Circadian rhythm response and its effect on photosynthetic characteristics of the Lhcb family genes in tea plant.

BACKGROUND: The circadian clock, also known as the circadian rhythm, is responsible for predicting daily and seasonal changes in the environment, and adjusting various physiological and developmental processes to the appropriate times during plant growth and development. The circadian clock controls the expression of the Lhcb gene, which encodes the chlorophyll a/b binding protein. However, the roles of the Lhcb gene in tea plant remain unclear. RESULTS: In this study, a total of 16 CsLhcb genes were identified based on the tea plant genome, which were distributed on 8 chromosomes of the tea plant. The promoter regions of CsLhcb genes have a variety of cis-acting elements including hormonal, abiotic stress responses and light response elements. The CsLhcb family genes are involved in the light response process in tea plant. The photosynthetic parameter of tea leaves showed rhythmic changes during the two photoperiod periods (48 h). Stomata are basically open during the day and closed at night. Real-time quantitative PCR results showed that most of the CsLhcb family genes were highly expressed during the day, but were less expressed at night. CONCLUSIONS: Results indicated that CsLhcb genes were involved in the circadian clock process of tea plant, it also provided potential references for further understanding of the function of CsLhcb gene family in tea plant.

Genome-wide identification and characterization of PIN-FORMED (PIN) and PIN-LIKES (PILS) gene family reveals their role in adventitious root development in tea nodal cutting (Camellia Sinensis).

Auxin affects all aspects of plant growth and development, including morphogenesis and adaptive responses. Auxin transmembrane transport is promoted by PIN formation (PIN) and a structurally similar PIN-like (PILS) gene family, which jointly controls the directional transport of the auxin between plant cells, and the accumulation of intracellular auxin. At present, there is no study investigating the roles of CslPIN and CslPILS gene family in root development in the tea plant (Camellia sinensis). In this study, 8 CslPIN and 10 CslPILS genes were identified in the tea plant, and their evolutionary relationships, physical and chemical properties, conserved motifs, cis-acting elements, chromosome location, collinearity, and expression characteristics were analyzed. The mechanism of CslPIN and CslPILS in the formation of tea adventitious roots (ARs) was studied by the AR induction system. Through functional verification, the regulation of CslPIN3 gene on root growth and development of tea plant was studied by over-expression of CslPIN3 in Arabidopsis thaliana and in situ hybridization in Camellia sinensis. The results confirmed CslPIN3 was involved in the regulation of root growth and development as well as auxin accumulation. This study provides a better insight into the regulatory mechanism of CslPIN and CslPILS gene family on the formation of AR in tea plant.

Reliable reference genes for normalization of gene expression data in tea plants (Camellia sinensis) exposed to metal stresses.

Tea plants [Camellia sinensis (L.) O. Kuntze] are an important leaf-type crop that are widely used for the production of non-alcoholic beverages in the world. Exposure to excessive amounts of heavy metals adversely affects the quality and yield of tea leaves. To analyze the molecular responses of tea plants to heavy metals, a reliable quantification of gene expression is important and of major importance herein is the normalization of the measured expression levels for the target genes. Ideally, stably expressed reference genes should be evaluated in all experimental systems. In this study, 12 candidate reference genes (i.e., 18S rRNA, Actin, CYP, EF-1α, eIF-4α, GAPDH, MON1, PP2AA3, TBP, TIP41, TUA, and UBC) were cloned from tea plants, and the stability of their expression was examined systematically in 60 samples exposed to diverse heavy metals (i.e., manganese, aluminum, copper, iron, and zinc). Three Excel-based algorithms (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of these genes. PP2AA3 and 18S rRNA were the most stably expressed genes, even though their expression profiles exhibited some variability. Moreover, commonly used reference genes (i.e., GAPDH and TBP) were the least appropriate reference genes for most samples. To further validate the suitability of the analyzed reference genes, the expression level of a phytochelatin synthase gene (i.e., CsPCS1) was determined using the putative reference genes for data normalizations. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in tea plants.

