Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants （Camellia sinensis （L.） O. Kuntze）

β-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity,with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50℃ and was stable at temperatures lower than 40℃. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.     

Detection of genetic variation among micropropagated tea [Camellia sinensis (L.) O. Kuntze] by RAPD analysis

Summary Randomly amplified polymorphic DNA (RAPD) techniques were applied to assess genetic instability among micropropagated tea [Camellia sinensis (L.) O. Kuntze] eultivar ‘T-78’. Out of 49 random 10-mer primers, 11 generated polymorphism in four out of 17 micropropagated plants and one mother plant. A total of 221 bands, ranging from 525 bp to 2.5 kb, were produced by the 49 primers. Twenty-four were polymorphic for those four plants. However, the remaining bands were monomorphic among all plants. Polymorphism among those four plants showed an identifical banding pattern suggesting the occurrence of a single mutation. Our results demonstrated that RAPD can be used successfully to determine the genetic instability among micropropagated plants which otherwise were morphologically indistinguishable.     

Characterization of fluoride uptake by roots of tea plants (Camellia sinensis (L.) O. Kuntze)

Backgrounds and aims

Tea plants (Camellia sinensis (L.) O. Kuntze) accumulate high fluoride in the leaves whereas the mechanism on its uptake is poorly understood. The measured F^? uptake was compared to calculated uptake from transpiration rates assumuing no discrimination between F^? and water to characterize the property of F^? absorption by tea plant roots.

Methods

The F^? uptake was examined by depletion method under variable external F^? concentrations, pH, temperature, relative air humidity, anion channel blockers and metabolism inhibitors in solution experiments.

Results

Measured F^? uptake rates were significantly larger than those calculated from transpiration rates regardless of external F^? concentrations, uptake durations, relative humidity, and solution pH. The measured and net F^? uptake (subtracting that calculated from transpiration rate from the measured uptake) were reduced by low temperature and inhibited by anion channel and metabolism inhibitors anthracene-9-carboxylic acid (A-9-C), niflumic acid (NFA), and carbonylcyanide m-chlorophenylhydrazone (CCCP) but not by dihydro-4, 4′ diisothiocyanostilbene-2, 2′-disulphonic acid (DIDS). The F^? uptake showed biphasic response patterns, following saturable Michaelis–Menten kinetics in the range of low external F^? (below 100 μmol?L^?1) while increased linearly with external supply in the range of high concentrations.

Conclusion

The uptake of F^? by roots of accumulator tea plants was likely an active process and energy-dependent. This helps to explain why tea plants are able to accumulate considerably high F^?.     

The ploidy of plants obtained from anther culture of cauliflowers (Brassica oleracea var. botrytis)

Anther culture of four cultivars of autumn cauliflower gave embryo yields of 100 embryos (per 100 anthers cultured) for one cultivar, eight embryos for a second cultivar, and almost zero for the two other cultivars. From 986 embryos which were grown on, 418 plants were regenerated. The ploidy of the regenerants was assessed by measuring guard cell lengths, pollen size, and by chromosome counts at meiosis. Only 1% of the regenerants were haploid, 41% diploid, 5% triploid and 53% tetraploid. One octoploid plant was also found. The results are discussed in relation to equivalent results for Brussels sprouts, and the use of anther culture in the breediing of cauliflowers.     

Metabolomic and Pathway Changes in Large-Leaf,Middle-Leaf and Small-Leaf Cultivars of Camellia sinensis (L.) Kuntze var. niaowangensis

As an economically important crop, tea is widely cultivated in more than 50 countries and has numerous health benefits. Metabolomics has considerable advantages in the analysis of small molecules and has been widely used in tea science. We applied a metabolomic method to evaluate the dynamic changes in metabolites and pathways in the large-, middle- and small-leaf cultivars of Camellia sinensis (L.) Kuntze var. niaowangensis grown in the same area from Yunwu Mountain. The results indicate that flavonoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis, citrate cycle (TCA cycle), and propanoate metabolism may play important roles in the differences among cultivars. The levels of tea polyphenols, flavonoids and amino acids may impact the sensory properties of teas of different cultivars. Our results may help to elucidate the mechanism underlying the difference in tea quality and offer references for the breeding of high-quality tea cultivars.     

