis on promoting the growth of adventitious roots (ARs). Despite its importance in tea cultivation, the mechanisms underlying AR development in tea remain unclear. In this study, we demonstrated the essential role of auxins in the initiation and progression of AR and established that the application of exogenous 1-naphthaleneacetic acid-enhanced AR formation in tissue-cultured seedlings and cuttings. Then, we found that the auxin-responsive transcription factor CsSPL9 acted as a negative regulator of AR development by reducing the levels of free indole-3-acetic acid (IAA) in tea plants. Furthermore, we identified CsGH3.4 as a downstream target of CsSPL9, which was activated by direct binding to its promoter. CsGH3.4 also inhibited AR development and maintained low levels of free IAA. Thus, these results revealed the inhibitory effect of the auxin-responsive CsSPL9-CsGH3.4 module on AR development by reducing free IAA levels in tea. These findings have significant theoretical and practical value for enhancing tea breeding practices.

Two galactinol synthases contribute to the drought response of Camellia sinensis.

MAIN CONCLUSION: CsGolS2-1 and CsGolS2-2 are involved in the transcriptional mechanism and play an important role in the drought response of tea plants. GolS is critical for the biosynthesis of galactinol and has been suggested to contribute to drought tolerance in various plants. However, whether GolS plays a role in drought response and the underlying transcriptional mechanism of GolS genes in response to drought stress in tea plants is still unclear. In this study, we found that drought stress promotes the accumulation of galactinol in tea leaves and that the expression of CsGolS2-1 and CsGolS2-2, which encode proteins capable of catalyzing galactinol biosynthesis, is continuously and dramatically induced by drought stress. Moreover, transgenic Arabidopsis plants expressing CsGolS2-1 and CsGolS2-2 were more drought-tolerant than WT plants, as evidenced by increased cell membrane stability. In addition, the drought-responsive transcription factor CsWRKY2 has been shown to positively regulate the expression of CsGolS2-1 and CsGolS2-2 by directly binding to their promoters. Furthermore, CsVQ9 was found to interact with CsWRKY2 and promote its transcriptional function to activate CsGolS2-1 and CsGolS2-2 expression. Taken together, our findings provide insights not only into the positive role played by CsGolS2-1 and CsGolS2-2 in the drought response of tea plants but also into the transcriptional mechanisms involved.

Genome-Wide Characterization and Expression Analysis of Pathogenesis-Related 1 (PR-1) Gene Family in Tea Plant (Camellia sinensis (L.) O. Kuntze) in Response to Blister-Blight Disease Stress.

Pathogenesis-related 1 (PR-1) proteins, which are defense proteins in plant-pathogen interactions, play an important role in the resistance and defense of plants against diseases. Blister blight disease is caused by Exobasidium vexans Massee and a major leaf disease of tea plants (Camellia sinensis (L.) O. Kuntze). However, the systematic characterization and analysis of the PR-1 gene family in tea plants is still lacking, and the defense mechanism of this family remains unknown. In this study, 17 CsPR-1 genes were identified from the tea plant genome and classified into five groups based on their signal peptide, isoelectric point, and C-terminus extension. Most of the CsPR-1 proteins contained an N-terminal signal peptide and a conserved PR-1 like domain. CsPR-1 genes comprised multiple cis-acting elements and were closely related to the signal-transduction pathways involving TCA, NPR1, EDS16, BGL2, PR4, and HCHIB. These characteristics imply an important role of the genes in the defense of the tea plant. In addition, the RNA-seq data and real-time PCR analysis demonstrated that the CsPR-1-2, -4, -6, -7, -8, -9, -10, -14, -15, and -17 genes were significantly upregulated under tea blister-blight stress. This study could help to increase understanding of CsPR-1 genes and their defense mechanism in response to tea blister blight.

The complete mitochondrial genome of Eterusia aedea (Lepidoptera, Zygaenidae) and comparison with other zygaenid moths.

