Ectopic Overexpression of Histone H3K4 Methyltransferase CsSDG36 from Tea Plant Decreases Hyperosmotic Stress Tolerance in Arabidopsis thaliana.

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.

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

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

Functional characterization of proanthocyanidin pathway enzymes from tea and their application for metabolic engineering.

Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (-)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (-)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols.

Identification and characterization of N9-methyltransferase involved in converting caffeine into non-stimulatory theacrine in tea.

Caffeine is a major component of xanthine alkaloids and commonly consumed in many popular beverages. Due to its occasional side effects, reduction of caffeine in a natural way is of great importance and economic significance. Recent studies reveal that caffeine can be converted into non-stimulatory theacrine in the rare tea plant Camellia assamica var. kucha (Kucha), which involves oxidation at the C8 and methylation at the N9 positions of caffeine. However, the underlying molecular mechanism remains unclear. Here, we identify the theacrine synthase CkTcS from Kucha, which possesses novel N9-methyltransferase activity using 1,3,7-trimethyluric acid but not caffeine as a substrate, confirming that C8 oxidation takes place prior to N9-methylation. The crystal structure of the CkTcS complex reveals the key residues that are required for the N9-methylation, providing insights into how caffeine N-methyltransferases in tea plants have evolved to catalyze regioselective N-methylation through fine tuning of their active sites. These results may guide the future development of decaffeinated drinks.

Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) and catechins (flavan-3-ols) accumulation in tea.

Phenylalanine ammonia-lyase and cinnamate 4-hydroxylase are important enzymes in allocating significant amounts of carbon from phenylalanine into the biosynthesis of several important secondary metabolites. Tea is an important crop of commerce known for its beverage and medicinally important flavonoid compounds, mainly catechins. As metabolic flux for the operation of the flavonoid pathway is maintained through the activities of PAL and C4H, thus, catechins biosynthesis in tea is critically dependent on the products of these enzymes. We examined the expression of PAL and C4H. Sequence encoding CsPAL was isolated from tea by polymerase chain reaction using sequence information available at the NCBI GenBank. Sequence encoding C4H was isolated from tea by using differential display of mRNA and rapid amplification of cDNA ends technology. CsC4H (AY641731) comprised of 1,352 bp full-length cDNA with open reading frame of 1,173 bp encoding 390 amino acids. Catechin contents decreased in response to drought stress (DS), abscisic acid (ABA), and gibberellic acid (GA(3)) treatments but increased in response to wounding. The expression of CsPAL and CsC4H showed the same behavior under the above treatments and was also in accordance with the catechin contents. A positive correlation between catechin contents and gene expression suggested a critical role of the enzymes in catechins biosynthesis and a crosstalk between phenylpropanoid and flavonoid pathways.

[Regulation of phenol oxidase activity with sulfur dioxide as the base for production of high-quality instant green tea].

The possibility to regulate phenoloxidase activity with sulfur dioxide was studied. It was found that this compound is a potent inhibitor of phenoloxidase of the reversible and mixed type. The inhibitory effect of sulfur dioxide on phenoloxidase provided grounds for a new biotechnological approach to the production of instant green tea. This approach allows increasing the yield of the extractive and the proportion of phenolics in the extractive, thereby improving the organoleptic quality of the product.

Ascorbate is the natural substrate for plant peroxidases.

Ascorbate-dependent detoxification of hydrogen peroxide by guaiacol-type peroxidases is increased considerably in the presence of 3,4-dihydroxyphenolic compounds, suggesting that ascorbate is the natural substrate for many types of peroxidase in situ and not just the ascorbate-specific peroxidases. The ascorbate-dependent destruction of hydrogen peroxide in the more acidic cellular compartments such as the vacuole may be an important function of such non-specific peroxidases. The stress-induced production of phenolic compounds would render the guaiacol peroxidases in other less acidic-cellular sites effective as ascorbate-dependent H2O2-detoxifying enzymes.

A new caffeine biosynthetic pathway in tea leaves: utilisation of adenosine released from the S-adenosyl-L-methionine cycle.

