Juan Li

Proteomic analysis of young leaves at three developmental stages in an albino tea cultivar

BackgroundWhite leaf No.1 is a typical albino tea cultivar grown in China and it has received increased attention in recent years due to the fact that white leaves containing a high level of amino acids, which are very important components affecting the quality of tea drink. According to the color of its leaves, the development of this tea cultivar is divided into three stages: the pre-albinistic stage, the albinistic stage and the regreening stage. To understand the intricate mechanism of periodic albinism, a comparative proteomic approach based on two-dimensional electrophoresis (2-DE) and mass spectrometry was adopted first time to identify proteins that changed in abundance during the three developmental periods.ResultsThe 2-DE results showed that the expression level of 61 protein spots varied markedly during the three developmental stages. To analyze the functions of the significantly differentially expressed protein spots, 30 spots were excised from gels and analyzed by matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry. Of these, 26 spots were successfully identified. All identified protein spots were involved in metabolism of carbon, nitrogen and sulfur, photosynthesis, protein processing, stress defense and RNA processing, indicating these physiological processes may play crucial roles in the periodic albinism. Quantitative real-time RT-PCR analysis was used to assess the transcriptional level of differentially expressed proteins. In addition, the ultrastructural studies revealed that the etioplast-chloroplast transition in the leaf cell of White leaf No. 1 was inhibited and the grana in the chloroplast was destroyed at the albinistic stage.ConclusionsIn this work, the proteomic analysis revealed that some proteins may have important roles in the molecular events involved in periodic albinism of White leaf No. 1 and identificated many attractive candidates for further investigation. In addition, the ultrastructural studies revealed that the change in leaf color of White leaf No. 1 might be a consequence of suppression of the etioplast-chloroplast transition and damage to grana in the chloroplast induced by temperature. These results provide much useful information to improve our understanding of the mechanism of albinism in the albino tea cultivar.

Isolation and identification of novel genes involved in artemisinin production from flowers of Artemisia annua using suppression subtractive hybridization and metabolite analysis.

Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Artemisinin is highly effective against multidrug-resistant Plasmodium falciparum and it has been widely used as part of the artemisinin-based combination therapies against malaria. To elucidate the biosynthetic pathway of artemisinin and to clone related genes in Artemisia annua, differentially expressed genes between blooming flowers and flower buds were isolated and characterized by a combined approach of suppression subtractive hybridization (SSH) and metabolite analysis. A total of 350 cDNA clones from a subtractive cDNA library were randomly picked, sequenced and analyzed and 253 high-quality sequences were obtained. BLASTX comparisons indicated that about 9.9 % of the clones encoded enzymes involved in isoprenoid (including artemisinin) biosynthesis. The expression of 4 gene transcripts involved in artemisinin biosynthesis was examined by RT-PCR and the results confirmed the higher expression of these transcripts in blooming flowers than in flower buds. In addition, 2 putative transcript factors transparenta testa glabra 1 (TTG1) and ENHANCER OF GLABRA3 (GL3), which promote trichome initiation, were presented in the library. Finally, this study demonstrated that the increase of expression level of the putative TTG1 gene correlated with the improvement of glandular trichome density and artemisinin production in A. annua leaves. The subtractive cDNA library described in the present study provides important candidate genes for future research in order to increase the artemisinin content in A. annua.

Affordable and sensitive determination of artemisinin in Artemisia annua L. by gas chromatography with electron-capture detection.

Artemisinin demand has increased sharply since the World Health Organization recommended its use as part of the artemisinin combination therapies in 2001. The area for the crop cultivation has expanded in Africa and Asia and simpler and affordable methods for artemisinin analysis are needed for crop quality control. This work presented a novel chromatographic method of artemisinin analysis using gas chromatography with electron-capture detection. The sample extraction and preparation involved a single-solvent one-step extraction, with samples being analyzed in the extraction solvent directly after extraction. This method was accurate and reproducible with over 97% recoveries. The limit of detection was less than 3 microg/mL and the limit of quantification was less than 9 microg/mL, allowing samples as low as 100mg dry weight to be analyzed for artemisinin. The method can be applied to quality control of commercial plant extracts and to artemisinin-derived pharmaceuticals.