Zygaenidae comprises >1036 species, including many folivorous pests in agriculture. In the present study, the complete mitochondrial genome (mitogenome) of a major pest of tea trees, Eterusia aedea was determined. The 15,196-bp circular genome contained the common set of 37 mitochondrial genes (including 13 protein-coding genes, two rRNA genes, and 22 tRNA genes) and exhibited the similar genomic features to reported Zygaenidae mitogenome. Comparative analyses of Zygaenidae mitogenomes showed a typical evolutionary trend of lepidopteran mitogenomes. In addition, we also investigated the gene order of lepidopteran mitogenomes and proposed that the novel gene order trnA-trnR-trnN-trnE-trnS-trnF from Zygaenidae and Gelechiidae and most other gene rearrangements of this tRNA cluster evolved independently. Finally, the mitogenomic phylogeny of Lepidoptera was reconstructed based on multiple mitochondrial datasets. And all the phylogenetic results revealed the sister relationships of Cossoidea and Zygaenoidea with both BI and ML methods, which is the first stable mitogenomic evidence for this clade.

Identification, characterization and expression analysis of the VQ motif-containing gene family in tea plant (Camellia sinensis).

BACKGROUND: VQ motif-containing (VQ) proteins are plant-specific proteins that interact with WRKY transcription factors and play important roles in plant growth, development and stress response. To date, VQ gene families have been identified and characterized in many plant species, including Arabidopsis, rice and grapevine. However, the VQ gene family in tea plant has not been reported, and the biological functions of this family remain unknown. RESULTS: In total, 25 CsVQ genes were identified based on the genome and transcriptome of tea plant, and a comprehensive bioinformatics analysis was performed. The CsVQ proteins all contained the typical conserved motif FxxhVQxhTG, and most proteins were localized in the nucleus. The phylogenetic analysis showed that the VQ proteins were classified into 5 groups (I, III-VI); the evolution of the CsVQ proteins is consistent with the evolutionary process of plants, and close proteins shared similar structures and functions. In addition, the expression analysis revealed that the CsVQ genes play important roles in the process of tea plant growth, development and response to salt and drought stress. Furthermore, a potential regulatory network including the interactions of CsVQ proteins with CsWRKY transcription factors and the regulation of upstream microRNA that is closely related to the above-mentioned processes is proposed. CONCLUSIONS: The results of this study increase our understanding and characterization of CsVQ genes and their encoded proteins in tea plant. This systematic analysis provided comprehensive information for further studies investigating the biological functions of CsVQ proteins in various developmental processes of tea plants.

CsINV5, a tea vacuolar invertase gene enhances cold tolerance in transgenic Arabidopsis.

BACKGROUND: Vacuolar invertases (VINs) have been reported to regulate plant growth and development and respond to abiotic stresses such as drought and cold. With our best knowledge, the functions of VIN genes little have been reported in tea plant (Camellia sinensis L.). Therefore, it is necessary to develop research in this field. RESULTS: Here, we identified a VIN gene, CsINV5, which was induced by cold acclimation and sugar treatments in the tea plant. Histochemical assays results showed that the 1154 bp 5'-flanking sequence of CsINV5 drove ß-glucuronidase (GUS) gene expression in roots, stems, leaves, flowers and siliques of transgenic Arabidopsis during different developmental stages. Moreover, promoter deletion analysis results revealed that an LTRE-related motif (CCGAAA) and a WBOXHVISO1 motif (TGACT) within the promoter region of CsINV5 were the core cis-elements in response to low temperature and sugar signaling, respectively. In addition, overexpression of CsINV5 in Arabidopsis promoted taproot and lateral root elongation through glucose-mediated effects on auxin signaling. Based on physiological and RNA-seq analysis, we found that overexpression of CsINV5 improved cold tolerance in transgenic Arabidopsis mainly by increasing the contents of glucose and fructose, the corresponding ratio of hexose to sucrose, and the transcription of osmotic-stress-related genes (P5CS1, P5CS2, AtLEA3, COR413-PM1 and COR15B) to adjust its osmotic potential. CONCLUSIONS: Comprehensive experimental results suggest that overexpression of CsINV5 may enhance the cold tolerance of plant through the modification of cellular sugar compounds contents and osmotic regulation related pathways.

Genome-Wide Identification, Classification and Expression Analysis of the HSP Gene Superfamily in Tea Plant (Camellia sinensis).

