The Mulberry SPL Gene Family and the Response of MnSPL7 to Silkworm Herbivory through Activating the Transcription of MnTT2L2 in the Catechin Biosynthesis Pathway.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, as unique plant transcription factors, play important roles in plant developmental regulation and stress response adaptation. Although mulberry is a commercially valuable tree species, there have been few systematic studies on SPL genes. In this work, we identified 15 full-length SPL genes in the mulberry genome, which were distributed on 4 Morus notabilis chromosomes. Phylogenetic analysis clustered the SPL genes from five plants (Malus × domestica Borkh, Populus trichocarpa, M. notabilis, Arabidopsis thaliana, and Oryza sativa) into five groups. Two zinc fingers (Zn1 and Zn2) were found in the conserved SBP domain in all of the MnSPLs. Comparative analyses of gene structures and conserved motifs revealed the conservation of MnSPLs within a group, whereas there were significant structure differences among groups. Gene quantitative analysis showed that the expression of MnSPLs had tissue specificity, and MnSPLs had much higher expression levels in older mulberry leaves. Furthermore, transcriptome data showed that the expression levels of MnSPL7 and MnSPL14 were significantly increased under silkworm herbivory. Molecular experiments revealed that MnSPL7 responded to herbivory treatment through promoting the transcription of MnTT2L2 and further upregulating the expression levels of catechin synthesis genes (F3'H, DFR, and LAR).

Transcriptomic analysis of tea plant (Camellia sinensis) revealed the co-expression network of 4111 paralogous genes and biosynthesis of quality-related key metabolites under multiple stresses.

The tea plant is an essential economic plant in many countries. However, its growing season renders them vulnerable to stresses. To understand the transcriptomic influences of these stresses on tea plants, we sequenced and analyzed the transcriptomes under drought, high-temperature, and pest. Paralogs were identified by comparing 14 evolutionarily close genomes. The differentially expressed paralog (DEPs) genes were analyzed regarding single or multiple stresses, and 1075 of the 4111 DEPs were commonly found in all the stresses. The co-expression network of the DEPs and TFs indicated that genes of catechin biosynthesis were associated with most transcription factors specific to each stress. The genes playing a significant role in the late response to drought and pest stress mainly functioned in the early response to high-temperature. This study revealed the relationship between stress and regulation of QRM synthesis and the role of QRMs in response to these (a)biotic stresses.

Exogenous melatonin improves tea quality under moderate high temperatures by increasing epigallocatechin-3-gallate and theanine biosynthesis in Camellia sinensis L.

Global warming has multifarious effects on crop growth and productivity. Nonetheless, the effects of moderate-high temperatures and melatonin on tea yield and quality remain unclear. In this study, we found that melatonin, a universal growth stimulatory molecule, not only promotes photosynthesis and biomass accumulation in tea plants (Camellia sinensis L.) but also improves tea quality under sub high temperature (SHT). SHT increased the dry biomass and photosynthesis by 40.8% and 28.1%, respectively, and exogenous melatonin caused a further improvement. Moreover, SHT increased the total polyphenol concentrations and decreased the free amino acid concentrations, leading to a significant increase (68.2%) in polyphenol to free amino acid ratio. However, melatonin decreased the polyphenol to free amino acid ratio by delicately improving the concentrations of polyphenols and amino acids. Consistent with the total polyphenol, melatonin increased the concentrations of (-)-catechin, (-)-gallocatechin (GC), and (-)-epigallocatechin-3-gallate (EGCG) in tea leaves. The qRT-PCR analysis revealed that melatonin increased the transcript levels of catechins biosynthesis genes, such as CsCHS, CsCH1, CsF3H, CsDFR, CsANS, CsLAR, and CsANR under SHT. Meanwhile, the theanine concentration was decreased by SHT, which was attributed to the attenuated expression of CsGS, CsGOGAT, CsGDH, and CsTS1. Nonetheless, melatonin significantly increased those transcripts and the content of theanine under SHT. Melatonin also increased the caffeine content by inducing the expression of CsTIDH, CssAMS, and CsTCS1. These results suggest that melatonin could positively alter tea growth and quality by modulating the photosynthesis and biosynthesis of polyphenols, amino acids, and caffeine in tea leaves under SHT.