Meta-Analysis of the Association between Tea Intake and the Risk of Cognitive Disorders.

BACKGROUND: Alzheimer's disease is a common neurodegenerative disorder in elderly. This study was aimed to systematically evaluate the association between tea intake and the risk of cognitive disorders by meta-analysis. METHODS AND FINDINGS: PubMed, Embase and Wanfang databases were systematically searched and a total of 26 observational studies were included in this study. Odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) were calculated and pooled by using fixed or random effects models according to the degree of heterogeneity. RESULTS: The overall pooled analysis indicated that tea intake could significantly reduce the risk of cognitive disorders (OR = 0.65, 95%CI = 0.58-0.73). Subgroup analyses were conducted based on study design, population, frequency of tea drinking and type of cognitive disorders. The results showed that tea drinking was significantly associated with the reduced incidence of cognitive disorders in all of subgroups based on study design and frequency of tea drinking. In particular, tea drinking was inversely associated with the risk of cognitive impairment (CoI), mild cognitive impairment (MCI), cognitive decline and ungrouped cognitive disorders. Moreover, for population subgroups, the significant association was only found in Chinese people. CONCLUSION: Our study suggests that daily tea drinking is associated with decreased risk of CoI, MCI and cognitive decline in the elderly. However, the association between tea intake and Alzheimer's disease remains elusive.

Isolation and dynamic expression of four genes involving in shikimic acid pathway in Camellia sinensis 'Baicha 1' during periodic albinism.

Flavonoids are the main flavor components and functional ingredients in tea, and the shikimic acid pathway is considered as one of the most important pathways in flavonoid biosynthesis, but little was known about the function of regulatory genes in the metabolism phenolic compounds in tea plant (Camellia sinensis), especially related genes in shikimic acid pathway. The dynamic changes of catechin (predominant flavonoid) contents were analyzed in this study, and four genes (CsPPT, CsDAHPS, CsSDH and CsCS) involving in shikimic acid pathway in C. sinensis albino cultivar 'Baicha 1' were cloned and characterized. The full-length cDNA sequences of these genes were obtained using reverse transcription-PCR and rapid amplification of cDNA ends. At the albinistic stage, the amounts of all catechins decreased to the lowest levels, when epigallocatechin gallate was the highest, whereas gallocatechin-3-O-gallate the lowest. Gene expression patterns analyzed by qRT-PCR showed that CsPPT and CsDAHPS were highly expressed in flowers and buds, while CsSDH and CsCS showed high expression levels in buds and leaves. It was also found that the transcript abundance of shikimic acid biosynthetic genes followed a tightly regulated biphasic pattern, and was affected by albinism. The transcript levels of CsPPT and CsDAHPS were decreased at albinistic stage followed elevated expression, whereas CsSDH and CsCS were increased only at re-greening stage. Taken together, these findings suggested that these four genes in C. sinensis may play different roles in shikimic acid biosynthesis and these genes may have divergent functions.

Selection of suitable reference genes for qRT-PCR normalization during leaf development and hormonal stimuli in tea plant (Camellia sinensis).

Tea plant (Camellia sinensis) leaf is an important non-alcoholic beverage resource. The application of quantitative real time polymerase chain reaction (qRT-PCR) has a profound significance for the gene expression studies of tea plant, especially when applied to tea leaf development and metabolism. In this study, nine candidate reference genes (i.e., CsACT7, CsEF-1α, CseIF-4α, CsGAPDH, CsPP2A, CsSAND, CsTBP, CsTIP41, and CsTUB) of C. sinensis were cloned. The quantitative expression data of these genes were investigated in five tea leaf developmental stages (i.e., 1st, 2nd, 3rd, 4th, and older leaves) and normal growth tea leaves subjected to five hormonal stimuli (i.e., ABA, GA, IAA, MeJA, and SA), and gene expression stability was calculated using three common statistical algorithms, namely, geNorm, NormFinder, and Bestkeeper. Results indicated that CsTBP and CsTIP41 were the most stable genes in tea leaf development and CsTBP was the best gene under hormonal stimuli; by contrast, CsGAPDH and CsTUB genes showed the least stability. The gene expression profile of CsNAM gene was analyzed to confirm the validity of the reference genes in this study. Our data provide basis for the selection of reference genes for future biological research in the leaf development and hormonal stimuli of C. sinensis.