Heat shock proteins (HSPs) function as molecular chaperones. These proteins are encoded by a multigene family whose members play crucial roles in plant growth, development and stress response. However, little is known about the HSP gene superfamily in tea plant. In this study, a total of 47 CsHSP genes were identified, including 7 CsHSP90, 18 CsHSP70, and 22 CssHSP genes. Phylogenetic and composition analyses showed that CsHSP proteins in the same subfamily have similar gene structures and conserved motifs, but significant differences exist in the different subfamilies. In addition, expression analysis revealed that almost all CsHSP genes were specifically expressed in one or more tissues, and significantly induced under heat and drought stress, implying that CsHSP genes play important roles in tea plant growth, development, and response to heat and drought stress. Furthermore, a potential interaction network dominated by CsHSPs, including HSP70/HSP90 organizing protein (HOP) and heat shock transcription factor (HSF), is closely related to the abovementioned processes. These results increase our understanding of CsHSP genes and their roles in tea plant, and thus, this study could contribute to the cloning and functional analysis of CsHSP genes and their encoded proteins in the future.

[Engineering of a flavonoid 3'-hydroxylase from tea plant (Camellia sinensis) for biosynthesis of B-3',4'-dihydroxylated flavones].

Objective: A flavonoid 3'-hydroxylase from tea plant was engineered to synthesize B-3',4'-dihydroxylated flavones such as eriodictyol, dihydroquercetin and quercetin. Methods: Four articifical P450 constructs harboring both flavonoid 3'-hydroxylase gene from Camellia sinensis (CsF3'H) and P450 reductase gene from Arabidopsis thaliana (ATR1 or ATR2) were introduced into Escherichia coli strains TOP10, DH5α and BL21, resultantly engineering strains S1 to S12. The plasmid pYES-Dest52-CsF3'H harboring CsF3'H gene was introduced into yeast Saccharomyces cerevisiae WAT11 designated as strain S13. The plasmid pES-HIS-CsF3H::AtFLS 9 AA was constructed through fusing flavanone 3-hydroxylase gene from Camellia sinensis (CsF3H) and flavonol synthase gene from Arabidopsis thaliana (AtFLS). Plasmid pES-URA-CsF3'H and pES-HIS-CsF3H::AtFLS 9 AA were then co-introduced into yeast S. cerevisiae WAT11 designated as strain S14. Results: Strain S6 generated highest bioconversion efficiency at 25℃ among all E. coli strains during 24 h fernentation. Supplemented with 1000 µmol/L naringenin, dihydrokaempferol and kaempferol, the maximum amounts of eriodictyol, dihydroquercetin and quercetin produced by strain S13 were 734.32 µmol/L, 446.07 µmol/L and 594.64 µmol/L respectively. Supplemented with 5 mmol/L naringenin, the maximum amounts of eriodictyol, kaempferol, quercetin, dihydroquercetin and dihydrokaempferol produced by strain S14 were 1412.16 µmol/L, 490.25 µmol/L, 445.75 µmol/L, 66.75 µmol/L and 73.50 µmol/L during 36-48 h fermentaion respectively. Conclusion: CsF3'H was engineered for biosynthesis of B-3',4'-dihydroxylated flavone.

Cloning and Characterization of a Flavonoid 3'-Hydroxylase Gene from Tea Plant (Camellia sinensis).

Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3'H, designated as CsF3'H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3'H was highly homologous with the characterized F3'Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3'H-specific conserved motifs were discovered in the protein sequence of CsF3'H. Enzymatic analysis of the heterologously expressed CsF3'H in yeast demonstrated that tea F3'H catalyzed the 3'-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 µM, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min(-1), respectively. Transcription level of CsF3'H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3',4'-flavan-3-ols, 3',4',5'-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3'H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3'H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3'H in the biosynthesis of 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols in tea leaves.

The CsmiR397a-CsLAC17 module regulates lignin biosynthesis to balance the tenderness and gray blight resistance in young tea shoots.

Lignin accumulation can enhance the disease resistance of young tea shoots (Camellia sinensis). It also greatly reduces their tenderness, which indirectly affects the quality and yield of tea. Therefore, the regulation of lignin biosynthesis appears to be an effective way to balance tenderness and disease resistance in young tea shoots. In this study, we identified a laccase gene, CsLAC17, that is induced during tenderness reduction and gray blight infection in young tea shoots. Overexpression of CsLAC17 significantly increased the lignin content in transgenic Arabidopsis, enhancing their resistance to gray blight and decreasing stem tenderness. In addition, we found that CsLAC17 was negatively regulated by the upstream CsmiR397a by 5'-RLM-RACE, dual-luciferase assay, and transient expression in young tea shoots. Interestingly, the expression of CsmiR397a was inhibited during tenderness reduction and gray blight infection of young tea shoots. Overexpression of CsmiR397a reduced lignin accumulation, resulting in decreased resistance to gray blight and increased stem tenderness in transgenic Arabidopsis. Furthermore, the transient overexpression of CsmiR397a and CsLAC17 in tea leaves directly confirms the function of the CsmiR397a-CsLAC17 module in lignin biosynthesis and its effect on disease resistance. These results suggest that the CsmiR397a-CsLAC17 module is involved in balancing tenderness and gray blight resistance in young tea shoots by regulating lignin biosynthesis.