Integrated transcriptomics and metabolomics analysis of catechins, caffeine and theanine biosynthesis in tea plant (Camellia sinensis) over the course of seasons.

BACKGROUND: Catechins, caffeine, and theanine as three important metabolites in the tea leaves play essential roles in the formation of specific taste and shows potential health benefits to humans. However, the knowledge on the dynamic changes of these metabolites content over seasons, as well as the candidate regulatory factors, remains largely undetermined. RESULTS: An integrated transcriptomic and metabolomic approach was used to analyze the dynamic changes of three mainly metabolites including catechins, caffeine, and theanine, and to explore the potential influencing factors associated with these dynamic changes over the course of seasons. We found that the catechins abundance was higher in Summer than that in Spring and Autumn, and the theanine abundance was significantly higher in Spring than that in Summer and Autumn, whereas caffeine exhibited no significant changes over three seasons. Transcriptomics analysis suggested that genes in photosynthesis pathway were significantly down-regulated which might in linkage to the formation of different phenotypes and metabolites content in the tea leaves of varied seasons. Fifty-six copies of nine genes in catechins biosynthesis, 30 copies of 10 genes in caffeine biosynthesis, and 12 copies of six genes in theanine biosynthesis were detected. The correlative analysis further presented that eight genes can be regulated by transcription factors, and highly correlated with the changes of metabolites abundance in tea-leaves. CONCLUSION: Sunshine intensity as a key factor can affect photosynthesis of tea plants, further affect the expression of major Transcription factors (TFs) and structural genes in, and finally resulted in the various amounts of catechins, caffeine and theaine in tea-leaves over three seasons. These findings provide new insights into abundance and influencing factors of metabolites of tea in different seasons, and further our understanding in the formation of flavor, nutrition and medicinal function.

Preferential assimilation of NH4+ over NO3- in tea plant associated with genes involved in nitrogen transportation, utilization and catechins biosynthesis.

Physiological effects of ammonium (NH4+) and nitrate (NO3-) on tea have confirmed that tea plants prefer NH4+ as the dominant nitrogen (N) source. To investigate the possible explanations for this preference, studies of 15NH4+ and 15NO3- assimilation using hydroponically grown tea plants were conducted. During the time course of 15NH4+ and 15NO3- assimilation, the absorption of 15N from 15NH4+ was more rapid than that from 15NO3-, as there was a more efficient expression pattern of NH4+ transporters compared with that of NO3- transporters. 15NH4+-fed tea plants accumulated more 15N than 15NO3- fed plants, which was demonstrated by that genes related to primary N assimilation, like CsNR, CsNiR, CsGDH and CsGOGAT, were more affected by 15NH4+ than 15NO3-. Markedly higher NH4+ concentrations were observed in 15NH4+-fed tea roots in comparison with NO3- treatment, whereas tea plants maintained a balanced concentration of NH4+ in tea leaves under both these two N forms. This maintenance was achieved through the increased expression of genes involved in theanine biosynthesis and the inhibition of genes related to catechins derived from phenylpropanoid pathway. The current results suggest that efficient NH4+ transportation, assimilation, and reutilization enables tea plant as an ammonium preferring plant species.

Overexpression of the GbF3'H1 Gene Enhanced the Epigallocatechin, Gallocatechin, and Catechin Contents in Transgenic Populus.

Ginkgo biloba L. leaves are a flavonoid resource for the pharmaceutical industry. The flavonoid 3'-hydroxylase (F3'H) is a key enzyme in the flavonoid biosynthesis pathway. However, the role of F3'H in flavonoid biosynthesis and metabolism is unclear. In this study, we characterized and functionally analyzed the ginkgo F3'H gene GbF3'H1 that encodes a protein of 520 amino acids. Expression profiling showed that GbF3'H1 was highly expressed in the leaves of ginkgo in September. Subcellular localization showed that GbF3'H1 occurred predominately in the cytoplasm. Transgenic poplars overexpressing GbF3'H1 had more red pigmentation in leaves than did wild-type (WT) plants. Furthermore, the concentrations of epigallocatechin, gallocatechin, and catechin in the downstream products synthesized by flavonoids were significantly higher in the transgenic plants than in the WT plants. These results indicate that the overexpression of GbF3'H1 enhances flavonoid production in transgenic plants and provides new insights into flavonoid biosynthesis and metabolism.