The four-step caffeine biosynthetic pathway includes three methylation steps that utilise S-adenosyl-L-methionine (SAM) as the methyl donor. In the process SAM is converted to S-adenosyl-L-homocysteine (SAH) which in turn is hydrolysed to L-homocysteine and adenosine. Significant amounts of radioactivity from [methyl-(14)C]methionine and [methyl-(14)C]SAM were incorporated into theobromine and caffeine in young tea leaf segments, and very high SAH hydrolase activity was found in cell-free extracts from young tea leaves. Substantial amounts of radioactivity from [adenosyl-(14)C]SAH were also recovered as theobromine and caffeine in tea leaf segments, indicating that adenosine derived from SAH is utilised for the synthesis of the purine ring of caffeine. From the profiles of activity of related enzymes in tea leaf extracts, it is proposed that the major route from SAM to caffeine is a SAM-->SAH-->adenosine-->adenine-->AMP-->IMP-->XMP-->xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway. In addition, direct adenosine kinase-catalysed formation of AMP from adenosine may participate as an alternative minor route. The activity of two of the three N-methyltransferase activities involved in caffeine biosynthesis and part of the activities of SAH hydrolase, adenosine nucleosidase, adenine phosphoribosyltransferase and adenosine kinase were located in tea chloroplasts. In contrast, no detectable activity of SAM synthetase was associated with the purified chloroplast fraction. This is a first demonstration that the purine skeleton of caffeine is synthesised from adenosine released from the SAM cycle.

Beta-glycosylamidine as a ligand for affinity chromatography tailored to the glycon substrate specificity of beta-glycosidases.

An affinity adsorbent for beta-glycosidases has been prepared by using beta-glycosylamidine as a ligand. beta-Glucosylamidine and beta-galactosylamidine, highly potent and selective inhibitors of beta-glucosidases and beta-galactosidases, respectively, were immobilized by a novel one-pot procedure involving the addition of a beta-glycosylamine and 2-iminothiolane.HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give an affinity adsorbent for beta-glucosidases and beta-galactosidases, respectively. This one-pot procedure enables various beta-glycosylamidine ligands to be formed and immobilized conveniently according to the glycon substrate specificities of the enzymes. A crude enzyme extract from tea leaves (Camellia sinensis) and a beta-galactosidase from Penicillium multicolor were chromatographed directly on each affinity adsorbent to give a beta-glucosidase and a beta-galactosidase to apparent homogeneity in one step by eluting the column with glucose or by a gradient NaCl elution, respectively. The beta-glucosidase and beta-galactosidase were inhibited competitively by a soluble form of the corresponding beta-glycosylamidine ligand with an inhibition constant (K(i)) of 2.1 and 0.80 microM, respectively. Neither enzyme was bound to the adsorbent with a mismatched ligand, indicating that the binding of the glycosidases was of specific nature that corresponds to the glycon substrate specificity of the enzymes. The ease of preparation and the selective nature of the affinity adsorbent should promise a large-scale preparation of the affinity adsorbent for the purification and removal of specific glycosidases according to their glycon substrate specificities.

Analysis of glycosidically bound aroma precursors in tea leaves. 2. Changes in glycoside contents and glycosidase activities in tea leaves during the black tea manufacturing process.

Glycosides are known to be precursors of the alcoholic aroma compounds of black tea. They are hydrolyzed by endogenous glycosidases during the manufacturing process. Changes in the amounts of these glycosides during the manufacturing process were investigated by using a capillary gas chromatographic--mass spectrometric analysis after trifluoroacetyl derivatization of the tea glycosidic fractions. Primeverosides were 3-fold more abundant than glucosides in fresh leaves, but they decreased greatly during the manufacturing process, especially during the stage of rolling. After the final stage of fermentation, primeverosides had almost disappeared, whereas glucosides were substantially unchanged. These results show that hydrolysis of the glycosides mainly occurred during the stage of rolling and confirm that primeverosides are the main black tea aroma precursors. This was also supported by the changes in the glycosidase activities in tea leaves. The glycosidase activities remained at a high level during withering but decreased drastically after rolling.

A study on tea aroma formation mechanism: alcoholic aroma precursor amounts and glycosidase activity in parts of the tea plant.

We have shown in molecular basis that alcoholic tea aroma is mainly formed by endogenous enzymatic hydrolysis of glycosidic aroma precursors during manufacturing. Amounts of alcoholic aroma precursor and glycosidase activity in each part of the tea shoot (Camellia sinensis var. sinensis cv Yabukita and a hybrid of var. assamica & var. sinensis cv Izumi) were indirectly measured by means of a crude enzyme assay. The aroma precursors were abundant in young leaves and decreased as the leaf aged. Glycosidase activity also decreased as leaves aged, but was high in stems.

Biochemical responses of Camellia sinensis (L.) O. Kuntze to heavy metal stress.

