Mutagenicity and carcinogenicity of tea, Camellia sinensis

Aqueous, caffeine free and tannin fractions of commercial tea and tannic acid were tested for mutagenicity in Ames test. Tea fractions of tannic acid were non mutagenic in strains TA 100, TA 98, TA 1535 and TA 1538 of Salmonella typhimurium with or without metabolic activation (rat-S9 mix) at different doses tested. In strain TA 98 the above tea fractions and tannic acid inhibited the S9 mix mediated mutagenicity of tobacco in a dose dependent manner. The different tea fractions at 60 degrees C, did not increase the tumor incidence in Swiss mice by gavage feeding. They also failed to produce tumors when injected subcutaneously. Caffeine free tea extract decreased the tobacco induced liver tumors but had no effect on lung tumors. The same fraction was ineffective in hexachlorocyclohexane induced liver tumors in Swiss mice.     

Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.)

Background

Despite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species.

Results

Considering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (H _E ) and observed (H _o ) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.

Conclusion

UGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.     

Development of microsatellite and amplicon length polymorphism markers for Camellia japonica L. from tea plant (Camellia sinensis) expressed sequence tags

Simple sequence repeats and amplicon length polymorphism markers for Camellia japonica were developed, based on Camellia sinensis sequences in the National Center for Biotechnology Information database. In total, 2495 gene sequences were used to design 216 primer pairs. To identify amplicon length polymorphism markers, 61 gene loci in 16 Camellia individuals were re-sequenced. In total, 10 markers (three expressed sequence tags-simple sequence repeats and seven amplicon length polymorphisms) yielded polymerase chain reaction products with clear polymorphic patterns and were used for genotyping 22 C. japonica individuals from a population. Numbers of alleles and expected heterozygosity ranged from two to 13 and from 0.28 to 0.90, respectively.     

Polyamines and senescence of maintenance foliage of tea,Camellia sinensis L.

In leaf discs of maintenance foliage of tea (Camellia sinensis) polyamines (PA_s) and kinetin retarded chlorophyll (Chl) loss, whereas inhibitors of PA biosynthesis [difluoromethyl arginine, difluoromethyl ornithine, methylglyoxal-bis(guanylhydrazone)] and abscisic acid (ABA) promoted senescence. The contents of RNA and protein were significantly higher in PA and kinetin treated leaf discs as compared to those treated with inhibitors and ABA. The contents of total and reducing saccharides declined with the progressive loss of Chl, and the concentration of starch increased in all the PA treated leaf discs. Free amino acid content also increased under all the treatments, but the increase was comparatively larger in case of inhibitors application. The authors thank Director, CSIR Complex, Palampur for providing necessary facilities and Merrel Dow Research Institute (Cincinnati, OH) for generous gift of DFMO and DFMA.     

Isotheasaponins B1-B3 from Camellia sinensis var. sinensis tea leaves

Three saponins, isotheasaponins B1-B3, were isolated from the leaves of the tea plant Camellia sinensis var. sinensis, and their structures were determined to be theasapogenol B [beta-D-galactopyranosyl(1-->2)][beta-D-xylopyranosyl(1-->2)-alpha-L-arabinopyranosyl(1-->3)]-beta-D-gulcopyranosiduronic acid with two acyl groups by spectroscopic analysis.     

Clonal variation of tea [Camellia sinensis (L.) O. Kuntze] in countering water deficiency

Various clones of tea [Camellia sinensis (L.) O. Kuntze] such as TTL-1, TTL-2, TTL-4, TTL-5, TTL-6, UPASI-2 and UPASI-3 planted in the field were subjected to soil moisture stress conditions by withholding irrigation. A control set of the same clones were maintained by watering regularly. The soil water content of the irrigated and non irrigated plants was monitored through the soil moisture status. The extent of effect of drought on tea plants were monitored through various physiological parameters such as shoot weight, leaf water potential, chlorophyll and carotenoid content, chlorophyll fluorescence (Fv/Fm), net photosynthetic rate, transpiration rate, stomatal conductance and biochemical parameters such as extent of proline accumulation and free radical generation. These parameters were studied on the 30 d of non irrigation and on the 5 d during recovery from drought. The plants recovered when re-irrigated after 30 d of non-irrigation, which suggests that permanent wilting did not occur due to non-irrigation up to 30 d. On the 30 d of non-irrigation the clones TTL-1, TTL-6 and UPASI-2 showed lesser reduction of shoot weight, leaf water potential, chlorophyll fluorescence, photosynthetic rate, transpiration rate and stomatal conductance and increased proline and lesser lipid peroxidation as compared to the other clones. From these results it can be concluded that the clones TTL-1, TTL-6 and UPASI-2 are comparatively more drought tolerant than the clones TTL-2, TTL-4, TTL-5 and UPASI-3.     