Genomic Variance and Transcriptional Comparisons Reveal the Mechanisms of Leaf Color Affecting Palatability and Stressed Defense in Tea Plant.

Leaves are one of the most important organs of plants, and yet, the association between leaf color and consumable traits remains largely unclear. Tea leaves are an ideal study system with which to investigate the mechanism of how leaf coloration affects palatability, since tea is made from the leaves of the crop Camellia sinensis. Our genomic resequencing analysis of a tea cultivar ZiJuan (ZJ) with purple leaves and altered flavor revealed genetic variants when compared with the green-leaf, wild type cultivar YunKang(YK). RNA-Seq based transcriptomic comparisons of the bud and two youngest leaves in ZJ and YK identified 93%, 9% and 5% expressed genes that were shared in YK- and ZJ-specific cultivars, respectively. A comparison of both transcript abundance and particular metabolites revealed that the high expression of gene UFGT for anthocyanin biosynthesis is responsible for purple coloration, which competes with the intermediates for catechin-like flavanol biosynthesis. Genes with differential expression are enriched in response to stress, heat and defense, and are casually correlated with the environmental stress of ZJ plant origin in the Himalayas. In addition, the highly expressed C4H and LDOX genes for synthesizing flavanol precursors, ZJ-specific CLH1 for degrading chlorophyll, alternatively spliced C4H and FDR and low photosynthesis also contributed to the altered color and flavor of ZJ. Thus, our study provides a better molecular understanding of the effect of purple coloration on leaf flavor, and helps to guide future engineering improvement of palatability.

Differentially Expressed Protein Are Involved in Dynamic Changes of Catechins Contents in Postharvest Tea Leaves under Different Temperatures.

In this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology were used to investigate three samples from postharvest tea leaves that were treated at room temperature (25 °C, control group), high temperature (38 °C), and low temperature (4 °C) for 4 h. In heat and cold treatments, a total of 635 and 566 differentially expressed proteins (DEPs) were determined, respectively. DEPs were annotated to GO and KEGG databases, which revealed that DEPs involved in various aspects of biological process. Three catechins-related DEPs, CsCHI, CsF3H, and CsANR, were identified. Both catechins contents and the expression profiles of catechins biosynthesis-related genes changed significantly under different temperature treatments. The correlations between catechins contents, gene expression profiles, and DEPs were analyzed. This study provides potential new insights into the molecular basis for tea production of postharvest leaves and catechins content changes at diverse temperature conditions and will guide the improvement of tea-processing technology.

VvLAR1 and VvLAR2 Are Bifunctional Enzymes for Proanthocyanidin Biosynthesis in Grapevine.

Proanthocyanidins (PAs) in grapevine (Vitis vinifera) are found mainly in berries, and their content and degree of polymerization are important for the mouth feel of red wine. However, the mechanism of PA polymerization in grapevine remains unclear. Previous studies in the model legume Medicago truncatula showed that 4ß-(S-cysteinyl)-epicatechin (Cys-EC) is an epicatechin-type extension unit for nonenzymatic PA polymerization, and that leucoanthocyanidin reductase (LAR) converts Cys-EC into epicatechin starter unit to control PA extension. Grapevine possesses two LAR genes, but their functions are not clear. Here, we show that both Cys-EC and 4ß-(S-cysteinyl)-catechin (Cys-C) are present in grapevine. Recombinant VvLAR1 and VvLAR2 convert Cys-C and Cys-EC into (+)-catechin and (-)-epicatechin, respectively, in vitro. The kinetic parameters of VvLARs are similar, with both enzymes being more efficient with Cys-C than with Cys-EC, the 2,3-cis conformation of which results in steric hindrance in the active site. Both VvLARs also produce (+)-catechin from leucocyanidin, and an inactive VvLAR2 allele reported previously is the result of a single amino acid mutation in the N terminus critical for all NADPH-dependent activities of the enzyme. VvLAR1 or VvLAR2 complement the M. truncatula lar:ldox double mutant that also lacks the leucoanthocyanidin dioxygenase (LDOX) required for epicatechin starter unit formation, resulting in increased soluble PA levels, decreased insoluble PA levels, and reduced levels of Cys-C and Cys-EC when compared to the double mutant, and the appearance of catechin, epicatechin, and PA dimers characteristic of the ldox single mutant in young pods. These data advance our knowledge of PA building blocks and LAR function and provide targets for grapevine breeding to alter PA composition.