Three heavy metals-mercury (II), copper (II) and nickel (II), each at a concentration of 10 and 100 micrograms/ml, were tested for their effects on various biochemical constituents of tea leaves. Both NI (II) and Hg (II) decreased the phenolic contents, while Cu (II) increased it to some extent. The metal treatments enhanced the activity of phenyl alanine ammonia lyase (PAL), while the activity of poly phenol oxidase (PPO) showed a decline. Heavy metal stress also decreased the chlorophyll content of the leaves, along with a significant reduction in Hill activity. Proline content increased significantly in all treatments.

[Laboratory studies of the antioxidant effect and free radical elimination of CoD tea]. / A CoD tea antioxidáns és gyökelimináló hatásainak laboratóriumi vizsgálata.

Free radical elimination properties of anticarcinogenic CoD tea were studied in vitro in our laboratory. Therefore its antioxidant effect, lipid-hydroperoxide elimination property and reductive capacity were measured. The isoenzyme composition of CoD tea lipoxygenases was deduced from pH dependence of its lipoxygenase activity. It was found that the antioxidant effect and lipid-hydroperoxide elimination property of CoD tea were comparable with those of ascorbic acid and the reductive capacity of a cup of CoD tea and 11-13 mg ascorbic acid were the same. In spite of the heat sensibility of lipoxygenases, a strong lipoxygenase activity (1102 U/g) of CoD tea was detected in acidic medium (pH 4.5). It is supposed that these free radical elimination properties support anticarcinogenic effect of CoD tea.

Tannase application in secondary enzymatic processing of inferior Tieguanyin oolong tea

Background: Inferior Tieguanyin oolong tea leaves were treated with tannase. The content and bioactivity of catechins in extracts from the treated tea leaves were investigated to assess the improvement in the quality of inferior Tieguanyin oolong tea. Results: Analysis showed that after treatment, the esterified catechin content decreased by 23.5%, whereas non-galloylated catechin and gallic acid contents increased by 15.3% and 182%, respectively. The extracts from tannase-treated tea leaves showed reduced ability to bind to BSA and decreased tea cream levels. The extracts also exhibited increased antioxidant ability to scavenge OH and DPPH radicals, increased ferric reducing power, and decreased inhibitory effects on pancreatic α-amylase and lipase activities. Conclusions: These results suggested that tannase treatment could improve the quality of inferior Tieguanyin oolong tea leaves.

Kinetic characterization of the enzymatic and chemical oxidation of the catechins in green tea.

The oxidation of green tea catechins by polyphenol oxidase/O2 and peroxidase/H2O2 gives rise to o-quinones and semiquinones, respectively, which inestability, until now, have hindered the kinetic characterization of enzymatic oxidation of the catechins. To overcome this problem, ascorbic acid (AH2) was used as a coupled reagent, either measuring the disappearance of AH2 or using a chronometric method in which the time necessary for a fixed quantity of AH2 to be consumed was measured. In this way, it was possible to determine the kinetic constants characterizing the action of polyphenol oxidase and peroxidase toward these substrates. From the results obtained, (-) epicatechin was seen to be the best substrate for both enzymes with the OH group of the C ring in the cis position with respect to the B ring. The next best was (+) catechin with the OH group of the C ring in the trans position with respect to the B ring. Epigallocatechin, which should be in first place because of the presence of three vecinal hydroxyls in its structure (B ring), is not because of the steric hindrance resulting from the hydroxyl in the cis position in the C ring. The epicatechin gallate and epigallocatechin gallate are very poor substrates due to the presence of sterified gallic acid in the OH group of the C ring. In addition, the production of H2O2 in the auto-oxidation of the catechins by O2 was seen to be very low for (-) epicatechin and (+) catechin. However, its production from the o-quinones generated by oxidation with periodate was greater, underlining the importance of the evolution of the o-quinones in this process. When the [substrate] 0/[IO4 (-)] 0 ratio = 1 or >>1, H2O2 formation increases in cases of (-) epicatechin and (+) catechin and practically is not affected in cases involving epicatechin gallate, epigallocatechin, or epigallocatechin gallate. Moreover, the antioxidant power is greater for the gallates of green tea, probably because of the greater number of hydroxyl groups in its structure capable of sequestering and neutralizing free radicals. Therefore, we kinetically characterized the action of polyphenol oxidase and peroxidase on green tea catechins. Furthermore, the formation of H2O2 during the auto-oxidation of these compounds and during the evolution of their o-quinones is studied.