Heterologous amplification and characterization of microsatellite markers in the Neotropical fish Leporinus friderici

Leporinus friderici, native to the Amazon Basin and popularly known as "piau-três-pintas", has great ecological and economic importance; it is widely fished and consumed throughout much of tropical South America. Knowledge of the genetic diversity of this native species is important to support management and conservation programs. We evaluated microsatellite loci amplification, using heterologous primers, in 31 individuals of L. friderici. These samples were collected from natural populations of the Araguaia River basin, in central Brazil, and the DNA was extracted from samples of muscle tissue. Eight loci were successfully analyzed. Six of them were polymorphic, and the number of alleles ranged from three to 10. Values of expected heterozygosities for these polymorphic loci ranged from 0.488 to 0.795. Exclusion probability (0.983), the identity probability (0.000073), and the mean genetic diversity values were high, showing that these microsatellite markers are suitable for assessing the genetic variability of L. friderici populations. There is a growing interest in studies that evaluate the genetic variability of natural populations for various purposes, such as conservation. Here, we showed that a viable alternative to the costly development of specific primers for fish populations is simply testing for heterologous amplification of microsatellite markers available from research on other species.     

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.     

Theanine transporters identified in tea plants (Camellia sinensis L.)

Theanine, a unique non‐proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H⁺‐dependent. The tissue‐specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root‐to‐bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root‐to‐shoot delivery in the tea plant.     

Anthocyanins from red flower tea (Benibana-cha), Camellia sinensis

Three anthocyanins were isolated from the leaves of red flower tea (Benibana-cha), Camellia sinensis, and their structures were determined by means of chemical and spectroscopic analyses. Two are the anthocyanins, delphinidin and cyanidin 3-O-beta-D-galactosides, respectively. Whereas the third, delphinidin 3-0-beta-D-(6-(E)-p-coumaryl)galactopyranoside. The anthocyanins were also contained in the flowers of Benibana-cha in different compositions.     

Anti-inflammatory activity of tea (Camellia sinensis) root extract

Pharmacological studies were carried out with methanol-water (1:1) extract of dried tea (Camellia sinensis) root extract (TRE). TRE was found to possess anti-inflammatory, analgesic and antipyretic activities at 1/10th of its LD50 dose of 100 mg/kg i.p. It was found that TRE inhibited the arachidonic acid-induced paw oedema in rats which indicated that TRE produced the anti-inflammatory activity by inhibiting both the cyclooxygenase and lypooxygenase pathways of arachidonic acid metabolism. TRE also enhanced peritoneal cell count and the number of macrophages in normal mice. It is plausible that the saponins present in TRE may be responsible for these activities of TRE.     

Studies with antitranspirants on tea (Camellia sinensis L.)

The antitranspirant effect of a number of substances on tea was studied and the effects of the most effective substance investigated in detail. The film type of antitranspirants reduced transpiration only at a considerably higher concentration than the stomatal type of antitranspirants. Of the stomatal type of antitranspirants phenyl mercuric acetate (PMA) was the most effective substance. Under actively transpiring conditions a 250 ppm solution of PMA reduced transpiration by about 30 per cent. The effect of PMA was localised and there was no translocation to untreated parts of the leaf blade. A spray of PMA reduced transpiration with gradually diminishing intensity for about 20 days, reduced vegetative growth of young plants grown both under non-water stress and water stressed conditions and reduced the yield of crop of mature plants during a drought. re]19760806     