Identification, classification, and expression profiles of heat shock transcription factors in tea plant (Camellia sinensis) under temperature stress.

In vascular plants, heat shock transcription factors (Hsfs) regulate heat stress response by regulating the expression of heat shock proteins. This study systematically and comprehensively analyzed the Hsf family in tea plant [Camellia sinensis (L.) O. Kuntze]. A total of 16 CsHsfs were identified from the transcriptome database of tea plant and analyzed for their phylogenetic relationships, motifs, and physicochemical characteristics. On the basis of the phylogenetic comparison of tea plant with Arabidopsis thaliana, Populus trichocarpa, Theobroma cacao, and Oryza sativa, the CsHsfs were classified into three classes, namely, A (56.25%), B (37.50%), and C (6.25%). Heat mapping showed that the expression profiles of CsHsf genes under non-stress conditions varied among four tea plant cultivars, namely, 'Yunnanshilixiang', 'Chawansanhao', 'Ruchengmaoyecha', and 'Anjibaicha'. Six CsHsf genes (CsHsfA1a, CsHsfA1b, CsHsfA6, CsHsfB1, CsHsfB2b, and CsHsfC1) were selected from classes A, B, and C to analyze the expression profiles of CsHsf genes through quantitative real-time PCR in 'Yingshuang', 'Anjibaicha', and 'Yunnanshilixiang' under high (38 °C) or low (4 °C) temperature stress. Temperature stress positively or negatively regulated all of the selected CsHsf genes, and the expression levels evidently varied even among CsHsf genes belonging to the same class. This study provided a relatively detailed summary of Hsfs in tea plant and may serve as a reference for further studies on the mechanism of temperature stress regulation by CsHsfs.

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-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.

De novo assembly and transcriptome characterization: novel insights into catechins biosynthesis in Camellia sinensis.

BACKGROUND: Tea is a popular natural non-alcoholic beverage consumed worldwide due to its bioactive ingredients, particularly catechins (flavan-3-ols). Catechins not only contribute to tea quality but also serve important functions in the anti-stress regulation of secondary metabolic pathways. However, the percentages of various catechins are different among tea plant [Camellia sinensis (L.) O. Kuntze] cultivars. This study aimed to elucidate the biosynthetic mechanism of catechins. Transcriptomes from leaf tissues of four tea plant cultivars, 'Yunnanshilixiang', 'Chawansanhao', 'Ruchengmaoyecha', and 'Anjibaicha', were sequenced using the high-throughput sequencing platform Illumina HiSeq™ 2000. De novo assemble were also performed. Catechins contents were measured through reversed-phase high-performance liquid chromatography (RP-HPLC), and the biosynthetic pathway was also surveyed. RESULTS: We constructed a unified unigene database. A total of 146,342 pairs of putative orthologs from the four tea plant cultivars, 'Yunnanshilixiang', 'Chawansanhao', 'Ruchengmaoyecha', and 'Anjibaicha' were generated. Approximately 68,890 unigenes (47.1%) were aligned to the sequences of seven public databases with a cut-off E-value of 1E-5. A total of 217 differentially expressed genes were found through RPKM values, and 150 unigenes were assigned to the flavonoid biosynthetic pathway using the integrated function annotation. The (-)-EGC and (-)-EC contents were significantly lower and the (+)-GC and (+)-C contents were abnormally higher in 'Ruchengmaoyecha' than in 'Yunnanshilixiang', 'Chawansanhao', and 'Anjibaicha'. The proportion of catechins was confirmed by selecting critical genes (ANS, ANR, and LAR) for qRT-PCR analysis. CONCLUSIONS: This study provided a global survey of transcriptomes from four tea plant cultivars and serves as an available resource of genetic diversity. The analyses of transcriptome profiles and physiological indicators not only identified the putative genes involved in the flavonoid biosynthetic pathway but also provided some novel insights for the mechanisms of catechins biosynthesis.

