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Magnesium (Mg) plays important roles in photosynthesis, sucrose partitioning, and biomass allocation in plants. However, the specific mechanisms of tea plant response to Mg deficiency remain unclear. In this study, we investigated the effects of Mg deficiency on the quality constituents of tea leaves. Our results showed that the short-term (7 days) Mg deficiency partially elevated the concentrations of polyphenols, free amino acids, and caffeine but decreased the contents of chlorophyll and Mg. However, long-term (30 days) Mg-deficient tea displayed decreased contents of these constituents. Particularly, Mg deficiency increased the index of catechins' bitter taste and the ratio of total polyphenols to total free amino acids. Moreover, the transcription of key genes involved in the biosynthesis of flavonoid, caffeine, and theanine was differentially affected by Mg deficiency. Additionally, short-term Mg deficiency induced global transcriptome change in tea leaves, in which a total of 2522 differentially expressed genes were identified involved in secondary metabolism, amino acid metabolism, and chlorophyll metabolism. These results may help to elucidate why short-term Mg deficiency partially improves the quality constituents of tea, while long-term Mg-deficient tea may taste more bitter, more astringent, and less umami.

Tea plant(Camellia sinensis) has very long history of cultivation and abundant germplasm resources in China. Purple bud is a characteristic variety, which has attracted the attention of breeding researchers because it accumulated a large number of anthocyanins naturally. In many species, R2R3-MYBtranscription factors (TFs)wereprovedto be involved in the regulation of anthocyanin biosynthesis.Research on anthocyanin metabolism has been relatively clear in some species, but that needs to be further elucidated in tea plants. In this research, anR2R3-MYB transcriptionfactor CsMYB113 relate to the anthocyanin accumulation regulation was identified from tea plants. Spatial and temporal expressionanalysis revealed differential expression of CsMYB113among different tissues and organs, with highest expression occurringin the roots.Subcellular localization assays showed that CsMYB113 localizedin the nucleus.Ectopic expression of CsMYB113increased pigmentation and anthocyanin contentsby the up-regulationof theexpression levelsof genes in anthocyanin biosynthesis pathwayamongdifferent tissues of Arabidopsis.Moreover, transient overexpressionof 35S::CsMYB113in tea plant increased the anthocyanin contents in the leaves.Our results indicated that CsMYB113 play important role in the anthocyaninbiosynthesis regulation in tea plants. It will also provide useful candidate gene for the modification of anthocyanin metabolism by genetic engineeringin plants.

Histone methylation plays an important regulatory role in the drought response of many plants, but its regulatory mechanism in the drought response of the tea plant remains poorly understood. Here, drought stress was shown to induce lower relative water content and significantly downregulate the methylations of histone H3K4 in the tea plant. Based on our previous analysis of the SET Domain Group (SDG) gene family, the full-length coding sequence (CDS) of CsSDG36 was cloned from the tea cultivar ‘Fuding Dabaicha’. Bioinformatics analysis showed that the open reading frame (ORF) of the CsSDG36 gene was 3138 bp, encoding 1045 amino acids and containing the conserved structural domains of PWWP, PHD, SET and PostSET. The CsSDG36 protein showed a close relationship to AtATX4 of the TRX subfamily, with a molecular weight of 118,249.89 Da, and a theoretical isoelectric point of 8.87, belonging to a hydrophilic protein without a transmembrane domain, probably located on the nucleus. The expression of CsSDG36 was not detected in the wild type, while it was clearly detected in the over-expression lines of Arabidopsis. Compared with the wild type, the over-expression lines exhibited lower hyperosmotic resistance by accelerating plant water loss, increasing reactive oxygen species (ROS) pressure, and increasing leaf stomatal density. RNA-seq analysis suggested that the CsSDG36 overexpression caused the differential expression of genes related to chromatin assembly, microtubule assembly, and leaf stomatal development pathways. qRT-PCR analysis revealed the significant down-regulation of stomatal development-related genes (BASL, SBT1.2(SDD1), EPF2, TCX3, CHAL, TMM, SPCH, ERL1, and EPFL9) in the overexpression lines. This study provides a novel sight on the function of histone methyltransferase CsSDG36 under drought stress.

