Putrescine modifies the pollen tube growth of tea (Camellia sinensis) by affecting actin organization and cell wall structure.

The aim of the current study was to examine the effect of different exogenous putrescine concentrations (200, 400, 600, and 800 µM) on the tea pollen performance. It was shown that putrescine has a dose-dependent effect on pollen performance. Results exhibited that pollen germination and tube elongation were induced by 200 and 400 µM putrescine treatment, especially, 400 µM putrescine-enhanced pollen performance. However, pollen performance was inhibited by higher concentrations of putrescine. Putrescine concentrations above 400 µM changed the actin filament distribution in pollen tubes by affecting the distribution of sucrose synthase enzyme. Alterations of the distribution on sucrose synthase enzyme also caused the alterations in the dispersion of cellulose and callose in the cell wall, and morphological alterations such as balloon-shaped and snake-shaped pollen tube tip accompanied them. Moreover, putrescine concentrations above 400 µM caused a decrease of ROS level in apex and led to chromatin condensation of the generative nucleus. In conclusion, exogenous putrescine application can be used as a pollen performance enhancer at low concentrations while the high concentrations cause adverse effects reducing fertilization success.

Structural and functional insights into the LBD family involved in abiotic stress and flavonoid synthases in Camellia sinensis.

Lateral organ boundaries domain (LBD) proteins are plant-specific transcription factors that play a crucial role in growth and development, as well as metabolic processes. However, knowledge of the function of LBD proteins in Camellia sinensis is limited, and no systematic investigations of the LBD family have been reported. In this study, we identified 54 LBD genes in Camellia sinensis. The expression patterns of CsLBDs in different tissues and their transcription responses to exogenous hormones and abiotic stress were determined by RNA-seq, which showed that CsLBDs may have diverse functions. Analysis of the structural gene promoters revealed that the promoters of CsC4H, CsDFR and CsUGT84A, the structural genes involved in flavonoid biosynthesis, contained LBD recognition binding sites. The integrative analysis of CsLBD expression levels and metabolite accumulation also suggested that CsLBDs are involved in the regulation of flavonoid synthesis. Among them, CsLOB_3, CsLBD36_2 and CsLBD41_2, localized in the nucleus, were selected for functional characterization. Yeast two-hybrid assays revealed that CsLBD36_2 and CsLBD41_2 have self-activation activities, and CsLOB_3 and CsLBD36_2 can directly bind to the cis-element and significantly increase the activity of the CsC4H, CsDFR and CsUGT84A promoter. Our results present a comprehensive characterization of the 54 CsLBDs in Camellia sinensis and provide new insight into the important role that CsLBDs play in abiotic and flavonoid biosynthesis.

Comparative transcriptomic analysis reveals gene expression associated with cold adaptation in the tea plant Camellia sinensis.

BACKGROUND: Low temperature restricts the planting range of all crops, but cold acclimation induces adaption to cold stress in many plants. Camellia sinensis, a perennial evergreen tree that is the source of tea, is mainly grown in warm areas. Camellia sinensis var. sinensis (CSS) has greater cold tolerance than Camellia sinensis var. assamica (CSA). To gain deep insight into the molecular mechanisms underlying cold adaptation, we investigated the physiological responses and transcriptome profiles by RNA-Seq in two tea varieties, cold resistant SCZ (classified as CSS) and cold susceptible YH9 (classified as CSA), during cold acclimation. RESULTS: Under freezing stress, lower relative electrical conductivity and higher chlorophyll fluorescence (Fv/Fm) values were detected in SCZ than in YH9 when subjected to freezing acclimation. During cold treatment, 6072 and 7749 DEGs were observed for SCZ and YH9, respectively. A total of 978 DEGs were common for both SCZ and YH9 during the entire cold acclimation process. DEGs were enriched in pathways of photosynthesis, hormone signal transduction, and transcriptional regulation of plant-pathogen interactions. Further analyses indicated that decreased expression of Lhca2 and higher expression of SnRK2.8 are correlated with cold tolerance in SCZ. CONCLUSIONS: Compared with CSA, CSS was significantly more resistant to freezing after cold acclimation, and this increased resistance was associated with an earlier expression of cold-induced genes. Because the greater transcriptional differentiation during cold acclimation in SCZ may contribute to its greater cold tolerance, our studies identify specific genes involved in photoinhibition, ABA signal conduction, and plant immunity that should be studied for understanding the processes involved in cold tolerance. Marker-assisted breeding focused on the allelic variation at these loci provides an avenue for the possible generation of CSA cultivars that have CSS-level cold tolerance.

Analysis of Differentially Expressed Genes in Tissues of Camellia sinensis during Dedifferentiation and Root Redifferentiation.