Nontargeted and targeted metabolomics analysis for evaluating the effect of "golden flora" amount on the sensory quality, metabolites, and the alpha-amylase and lipase inhibitory activities of Fu brick tea.

To investigate the effects of "golden flora" amount on the sensory quality, metabolites and bioactivities of Fu brick tea (FBT), FBT samples with different "golden flora" amounts were prepared from the same materials by adjusting the water content before pressing. With the increase of "golden flora" in samples, the tea liquor color changed from yellow to orange red and the astringent taste gradually diminished. Targeted analysis demonstrated that (-)-epigallocatechin gallate, (-)-epicatechin gallate, and most amino acids gradually decreased as the increase of "golden flora". Seventy differential metabolites were identified by untargeted analysis. Among them, sixteen compounds including two Fuzhuanins and four EPSFs were positively correlated with "golden flora" amount (P < 0.05). The FBT samples with "golden flora" exhibited significantly higher inhibitory potency on α-amylase and lipase than the samples without "golden flora". Our results provide a theoretical basis of guiding FBT processing based on desired sensory quality and metabolites.

Determination of Tea Aroma Precursor Glycosides: An Efficient Approach via Liquid Chromatography-Tandem Mass Spectrometry.

Tea aroma components are often stored as glycosidically bound forms in the tea plant (Camellia sinensis). However, the determination of these glycosides in tea samples is far from optimal. In the present study, we developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous quantification of eight primary aroma glycosides within 10 min. After systematic optimization of multiple reaction monitoring (MRM) parameters, the proposed method was highly sensitive and accurate. Optimization of the method permitted the efficient extraction of aroma glycosides. The developed method was applied to analyze the contents of aroma glycosides in different organs of tea plants, including the bud, leaves, and stem. Contents of aroma glycosides in the harvested 'Shaancha 1' ranged from 36.1 to 40454.4 µg kg-1. Geranyl glucoside and primeveroside mainly accumulated in young leaves, while other glycosides mainly accumulated in mature leaves. The findings document a rapid, reliable, and efficient analysis method. This method will be helpful in elucidating the biosynthesis and biotransformation mechanism of tea aroma glycosides and in promoting the development of the tea industry using advanced technological control approaches during the cultivation of tea plants and tea manufacture.

Identification and profile of phenolamides with anthracnose resistance potential in tea (Camellia sinensis).

Tea anthracnose is a prevalent disease in China that can lead to reduced tea production and lower quality, yet there is currently a lack of effective means for controlling this disease. In this study, we identified 46 phenolamides (including 27 isomers) in different tissues and organs of tea plants based on a developed workflow, and the secondary mass spectra of all these compounds have been documented. It was revealed that tea plants predominantly accumulate protonated aliphatic phenolamides, rather than aromatic phenolamides. The profile of phenolamides indicate that their buildup in tea plants is specific to certain tissues and acyl-acceptors, and this distribution is associated with the extent of phenolamide acyl-modification. Additionally, it was observed that N-Feruloylputrescine (Fer-Put, a type of phenolamides) was responsive to the stimulated accumulation of the tea anthracnose pathogen. The findings of anti-anthracnose experiments in vitro and on tea leaf demonstrated that Fer-Put was capable of significantly inhibiting the growth of anthracnose pathogen colony, effectively prevented tea leaf disease. Furthermore, it was observed that Fer-Put treatment can enhance the antioxidant enzyme activity of tea leaves. TEA002780.1 and TEA013165.1 gene may be responsible for the biosynthesis of Fer-Put in the disease resistance process in tea plants. Through these studies, the types and distribution of phenolamides in tea plants have been elucidated, and Fer-Put's ability to resist anthracnose has been established, providing new insights into the resistance of tea anthracnose.