This study investigated the regulatory relationship between important flavor compounds and microRNA (miRNA) in nine different tissues of tea plant by analyzing the related metabolites, small RNAs (sRNAs), degradome, and coexpression network. A total of 272 differential expressed miRNAs (DEmiRNAs) were obtained, including 198 conserved miRNAs and 74 novel miRNAs. Meanwhile, the expression patterns of miR159

The vigor of tea plants (Camellia sinensis) and tea quality are strongly influenced by the abundance and forms of nitrogen, principally NO3&minus, NH4+, and amino acids. Mechanisms to access different nitrogen sources and the regulatory cues remain largely elusive in tea plants. A transcriptome analysis was performed to categorize differentially expressed genes (DEGs) in roots and young leaves during the early response to four nitrogen treatments. Relative to the continuously nitrogen-replete control, the three nitrogen-deprived and resupplied treatments shared 237 DEGs in the shoots and 21 DEGs in the root. Gene-ontology characterization revealed that transcripts encoding genes predicted to participate in nitrogen uptake, assimilation, and translocation were among the most differentially expressed after exposure to the different nitrogen regimes. Because of its high transcript level regardless of nitrogen condition, a putative amino acid transporter, TEA020444/CsCAT9.1, was further characterized in Arabidopsis and found to mediate the acquisition of a broad spectrum of amino acids, suggesting a role in amino acid uptake, transport, and deposition in sinks as an internal reservoir. Our results enhance our understanding of nitrogen-regulated transcript level patterns in tea plants and pinpoint candidate genes that function in nitrogen transport and metabolism, allowing tea plants to adjust to variable nitrogen environments.

Cation diffusion facilitators (CDFs) play central roles in metal homeostasis and tolerance in plants, but the specific functions of Camellia sinensis CDF-encoding genes and the underlying mechanisms remain unknown. Previously, transcriptome sequencing results in our lab indicated that the expression of CsMTP8.2 in tea plant shoots was down-regulated exposed to excessive amount of Mn2+ conditions. To elucidate the possible mechanisms involved, we systematically identified 13 C. sinensis CsMTP genes from three subfamilies and characterized their phylogeny, structures, and the features of the encoded proteins. The transcription of CsMTP genes was differentially regulated in C. sinensis shoots and roots in responses to high concentrations of Mn, Zn, Fe, and Al. Differences in the cis-acting regulatory elements in the CsMTP8.1 and CsMTP8.2 promoters suggested the expression of these two genes may be differentially regulated. Transient expression analysis indicated that CsMTP8.2 was localized to the plasma membrane in tobacco and onion epidermal cells. Moreover, when heterologously expressed in yeast, CsMTP8.2 conferred tolerance to Ni and Mn but not to Zn. Additionally, heterologous expression of CsMTP8.2 in Arabidopsis thaliana revealed that CsMTP8.2 positively regulated the response to manganese toxicity by decreasing the accumulation of Mn in plants. However, there was no difference in the accumulation of other metals, including Cu, Fe, and Zn. These results suggest that CsMTP8.2 is a Mn-specific transporter that contributes to the efflux of excess Mn2+ from plant cells.