Tea polyphenol epigallocatechin gallate inhibits Escherichia coli by increasing endogenous oxidative stress.

The antibacterial effects of tea polyphenol epigallocatechin gallate (EGCG), a common phytochemical with a number of potential health benefits, are well known. However, the mechanism of its bactericidal action remains unclear. Using E. coli as a model organism, it is argued here that H2O2 synthesis by EGCG is not attributed to its inhibitory effects. In contrast, the bactericidal action of EGCG was a result of increased intracellular reactive oxygen species and blunted adaptive oxidative stress response in E. coli due to the co-administration of antioxidant N-acetylcysteine, and not on account of exogenous catalase. Furthermore, we noted a synergistic bactericidal effect for EGCG when combined with paraquat. However, under anaerobic conditions, the inhibitory effect of EGCG was prevented. In conclusion, EGCG caused an increase in endogenous oxidative stress in E. coli, thereby inhibiting its growth, and hence the use of EGCG as a prooxidant is supported by this study.

Simultaneous isolation of artemisinin and its precursors from Artemisia annua L. by preparative RP-HPLC.

It is still a major challenge to simultaneously isolate artemisinin and its precursors, especially dihydroartemisinic acid and artemisinic acid, from herbal Artemisia annua. A rapid, economical and automatical chromatographic separation process to isolate and purify artemisinin, dihydroartemisinic acid and artemisinic acid at the same time on a preparative scale was developed. The procedure included solvent extraction of ground Artemisia annua leaves by refluxing and purification of crude extract by preparative reverse-phase high-performance liquid chromatography (RP-HPLC). Fractions containing artemisinin and its precursors were collected and identified by gas chromatography and mass spectrometry. High purity of artemisinin, dihydroartemisinic acid and artemisinic acid was obtained by preparative HPLC with a C(18) column and 60% acetonitrile in water as the mobile phase. The techniques described here are useful tools for the preparative-scale isolation of artemisinin and its precursors in a fast, cost-effective and environmental friendly manner.

Simple and rapid micro‐scale quantification of artemisinin in living Artemisia annua L. by improved gas chromatography with electron‐capture detection

Malaria threatens 300-500 million people and kills more than one million people annually. Artemisinin has been widely used as part of the artemisinin-based combination therapies against malaria. However, its supply is seriously short due to very small amounts of production of artemisinin in Artemisia annua. Molecular biologic researches aimed at increasing the artemisinin yield in plant have received more and more attention and therefore corresponding quantification methods for artemisinin analysis are urgently needed. A variety of methods for determination of artemisinin have been developed but they cannot be applied when only very little plant material is available or the material should be kept live, which often occurs in molecular biologic researches. The present work developed a simple, fast and low toxic micro-scale analysis procedure for determination of artemisinin in a single leaf or flower of living Artemisia annua using improved gas chromatography with electron-capture detection. The recovery of >95% was achieved by vortex of a piece of fresh leaf in 1 mL ethyl acetate for 2 min at room temperature. This method provides a powerful tool for biosynthesis study of artemisnin, high-throughput screening high-yield clone in an early stage, or real-time quality control of Artemisia annua crop.

Comparative profiling of gene expression in Camellia sinensis L. cultivar AnJiBaiCha leaves during periodic albinism.