Comparative Assessment of the Antibacterial Efficacies and Mechanisms of Different Tea Extracts.

Tea is a popular beverage known for its unique taste and vast health benefits. The main components in tea change greatly during different processing methods, which makes teas capable of having different biological activities. We compared the antibacterial activity of four varieties of tea, including green, oolong, black, and Fuzhuan tea. All tea extracts showed antibacterial activity and Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) were more susceptible to tea extracts than Gram-negative bacteria (Escherichia coli and Salmonella typhimurium). Green tea extracts inhibited bacterial pathogens much more effectively in all four varieties of tea with the minimum inhibitory concentration (MIC) values at 20 mg/mL, 10 mg/mL, 35 mg/mL, and 16 mg/mL for E. faecalis, S. aureus, E. coli, and S. typhimurium, respectively. Catechins should be considered as the main antibiotic components of the four tea extracts. Total catechins were extracted from green tea and evaluated their antibacterial activity. Additional studies showed that the catechins damaged the cell membrane and increased cell membrane permeability, leading to changes in the relative electrical conductivity and the release of certain components into the cytoplasm. Tea extracts, especially green tea extracts, should be considered as safe antibacterial food additives.

Widely targeted metabolomics using UPLC-QTRAP-MS/MS reveals chemical changes during the processing of black tea from the cultivar Camellia sinensis (L.) O. Kuntze cv. Huangjinya.

Huangjinya is a light-sensitive mutant tea cultivar that produces fresh leaves with a yellow phenotype, and the leaves also be used to produce black tea with special sensory characteristics. To thoroughly explore the chemical changes that occur during the processing of Huangjinya black tea, tea samples were collected from each processing step to perform quantitative and qualitative analyses by high-performance liquid chromatography and ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS). Compared to fresh tea leaves, only approximately 20% of the catechins remained at the end of processing, while theaflavins levels peaked at the rolling step and were slightly reduced in the fermentation and drying processes. The levels of amino acids derived from protein hydrolysis increased significantly in the withering and rolling processes. Altogether, 620 differential metabolites were identified from 11 subclasses using widely targeted metabolomics based on UPLC-HRMS for the four steps used to process Huangjinya black tea. Flavonoids, phenolic acids, and lipids were the three major classes of differential metabolites, accounting for 52.4% of the differential compounds. The greatest changes in the metabolite profile occurred during the rolling step, with 292 metabolites showing increases or decreases. Two glycoconjugates of the amino acid were first identified in tea, which was sharply increased in the drying stage. The present study provides comprehensive information on the chemical changes during the processing of Huangjinya black tea, and this information is valuable for optimizing manufacturing process and utilization of the Huangjinya tea plant.

Accumulation of Galactinol and ABA Is Involved in Exogenous EBR-Induced Drought Tolerance in Tea Plants.

Drought stress severely limits growth and causes losses in the yield of tea plants. Exogenous application of 24-epibrassinolide (EBR) positively regulates drought responses in various plants. However, whether EBR could contribute to drought resistance in tea plants and the underlying mechanisms has not been investigated. Here, we found that EBR application is beneficial for the drought tolerance of tea plants. The transcriptome results revealed that EBR could contribute to tea plant drought resistance by promoting galactinol and abscisic acid (ABA) biosynthesis gene expression. The content of galactinol was elevated by EBR and EBR-responsive CsDof1.1 positively regulated the expression of the galactinol synthase genes CsGolS2-1 and CsGolS2-2 to contribute to the accumulation of galactinol by directly binding to their promoters. Moreover, exogenous EBR was found to elevate the expression of genes related to ABA signal transduction and stomatal closure regulation, which resulted in the promotion of stomatal closure. In addition, EBR-responsive CsMYC2-2 is involved in ABA accumulation by binding to the promoters CsNCED1 and CsNCED2 to activate their expression. In summary, findings in this study provide knowledge into the transcriptional regulatory mechanism of EBR-induced drought resistance in tea plants.

Hormone Orchestrates a Hierarchical Transcriptional Cascade That Regulates Al-Induced De Novo Root Regeneration in Tea Nodal Cutting.