Recombinant polyphenol oxidases for production of theaflavins from tea polyphenols.

Theaflavins (TFs) have attracted much attention due to their various bioactivities in black tea. This paper describes the first trial for enzymatic production of TFs by recombinant polyphenol oxidases (PPOs). PPO genes were cloned from nine species and expressed in E. coli. Crude enzyme assays by LC-MS revealed that eight recombinant PPOs were active for TFs production from tea polyphenols as substrates. Much higher activities were observed for crude enzymes of Md2 from Malus domestica (apple), Pp4 from Pyrus pashia (pear), and Ej2 from Eriobotrya japonica (loquat). When immobilized on mesoporous silica, crude Md2 was most active. The purified Md2 was immobilized and showed almost twice activity as high as its free enzyme. While the maximum activity of free enzyme was found at pH 5 and 10-30 °C, the immobilized enzyme had broader range of pH 4-6 and 10-40 °C. The activity of immobilized enzyme was relatively constant during the pH and thermal stability test. When used at 0.2 mg/ml in the beginning, the immobilized enzyme retained approximately 40% of its initial activity after 8 cycles of operation.

Habitat management as a safe and effective approach for improving yield and quality of tea (Camellia sinensis) leaves.

Tea (Camellia sinensis) leaves are used to make the most widely consumed beverage globally after water. Therefore, the safety and quality of raw tea leaves are important indices for making tea and related products. Habitat management has been widely used as an environmentally friendly method to control pests in agroecosystems. To investigate the impact of habitat management on tea plantation ecosystems, a habitat management approach with intercropping was established. The function of habitat management on pest control was evaluated. Furthermore, metabolome and transcriptome analysis were applied to assay changes in quality-related metabolites. The habitat management approach was found to maintain arthropod biodiversity and develop natural arthropod enemies in the tea plantation. Therefore, the yield of the habitat management-treated tea plantation was increased. Metabolome analysis showed that epigallocatechin-3-gallate, the major catechin in tea leaves, has a significantly increased content in leaves of tea plants under habitat management compared with those in the control tea plantation. The content of L-theanine, the major amino acid in tea leaves, was not significantly changed in tea plants under habitat management. Furthermore, aroma compounds were more abundant in tea leaves from the habitat management-treated tea plantation than those from the chemical pesticide-treated tea plantation. Therefore, habitat management is reported for the first time as a safe and effective approach to improving the yield and quality of tea leaves.

Molecular Characterization of WRKY Transcription Factors That Act as Negative Regulators of O-Methylated Catechin Biosynthesis in Tea Plants ( Camellia sinensis L.).

Tea O-methylated catechins, especially (-)-epigallocatechin 3- O-(3- O-methyl)gallate (EGCG3″Me), have been attracting much attention as a result of their positive health effects. The transcription regulators of O-methylated catechin biosynthesis remain elusive. In this study, the expression pattern of genes related to O-methylated catechin biosynthesis, including CsLAR, CsANS, CsDFR, CsANR, and CCoAOMT, in three tea cultivars with different contents of EGCG3″Me was investigated. Two WRKY transcription factors (TFs), designated as CsWRKY31 and CsWRKY48, belonging to groups IIb and IIc of the WRKY family, respectively, were further identified. CsWRKY31 and CsWRKY48 were nuclear-localized proteins and possessed transcriptional repression ability. Furthermore, expression of CsWRKY31 and CsWRKY48 showed negative correlation with CsLAR, CsDFR, and CCoAOMT during EGCG3″Me accumulation in tea leaves. More importantly, W-box (C/T)TGAC(T/C) elements were located in the promoter of CsLAR, CsDFR, and CCoAOMT, and further assays revealed that CsWRKY31 and CsWRKY48 were capable of repressing the transcription of CsLAR, CsDFR, and CCoAOMT via the attachment of their promoters to the W-box elements. Collectively, our findings identify two novel negative regulators of O-methylated catechin biosynthesis in tea plants, which might provide a potential strategy to breed high-quality tea cultivar.

