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.     

In vitro regeneration of Camellia sinensis (L.) O. Kuntze by somatic embryogenesis

Within 3 weeks of culture, excised cotyledon expiants of Camellia sinensis (L.) O. Kuntze produced somatic embryos without intermediate callus when cultured in Murashige and Skoog's basal medium with 30 g^–1 sucrose. In medium without plant growth regulators, up to 60% of the cultures developed somatic embryos. Embryogenic competence was reduced by increasing concentrations of plant growth regulators tested (i.e. kinetin, 6-benzylaminopurine, and indole butyric acid). The somatic embryos developed, grew to maturity without being subcultured within 6–8 weeks. Secondary embryogenesis was not observed. Germination of isolated mature somatic embryos was low in medium without plant growth regulators. Up to 53% and 60% germination occurred when medium impregnated with kinetin at 1.8 mgl^–1 or 1.0 mgl^–1 6-benzylaminopurine were used respectively. Callus was also routinely produced when cotyledons were cultured in MS basal medium with auxins (2,4-dichlorophenoxyacetic acid and indole acetic acid). Callus induction was however, also achieved in plant growth regulator free medium. Indirect somatic embryogenesis was not induced in the present study.Abbreviations K kinetin - BAP 6-benzylaminopurine - IBA indole butyric acid - IAA indole acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthalene acetic acid - Fe-EDTA Ethylenediaminetetra-acetic acid (Ferric monosodium salt)     

In vitro clonal propagation of tea (Camellia sinensis (L.) O. Kuntze)

A system for in vitro clonal propagation has been developed in tea plants. Shoots obtained from primary explants were induced from terminal buds and axillary buds of mature field-grown plants. Cultures were initiated from both types of explants on Murashige and Skoog (MS) medium supplemented with 10% coconut milk (CM), 200 mg l^-1 of yeast extract (YE), 1.4 M indoleacetic acid (IAA) and 17.8 M benzyladenine (BA). The shoot tips were multiplied on 1/2 strength MS medium containing 10% CM, 2.9 M IAA and 17.8 M BA. The larger shoots were separated after multiplication and rooted on 1/2 MS medium supplemented with 11.4 M ascorbic acid and 34.5 M indolebutyric acid (IBA). A pretreatment of the plants with an aqueous solution of 493 M IBA greatly increased the frequency of rooting. More than 60% of the rooted plants have been transferred to soil successfully.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - YE yeast extract - CM coconut milk - MS Murashige and Skoog medium (1962)     

Molecular regulation of catechins biosynthesis in tea [Camellia sinensis (L.) O. Kuntze

Catechins are bioprospecting molecules present in tea and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. These are synthesized through the activities of phenylpropanoid and flavonoid pathways. Expression regulation of various genes of these pathways namely phenylalanine ammonia-lyase (CsPAL), cinnamate 4-hydroxylase (CsC4H), p-coumarate:CoA ligase (Cs4CL), flavanone 3-hydroxylase (CsF3H), dihydroflavonol 4-reductase (CsDFR) and anthocyanidin reductase (CsANR) was accomplished previously. In depth analyses of the remaining genes namely, chalcone synthase (CsCHS), chalcone isomerase (CsCHI), flavonoid 3'5'-hydroxylase (CsF3'5'H) and anthocyanidin synthase (CsANS) were lacking. The objective of the work was to clone and analyze these genes so as to generate a comprehensive knowledge on the critical genes of catechins biosynthesis pathway. Gene expression analysis was carried out in response to leaf age and external cues (drought stress, abscisic acid, gibberellic acid treatments and wounding). A holistic analysis suggested that CsCHI, CsF3H, CsDFR, CsANS and CsANR were amongst the critical regulatory genes in regulating catechins content.     