The aluminum in acid soils is very rhizotoxic to most plant species, but it is essential for root growth and development in Camellia sinensis. However, the molecular basis of Al-mediated signaling pathways in root regeneration of tea plants is largely unclear. In this study, we profiled the physiological phenotype, transcriptome, and phytohormones in the process using stems treated with Al (0.3 mM) and control (0.02 mM). The anatomical analysis showed that the 0.3 mM Al-treated stem began to develop adventitious root (AR) primordia within 7 days, ARs occurred after 21 days, while the control showed a significant delay. We further found that the expression patterns of many genes involved in the biosynthesis of ZT, ACC, and JA were stimulated by Al on day 3; also, the expression profiles of auxin transporter-related genes were markedly increased under Al during the whole rooting process. Moreover, the expression of these genes was strongly correlated with the accumulation of ZT, ACC, JA, and IAA. CsERFs, CsMYBs, and CsWRKYs transcription factor genes with possible crucial roles in regulating AR regeneration were also uncovered. Our findings suggest that multiple phytohormones and genes related to their biosynthesis form a hierarchical transcriptional cascade during Al-induced de novo root regeneration in tea nodal cuttings.

CsGSTU8, a Glutathione S-Transferase From Camellia sinensis, Is Regulated by CsWRKY48 and Plays a Positive Role in Drought Tolerance.

Glutathione S-transferases (GSTs) constitute a large family of enzymes with a wide range of cellular functions. Recently, plant GSTs have gained a great deal of attention due to their involvement in the detoxification of electrophilic xenobiotics and peroxides under adverse environmental conditions, such as salt, cold, UV-B and drought stress. A previous study reported that a GST gene (CsGSTU8) in tea plant was distinctly induced in response to drought, suggesting this gene plays a critical role in the drought stress response. In this study, by using quantitative real-time PCR (qRT-PCR) and ß-glucuronidase (GUS) reporter lines, we further demonstrated that CsGSTU8 was upregulated in response to drought stress and exogenous abscisic acid (ABA) treatments. Overexpression of CsGSTU8 in Arabidopsis resulted in enhanced drought tolerance as indicated by the improved scavenging of excess amounts of reactive oxygen species (ROS) under drought conditions. Furthermore, we found that CsWRKY48 acts as a transcriptional activator and that its expression is induced in response to drought stress and ABA treatment. Electrophoretic mobility shift assays (EMSAs), dual-luciferase (LUC) assays and transient expression assays in tea plant leaves revealed that CsWRKY48 directly binds to the W-box elements in the promoter of CsGSTU8 and activates its expression. Taken together, our results provide additional knowledge of drought stress responses in tea plant.

CsUGT78A15 catalyzes the anthocyanidin 3-O-galactoside biosynthesis in tea plants.

Anthocyanins are a group of natural water-soluble pigments in plants that contribute to the pink-purple color of a range of tissues. Because anthocyanins have various biological activities in human health, there is great research interest in the development of anthocyanin-rich foods and beverages, including purple shoot tea. Anthocyanidin 3-O-galactosides have been identified as one of the main anthocyanin components in purple shoot tea, but the enzyme responsible for their biosynthesis remains unclear. UDP-galactose anthocyanidin 3-O-galactosyltransferase (UA3GalT) is presumed to catalyze the galactosylation of anthocyanidin. Therefore, we assayed the UA3GalT activity in five tea samples with varying degrees of purple color and found that its activity was strongly positively correlated (r = 0.929, p < 0.05) with anthocyanin content. Phylogenetic analysis and sequence alignment suggested that CsUGT78A15 encoded a UA3GalT enzyme. Enzymatic assays indicated that rCsUGT78A15 could catalyze the synthesis of cyanidin 3-O-galactoside and delphinidin 3-O-galactoside using UDP-galactose as a sugar donor, and it showed higher catalytic efficiency towards delphinidin than cyanidin. These results indicate that CsUGT78A15 acts as a UA3GalT in vitro. Subcellular localization showed that CsUGT78A15 was located in the endoplasmic reticulum (ER) and nucleus, consistent with the location of anthocyanin synthesis. Transient overexpression of CsUGT78A15 in the fruit of mature 'Granny Smith' apples showed that the upregulation of CsUGT78A15 promoted cyanidin 3-O-galactoside accumulation in apple skins. These results suggested that CsUGT78A15 could catalyze galactosylation of anthocyanidins in planta. Our findings provide insight into the biosynthesis of anthocyanins in tea plants.