Betaine a novel candidate for rapid induction of somatic embryogenesis in tea (Camellia sinensis (L.) O. Kuntze)

Addition of betaine to the inductionmedium significantly enhanced the rapid formation ofsomatic embryos directly without callusing from maturefresh seeds of tea within two weeks of cultureinitiation. The induction response was furtherenhanced when ABA (7.5 mgl^–1) was co-supplementedwith betaine in the induction medium. The rate ofinduction of somatic embryogenesis increased linearlywith external betaine concentration. Globular somaticembryo-like structures (embryoids) were observed in 4-week old cultures when inoculated on the inductionmedium without ABA and betaine. The positive effectof ABA on the induction process was found to bedependent on the presence of betaine in the medium. ABA alone in the medium could not bring the inductionstimulus in the explants; on the contrary, it provedinhibitory. The optimum response of ABA was observedwhen the medium was supplemented with 500 to1000 mgl^–1 of betaine. Primary somatic embryosobtained in the presence of ABA and betaine were ableto produce secondary embryos. A conversion rate of15–20% was achieved upon transfer of somatic embryosof size 3–5 mm in diameter to the basal medium consistof half strength of macro nutrients, full strength ofmicro nutrients and vitamins of MS. Medium wasfurther supplemented with 100 mg l^–1 each ofadenine hemisulfate sulphate and L-glutamine, 30 gl^–1 sucrose, gelled with 7 gl^–1 bitek agar. The plantlets regenerated by this procedure did notshow any visible abnormalities. This report for thefirst time details the potential use of betaine inplant tissue culture.     

Diversity analysis of diazotrophic bacteria associated with the roots of tea (Camellia sinensis (L.) O. Kuntze)

The diversity elucidation by amplified ribosomal DNA restriction analysis and 16S rDNA sequencing of 96 associative diazotrophs, isolated from the feeder roots of tea on enriched nitrogen-free semisolid media, revealed the predominance of Gram-positive over Gram-negative bacteria within the Kangra valley in Himachal Pradesh, India. The Gram-positive bacteria observed belong to two taxonomic groupings; Firmicutes, including the genera Bacillus and Paenibacillus; and Actinobacteria, represented by the genus Microbacterium. The Gram-negative bacteria included alpha-Proteobacteria genera Brevundimonas, Rhizobium, and Mesorhizobium; gamma-Proteobacteria genera Pseudomonas and Stenotrophomonas; and beta-Proteobacteria genera Azospira, Burkholderia, Delftia, Herbaspirillum and Ralstonia. The low level of similarity of two isolates, with the type strains Paenibacillus xinjiangensis and Mesorhizobium albiziae, suggests the possibility of raising species novum. The bacterial strains of different phylogenetic groups exhibited distinct carbon-source utilization patterns and fatty acid methyl ester profiles. The strains differed in their nitrogenase activities with relatively high activity seen in the Gramnegative strains exhibiting the highest similarity to Azospira oryzae, Delftia lacustris and Herbaspirillum huttiense.     

Expression and biochemical analysis of codon-optimized polyphenol oxidase from Camellia sinensis (L.) O. Kuntze in E. coli

Polyphenol oxidases (PPOs) are copper-containing industrially important enzymes that catalyze the synthesis of many commercially important products by using polyphenols as substrate. Camellia sinensis polyphenol oxidase (CsPPO) is interesting because it oxidizes epicatechins to yield theaflavins and thearubigins. The present study aimed to optimize the expression of CsPPO in Escherichia coli. Because CsPPO had a large number of E. coli rare codons, it yielded a poor quantity of protein in E. coli Rosetta™ 2 cells, which have additional tRNAs for E. coli rare codons. Thus, synthetically constructed codon-optimized CsPPO was cloned into pET-47b(+) vector and expressed in a bacterial host. Ectopic expression led to the formation of inclusion bodies. However, extensive standardization of buffers and methods of refolding such as dialysis, on-column refolding, and rapid dilution yielded active PPO from solubilized inclusion bodies with copper content of 0.880 ± 0.095 atom/molecule of protein.Experimental data produced maximum PPO activity in a rapid dilution buffer containing 0.5 M L-arginine. Refolded CsPPO had an optimum pH of 5.0 and K_m values of 3.10, 0.479, and 0.314 mM, and a V_max of 163.9, 82.64, and 142.8 U/mg of protein for catechol, catechin, and epicatechin, respectively.     