Transcriptomic analyses of cacao cell suspensions in light and dark provide target genes for controlled flavonoid production.

Catechins, including catechin (C) and epicatechin (E), are the main type of flavonoids in cacao seeds. They play important roles in plant defense and have been associated with human health benefits. Although flavonoid biosynthesis has been extensively studied using in vitro and in vivo models, the regulatory mechanisms controlling their accumulation under light/dark conditions remain poorly understood. To identify differences in flavonoid biosynthesis (particularly catechins) under different light treatments, we used cacao cell suspensions exposed to white-blue light and darkness during 14 days. RNA-Seq was applied to evaluate differential gene expression. Our results indicate that light can effectively regulate flavonoid profiles, inducing a faster accumulation of phenolic compounds and shifting E/C ratios, in particular as a response to switching from white to blue light. The results demonstrated that HY5, MYB12, ANR and LAR were differentially regulated under light/dark conditions and could be targeted by overexpression aiming to improve catechin synthesis in cell cultures. In conclusion, our RNA-Seq analysis of cacao cells cultured under different light conditions provides a platform to dissect key aspects into the genetic regulatory network of flavonoids. These light-responsive candidate genes can be used further to modulate the flavonoid production in in vitro systems with value-added characteristics.

Metabolic Flux Enhancement and Transcriptomic Analysis Displayed the Changes of Catechins Following Long-Term Pruning in Tea Trees ( Camellia sinensis).

The tea tree is a perennial woody plant, and pruning is one of the most crucial cultivation measurements for tea plantation management. To date, the relationship between long-term pruning and metabolic flux enhancement in tea trees has not been studied. In this research, 11-year-old pruned tea trees from four different cultivars were randomly selected for transcriptome analysis and characteristic secondary metabolite analysis together with controls. The findings revealed that epigallocatechin gallate (EGCG) accumulation in pruned tea trees was significantly higher than that in unpruned tea trees. SCPL1A expression (encoding a class of serine carboxypeptidase), which has been reported to have a catalytic ability during EGCG biosynthesis, together with LAR, encoding leucoanthocyanidin reductase, was upregulated in the pruned tea trees. Moreover, metabolic flux enhancement and transcriptome analysis revealed low EGCG accumulation in the leaves of unpruned tea trees. Because of the bitter and astringent taste of EGCG, these results provide a certain understanding to the lower bitterness and astringency in teas from "ancient tea trees", growing in the wild with no trimming, than teas produced from pruned plantation trees.

Synthesis of Theaflavins and Their Functions.

Numerous epidemiological and interventional clinical studies have consistently reported that black tea is good for human health. The polyphenolic compound, theaflavin, and its galloyl esters (theaflavins) are the primary red pigments in black tea that possess several health benefits, including fat-reducing and glucose-lowering capabilities and lifestyle-related disease prevention related to anti-obesity, anticancer, anti-atherosclerotic, anti-inflammatory, antiviral, antibacterial, anti-osteoporotic, and anti-dental caries properties. These compounds are produced by key enzymes, such as polyphenol oxidase and peroxidase, from parent green tea catechins present in fresh green tea leaves during the production of black tea leaves or the fermentation of green tea. However, theaflavins are only present in low concentrations in black tea; thus, their extraction from black tea leaves at sufficient levels for use in medical studies has been difficult. To circumvent this issue, different procedures for the synthesis of theaflavins using chemical oxidizing reagents or enzymes have been studied; however, low yields have limited their utility. Recently, however, several biosynthetic methods have been developed for the mass production of theaflavins. Using these methods, the physiological functions of theaflavins in lifestyle-related diseases in mice and humans have also been studied. In this review, we present the synthesis of theaflavins and their health benefits.