Exposure assessment of lovastatin in Pu-erh tea.

This paper reports the results of an extensive survey on the levels of lovastatin in Pu-erh tea samples. The microbial source of lovastatin was assessed by testing the ability of fungi with higher isolation frequency in the Pu-erh tea samples to produce lovastatin on Czapek yeast extract agar (CYA). Lovastatin was not detected in any of the raw Pu-erh tea samples without storage but was found in almost all the ripe Pu-erh tea samples, with lovastatin contents ranging from 20.61 ng/gdw to 226.38 ng/gdw. After five years' storage, the lovastatin levels increased obviously in ripe Pu-erh tea samples and 55% of raw Pu-erh tea samples from 2007 were found to contain lovastatin with concentrations ranging between 28.41 ng/gdw and 228.61 ng/gdw. With increasing storage time, lovastatin concentration in ripe Pu-erh tea, and the occurrence and concentration of lovastatin for raw Pu-erh tea increased significantly. Three genera of fungi: Aspergillus, Penicillium and Trichoderma were often isolated from Pu-erh tea samples. A total of 40 strains from 3 fungal genera were selected to test their ability to produce lovastatin. Only 6 strains, Aspergillus tubingensis, Aspergillus wentii, Aspergillus fumigatus, Penicillium chrysogenum, Trichoderma asperellum and Trichoderma citrinoviride, were able to produce lovastatin reaching concentrations of 9.59 ± 0.42 ng/g CYA, 2.33 ± 0.21 ng/g CYA, 2.77 ± 0.13 ng/g CYA, 3.36 ± 0.69 ng/g CYA, 4.8 ± 0.17 ng/g CYA, and 1.47 ± 0.36 ng/g CYA respectively in Czapek yeast extract agar.

Nitric oxide participates in cold-inhibited Camellia sinensis pollen germination and tube growth partly via cGMP in vitro.

Nitric oxide (NO) plays essential roles in many biotic and abiotic stresses in plant development procedures, including pollen tube growth. Here, effects of NO on cold stress inhibited pollen germination and tube growth in Camellia sinensis were investigated in vitro. The NO production, NO synthase (NOS)-like activity, cGMP content and proline (Pro) accumulation upon treatment with NO scavenger cPTIO, NOS inhibitor L-NNA, NO donor DEA NONOate, guanylate cyclase (GC) inhibitor ODQ or phosphodiesterase (PDE) inhibitor Viagra at 25°C (control) or 4°C were analyzed. Exposure to 4°C for 2 h reduced pollen germination and tube growth along with increase of NOS-like activity, NO production and cGMP content in pollen tubes. DEA NONOate treatment inhibited pollen germination and tube growth in a dose-dependent manner under control and reinforced the inhibition under cold stress, during which NO production and cGMP content promoted in pollen tubes. L-NNA and cPTIO markedly reduced the generation of NO induced by cold or NO donor along with partly reverse of cold- or NO donor-inhibited pollen germination and tube growth. Furthermore, ODQ reduced the cGMP content under cold stress and NO donor treatment in pollen tubes. Meanwhile, ODQ disrupted the reinforcement of NO donor on the inhibition of pollen germination and tube growth under cold condition. Additionally, Pro accumulation of pollen tubes was reduced by ODQ compared with that receiving NO donor under cold or control condition. Effects of cPTIO and L-NNA in improving cold-treated pollen germination and pollen tube growth could be lowered by Viagra. Moreover, the inhibitory effects of cPTIO and L-NNA on Pro accumulation were partly reversed by Viagra. These data suggest that NO production from NOS-like enzyme reaction decreased the cold-responsive pollen germination, inhibited tube growth and reduced Pro accumulation, partly via cGMP signaling pathway in C. sinensis.