Boron re-translocation in tea (Camellia sinensis (L.) O. Kuntze) plants

Boron (B) re-translocation is an important factor determining tolerance to B deficiency in plants. In this work growth, B content of leaves with different ages, B partitioning between soluble and cell wall (CW) fractions, and B re-translocation were investigated in tea (Camellia sinensis (L.) O. Kuntze) plants grown hydroponically without (<2.5 μM) and with adequate (46 μM) B supply. Under B deficiency, the proportion of CW bound B increased in the old leaves but decreased in roots. Contrastingly, the proportion of CW bound B was not influenced by B supply in the young leaves. A continuous reduction of B content was observed in all fully expanded leaves as well as in roots of low B plants. Taken together, these results revealed considerable re-translocation of B from mature to growing leaves. Leaf extract and phloem exudate samples were analyzed and sucrose, glucose, and fructose were detected while xylitol, sorbitol, mannitol, maltose, galactose, cellobiose or rafinose were not found in these samples. In the leaf extracts, concentration of sucrose increased under B deficiency conditions, concentration of glucose decreased, while that of fructose remained unchanged. Our results provide circumstantial evidence for a considerable re-translocation of B in tea plants despite lacking polyol compounds.     

Isolation and characterization of highly polymorphic microsatellites in tea (Camellia sinensis)

Relatively little is known about the diversity and origins of tea. The highest value tea products are sold on the basis of their region of origin but there are currently no methods available to verify the claims made on packages. We have developed 15 microsatellite loci for tea. These have been evaluated for polymorphism in a set of tea clones to determine their usefulness for authentication purposes. The majority of the microsatellites developed proved to be highly polymorphic both between and within different geographical origins and offer the potential to investigate the population genetics and genetic origins of tea.     

Characterization of Armillaria isolates from tea (Camellia sinensis) in Kenya

Armillaria is a primary root rot pathogen of tea (Camellia sinensis) in Kenya. The main species presently described in this country are A. mellea and A. heimii. A survey covering fourteen districts of Kenya was carried out and forty-seven isolates of Armillaria collected. Cultural morphology, rhizomorph characteristics, somatic incompatibility and features of basidiomata were used to characterize the isolates, together with molecular analysis based on randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) and the intergenic spacer (IGS) regions and sequence of the IGS region. It can be concluded that two Armillaria species were present and they were different from A. mellea. The first group was morphologically similar to A. heimii but this was contradicted by the molecular data, suggesting that A. heimii could be a complex of several species. The second group was different from the first and morphological and molecular data strongly suggest that it could be a new Armillaria species.     

Water deficit induced oxidative damage in tea (Camellia sinensis) plants

When the tea (Camellia sinensis) leaf water potential was -1.1 MPa (Moderate water deficit), there was 58% inhibition of photosynthesis accompanied by increased zeaxanthin, malondialdehyde, oxidized proteins and superoxide dismutase activity. When the leaf water potential was -2MPa (severe water deficit), there was nearly complete inhibition of photosynthesis apart from a decrease in chlorophylls, beta-carotene, neoxanthin and lutein. Water deficit at this level caused further conversion of violaxanthin to zeaxanthin, suggesting damage to the photosynthetic apparatus. There were consistent decreases in antioxidants and pyridine nucleotides, and accumulation of catalytic Fe, malondialdehyde and oxidized proteins. It is inferred that, in tea plants, the increase in catalytic Fe and the decrease in antioxidant protection may be involved in the oxidative damage caused by severe water deficit, but not necessarily in the incipient stress induced by moderate water deficit.     

Clonal variability in root growth and drought resistance in tea (Camellia sinensis)

Summary An investigation was carried out to study whether differences in rooting depth, root weight and vertical distribution of roots in the soil were some of the factors responsible for clonal variation in drought resistance in tea. The results showed that of these factors only rooting depth influenced drought resistance. Shallow rooted clones were drought susceptible and deep rooted clones drought resistant. In shallow rooted clones drought resistance increased with rooting depth. However in deep rooted clones drought resistance was not related to rooting depth.