Oxidases from mate tea leaves (Ilex paraguariensis): extraction optimization and stability at low and high temperatures.

Mate (Ilex paraguariensis) is an important natural product in the economic and cultural context of Brazil. Peroxidase and polyphenol oxidase have been responsible for quality deterioration and browning in mate. The objective of this work was to investigate a methodology of extraction and enzymatic activity determination of oxidases present in mate tea leaves and to evaluate the oxidases stability. The effects of raw-material mass, buffer molar concentration, Triton X-100 addition, extraction pH, pH activity measurement, polyvinylpyrrolidone K90 addition, and centrifugation time were evaluated by the experimental planning methodology. The storage of the oxidases along 150 days at low temperatures showed that no significant difference was found at -4 and -80 degrees C but significant difference was observed when compared to 4 degrees C. The results showed that higher activities of oxidases are obtained at similar conditions. The exposition to high-temperatures and the variation of the time of exposition affected the enzymatic activity significantly.

[Intracellular localization and characteristics of monophenol monooxygenase in the leaves of tea plants]. / Vnutrikletochnaia lokalizatsiia i kharakteristika monofenol-monooksigenazy list'ev chainogo rasteniia.

The intracellular localization and some properties of monophenol monooxygenase (MPMO) from fresh tea leaves have been studied. It has been demonstrated that MPMO activity is located in cytosole and chloroplasts. These two forms have different properties. Molecular weights of cytosole and chloroplasts MPMO are 41 and 28 kD respectively. The chloroplasts and cytosole forms of MPMO reveal maximum activity at pH 5.3 and 7.1 respectively.

5-Dehydroshikimate reductase in the tea plant (Camellia sinensis L.). Properties and distribution.

1. A method for the extraction of 5-dehydroshikimate reductase (EC 1.1.1.25) from tea plant tissues in an active soluble state has been developed. It is dependent on the use, in the extraction medium, of an insoluble polyphenol adsorbent (Polyclar AT), which prevents the polyphenols present from precipitating all the proteins. 2. The enzyme has the following properties: pH optima at pH10.1 in glycine-sodium hydroxide buffer and at pH7.7 in tris-hydrochloric acid buffer; K(m) (NADP) 32mum and K(m) (shikimate) 0.43mm; and NADP-specificity. It was completely inhibited by 0.33mm-p-chloromercuribenzoate and this inhibition was completely reversed by 10mm-cysteine. Iodoacetate and arsenite inhibited the enzyme to a smaller extent. 3. The specific activity of the enzyme was higher in the parts of the actively growing shoot tips (third leaf>stem>second leaf>first leaf>bud) than in the mature leaves. However, the mature leaves had the greatest total activity. 4. The importance of these findings with respect to flavanol biosynthesis in tea plants is discussed.

Hydroxyurea and p-aminophenol are the suicide inhibitors of ascorbate peroxidase.

Guaiacol peroxidase from spinach catalyzes the oxidation of p-aminophenol to produce the aminophenoxy radical as the primary product which is converted further into a stable oxidation product with an absorption peak at 470 nm. The p-aminophenol radicals oxidize ascorbate (AsA) to produce monodehydroascorbate radicals. Kinetic analysis indicates that p-aminophenol radicals also oxidize monodehydroascorbate to dehydroascorbate. Incubation of AsA peroxidase from tea leaves and hydrogen peroxide with p-aminophenol, p-cresol, hydroxyurea, or hydroxylamine results in the inactivation of the enzyme. No inactivation of the enzyme was found upon incubation of the enzyme with these compounds either in the absence of hydrogen peroxide or with the stable oxidized products of these compounds. The enzyme was protected from inactivation by the inclusion of AsA in the incubation mixture. The radicals of p-aminophenol and hydroxyurea were produced by AsA peroxidase as detected by their ESR signals. These signals disappeared upon the addition of AsA, and the signal characteristic of monodehydroascorbate was found. Thus, AsA peroxidase is inactivated by the radicals of p-aminophenol, p-cresol, hydroxyurea, and hydroxylamine which are produced by the peroxidase reaction, and it is protected from inactivation by AsA via the scavenging of the radicals. Thus, these compounds are the suicide inhibitors for AsA peroxidase. Isozyme II of AsA peroxidase, which is localized in chloroplasts, is more sensitive to these compounds than isozyme I. In contrast to AsA peroxidase, guaiacol peroxidase was not affected by these various compounds, even though each was oxidized by it and the corresponding radicals were produced.