Molecular Evidence for Catechin Synthesis and Accumulation in Tea Buds (Camellia sinensis).

Early spring buds of the Camellia sinensis variety Shuchazao were separated into two parts, including the shoot tip (ST) and non-expanded young leaves (YL), in which the synthesis and accumulation of catechins in the two parts were assessed by high-performance liquid chromatography (HPLC), p-dimethylaminocinnamaldehyde (DMACA) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and in situ hybridization. HPLC showed that (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) amounts in YL were increased significantly by 74.0 and 71.8%, respectively. The results of DMACA staining indicated that catechins in buds accumulated mainly in mesophyll cells and the bud shaft of YL. Meanwhile, qRT-PCR demonstrated that the relative expression levels of genes related to flavonoid metabolism, including CsPAL1, CsC4H1, CsC4H2, CsCHS2, CsF3'5'H1, CsDFR1, CsDFR2, and CsANR1, were significantly higher in YL than in the ST. In situ hybridization revealed that CsDFR1, CsDFR2, CsLAR, and CsANR1 were expressed in leaf primordia and YL but not in the apical meristem. These findings highlight the synthesis and accumulation patterns of catechins in different parts of the ST in C. sinensis, providing a theoretical basis for the assessment of synthesis, accumulation, and transfer patterns of catechins in tea plants.

Transcriptional profiling of catechins biosynthesis genes during tea plant leaf development.

MAIN CONCLUSION: A total of 299,113 unigenes were generated and 15,817 DEGs were identified. We identified candidate genes associated with the regulation of catechins biosynthesis during leaf development in tea plant. The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most economically significant crops worldwide because of its positive effects on human health. The health benefits of tea are mainly attributed to catechins, which are the predominant polyphenols that accumulate in tea. Catechins are products of the phenylpropanoid and flavonoid biosynthetic pathways. Although catechins were identified in tea leaves long ago, the molecular mechanisms regulating catechins biosynthesis remain unclear. To identify candidate genes involved in catechins biosynthesis, we analyzed the transcriptomes of tea leaves during five different leaf stages of development using RNA-seq. Approximately 809 million high-quality reads were obtained, trimmed, and assembled into 299,113 unigenes with an average length of 565 bp. A total of 15,817 unigenes were differentially expressed during the different stages of leaf development. These differentially expressed genes were enriched in a variety of processes such as the regulation of the cell cycle, starch and sucrose metabolism, photosynthesis, phenylpropanoid biosynthesis, phenylalanine metabolism, and flavonoid biosynthesis. Based on their annotations, 51 of these differentially expressed unigenes are involved in phenylpropanoid and flavonoid biosynthesis. Furthermore, transcription factors such as MYB, bHLH and MADS, which may involve in the regulation of catechins biosynthesis, were identified through co-expression analysis of transcription factors and structural genes. Real-time PCR analysis of candidate genes indicated a good correlation with the transcriptome data. These findings increase our understanding of the molecular mechanisms regulating catechins biosynthesis in the tea plant.

The proposed biosynthesis of procyanidins by the comparative chemical analysis of five Camellia species using LC-MS.

The genus Camellia (C.) contains many species, including C. sinensis, C. assamica, and C. taliensis, C. gymnogyna and C. tachangensis. The polyphenols of C. sinensis and C. assamica are flavan-3-ols monomers and their dimers and trimmers. However, the biosynthesis of procyanidins in Camellia genus remains unclear. In the present study, a comparative chemical analysis of flavan-3-ols, flavan-3-ols glycoside and procyanidins was conducted by high performance liquid chromatography (HPLC) and liquid chromatography diode array detection coupled with triple-quadrupole mass-spectrometry (LC-DAD-QQQ-MS). The results showed that C. tachangensis had a significant higher contents of (-)-epicatechin (EC) and (-)-epigallocatechin (EGC) compared with C. sinensis (p < 0.001). By contrast, higher levels of galloylated catechins were detected in C. sinensis. LC-DAD-MS/MS indicated that the main secondary metabolites of C. tachangensis were non-galloylated catechins, procyanidin dimers and trimers. Furthermore, (-)-epicatechin glucose (EC-glucose) and (-)-epigallocatechin glucose (EGC-glucose) were also abundant in C. tachangensis. A correlation analysis of EC-glucose and procyanidins dimers was conducted in five Camellia species. The levels of EC-glucose were closely related to the procyanidin dimers content. Thus, it was suggested that EC-glucose might be an important substrate for the biosynthesis of procyanidins.