The molecular mechanisms regulating calcium-mediated thermotolerance in Camellia sinensis were revealed by RNA-Sequencing. Heat stress is one of the most remarkable abiotic factors limiting the growth and productivity of Camellia sinensis plants. Calcium helps regulate plant responses to various adverse environmental conditions, including heat stress. In this study, the effects of exogenous calcium on the physiological characteristics of heat-stressed C. sinensis were investigated. A calcium pretreatment increased the proline, soluble sugar, Ca2+, and chlorophyll contents, but decreased the malondialdehyde content and relative electrical conductivity in C. sinensis leaves under heat stress. Further analysis of the ultra-structure of chloroplasts indicated that heat stress induced accumulation of starch granules and destruction of the stroma lamella in C. sinensis. However, calcium pretreatment counteracted the adverse effects of heat stress on the structure of the photosynthetic apparatus. These results imply that the calcium pretreatment increased C. sinensis thermotolerance. Moreover, RNA-sequencing was applied to characterize the calcium-mediated transcript-level responses to heat stress. A total of 923 differentially expressed genes (DEGs) including 299 up-regulated and 624 down-regulated genes were identified. Functional annotations indicated that these DEGs were primarily related to signal transduction, transcriptional regulation, and post-translational modification. In addition, a C. sinensis gene [CsCML45 (GenBank: KY652927)] encoding a calmodulin-like protein was isolated. The heterologous expression of CsCML45 enhanced the thermotolerance of transgenic Arabidopsis thaliana plants. These results may be useful for characterizing the calcium-mediated molecular mechanism responsible for C. sinensis thermotolerance.

Calcium-dependent protein kinases (CDPKs) play critical roles in the regulation of plant growth, development, and stress response. In this study, Camellia sinensis CsCDPK1, CsCDPK3, CsCDPK20, and CsCDPK26 were cloned and functionally characterized. Bioinformatics analyses showed that all analyzed CsCDPK genes encoded the expected CDPK structures. Subcellular localization indicated that the four CsCDPK proteins were localized to the cytoplasm and nucleus in onion epidermal cells. The quantitative real-time PCR results indicated that the four CsCDPK genes exhibited tissue-specific expression patterns. Exposure to heat stress and exogenous abscisic acid induced increases in CsCDPK20 and CsCDPK26 transcript abundance at different time points. Furthermore, overexpression of CsCDPK20 and CsCDPK26 increased the thermotolerance in transgenic Arabidopsis thaliana plants. The proline content in leaves was significantly higher in transgenic plants than that in wild-type under heat stress, whereas malondialdehyde content was lower in transgenic plants. Additionally, expression of stress-responsive genes (i.e., AtAPX1, AtPOD, AtProT1, AtP5CS2, AtHSFA2, AtHSP70, AtHSP101, AtRD29B, AtRAB18, AtABI1, AtRBOHD, and AtRBOHF) was increased in the CsCDPK20 and CsCDPK26 transgenic A. thaliana plants compared with that in the wild type. Collectively, our results suggest that CsCDPK20 and CsCDPK26 may act as positive regulator in C. sinensis response to heat stress.

Dehydration-responsive element-binding protein (DREB) transcription factors play key roles in plant stress signal transduction pathways. We herein describe the functions of a Camellia sinensis DREB transcription factor (CsDREB) in response to abiotic stress. Subcellular localization analyses indicated that the CsDREB localizes to the nucleus. CsDREB expression in C. sinensis leaves was induced by heat, cold, drought, high salinity, H2O2, and exogenous abscisic acid (ABA). Additionally, CsDREB showed no transcriptional activation in Saccharomyces cerevisiae. Transgenic Arabidopsis thaliana plants overexpressing CsDREB exhibited enhanced tolerance to salt and drought stresses. The overexpression of CsDREB in A. thaliana plants resulted in the up-regulated expression of ABA-dependent stress-induced genes (i.e., AtRD29B, AtRAB18, AtABI1, and AtABI2) and ABAindependent stress-induced genes (i.e., AtCOR15a and AtRD29A). Furthermore, an analysis of the CsDREB promoter sequence revealed the presence of several abiotic and biotic stress-related motifs, along with the developmental stageand tissue-specific elements. An examination of the transient expression of the CsDREB promoter in Nicotiana benthamiana leaves revealed that the promoter is highly responsive to ABA and methyl jasmonate. Collectively, these results suggest that CsDREB may increase plant tolerance to salt and drought stresses via both ABA-dependent and ABAindependent pathways.