The AnJiBaiCha albino mutant tea cultivar has a reversible albino phenotype at low temperatures. Albino AnJiBaiCha leaves contain high levels of amino acids, which are important components affecting the quality of tea as a beverage. To examine the molecular mechanism of albinism and amino acid enrichment in AnJiBaiCha, we used the amplified fragment length polymorphism (cDNA-AFLP) technique to isolate genes that are differentially expressed during periodic albinism in AnJiBaiCha. A total of 127 transcript-derived fragments (TDFs) were successfully sequenced; among those, 60 TDFs showed high similarity to sequences with known functions, but 67 TDFs were not similar to any known genes. The identified transcripts include transcription factors, ubiquitination-related genes, chloroplast biogenesis genes, signal transduction genes, stress-related genes, cell cycle genes, and carbohydrate and energy metabolism genes. To validate the cDNA-AFLP results, quantitative real-time PCR was used to confirm the differential expression of six of the identified genes. The cDNA-AFLP and quantitative real-time PCR results correlated well, indicating that the cDNA-AFLP results are reliable. This study provides insights into the molecular mechanisms by which periodic albinism and amino acid accumulation take place in AnJiBaiCha.

Water extract of the fungi from Fuzhuan brick tea improves the beneficial function on inhibiting fat deposition

Abstract Fuzhuan brick tea (FBT) is traditionally consumed by the ethnic group in the border region of northwest China. The unique yellow fungal (Eurotium cristatum) growth phase is considered to be the key process point in the manufacture of the brick tea. The fungi from FBT are not only strongly correlated to the quality of brick tea, but also have the potential function of preventing obesity. The water extract of fungi (100 μg/mL) can significantly inhibit fat deposition in 3T3-L1 adipocyte and Caenorhabditis elegans. Furthermore, the inhibition of 3T3-L1 adipocyte formation was not due to the suppression on cell viability.

A Comparative Proteomic Analysis of the Buds and the Young Expanding Leaves of the Tea Plant (Camellia sinensis L.)

Tea (Camellia sinensis L.) is a perennial woody plant that is widely cultivated to produce a popular non-alcoholic beverage; this beverage has received much attention due to its pleasant flavor and bioactive ingredients, particularly several important secondary metabolites. Due to the significant changes in the metabolite contents of the buds and the young expanding leaves of tea plants, high-performance liquid chromatography (HPLC) analysis and isobaric tags for relative and absolute quantitation (iTRAQ) analysis were performed. A total of 233 differentially expressed proteins were identified. Among these, 116 proteins were up-regulated and 117 proteins were down-regulated in the young expanding leaves compared with the buds. A large array of diverse functions was revealed, including roles in energy and carbohydrate metabolism, secondary metabolite metabolism, nucleic acid and protein metabolism, and photosynthesis- and defense-related processes. These results suggest that polyphenol biosynthesis- and photosynthesis-related proteins regulate the secondary metabolite content of tea plants. The energy and antioxidant metabolism-related proteins may promote tea leaf development. However, reverse transcription quantitative real-time PCR (RT-qPCR) showed that the protein expression levels were not well correlated with the gene expression levels. These findings improve our understanding of the molecular mechanism of the changes in the metabolite content of the buds and the young expanding leaves of tea plants.

Metabolic analysis of the increased adventitious rooting mutant of Artemisia annua reveals a role for the plant monoterpene borneol in adventitious root formation

Adventitious root (AR) formation is a critical process for plant clonal propagation. The role of plant secondary metabolites in AR formation is still poorly understood. Chemical and physical mutagenesis in combination with somatic variation were performed on Artemisia annua in order to obtain a mutant with changes in adventitious rooting and composition of plant secondary metabolites. Metabolic and morphological analyses of the iar (increased adventitious rooting) mutant coupled with in vitro assays were used to elucidate the relationship between plant secondary metabolites and AR formation. The only detected differences between the iar mutant and wild-type were rooting capacity and borneol/camphor content. Consistent with this, treatment with borneol in vitro promoted adventitious rooting in wild-type. The enhanced rooting did not continue upon removal of borneol. The iar mutant displayed no significant differences in AR formation upon treatment with camphor. Together, our results suggest that borneol promotes adventitious rooting whereas camphor has no effect on AR formation.