Metabolic Flux Redirection and Transcriptomic Reprogramming in the Albino Tea Cultivar 'Yu-Jin-Xiang' with an Emphasis on Catechin Production.

In this study, shade-induced conversion from a young pale/yellow leaf phenotype to a green leaf phenotype was studied using metabolic and transcriptomic profiling and the albino cultivar 'Yu-Jin-Xiang' ('YJX') of Camellia sinensis for a better understanding of mechanisms underlying the phenotype shift and the altered catechin and theanine production. Shaded leaf greening resulted from an increase in leaf chlorophyll and carotenoid abundance and chloroplast development. A total of 1,196 differentially expressed genes (DEGs) were identified between the 'YJX' pale and shaded green leaves, and these DEGs affected 'chloroplast organization' and 'response to high light' besides many other biological processes and pathways. Metabolic flux redirection and transcriptomic reprogramming were found in flavonoid and carotenoid pathways of the 'YJX' pale leaves and shaded green leaves to different extents compared to the green cultivar 'Shu-Cha-Zao'. Enhanced production of the antioxidant quercetin rather than catechin biosynthesis was correlated positively with the enhanced transcription of FLAVONOL SYNTHASE and FLAVANONE/FLAVONOL HYDROXYLASES leading to quercetin accumulation and negatively correlated to suppressed LEUCOANTHOCYANIDIN REDUCTASE, ANTHOCYANIDIN REDUCTASE and SYNTHASE leading to catechin biosynthesis. The altered levels of quercetin and catechins in 'YJX' will impact on its tea flavor and health benefits.

Combined small RNA and degradome sequencing reveals complex microRNA regulation of catechin biosynthesis in tea (Camellia sinensis).

MicroRNAs are endogenous non-coding small RNAs playing crucial regulatory roles in plants. Tea, a globally popular non-alcoholic drink, is rich in health-enhancing catechins. In this study, 69 conserved and 47 novel miRNAs targeting 644 genes were identified by high-throughout sequencing. Predicted target genes of miRNAs were mainly involved in plant growth, signal transduction, morphogenesis and defense. To further identify targets of tea miRNAs, degradome sequencing and RNA ligase-mediated rapid amplification of 5'cDNA ends (RLM-RACE) were applied. Using degradome sequencing, 26 genes mainly involved in transcription factor, resistance protein and signal transduction protein synthesis were identified as potential miRNA targets, with 5 genes subsequently verified. Quantitative real-time PCR (qRT-PCR) revealed that the expression patterns of novel-miR1, novel-miR2, csn-miR160a, csn-miR162a, csn-miR394 and csn-miR396a were negatively correlated with catechin content. The expression of six miRNAs (csn-miRNA167a, csn-miR2593e, csn-miR4380a, csn-miR3444b, csn-miR5251 and csn-miR7777-5p.1) and their target genes involved in catechin biosynthesis were also analyzed by qRT-PCR. Negative and positive correlations were found between these miRNAs and catechin contents, while positive correlations were found between their target genes and catechin content. This result suggests that these miRNAs may negatively regulate catechin biosynthesis by down-regulating their biosynthesis-related target genes. Taken together, our results indicate that miRNAs are crucial regulators in tea, with the results of 5'-RLM-RACE and expression analyses revealing the important role of miRNAs in catechin anabolism. Our findings should facilitate future research to elucidate the function of miRNAs in catechin biosynthesis.