Small heat shock proteins (sHSPs) play important roles in responses to heat stress. However, the functions of sHSPs in tea plants (Camellia sinensis) remain uncharacterized. A novel sHSP gene, designated CsHSP17.2, was isolated from tea plants. Subcellular localization analyses indicated that the CsHSP17.2 protein was present in the cytosol and the nucleus. CsHSP17.2 expression was significantly up-regulated by heat stress but was unaffected by low temperature. The CsHSP17.2 transcript levels increased following salt and polyethylene glycol 6000 treatments but decreased in the presence of abscisic acid. The molecular chaperone activity of CsHSP17.2 was demonstrated in vitro. Transgenic Escherichia coli and Pichia pastoris expressing CsHSP17.2 exhibited enhanced thermotolerance. The transgenic Arabidopsis thaliana exhibited higher maximum photochemical efficiencies, greater soluble protein proline contents, higher germination rates and higher hypocotyl elongation length than the wild-type controls. The expression levels of several HS-responsive genes increased in transgenic A. thaliana plants. Additionally, the CsHSP17.2 promoter is highly responsive to high-temperature stress in A. thaliana. Our results suggest that CsHSP17.2 may act as a molecular chaperone to mediate heat tolerance by maintaining maximum photochemical efficiency and protein synthesis, enhancing the scavenging of reactive oxygen species and inducing the expression of HS-responsive genes.

Histone methylation mediated by SET Domain Group (SDG) proteins plays an important role in plant response to various abiotic stresses, regulating the expressions of many stress-inducible genes. However, little is known about the SDG genes in Camellia sinensis. In this study, we identified 46 CsSDGs by genome-wide search with the conserved SET domain. Phylogenetic tree, domain composition and gene structure analyses revealed that 46 CsSDGs were classified into five groups, including E(z), ASH1, TRX, SU(VAR) and class VI/VII genes. Furthermore, In silico and qRT-PCR analyses suggested the diverse expression patterns of the 46 CsSDGs in different tissues and in response to various abiotic stresses. To reveal the vital role of CsSDGs in plant responses to abiotic stresses, CsSDG43 was chosen for the functional analysis in hyperosmosis in Arabidopsis. Importantly, CsSDG43 significantly weakened the hyperosmotic tolerance by increasing water loss, reactive oxygen species (ROS) pressure and AHG3 expression in the overexpression lines compared with the wild type of Arabidopsis.

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.

This paper presents a new simulation framework for liquid wetting across porous textiles with anisotropic inner structure. The influence of the textile's properties on the wetting process, such as contact angle, hygroscopicity and porosity, is considered into the liquid wetting process in detail. By liquid-textile coupling, the wetting process is simulated through liquid absorption/desorption by fiber and liquid diffusion in the means of inner fiber, intersected fibers and capillary action. This framework can simulate the liquid wetting across the porous anisotropic textile by dripping single or multiple drops of water with realistic results.

CsHSP17.7, CsHSP18.1, and CsHSP21.8 expressions are induced by heat and cold stresses, and CsHSP overexpression confers tolerance to heat and cold stresses in transgenic Pichia pastoris and Arabidopsis thaliana. Small heat shock proteins (sHSPs) are crucial for protecting plants against biotic and abiotic stresses, especially heat stress. However, knowledge concerning the functions of Camellia sinensis sHSP in heat and cold stresses remains poorly understood. In this study, three C. sinensis sHSP genes (i.e., CsHSP17.7, CsHSP18.1, and CsHSP21.8) were isolated and characterized using suppression subtractive hybridization (SSH) technology. The CsHSPs expression levels in C. sinensis leaves were significantly up-regulated by heat and cold stresses. Phylogenetic analyses revealed that CsHSP17.7, CsHSP18.1, and CsHSP21.8 belong to sHSP Classes I, II, and IV, respectively. Heterologous expression of the three CsHSP genes in Pichia pastoris cells enhanced heat and cold stress tolerance. When exposed to heat and cold treatments, transgenic Arabidopsis thaliana plants overexpressing CsHSP17.7, CsHSP18.1, and CsHSP21.8 had lower malondialdehyde contents, ion leakage, higher proline contents, and transcript levels of stress-related genes (e.g., AtPOD, AtAPX1, AtP5CS2, and AtProT1) compared with the control line. In addition, improved seed germination vigor was also observed in the CsHSP-overexpressing seeds under heat stress. Taken together, our results suggest that the three identified CsHSP genes play key roles in heat and cold tolerance.