Mycorrhizal differences between genuine roots and tuberous roots of adult plants ofSpiranthes sinensis var.amoena (Orchidaceae)

Genuine roots ofSpiranthes sinensis var.amoena were infected with the mycorrhizal fungusRhizoctonia repens immediately after root formation in autumn. Infection by the mycorrhizal fungus extended, reaching a maximum the following early summer. The amount of living mycorrhizal fungus in the genuine roots dramatically declined in the flowering season, and then the roots decomposed. Tuberous roots were formed in spring. Mycorrhizas were limited to local infections and did not spread along the roots. The infection level of living mycorrhizal fungus in the tuberous roots was less than in the genuine roots throughout the year. The amount of dead fungal coils in the tuberous roots increased as the tuberous roots aged. The mycorrhizal characteritics of tuberous roots ofS. sinensis var.amoena were totally different from those of genuine roots although the tuberous roots morphologically resembled the genuine roots. Contribution No. 96, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Triterpenoid saponins from the roots of tea plant (Camellia sinensis var. assamica)

Three olean-12-ene type triterpenoid saponins, named TR-saponins A, B and C, were isolated as methyl esters from tea roots (Camellia sinesis var. assamica) after treatment with diazomethane. Their structures were established as the methyl esters of 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-21, 22-di-O-angeloyl-R1-barrigenol-23-oic acid, 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-21-O-angeloyl-22-O-2-me thylbutanoyl-R1- barrigenol-23-oic acid and 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-16 alpha-O-acetyl-21-O-angeloyl-22-O-2-methylbutanoyl-R1-bar rigenol-23-oic acid, by extensive 1D and 2D-NMR as well as FABMS and HR-MS analyses.     

RAPD analysis of a variant of banana (Musa sp.) cv. grande naine and its propagation via shoot tip culture

Summary A morphological variant obtained from in vitro corm-derived plants of banana (Musa sp.) cv. Grande Naine (AAA) was evaluated up to harvest and the genetic basis of variation was confirmed by the random amplified polymorphic DNA (RAPD). The corms formed during the multiplication phase of shoot tip-derived cultures of the cv. Grande Naine grown on Murashige and Skoog (MS) medium enriched with 13.3 μM N ⁶-benzyladenine (BA) developed numerous morphological variants after transfer to MS medium with 6.66 μM BA. The variant designated as CUDBT-B1, with distinct morphological features, was further evaluated. The morphological features of CUDBT-B1 were variegated leaf, pseudostem, bracts, ovary of the male flower and fruits, reduced height, decreased lamina length and breadth, and early flowering. These features were also manifested in the second-cycle progeny of CUDBT-B1. RAPD assay showed a marker DNA band of 1650 bp, and differential band intensity between the CUDBT-B1 and normal clone. CUDBT-B1 was multiplied using shoot tip culture, and the shoots were rooted on half-strength MS medium supplemented with 2.69 μM α-naphthaleneacetic acid. All plantlets showed variegated leaves under field conditions.     

An Improved Protocol for the Isolation of RNA from Roots of Tea (Camellia sinensis (L.) O. Kuntze)

Tea, a beverage crop, is a rich source of polyphenols and polysaccharides which greatly attribute to its importance. However, oxidation and precipitation of these compounds during nucleic acids extraction is a limitation to molecular biology and genomic studies. On isolation of total RNA from root tissue using established protocols, difficulties were encountered in terms of purity and quantity of isolated RNA or some of the methods were time-consuming and also yields were low. The present communication combines a phenol-based RNA isolation protocol with a cetyltrimethylammonium bromide-based procedure with appropriate modifications. This protocol successfully isolated RNA from tap root tissue in 2-3 h as compared with 16 h reported by the previous method. Also, RNA yield was higher by more than fourfold. The RNA isolated by this protocol was successfully used for downstream applications such as RT-PCR and the construction of suppression subtractive hybridization library. The developed protocol worked well with other plant tissue with high polyphenols and polysaccharides contents.     

RAPD analysis of seed purity in a commercial hybrid cabbage (Brassica oleracea var. capitata) cultivar.

The use of random amplified polymorphic DNA (RAPD) markers for evaluating seed purity in a commercial F1-hybrid cabbage (Brassica oleracea var. capitata) cultivar is demonstrated. Genomic DNA isolated from single ungerminated seed was found to be suitable for RAPD analysis. DNA from F1-hybrid and its parental lines was subjected to RAPD screening with 36 random decamer arbitrary primers. A total of 241 scorable products were observed with 54 (22%) being polymorphic. The RAPD data showed that the parental lines of this commercial cabbage cultivar were not very closely related. Two primers were chosen for purity testing of the F1-hybrid seeds. The sib (inbred seed; seed from self-pollination of parental lines) contamination results obtained by RAPD analysis were comparable to the commonly used grow-out trial and isozyme analysis, hence showing that RAPD analysis can be used for seed purity testing of commercial hybrid cabbage seeds.     