Tissue culture is very important for identifying the gene function of Camellia sinensis (L.) and exploiting novel germplasm through transgenic technology. Regeneration system of tea plant has been explored but not been well established since the molecular mechanism of tea plant regeneration is not clear yet. In this study, transcriptomic analysis was performed in the initial explants of tea plant and their dedifferentiated and redifferentiated tissues. A total of 93,607 unigenes were obtained through de novo assembly, and 7,193 differentially expressed genes (DEGs) were screened out from the 42,417 annotated unigenes. Much more DEGs were observed during phase transition rather than at growth stages of callus. Our KOG and KEGG analysis, and qPCR results confirmed that phase transition of tea plant was closely related to the mechanism that regulate expression of genes encoding the auxin- and cytokinin-responsive proteins, transcription factor MYB15 and ethylene-responsive transcription factor ERF RAP2-12. These findings provide a reliable foundation for elucidating the mechanism of the phase transition and may help to optimize the regeneration system by regulating the gene expression pattern.

Sclereids are strong enough to support the delicate corollas: experimental and computational data evidence from Camellia sinensis (L.).

Sclereids are a fundamental cell type that widely exist in higher plants and are generally thought to have a mechanical function. However, the occurrence of sclereids in the ephemeral corolla has rarely been documented and their biological significance is poorly understood. In this study, flower buds from Camellia sinensis at various ontogenetic stages were sampled, cleared, sectioned, stained, and examined using light microscopy to ascertain the morphology and distribution of sclereids and their variation. In addition, Camellia japonica plants with distinctive floral structures were investigated and compared to explore whether sclereid occurrence is associated with floral form. In particular, a computational simulation using finite element analysis was undertaken to investigate how corollas, with and without sclereids, responded to wind and rain. The results showed that sclereids have some mechanical properties that are based on their shape and distribution, which make the soft corolla strong enough to protect the inner ovary. Thus, corolla sclereids may explain how the seemingly delicate corolla performs its protective function in response to environmental stresses. These findings provide further evidence for the hypothesis that flower traits exhibit adaptive responses to abiotic factors in addition to their traditionally recognized pollinator-mediated selection.

A kinetic model for flavonoid production in tea cell culture.

As one of the strategies for efficient production of a metabolite from cell cultures, a kinetic model is very useful tool to predict productivity under various culture conditions. In this study, we propose a kinetic model for flavonoid production in tea cell culture based on the cell life cycle and expression of PAL, the gene encoding phenylalanine ammonia-lyase (PAL)-the key enzyme in flavonoid biosynthesis. The flavonoid production rate was considered to be related to the amount of active PAL. Synthesis of PAL was modelled based on a general gene expression/translation mechanism, including the transcription of DNA encoding PAL into mRNA and the translation of PAL mRNA into the PAL protein. The transcription of DNA was assumed to be promoted at high light intensity and suppressed by a feedback regulatory mechanism at high flavonoid concentrations. In the model, mRNA and PAL were considered to self-decompose and to be lost by cell rupture. The model constants were estimated by fitting the experimental results obtained from tea cell cultures under various light intensities. The model accurately described the kinetic behaviors of dry and fresh cell concentrations, glucose concentration, cell viability, PAL specific activity, and flavonoid content under a wide range of light intensities. The model simulated flavonoid productivity per medium under various culture conditions. Therefore, this model will be useful to predict optimum culture conditions for maximum flavonoid productivity in cultured tea cells.

Theaflavin Synthesized in a Selective, Domino-Type, One-Pot Enzymatic Biotransformation Method with Camellia sinensis Cell Culture Inhibits Weight Gain and Fat Accumulation to High-Fat Diet-Induced Obese Mice.

The polyphenolic compound theaflavin, which is the main red pigment present in black tea, is reported to elicit various physiological effects. Because of the extremely low concentration of theaflavin present in black tea, its extraction from black tea leaves in quantities sufficient for use in medical studies has been difficult. We have developed a simple, inexpensive, selective, domino-type, one-pot enzymatic biotransformation method for the synthesis of theaflavin that is suitable for use in medical studies. Subsequent administration of this synthetic theaflavin to high-fat diet-induced obese mice inhibited both body weight gain and visceral fat accumulation, with no significant difference in the amount of faeces between the experimental and control mice.

Anion Channel Inhibitor NPPB-Inhibited Fluoride Accumulation in Tea Plant (Camellia sinensis) Is Related to the Regulation of Ca²âº, CaM and Depolarization of Plasma Membrane Potential.

Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca(2+) efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca(2+) chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca(2+) fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca(2+) chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca(2+) and H⁺ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca(2+)-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants.