The tea plant [Camellia sinensis (L.) O. Kuntze] is an important commercial crop rich in bioactive ingredients, especially catechins, caffeine, theanine and other free amino acids, which the quality of tea leaves depends on. Drought is the most important environmental stress affecting the yield and quality of this plant. In this study, the effects of drought stress on the phenotype, physiological characteristics and major bioactive ingredients accumulation of C. sinensis leaves were examined, and the results indicated that drought stress resulted in dehydration and wilt of the leaves, and significant decrease in the total polyphenols and free amino acids and increase in the total flavonoids. In addition, HPLC analysis showed that the catechins, caffeine, theanine and some free amino acids in C. sinensis leaves were significantly reduced in response to drought stress, implying that drought stress severely decreased the quality of C. sinensis leaves. Furthermore, differentially expressed genes (DEGs) related to amino acid metabolism and secondary metabolism were identified and quantified in C. sinensis leaves under drought stress using high-throughput Illumina RNA-Seq technology, especially the key regulatory genes of the catechins, caffeine, and theanine biosynthesis pathways. The expression levels of key regulatory genes were consistent with the results from the HPLC analysis, which indicate a potential molecular mechanism for the above results. Taken together, these data provide further insights into the mechanisms underlying the change in the quality of C. sinensis leaves under environmental stress, which involve changes in the accumulation of major bioactive ingredients, especially catechins, caffeine, theanine and other free amino acids.

Permeation characteristics of an azobenzene-containing liquid crystalline (LC) non-porous film are investigated using a metallic corrosion method. Thin films (300 nm) are fabricated by the solution casting of an azobenzene side-chain LC polymer on freshly polished carbon steel coupons. Coated coupons are treated under the following conditions: a) gradual annealing at a cooling rate lower than 1 °C · min(-1) from 150 °C (above its Tg ) to room temperature, and b) irradiation at 465 nm (20 mW · cm(-2) ) with either circularly polarized light (CPL) or non-polarized light (NPL). The morphology of these films is characterized using X-ray diffraction, polarized optical microscopy, and transmission measurements. The results suggest that the annealing treatment resulted in the formation of a polydomain structure consisting of locally ordered small smectic domains that lack mutual orientation. Ordered micro domains are surrounded by disordered phases. CPL and NPL irradiation generates a monodomain orientated structure and an isotropic liquid crystal glass, respectively. The permeability of these non-porous films treated by CPL, NPL, and annealing are found to be 6.14 × 10(-4) , 1.92 × 10(-2) , and 1.56 × 10(-3) cm(3) · m(-2) · d(-1) . An orientation-dependent structure model is constructed to explain the permeation phenomenon, considering the ordered phase is impermeable, only the disordered phase is accessible to penetrating molecules. Fast switching of gas permeation is demonstrated by alternative irradiation of the film with CPL and NPL, which results in an approximately 30-fold difference in the permeability of the non-porous film.

Assembly of ordered structures by an external stimulus allows for design of functional materials with enhanced physical and chemical properties. A new side-chain liquid crystal polymer containing strong polar azobenzene mesogens was synthesised. A macroscopically ordered monodomain smectic-like lamellar structure having orientational order and positional order was immediately assembled by linear polarised light irradiation (473 nm, 20 mW/cm2) at room temperature. The lamellar layer with its periodic d-spacing of 1.9 nm and mesogens arranged at an inclination angle of about 75° were characterised by X-ray diffraction and polarising optical microscopy which showed a diffraction peak at 2θ = 4.53° and an off-centred interference figure. Reversible assembly and disassembly of the lamellar phase were achieved by alternative irradiation with polarised light and non-polarised light. Potential factors influencing the assembly of the ordered lamellar structure were investigated by controlling the mesogens out-of-p...