Direct somatic embryogenesis in a selected tea clone, `TRI-2025' (Camellia sinensis (L.) O. Kuntze) from nodal explants

An efficient method for the in vitro propagation of a tea (Camellia sinensis (L) O. Kuntze) clone, `TRI-2025', from somatic embryos is described. This technique involves two phases; the induction of adventitious buds from nodal cuttings followed by the development of somatic embryos. Single nodal cuttings were excised from 1-year-old in vitro axenic cultures and inoculated on MS medium with different combinations of IBA/BAP/GA₃. Induction of multiple shoots from nodal explants occurred on MS medium with 0.5 mg l^–1 BAP, 0.1 mg l^–1 IBA and 0.0 mg l^–1 GA₃ within 6 weeks of incubation. The cultures with multiple shoots were transferred to fresh medium, incubated for 120 days and transferred to MS medium with half-strength macro nutrients, full-strength micronutrients and vitamins and no growth regulator. The direct induction of somatic embryos without callus formation occurred on this medium at 60% frequency within 4 weeks. The production of embryos continued upon transfer of the cultures to fresh medium and a four- to eightfold multiplication rate was obtained during each 6-week culture cycle. The plantlets from these embryos were acclimatised with a 90% success rate. All plants were vigorous and hardy, with well-developed tap-root systems. Received: 20 July 1996 / Revision received: 2 January 1998 / Accepted: 19 January 1998     

Shoot meristem differentiation in shoot primordia culture (SPC) obtained from root primordia culture (RPC) of Solanum lycopersicoides Dun.

We present a unique example of conversion of the morphogenesis type (from rhisogenesis to shoot organogenesis) in in vitro cultures of Solanum lycopersicoides. Liquid shoot primordia cultures (SPC) were obtained from root primordia culture (RPC) on two kinds of MS-based media with BA. The first SMS₈, contained a full set of organic compounds; the second, ²SMS₈, was devoid of organic compounds except sucrose and edamine. Two weeks after passage of RPC onto both the media, disintegration of root primordia and cell aggregates began. After 8 weeks of cultivation on SMS₈ and ²SMS₈ media, SPC aggregates developed from meristematic cells located near the vascular tissue of disintegrating RPC aggregates. Initiation of shoot meristems started from meristematic cells centers which were localised under the surface of the newly formed SPC aggregates. The change in the type of morphogenesis occurred faster on medium SMS₈, but the SPC on medium ²SMS₈ was characterized by more frequent formation of shoots and plants (45% of aggregates, in the case of SPC on medium SMS₈, and 90% in the case of SPC on medium ²SMS₈). This fact was correlated with the structural organization of the meristematic centers. Our results indicate that an RPC system has high morphogenetic potential due to the continual presence of meristematic cells. The change in the type of morphogenesis was followed by a rebuilding of the aggregate structure on the basis of the meristematic cells already existing in RPC, which gave rise to SPC aggregates from which shoot meristems developed.     

Biotransformation of geranyl acetate to geraniol during palmarosa (Cymbopogon martinii, Roxb. wats. var. motia) inflorescence development

Only immature palmarosa (Cymbopogon martinii, Roxb. wats. var. motia) inflorescence with unopened spikelets accumulated essential oil substantially. Geraniol and geranyl acetate together constituted about 90% of the palmarosa oil. The proportion of geranyl acetate in the oil decreased significantly with a corresponding increase of geraniol, during inflorescence development. An esterase enzyme activity, involved in the transformation of geranyl acetate to geraniol, was detected from the immature inflorescence using a gas chromatographic procedure. The enzyme, termed as geranyl acetate cleaving esterase (GAE), was found to be active in the alkaline pH range with the optimum at pH 8.5. The catalysis of geranyl acetate was linear up to 6 h, and after 24 h of incubation, 75% of the geranyl acetate incubated was hydrolyzed. The GAE enzymic preparation, when stored at 4 degrees C for a week, was quite stable with only 40% loss of activity. The physiological role of GAE in the production of geraniol during palmarosa inflorescence development has been discussed.