Isolation and Characterization of Brassinosteroids from Leaves of Camellia sinensis (L.) O. Kuntze

Brassinosteroids are of ubiquitous occurrence in plants and elicit a wide spectrum of physiological responses. In our study, brassinosteroids were isolated and identified in topmost dormant leaves of tea plants. Six brassinosteriods, i.e. 6-deoxocastasterone, 24-epibrassinolide,3-dehydroteasterone, typhasterol, 3-deoxotyphasterol and 28-homodolicholide, were isolated and identified by GC–MS. All the brassinosteroids identified belong to important components of early and late C6 oxidation pathways proposed for brassinosteroids biosynthesis in plants. It suggests that both pathways are operating in tea to produce brassinolide, the most active brassinosteroid biologically.     

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

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

Changes in endogenous auxins during winter dormancy in tea (Camellia sinensis L.) O. Kuntze

Changes in endogenous auxin (IAA) were determined in tea shoots with the onset of winter dormancy (30 October) and subsequent to dormancy release (15 April). Very low levels of free IAA were detected in dormant shoots when they were still in deep dormancy. The levels increased after 30 January and reached highest value after dormancy release. Conjugated IAA levels increased with onset of dormancy reaching its maximum value when free IAA levels were lowest. With increase in free IAA levels, the conjugated IAA levels decreased in shoots prior to dormancy release suggesting of metabolic interconversion of IAA during these periods. The possible roles of free and conjugated IAA in dormancy and following its release are discussed in relation to winter dormancy in tea shoots.     

ABA associated biochemical changes during somatic embryo development in Camellia sinensis (L.) O. Kuntze

The effect of ABA on reserve accumulation in maturing somatic embryos of tea was compared with and without ABA treatment. Changes in the levels of starch, total soluble sugars (TSS), proteins, and phenols were studied in the somatic embryos at different stages of development (globular, heart, torpedo and germinating embryos) in order to investigate whether ABA could trigger accumulation of storage reserves and thereby overcome the problem of poor germination. After ABA treatment (5.0 mg l(-1)) for 14 days, the starch and protein contents that were negligible in the untreated embryos increased by several fold with a simultaneous increase in TSS. When ABA treatment occurred at the heart stage, the germination of the embryos also improved, relative to untreated controls, after ABA treatment. ABA treatment prior to or after heart stage did not improve somatic embryo germination.     

Agrobacterium tumefaciens-mediated transformation of embryogenic tissues of tea (Camellia sinensis (L.) O. Kuntze)

Embryogenic tissues of tea were cocultivated withAgrobacterium tumefaciens LBA4404. The plasmid pBi121, which contains the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the β-glucuronidase (uidA) reporter gene, was used as binary vector. The highest transformation frequency (12 transformants/g fresh weight [FW] of treated embryogenic tissue) was obtained with 5-day-old tissues grown in liquid medium and cocultivated withAgrobacterium for 2 d in the same medium but containing 50 μM acetosyringone. There was improvement in the recovery of kanamycin-resistant tissues when tissues were first grown for 10 d on a medium containing 350 mg/L Timentin to prevent bacterial overgrowth, before application of the selection pressure. Resistant tissues obtained after 6 wk on kanamycin-selection medium showed stableuidA expression. Polymerase chain reaction demonstrated the presence of the transgenes, while Southern hybridization confirmed their integration into the genome. Transgenic plants were regenerated from transformed tissues within 4 mo after coculture.     

Inhibition of glycerol-3-phosphate acyltransferase by analogs of glycerol-3-phosphate.