Spermine synthase (SPMS, EC 2.5.1.22), enzyme of spermine (Spm) biosynthesis, has been shown to be related to stress response. In this study, attempts were made to clone and characterize a gene encoding SPMS from tea plant (Camellia sinensis). The effect of exogenous application of Spm in C. sinensis subjected to low-temperature stress was also investigated. A full-length SPMS complementary DNA (cDNA) (CsSPMS) with an open reading frame of 1113 bp was cloned using reverse transcription-PCR and rapid amplification of cDNA ends (RACE) techniques from cultivar "Yingshuang". The CsSPMS gene, which encoded a 371 amino acid polypeptide, in four cultivars is highly homologous. Quantitative real-time PCR indicated that the CsSPMS gene shows tissue-specific expression, mainly in the leaf and root of tea plant. The expression analysis demonstrated that the CsSPMS gene is quickly induced by cold stress and had similar trends in four cultivars. Spm-supplemented "Baicha" cultivar contains higher endogenous polyamines compared to the control, coupling with higher expression levels of ADC and SPMS. In addition, activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), as well as free proline content in the Spm-supplemented samples were higher than the control during the experiment course or at a given time point, indicating that Spm exerted a positive effect on antioxidant systems. Moreover, Agrobacterium-mediated expression of CsSPMS in tobacco leaves showed relatively higher cold tolerance. Taken together, these findings will enhance the understanding of the relationships among CsSPMS gene regulatory, polyamines accumulation, and cold tolerance in tea plant.

Background MicroRNAs (miRNAs) are approximately 19 ~ 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs have critical functions in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interaction with specific target mRNAs. Camellia sinensis is one of the most important commercial beverage crops in the world. However, miRNAs associated with cold stress tolerance in C. sinensis remains unexplored. The use of high-throughput sequencing can provide a much deeper understanding of miRNAs. To obtain more insight into the function of miRNAs in cold stress tolerance, Illumina sequencing of C. sinensis sRNA was conducted. Result Solexa sequencing technology was used for high-throughput sequencing of the small RNA library from the cold treatment of tea leaves. To align the sequencing data with known plant miRNAs, we characterized 106 conserved C. sinensis miRNAs. In addition, 215 potential candidate miRNAs were found, among, which 98 candidates with star sequences were chosen as novel miRNAs. Both congruously and differentially regulated miRNAs were obtained, and cultivar-specific miRNAs were identified by microarray-based hybridization in response to cold stress. The results were also confirmed by quantitative real-time polymerase chain reaction. To confirm the targets of miRNAs, two degradome libraries from two treatments were constructed. According to degradome sequencing, 455 and 591 genes were identified as cleavage targets of miRNAs from cold treatments and control libraries, respectively, and 283 targets were present in both libraries. Functional analysis of these miRNA targets indicated their involvement in important activities, such as development, regulation of transcription, and stress response. Conclusions We discovered 31 up-regulated miRNAs and 43 down-regulated miRNAs in ‘Yingshuang’, and 46 up-regulated miRNA and 45 down-regulated miRNAs in ‘Baiye 1’ in response to cold stress, respectively. A total of 763 related target genes were detected by degradome sequencing. The RLM-5′RACE procedure was successfully used to map the cleavage sites in six target genes of C. sinensis. These findings reveal important information about the regulatory mechanism of miRNAs in C. sinensis, and promote the understanding of miRNA functions during the cold response. The miRNA genotype-specific expression model might explain the distinct cold sensitivities between tea lines. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0271-x) contains supplementary material, which is available to authorized users.