Application of QuEChERS based method for the determination of pesticides in nutraceutical products (Camellia sinensis) by liquid chromatography coupled to triple quadrupole tandem mass spectrometry.

A QuEChERS (quick, easy, cheap, effective, rugged, and safe) based method has been evaluated and validated for the determination and quantification of approximately 100 LC-amenable pesticides in nutraceutical products obtained from green tea (Camellia sinensis). Extraction was performed with acidified acetonitrile (acetic acid 1% (v/v)), and additional clean-up steps were not necessary. Pesticides determination was achieved using ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Total running time was 11 min. Pesticides were quantified using matrix-matched calibration. Recoveries ranged from 70% to 117% and relative standard deviation (RSD) was lower than 20% at concentration levels of 25, 50 and 100 µg/kg for intra-day precision and equal or lower than 25% for inter-day precision. Limits of quantification (LOQ) were equal or lower than 25 µg/kg. The validated method was applied to commercial nutraceutical products, detecting acetamiprid (56 µg/kg) and carbendazim (13 µg/kg) in two samples.

[Revealing the chemical changes of tea cell wall induced by anthracnose with confocal Raman microscopy].

Healthy tea and tea infected by anthracnose were first studied by confocal Raman microscopy to illustrate chemical changes of cell wall in the present paper. Firstly, Raman spectra of both healthy and infected sample tissues were collected with spatial resolution at micron-level, and ultrastructure of healthy and infected tea cells was got from scanning electron microscope. These results showed that there were significant changes in Raman shift and Raman intensity between healthy and infected cell walls, indicating that great differences occurred in chemical compositions of cell walls between healthy and infected samples. In details, intensities at many Raman bands which were closely associated with cellulose, pectin, esters were reduced after infection, revealing that the content of chemical compounds such as cellulose, pectin, esters was decreased after infection. Subsequently, chemical imaging of both healthy and infected tea cell walls were realized based on Raman fingerprint spectra of cellulose and microscopic spatial structure. It was found that not only the content of cellulose was reduced greatly after infection, but also the ordered structure of cellulose was destroyed by anthracnose infection. Thus, confocal Raman microscopy was shown to be a powerful tool to detect the chemical changes in cell wall of tea caused by anthracnose without any chemical treatment or staining. This research firstly applied confocal Raman microscopy in phytopathology for the study of interactive relationship between host and pathogen, and it will also open a new way for intensive study of host-pathogen at cellular level.

Global transcriptome profiles of Camellia sinensis during cold acclimation.

BACKGROUND: Tea is the most popular non-alcoholic health beverage in the world. The tea plant (Camellia sinensis (L.) O. Kuntze) needs to undergo a cold acclimation process to enhance its freezing tolerance in winter. Changes that occur at the molecular level in response to low temperatures are poorly understood in tea plants. To elucidate the molecular mechanisms of cold acclimation, we employed RNA-Seq and digital gene expression (DGE) technologies to the study of genome-wide expression profiles during cold acclimation in tea plants. RESULTS: Using the Illumina sequencing platform, we obtained approximately 57.35 million RNA-Seq reads. These reads were assembled into 216,831 transcripts, with an average length of 356 bp and an N50 of 529 bp. In total, 1,770 differentially expressed transcripts were identified, of which 1,168 were up-regulated and 602 down-regulated. These include a group of cold sensor or signal transduction genes, cold-responsive transcription factor genes, plasma membrane stabilization related genes, osmosensing-responsive genes, and detoxification enzyme genes. DGE and quantitative RT-PCR analysis further confirmed the results from RNA-Seq analysis. Pathway analysis indicated that the "carbohydrate metabolism pathway" and the "calcium signaling pathway" might play a vital role in tea plants' responses to cold stress. CONCLUSIONS: Our study presents a global survey of transcriptome profiles of tea plants in response to low, non-freezing temperatures and yields insights into the molecular mechanisms of tea plants during the cold acclimation process. It could also serve as a valuable resource for relevant research on cold-tolerance and help to explore the cold-related genes in improving the understanding of low-temperature tolerance and plant-environment interactions.

Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves.