Analogs of glycerol-3-phosphate were tested as substrates or inhibitors of the glycerol-3-phosphate acyltransferases of mitochondria and microsomes. (rac)-3,4-Dihydroxybutyl-1-phosphonate, (rac)-glyceraldehyde 3-phosphate, (rac)-3-hydroxy-4-oxobutyl-1-phosphonate, (1S,3S)-1,3,4-trihydroxybutyl-1-phosphonate, and (1R,3S)-1,3,4 trihydroxybutyl-1-phosphonate were competitive inhibitors of both mitochondrial and microsomal sn-glycerol-3-phosphate acyltransferase activity. An isosteric analog of dihydroxyacetone phosphate, 4-hydroxy-3-oxobutyl-1-phosphonate, was a much stronger competitive inhibitor of the microsomal than the mitochondrial enzyme. Phenethyl alcohol was a noncompetitive inhibitor of both the microsomal and the mitochondrial acyltransferases. The product of the mitochondrial acyltransferase reaction with (rac)-3,4-dihydroxybutyl-1- phosphonate was almost exclusively (rac)-4-palmitoyloxy-3-hydroxybutyl-1-phosphonate. The microsomal acylation reaction generated both the monoacyl product and (S)-3,4-dipalmitoyloxybutyl-1-phosphonate. The apparent Km for (S)-3,4-dihydroxybutyl-1-phosphonate was 2.50 and 1.38 mM for the mitochondrial and microsomal enzymes, respectively.     

5S rDNA gene diversity in tea (Camellia sinensis (L.) O. Kuntze) and its use for variety identification.

Variability in the organization of repeats of 5S rDNA is useful for phylogenetic studies in various crops. We found variable repeats of 5S rDNA gene in the genome of tea (Camellia sinensis (L.) O. Kuntze) during Southern hybridization. Variability in the repeats of 5S rDNA with specific restriction endonucleases (Sau3AI, BamHI, and ApoI) was analyzed in 28 different tea clones representing 3 types of tea. Our results clearly show that the 5S rDNA gene in tea could be used as a molecular marker to distinguish C. sinensis Chinary tea from the other important types of tea, namely Assamica and Cambod. Upon analysis with restriction endonucleases, the 5S rDNA gene in the tea genome was found to be heavily methylated.     

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.     

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 Role of Context-Dependent Mutations in Generating Compositional and Codon Usage Bias in Grass Chloroplast DNA

Abstract The influence of local base composition on mutations in chloroplast DNA (cpDNA) is studied in detail and the resulting, empirically derived, mutation dynamics are used to analyze both base composition and codon usage bias. A 4 × 4 substitution matrix is generated for each of the 16 possible flanking base combinations (contexts) using 17,253 noncoding sites, 1309 of which are variable, from an alignment of three complete grass chloroplast genome sequences. It is shown that substitution bias at these sites is correlated with flanking base composition and that the A+T content of these flanking sites as well as the number of flanking pyrimidines on the same strand appears to have general influences on substitution properties. The context-dependent equilibrium base frequencies predicted from these matrices are then applied to two analyses. The first examines whether or not context dependency of mutations is sufficient to generate average compositional differences between noncoding cpDNA and silent sites of coding sequences. It is found that these two classes of sites exist, on average, in very different contexts and that the observed mutation dynamics are expected to generate significant differences in overall composition bias that are similar to the differences observed in cpDNA. Context dependency, however, cannot account for all of the observed differences: although silent sites in coding regions appear to be at the equilibrium predicted, noncoding cpDNA has a significantly lower A+T content than expected from its own substitution dynamics, possibly due to the influence of indels. The second study examines the codon usage of low-expression chloroplast genes. When context is accounted for, codon usage is very similar to what is predicted by the substitution dynamics of noncoding cpDNA. However, certain codon groups show significant deviation when followed by a purine in a manner suggesting some form of weak selection other than translation efficiency. Overall, the findings indicate that a full understanding of mutational dynamics is critical to understanding the role selection plays in generating composition bias and sequence structure.     

Changes in endogenous gibberellin activity during winter dormancy in tea (Camellia sinensis (L.) O. Kuntze)

Changes in gibberellin (GAs) activities were determined in tea shoots during winter dormancy and subsequent to dormancy release. Free GA-like activity was extremely low at the initiation of dormancy and remained so during the dormancy period. Conjugated GA-like activity (ficin hydrolyzable and β-glucosidase hydrolyzable compounds) remained high during the dormancy period. With an increase in free GA activity, conjugated GA activity decreased in tea shoots prior to dormancy release. The possible role of free and conjugated GAs in dormancy and following its release is discussed in relation to winter dormancy in tea shoots. IHBT Communication number 9846     

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.