The anatomical localization of caffeine within young Camellia sinensis leaves was investigated using immunohistochemical methods and confocal scanning laser microscopy. Preliminary fixation experiments were conducted with young C. sinensis leaves to determine which fixation procedure retained caffeine the best as determined by high-performance liquid chromatography analysis. High pressure freezing, freeze substitution, and embedding in resin was deemed the best protocol as it retained most of the caffeine and allowed for the samples to be sectioned with ease. Immunohistochemical localization with primary anti-caffeine antibodies and conjugated secondary antibodies on leaf sections proved at the tissue level that caffeine was localized and accumulated within vascular bundles, mainly the precursor phloem. With the use of a pressure bomb, xylem sap was collected using a micro syringe. The xylem sap was analyzed by thin-layer chromatography and the presence of caffeine was determined. We hypothesize that caffeine is synthesized in the chloroplasts of photosynthetic cells and transported to vascular bundles where it acts as a chemical defense against various pathogens and predators. Complex formation of caffeine with chlorogenic acid is also discussed as this may also help explain caffeine's localization.

Localization of aluminium in tea (Camellia sinensis) leaves using low energy X-ray fluorescence spectro-microscopy.

Information on localization of Al in tea leaf tissues is required in order to better understand Al tolerance mechanism in this Al-accumulating plant species. Here, we have used low-energy X-ray fluorescence spectro-microscopy (LEXRF) to study localization of Al and other low Z-elements, namely C, O, Mg, Si and P, in fully developed leaves of the tea plant [Camellia sinensis (L.) O. Kuntze]. Plants were grown from seeds for 3 months in a hydroponic solution, and then exposed to 200 microM AlCl(3) for 2 weeks. Epidermal-mesophyll and xylem phloem regions of 20 microm thick cryo-fixed freeze-dried tea-leaf cross-sections were raster scanned with 1.7 and 2.2 keV excitation energies to reach the Al-K and P-K absorption edges. Al was mainly localized in the cell walls of the leaf epidermal cells, while almost no Al signal was obtained from the leaf symplast. The results suggest that the retention of Al in epidermal leaf apoplast represent the main tolerance mechanism to Al in tea plants. In addition LEXRF proved to be a powerful tool for localization of Al in plant tissues, which can help in our understanding of the processes of Al uptake, transport and tolerance in plants.

Flavanols in somatic cell division and male meiosis of tea (Camellia sinensis) anthers.

Young anthers excised from closed tea flower buds ( Camellia sinensis L.) were stained as fresh tissues with p-dimethylaminocinnamaldehyde reagent to localize flavanols associated with nuclei and chromosomes, apart from those flavanols stored in vacuoles. This staining reagent yields a blue colour for flavanols. In the nonsporogenic somatic cells of developing anthers, flavanols were found to be attached to chromosomes at all mitotic stages. Male meiosis started at a bud size of about 3.5 mm in diameter in pollen mother cells which displayed generally more or less pronounced blue nuclei and cytoplasm. The meiotic divisions from prophase I to telophase II were characterized by blue stained nuclei and chromosomes, but within the cytoplasm there was, if any, a random and very poor reaction for flavanols. Metaphase and telophase of meiotic divisions showed maximally condensed chromosomes staining dark blue. Early in telophase II, the cytoplasm was again stained blue; this faded at late tetrad stage. Flavanols of young mitotic and older non-mitotic anthers were determined using high pressure liquid chromatography--chemical reaction detection (HPLC-CRD). Catechin, epicatechin, B2, and epigallocatechin were minor compounds, whereas epicatechin gallate and epigallocatechin gallate were found in higher amounts. The major flavanol compound of the anthers, epicatechin gallate, exhibited a significant affinity to histone sulphate, as shown by UV-VIS spectroscopic titration.

Improvement of catechin productivity in suspension cultures of tea callus cells.

In the suspension cultures of tea callus cells, C.sinensis cv. Yabukita, the effects of the culture conditions, such as culture period and light irradiation, on cell growth and catechin production were investigated. The production of flavonoids (catechins + proanthocyanidins) was promoted by inoculating the cells into the fresh medium at the culture period giving the maximum flavonoid content in the cells. The cultivation under light irradiation was repeated several times by inoculating the cells with the maximum flavonoid content. The flavonoid production was significantly increased without inhibiting the cell growth. We obtained the maximum flavonoid production, 1.5 g/dm(3) medium, and the maximum content, 150 mg/(g of dry cell weight (DCW)). The latter value was larger than that in the leaves of the tea plant.

Tissue distribution and intracellular localization of catechins in tea leaves.

We investigated the leaf tissue and cellular morphology of tea (Camellia sinensis). Osmiophilic material, presumably catechins, was present in mesophyll cells, but not in epidermal cells. Electron microscopy showed that catechins were localized to restricted regions within the central vacuoles. In addition, two kinds of small vacuoles of 0.5-3 microm were present in mesophyll cells. One vacuole had catechins within its whole lumen, while the other had an electron-lucent lumen. We found fusion profiles between a large central vacuole and these small vacuoles. We propose that after catechins are synthesized, they are incorporated into small vacuoles and transported to the large central vacuoles.